U.S. patent application number 13/021319 was filed with the patent office on 2011-09-29 for methods and compositions for analyte detection.
This patent application is currently assigned to Nexus, DX, Inc.. Invention is credited to David Dickson Booker, Richard Laswell Egan, Christopher Johann Johnson, Graham Peter Lidgard.
Application Number | 20110236879 13/021319 |
Document ID | / |
Family ID | 38437962 |
Filed Date | 2011-09-29 |
United States Patent
Application |
20110236879 |
Kind Code |
A1 |
Egan; Richard Laswell ; et
al. |
September 29, 2011 |
METHODS AND COMPOSITIONS FOR ANALYTE DETECTION
Abstract
The present invention is directed to methods and apparatus for
detection of one or more analytes. Analytes include agents or
components of infectious agents such as pathogenic virus, as well
as enzymes, proteins and biomarkers.
Inventors: |
Egan; Richard Laswell;
(Oceanside, CA) ; Lidgard; Graham Peter; (La
Jolla, CA) ; Booker; David Dickson; (Oceanside,
CA) ; Johnson; Christopher Johann; (San Diego,
CA) |
Assignee: |
Nexus, DX, Inc.
San Diego
CA
|
Family ID: |
38437962 |
Appl. No.: |
13/021319 |
Filed: |
February 4, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11677559 |
Feb 21, 2007 |
7932099 |
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13021319 |
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60775649 |
Feb 21, 2006 |
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60789345 |
Apr 5, 2006 |
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60866932 |
Nov 22, 2006 |
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Current U.S.
Class: |
435/5 ; 422/68.1;
435/283.1; 436/518 |
Current CPC
Class: |
G01N 33/558 20130101;
B01L 2300/0816 20130101; B01L 2400/0478 20130101; B01L 3/5029
20130101; B01L 2400/0683 20130101; G01N 2469/10 20130101; B01L
2300/0681 20130101; B01L 2300/0636 20130101; G01N 2333/11 20130101;
B01L 3/5023 20130101; B01L 2300/087 20130101; Y10S 435/81 20130101;
Y10S 436/81 20130101; G01N 33/56983 20130101; C12Q 1/701 20130101;
Y10S 436/808 20130101; B01L 2300/0832 20130101 |
Class at
Publication: |
435/5 ;
435/283.1; 436/518; 422/68.1 |
International
Class: |
C12Q 1/70 20060101
C12Q001/70; C12M 1/00 20060101 C12M001/00; G01N 33/543 20060101
G01N033/543; G01N 33/50 20060101 G01N033/50 |
Goverment Interests
STATEMENT AS TO GOVERNMENT SUPPORTED RESEARCH
[0002] Portions of this invention may have been made with the
support of the United States government under contract number
200-2007-19345 granted by the Center for Disease Control. The
Government may have certain rights to portions of this invention.
Claims
1. A sample collection device comprising a body comprising: (a) an
upper sealed chamber containing one or more solutions, and one or
more breakable seals; (b) a lower chamber in fluid communication
with the upper chamber containing an extraction buffer and/or
reagents; (c) a sample collection implement in fluid communication
with said upper scaled chamber; and (d) wherein said reagents
comprise a plurality of specific binding agent (SBA) sandwich
pairs, each pair comprising: (i) an SBA-capture moiety conjugate
comprising an SBA which specifically binds a target antigen and a
capture moiety; (ii) an SBA-label conjugate, comprising an SHA
which also specifically binds said target antigen, and wherein the
pair can form a sandwich with said target antigen; and wherein the
plurality of pairs arc directed against different antigens.
2. The sample collection device of claim 1 wherein the capture
moiety is an oligonucleotide, avidin, streptavidin, pyranosyl RNA
(pRNA), aptamer or a combination thereof.
3. The sample collection device of claim 2 wherein said
oligonucleotide is DNA or RNA.
4. The sample collection device of claim 1 wherein the label is a
fluorophore, chromophore, metal or a combination thereof.
5. The device of claim 1, wherein said capture moiety is pRNA and
said label is Europium.
6. The sample collection device of claim 1 wherein the target
antigen is one or more influenza viruses.
7-23. (canceled)
24. A sample collection device comprising a body comprising: (a) an
upper sealed chamber containing buffer, and a valve; (b) a lower
chamber in fluid communication with the upper chamber containing
(c) a swab contained within said body; (d) a mixing area downstream
of said swab comprising an adsorbent or absorbent substrate, and at
least one pair of reagents comprising a label and binding
moiety.
25. A test device comprising a body comprising: (a) a lateral flow
membrane in the body; and (b) upstream of the direction of lateral
flow, a chamber comprising fluid, wherein the chamber is capable of
controllably releasing the fluid into the lateral flow material,
wherein there is a gap between said chamber and said lateral flow
membrane.
26-43. (canceled)
44. A method comprising: (a) forming a mixture by mixing a sample
being tested for the presence of at least one target antigen with a
solution comprising a plurality of reagents comprising: (i) an
antibody-capture moiety conjugate comprising an antibody which
specifically binds a target antigen and a capture moiety, and (ii)
a labeled antibody, wherein the antibody specifically binds the
same target antigen, wherein said reagents are directed against a
plurality of different target antigens and form a complex; (b)
applying the mixture to a lateral flow membrane comprising a
plurality of detection regions, each of said detection region
having immobilized thereto a capture partner, each capture partner
capable of specifically binding a different capture moiety; (c)
flowing the mixture across the membrane, whereby the capture moiety
captures a complex having a capture moiety to which it is directed;
and (d) detecting label in at least one of said detection region,
whereby detection of label indicates the presence of a target
antigen in said sample.
45-85. (canceled)
86. A method for determining whether a subject is infected with one
or more types of influenza virus and/or subtypes and/or strains,
comprising: (a) contacting a sample from said subject with a system
capable of detecting multiple analytes, wherein said system
comprises components that are capable of specifically binding a
component of one or more of said influenza virus strains and/or
subtypes thus forming one or more complexes; wherein complexes
formed from said -sample travel through said system via a lateral
flow path, wherein said one or more complexes are capable of
specifically binding an immobilized capture reagent present in two
or more distinct test regions along said lateral flow path, and
wherein each of said two or more test regions comprises a different
said immobilized capture reagent capable of capturing a different
analyte complex; (b) determining whether said one or more influenza
virus strains and subtypes are present in said fluid sample by
observing said two or more test regions, wherein a positive result
indicates presence of a specific Influenza virus type and/or
subtype and said positive result is stable for a period greater
than 30 minutes without change.
87-154. (canceled)
Description
RELATED APPLICATIONS
[0001] This application is a divisional of U.S. application Ser.
No. 11/677,559, filed Feb. 21, 2007, and claims priority to U.S.
provisional application No. 60/775,649, filed Feb. 21, 2006, U.S.
Provisional Application No. 60/789,345, filed Apr. 5, 2006, and
U.S. Provisional Application No. 60/866,932 filed Nov. 22, 2006,
pursuant 35 U.S.C. 119(e), and the disclosure for each of which is
incorporated by reference herein in its entirety.
BACKGROUND OF THE INVENTION
[0003] This invention relates to assays for analyte(s), e.g.,
antigens, in a sample such as a biological sample obtained from an
animal. In particular, the invention relates to a method and
device(s) for the detection of an analyte(s) utilizing binding
moieties specifically targeting a selected analyte. More
particularly, the analytes to be detected include infectious agents
and/or components thereof.
[0004] Many types of assays have been used to detect the presence
of various substances, often generally called analytes or ligands,
in bodily samples. These assays typically involve antigen antibody
reactions, ligand, anti-ligand, ligand receptor and utilize,
synthetic conjugates comprising radioactive, enzymatic,
fluorescent, or visually observable metal soluble tags, and
specially designed reactor chambers. Most current tests are
designed to make a quantitative determination, but in many
circumstances all that is required is qualitative or
positive/negative indication. Assays have been utilized to detect
infectious agents, such as influenza.
[0005] Even the positive/negative assays must be very sensitive
because of the often small concentration of the analyte of interest
in the test fluid. False positives can also be troublesome,
particularly with agglutination and other rapid detection methods
such as dipstick and color change tests. Because of these problems,
sandwich assays and other sensitive detection methods which use
metal sols or other types of colored particles have been developed.
These techniques have not solved all of the problems encountered in
these rapid detection methods. Moreover, with the emergence of
highly pathogenic agents such as influenza virus, there is a need
to develop effective laboratory or point-of-care methods and
systems that can effectively and accurately detect one or more
infectious agents, such as influenza Types or strains within
subtypes.
[0006] Influenza is commonly seen in local outbreaks or epidemics
throughout the world. Epidemics may appear at any time and can
occur explosively with little or no warning. The number of people
affected can vary from a few hundred to hundreds of thousands.
Epidemics may be short-lived, lasting days or weeks but larger
epidemics may last for months. Although influenza is a mild disease
in most individuals, it is life threatening to elderly, the very
young or debilitated individuals. Epidemics are responsible for
large losses in productivity. Therefore, there is a need to develop
devices and methods to effectively detect what Types and subtypes
of a pathogen, such as influenza, present in samples obtained from
subjects in order to determine whether the infection is caused by a
typical or expected subtype of Influenza (seasonal flu) or a
subtype that is the causative agent of an epidemic or pandemic.
[0007] It is an object of this invention to provide a rapid,
sensitive method for detecting analytes in a biological sample.
Another object is to provide an assay which has high sensitivity
and fewer false positives than conventional assays. A further
object is to provide an apparatus or system for detection of low
levels of analytes present in biological samples. Another object is
to provide an assay system which involves a minimal number of
procedural steps, and yields reliable results even when used by
untrained persons. An additional object is to provide a system for
testing infectious agents that provides results identifying one or
more infectious agents in a matter of minutes. A further object
provides a system where results on a testing implement are equally
specific and sensitive for the target analytes, notwithstanding
that results can be read one to several hours after completion of a
reaction necessary to obtain a result. These and other objects and
features of the invention will be apparent from the following
description, drawing, and claims.
SUMMARY OF THE INVENTION
[0008] In certain aspects of the invention a sample collection
device (SCD) is provided for use in detection of one or more target
antigens or analytes that may be present in a sample. The sample
collection device can be utilized in conjunction with a test
device. In one aspect, a system is provided for detection of one or
more analyte comprising a SCD, a Test Device and a Reader, as
further described herein.
[0009] In various embodiments, a SCD comprises one or more upper
sealed chambers, which can contain the same or different solutions.
In one embodiment, the upper sealed chamber comprises at least two
compartments or subchambers each comprising. In other embodiments,
the upper chamber can comprise puncturable, breakable or rupturable
ampoules. In some embodiments, a SCD provides the necessary
reagents to form a complex with one or more different target
antigens that may be present in a sample, wherein the complex
comprises a capture moiety and a detectable label, a Test Device
provides the necessary means to addressably capture one or more
complexes so formed and a Reader which provides a means to detect
one or more signals from addressably captured complexes.
[0010] In various embodiments, an upper sealed chamber comprising
extraction buffer and/or reagents, a sample collection implement, a
sample collection implement holder and a plurality of reagents,
wherein the reagents comprise a plurality of specific binding
pairs, where each pair comprises a label conjugated to first
specific binding agent and a capture moiety conjugated to a second
specific binding agent, where the first and second specific binding
agents specifically bind a target antigen to form a complex. The
capture moiety can be "captured" or immobilized where a partner
capture moiety disposed on a substrate binds to the capture
moiety-specific binding agent conjugate.
[0011] In various embodiments, the plurality of specific binding
agents comprise a multitude of groups of specific binding pairs,
wherein each group comprises specific binding agents that
specifically bind one target antigen, and a second group of
specific binding pairs specifically bind a second different
antigen. Thus a plurality of specific binding pairs comprised in a
SCD is capable of detecting a plurality of different antigens.
[0012] In various embodiments, the capture moiety is an
oligonucleotide, avidin, streptavidin, pyranosyl RNA (pRNA),
aptamer, or a combination thereof. In various embodiments, the
label is a metal, a fluorophore, a chromophore or a combination
thereof. In some embodiments, the plurality of specific binding
pairs comprised in a SCD can contain one type of capture moiety but
with different capture moiety partners, e.g., each specific binding
agent conjugated to pRNA, where each group that is specific to a
different antigen comprises different pRNAs. In other embodiments,
the plurality of specific binding pairs comprises one or more
different capture moieties, e.g., pRNA for one group of specific
binding pairs, while streptavidin for another, or a combination of
different types of capture moieties.
[0013] In some embodiments, the plurality of specific binding pairs
comprised in a SCD can contain one type of label (e.g., specific
binding pairs where each group is conjugated to fluorophores having
the same or fluorophores having different wavelength signals). In
other embodiments, specific binding pairs can comprise a
combination of different types of labels (e.g., combination of
metals and fluorophores). In one embodiment, the capture moiety is
pRNA and the label is Europium.
[0014] In various embodiments, the specific binding agents are
antibodies, thus a specific binding pair comprises an
antibody-label conjugate ("label probe") or antibody-capture moiety
("capture probe"). In such embodiments, a "partner capture moiety"
is comprised on a test membrane disposed in a Test Device, which
partner binds a specific capture probe, e.g., pRNA partner
specifically binding a pRNA (i.e., capture moiety) contained on a
capture probe (i.e., antibody specific for a target antigen).
[0015] In some embodiments, the different analytes detected are
virus or components of virus (e.g., polypeptides). In various
embodiments, the different antigens are from influenza virus and
subtypes of influenza virus. In one embodiment, the influenza virus
that can be detected is influenza virus A and B as well as subtypes
of influenza virus. One embodiment is directed to detection of
influenza virus strains A and B and subtypes of the formula HxNy,
wherein x can be 1-16 and y can be 1-9, or any combination of xy
thereof.
[0016] In yet other embodiments, the different analytes detected
are one or more different infectious agents and/or one or more
different subtypes of an infectious agents. Such infectious agents
include HIV, HCV, and myobacterium, tuberculosis, bacteria, fungi,
yeast, HSV, HPV or a combination thereof.
[0017] In various embodiments, an SCD comprises a sampling
implement that provides a means to collect a sample from a subject,
wherein the sampling implement is comprised connected to the upper
chamber via a sampling implement holder. The sampling implement is
disposed at the distal end of a shaft, which shaft can be solid,
hollow or semi-permeable. In some embodiments, the sampling
implement is a swab, a comb, a brush, a spatula, a rod, a foam, a
flocculated substrate or a spun substrate.
[0018] In various embodiments, an SCD comprises one or more sealed
upper chambers wherein the seal functions as a valve to control
fluid communication between the upper chamber and lower chamber of
an SCD. In some embodiments, the valve can be a break-away valve, a
flapper valve, a twist, screw, rupturable, puncturable or breakable
valve. In other embodiments, the upper chamber can contain one or
more ampoules which prevent solutions contained therein to flow to
the lower chamber, unless pressure is exerted to rupture, puncture
or break the ampoule so as to release any contents therein.
[0019] One aspect of the invention is directed to a SCD comprising
a sample reservoir upstream of a plunger implement, a plurality of
sealable apertures for delivery of one or more solutions, a
substrate for filtering one or more compounds from a sample
administered to the sample reservoir and reagents that are capable
of specifically binding at least one analyte in said sample.
[0020] In another aspect of the invention, a Test Device is
provided for detection of one or more analytes, wherein the device
comprises a lateral flow membrane in a body, a chamber upstream of
the lateral flow membrane containing a fluid or solution, wherein a
gap is disposed between said chamber and said lateral flow membrane
thus precluding fluid communication between the chamber and the
lateral flow membrane. In one embodiment, the pressure exerted on
the chamber pushes close the gap thus forming fluid communication
between the chamber and the lateral flow membrane. In one
embodiment, an opening into which a distal end of an SCD fits, is
disposed directly above a wicking pad that is disposed downstream
of the gap, but upstream of the lateral flow membrane.
[0021] In one embodiment, the Test Device chamber comprises one or
more subchambers containing the same or different solutions. In
other embodiments, the chamber of subchambers comprise one or more
ampoules that are breakable, puncturable or rupturable. Thus, where
pressure is exerted on such ampoules the contents are controllably
released. As described herein, a Test Device can comprise a gap
means or not comprise a gap means for disrupting fluid
communication from the chamber to the lateral flow membrane. A Test
Device gap can be from zero to 3.0, 0.5, to 3.5, 1.0 to 2.5, 1.0 to
3.0, or 2.0 to 4.0 mm.
[0022] In some embodiments, a Test Device can comprise a body
housing the lateral flow membrane, wherein the body provides one or
a plurality of windows through which the lateral flow membrane is
visible. In various embodiments described herein, a Test Device
comprises a lateral flow membrane that comprises a wicking
substrate and an absorbent substrate upstream or downstream of the
test zones disposed on said lateral flow membrane. In some
embodiments, a substrate for collecting a small volume of sample
for archiving is provided in a SCD or Test Device. In one
embodiment, the substrate providing such archiving means is a
filter, membrane or paper that collects a small volume of sample
and said substrate is subsequently removed from the device.
[0023] In various embodiments, an SCD and/or Test Device comprises
one or more identical identifiable tags, which can be removed from
one device and placed on another device.
[0024] In some embodiments, the Test Device is shaped to only fit
(specialized adaptor shape) into the receiving port of a reader if
the upstream chamber has been depressed thus indicating that wash
buffer or chase buffer contained therein has been released through
the lateral flow membrane. In such embodiments, the Test Device and
Reader specialized adaptor provides a means to verify that chase
buffer or solution in the upstream chamber of the Test Device has
been released and thus washed any sample present upstream of the
lateral flow membrane through the lateral flow membrane. Thereby,
the specialized adaptor provides a "safety means" to prevent
reading of unprocessed samples.
[0025] In another aspect of the invention, the processed samples
are run through the Test Device's lateral flow membrane, but can be
placed aside from 30 minutes to several hours. In various
embodiments, a plurality of samples can be run through the Test
Device but read at 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12
hours later, with consistent and accurate signals.
[0026] In certain aspects of the invention, the devices disclosed
herein are utilized in methods for detection of one or more analyte
that may be present in a sample. In some embodiments, methods are
directed to detecting one or more strain of an infectious agent. In
one embodiment, a method is directed to utilizing the devices of
the invention to detect one or more influenza virus and subtypes
thereof. For example, methods are provided for detection of
influenza virus A and B, and subtype of influenza A that may be
present in a single sample.
[0027] In one embodiment, a method is provided for determining
whether a subject is infected with a pandemic, non-pandemic or
strain of influenza virus for which vaccine is available.
[0028] In some embodiments, the Test Device excludes any reagent or
binding agent that is capable of specifically binding a target
antigen.
[0029] In one aspect of the invention, a reader is providing to
detect a signal from a Test Device wherein said reader is a UV LED
reader. In various embodiments, the signal detected is a
fluorescence signal from a detector molecule. In further
embodiments, the detector molecule is a lanthanide. In yet a
further embodiment, the lanthanide is Europium.
[0030] In one embodiment, the reader comprise a UV photodiode. In
another embodiment, the reader comprises a UV laser diode.
[0031] In another aspect of the invention, a reader is configured
to comprise at least one hard standard. In another embodiment, a
reader is configured to comprise at least two or more hard
standards. In various embodiments, a hard standard comprises a
label molecule emitting a detectable signal. In further
embodiments, the label is a fluorescence label. In yet further
embodiments, the fluorescence label is a lanthanide. In yet a
further embodiment, the lanthanide is Europium.
[0032] In another aspect of the invention, an SCD and Test device
of the invention are used in a method to detect one or more
analytes, wherein such an analyte is associated with a disease,
pathologic or other physiological condition. In various
embodiments, such analytes are biomarkers associated with a
condition related to the heart, liver, kidney, intestine, brain,
fetus, or pancreas. In one embodiment, such analytes are associated
with a cardiac condition (e.g., myocardial infarction).
[0033] In various embodiments, the devices of the invention can be
utilized in any method to detect an antigen or protein (analytes)
in a sample obtained from a subject to detect any such analytes,
through utilization of a particular panel of immunoreactive or
specific binding reagents that are specific for the desired
analytes.
[0034] In several aspects of the invention, the Test Device
comprises an upstream chamber that contains a means for providing a
wash/running buffer or liquid. In various embodiment, such a buffer
or liquid comprises additional agents such as signal/detector
molecules that can be read by an optical reader or by unaided
visualization. In certain embodiments, the buffer or liquid is
comprised in a compartment that comprised of a glass ampoule or
membrane pouch, sac or formed filled pouch. In further embodiments,
such compartments are ruptured, broken or otherwise released of
their contents by exerting pressure on said compartments. In other
embodiments, such compartments are punctured or lanced by an
appendage or needle. In yet further embodiments, such compartments
are protected by a safeguard means that precludes accidental or
unintentional release of their contents.
INCORPORATION BY REFERENCE
[0035] All publications and patent applications mentioned in this
specification are herein incorporated by reference to the same
extent as if each individual publication or patent application was
specifically and individually indicated to be incorporated by
reference.
BRIEF DESCRIPTION OF THE DRAWINGS
[0036] The novel features of the invention are set forth with
particularity in the appended claims. A better understanding of the
features and advantages of the present invention will be obtained
by reference to the following detailed description that sets forth
illustrative embodiments, in which the principles of the invention
are utilized, and the accompanying drawings of which:
[0037] FIG. 1 illustrates a sample collection device.
[0038] FIG. 2A illustrates a sample collection device with a blow
up view of a mixing compartment.
[0039] FIG. 2B illustrates a sampling assembly.
[0040] FIG. 3 illustrates a test device.
[0041] FIG. 4 illustrates a reader.
[0042] FIG. 5 illustrates a sample collection device.
[0043] FIG. 6 illustrates a sample collection device.
[0044] FIG. 7 illustrates a test device comprising a gap means.
[0045] FIG. 8 illustrates a sample collection device and test
device.
[0046] FIGS. 9A and 9B illustrates test strips tested with
different volume of extraction buffer.
[0047] FIGS. 10A and 10B illustrate effects of background clean-up
and various amounts of extraction buffer.
[0048] FIG. 11 illustrates two different tests for detection of
influenza A.
[0049] FIG. 12 illustrates two different tests for detection of
influenza A and influenza B.
[0050] FIG. 13 illustrates the detection of influenza A using gold
as label.
[0051] FIG. 14 illustrates the detection of influenza B using gold
as label.
[0052] FIG. 15 illustrates the detection of influenza A using
Europium conjugate.
[0053] FIG. 16 illustrates the detection of influenza B using
Europium conjugate.
[0054] FIG. 17 illustrates sensitivity of Europium conjugate in
detection of influenza B.
[0055] FIG. 18 illustrates strips tested with influenza A
subtypes.
[0056] FIG. 19 illustrates buffer only control.
[0057] FIG. 20 illustrates test for influenza A and subtype
H3N2.
[0058] FIG. 21 illustrates test for influenza B.
[0059] FIG. 22 illustrates test for influenza A and subtype
H5N1.
[0060] FIG. 23 illustrates test for influenza A and subtype
H1N1.
[0061] FIG. 24 illustrates test for influenza B and influenza A
subtype H5N1.
[0062] FIG. 25 illustrates test for Epstein-Barr virus
[0063] FIG. 26 illustrates a test device comprising multiple test
lines for different type and subtypes of an analyte; 901 filter or
sample pad onto which a sample is applied; 902 reagent pad; 903
test line for type 1 of an analyte/infectious agent; 904 type 2 of
an analyte/infectious agent; 905 subtype of type 1 or type 2; 906
subtype of type 1 or type 2; 907 control line.
[0064] FIG. 27 illustrates various aspects of one Test Device; (A)
an assembled Test Device 1001; (B) an unassembled Test Device with
upper 1002, lower 1004 and test strip 1003, as well as the
aperture/port to receive a sample 1005; (c) closer view of the
spring/button compartment which can be pushed down to rupture,
break or puncture a buffer/liquid compartment disposed above the
membrane/pad 1006 that is in fluid communication with the test
strip 1003.
[0065] FIG. 28(A) illustrates an embodiment wherein a collar lock
is provided that connects an SCD 1101 and Test Device 1105
component, wherein the collar lock 1104 provides channels/pores
1103 in a geometrical configuration that is open to an upstream
compartment 1102 only when turned in the lock position (i.e.,
allowing fluid flow from the upstream buffer/liquid compartment
laterally through the Test Device. The upstream compartment 1102
can be an ampoule or formed filled sac.
[0066] FIG. 28(B) illustrates an embodiment of a Test Device
whereby the device comprises a safeguard cover 1107 over the
upstream compartment 1106 containing a liquid (e.g., wash/running
buffer). Furthermore, the device comprises a reservoir 1109 in
fluid communication with the sample aperture/port 1108 which
reservoir can comprise a membrane or compartment for retaining an
archive sample.
[0067] FIG. 28: (C) illustrates a compartment (e.g., ampoule) 1111
disposed in a Test Device with a break point 1110, wherein the Test
Device is configured to provide a trough/channel 1112 and
supporting substrate 1113 which directs the liquid contents of the
ampoule downstream to the test strip; (D) provides an overhead view
of the compartment comprising a sac/ampoule 1114 positioned
upstream of where sample introduction occurs 1115, configured to
provide a fluid ramp 1119 and supports 1118 through which the
contents of the compartment to a space 1117 that is in fluid
communication with a wicking pad 1116.
[0068] FIG. 29 provides one illustration of an sampling device with
an archival component; the sampling swab 1205 is attached to a
hollow shaft 1201 and is flanked by sleeves 1202 upstream of a
hydrophobic frit (10+u) 1206 and 1203 followed by a (FIG. 29B)
filter paper that can be in various shapes with regions for
retaining an archive sample 1209 and three dimensional regions 1210
that can for example retain a reagent pill/bead. In addition,
reagent pills/beads can also be disposed in the compartment
downstream of the filter paper 1208 which can allow mixing before
expelling the sample through the tip 1204.
[0069] FIG. 30 illustrates (A) striped test strip results utilizing
pRNA and (B) monoclonal antibodies.
[0070] FIG. 31 illustrates a comparison of sensitivity using pRNA
and antibody.
[0071] FIG. 32 provides a schematic of pRNA binding of multiple
analytes on a test strip.
[0072] FIG. 33. Hard Standards; (A) graph of six different
concentrations of Europium as read of an UV LED reader; (B) photo
of the six hard standards; (C) graph for concentration #1; (D)
graph for concentration of #5; (E) graph for concentration #3; (F)
graph for concentration of #6; (G) graph for concentration of #2;
(H) graph for concentration of #4.
[0073] FIG. 34. Hard Standards; (A) provides a graph for 7
different hard standards as read by an UV LED reader; (B) provides
a Test Device with multiple windows through which the signal is
detected when the Test Device is placed in a reader.
[0074] FIG. 35 illustrates an embodiment where an upstream
compartment 1401 (e.g., ampoule) containing a liquid is in fluid
communication with a dry wicking pad 1402 which swells when said
compartment is manipulated to release its liquid content, wherein
said swelling increases the size of the wicking pad 1403 so that it
becomes in fluid communication with a sample 1405 disposed on a
test strip 1406. Therefore, based on the density/type of wicking
material 1402 a predetermined time delay can be built into the
system from the point of release to point of initiating flow of the
sample 1405 through the test strip 1406.
[0075] FIG. 36 illustrates fluorescence intensity of subtype H5N1
viruses.
[0076] Figure depicts a graph of fluorescence intensity of subtype
H1N1 viruses.
[0077] FIG. 38 depicts a graph of fluorescence intensity of Flu B
viruses.
[0078] FIG. 39 illustrates the sensitivity using a MAB system.
[0079] FIG. 40 illustrates the sensitivity using a pRNA system.
[0080] FIG. 41 illustrates the sensitivity using a pRNA system and
membrane blocking.
[0081] FIG. 42 provides a comparison of sensitivity for MAB, pRNA
and pRNA with blocking.
DETAILED DESCRIPTION OF THE INVENTION
[0082] While preferred embodiments of the present invention have
been shown and described herein, it will be obvious to those
skilled in the art that such embodiments are provided by way of
example only. Numerous variations, changes, and substitutions will
now occur to those skilled in the art without departing from the
invention. It should be understood that various alternatives to the
embodiments of the invention described herein may be employed in
practicing the invention. It is intended that the following claims
define the scope of the invention and that methods and structures
within the scope of these claims and their equivalents be covered
thereby.
[0083] Various aspects of the present invention are directed to
devices and binding-pair assays that utilize specific binding
moieties and capture moieties for the qualitative and/or
quantitative analysis of selected analytes in samples. The
invention is useful in a variety of assays that are utilized to
detect one or more infectious agents present in a sample. Assays
useful in the invention include, but are not limited to,
competitive immunoassays, non-competitive immunoassays, sandwich
immunoassays and blocking assays.
[0084] However, the use of the invention is not limited to
immunoassays, as other assays including ligand-receptor,
ligand-binding protein, aptamer-binding agent, enzyme-substrate,
chemical or biochemical reactions are detected by utilizing a
device or method of the invention. In one aspect of the invention,
methods and systems of the invention utilize a sample collection
device, a test device and a reader. Various specific assay
protocols, reagents, devices or analytes useful in the practice of
the invention are known, see, e.g., U.S. Pat. Nos. 4,313,734;
4,366,241; 5,266,266; 6,235,539; 6,468,474; 6,565,808; 6,448,001;
or 5,415,994, as well as U.S. Pub. Nos. 2006/0040405; 2004/0014094;
2004/0048395; or 2005/0130120.
[0085] In one aspect of the invention, a sample collection device
is utilized to collect a sample and process a sample with
immunoreactive reagents that provide a detection means and a
capture means, which processed sample is subsequently provided to a
test device, which provides a means for reading/viewing signals
provided by said detection means. The signals can be read by the
naked eye or with an instrument depending on the combination of
detection means (e.g., conjugate labels) utilized. Furthermore, the
test device can be configured to allow detection of multiple
analytes. Such analytes can be from one or more infectious agents,
including different strains and/or subtypes. Detection can include
qualitative and/or quantitative measurements of one or more
analytes.
[0086] Sample Collection Device. One aspect of the invention is
directed to a sample collection device ("SCD") that comprises the
necessary means to collect a biological sample, as well as the
reagents and buffers necessary to process the sample and react to
the binding reagents with one or more target analytes. As shown in
FIG. 2B, an exemplary SCD comprises an upper chamber 210 to which
is attached a sampling implement holder 209 a stem 208 a sampling
implement 207, collectively forming a sampling assembly-.
[0087] The sampling assembly FIG. 1 is removable from a housing
comprising a sample receiving tube 103, a lower chamber mixing or
reagent area 104, which can contain reagents that specifically bind
to one or more target antigens. The lower chamber 104 can comprise
one or more compartments. For example, two compartments can be
arranged in series in the lower chamber. The sampling assembly is
placed into the sample receiving tube 103 portion of the housing to
provide an integrated configuration. In such a configuration a
sampling implement 107 is upstream of and in fluid communication
with the lower chamber. The length the sampling assembly can be
optimized for sample collection, e.g., throat and nasal sample
collection. For example, the length of the device (e.g., integrated
configuration) can be about 1 to 9 inches, or about 3, 4, 5, 6, 7,
8 or 9 inches.
[0088] The stem 102 can be hollow, solid or semi-porous. Therefore,
in some embodiments, the stem actually provides a path of fluid
communication from the upper chamber 100 to the sampling implement
107 (e.g., swab). The stem is held by the sample holder 101 which
fits into a receiving end of the upper chamber 100. For example,
the stem may be hollow or semi-permeable and a portion of the stem
which may extend into the upper chamber has a terminal end that is
closed, so that if the stem portion in the upper chamber is snapped
or broken, then fluid communication (i.e., fluid flow) is provided
between the upper chamber down through the distal end of the stem
to a sampling implement (e.g., swab) comprising a sample (e.g.,
biological sample).
[0089] An upper chamber can comprise one or more compartments-. The
upper chamber can be comprised of a semi-rigid or depressible
material, and shaped as a bulb 99. Such a bulb can comprise a
solution.
[0090] The upper chamber can be sealed. Furthermore such a seal can
be punctured, broken or opened via a valve structure, so as to
provide fluid communication between the upper chamber and lower
chamber. In some embodiments, the solution in the upper sealed
chamber is a buffer solution. In various embodiments, the volume
for a solution in the upper chamber is from about 10-500 .mu.l, or
from about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130,
140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260,
270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390,
400, 410, 420, 430, 440, 450, 460, 470, 480, 490 or 500. In one
embodiment, the solution volume is up to 150 .mu.l.
[0091] In some embodiments, a liquid solution comprising the
necessary reagents (e.g., detection/capture specific binding
agents, etc.) can be disposed in the reagent area of the lower
chamber in liquid communication with the upper chamber. As
exemplified in FIG. 2A, fluid from the upper chamber flows down to
the sampling implement to extract sample and--the extracted sample
passes through an aperture that may restrict/control the liquid
flow from the upper chamber to the lower chamber comprising, for
example, an aperture to control flow by size, e.g., size of
perforations or type of substrate or filter that is disposed on the
proximal end 201 of a compartment in the lower chamber-. The lower
chamber may contain a reagent area. In one embodiment, the reagent
area (e.g., 203) contains a solid substrate that includes the
necessary reagents 202 (e.g., immunoassay reagents, such as
detection and capture probes, etc.), formed as a dried solid,
separately disposed or in a unified solid.
[0092] Therefore, where a sample is washed downward via the
solutions (e.g., buffer) in the upper chamber, a mixture is
produced carrying the sample that travels down to the lower chamber
reagent area, which chamber comprises the solid reagent-containing
substrate 202. The solid reagent is dissolved rapidly by the buffer
and the resultant solution is a mixture of sample that may contain
analyte(s) of interest, and the immunoassay reagents (e.g.,
specific binding agents, label conjugate and capture probes, etc.).
For example, a solid reagent 202 can include both label and capture
probes used in the assay that are capable of specifically binding a
target analyte. The integrated device 200 includes the upper and
lower chambers, the sample collection device and the luer lock 206
which locks into the lateral flow device for delivery of the
reaction mixture for subsequent detection. Furthermore, as noted
the upper chamber can be designed with a depressible (e.g.,
plastic) bulb 205 so that if desired additional pressure is applied
to force fluid flow from an upper chamber into a lower chamber.
[0093] In one embodiment, the upper chamber comprises a valve that
allows controllable release of a solution comprised in the upper
chamber. For example, where the valve is a snap-valve, the user
applies force to the valve stem to break the stem, whereby the
breakaway feature allows buffer to enter the lower chamber via the
stem. Furthermore, the sealed chamber can be a squeezable bulb,
which is capable of being compressed (e.g., user applies pressure
to the bulb), thus controlling the flow rate of the solution (e.g.,
buffer) to the sampling implement.
[0094] In some embodiments, the upper chamber is comprised of a
bulb component that is a self-contained compartment that includes a
solution. Such solutions include extraction, lysis, reagent, buffer
or preservative solutions. In one embodiment, the solution is a
buffer solution that is utilized to transfer the biological sample
from the sampling implement down to the lower chamber.
[0095] Furthermore, such an upper chamber can comprise one or more
compartments, such as depicted in FIG. 5. Each compartment can
comprise a solution that is the same or different 501 and 502.
[0096] Furthermore, such solutions can comprise reagents as desired
including but not limited to extraction buffers, reducing agents,
immunoreactive agents, such as, anti-analyte specific binding
agents comprising detection labels (e.g., conjugates) and capture
moieties. The reagents for reactions are depicted in 503 and 504
shows attachment of the luer to the test strip 505.
[0097] In another embodiment, the sampling assembly is not
integrated with the housing containing a sample receiving tube. See
FIG. 6. In such a configuration, the sampling assembly 601 is
utilized to collect and deliver a sample to a sample receiving
chamber 609. The sample receiving chamber can be open or closed to
allow a sample to be introduced into sample receiving tube. It
should be understood that any sample receiving tube disclosed
herein can be of a variety of geometric shapes, including cylinder,
square, triangular or any polygonal, as desired. In some
embodiments, the housing can comprise one or more sealable
apertures 603 that can be opened to add one or more selected
reagents 607, buffers or wash fluids.
[0098] For example, in one embodiment, whole blood is drawn into
the sample receiving chamber 609. Subsequently, the sample passes
through a membrane 602 (e.g., a membrane to separate red blood
cells from plasma, allowing the plasma to pass through) into a
lower portion of the sample receiving tube to mix with various
reagents, for example, necessary for an immunoassay. Immunoreagents
-necessary to target specific analytes can be pre-selected and
disposed as a solid substrate 607 in the SCD or added through an
aperture 603, or can be disposed on a membrane 604.
[0099] As the whole blood sample is discharged, the membrane 604
may act as a filter to precludes passage of blood components, thus
allowing only plasma to pass through the distal end of the sample
receiving tube 605, which will fit into the Test Device 606.
Additional valves that can be utilized include a rotary, breakable,
stopcock, gate, ball, flapper, needle, butterfly, pinch, bellows,
piston, slide, plug, diverter, or actuator valve.
[0100] As used herein, a "capture probe" refers to a conjugate of a
binding agent linked to a capture moiety and a "detection probe"
refers to a conjugate of a binding agent linked to a label or
signal producing moiety, wherein each is capable of specifically
binding to a target analyte. Furthermore, for clarity, a "capture
moiety partner" in the context of the Test Device (described below)
refers to a complement, cognate or partner molecule that
specifically binds to a capture moiety comprised on a capture
probe.
[0101] Solid reagent components include, a powder, pill, bead,
lyophilized pellet, pressed lyophilized power, dried on solid
support (e.g., glass/plastic bead), lyophilized on or in
association with a solid support or dried directly in the mixing or
lower chamber. Such reagents are known in the art such as disclosed
in CURRENT PROTOCOLS IN IMMUNOLOGY (Coligan, John E. et. al., eds.
1999).
[0102] In some embodiments, as the solution passes through the
sampling implement, an extraction step of a sample occurs (e.g.,
where solution includes an extraction buffer). Furthermore, the
lower chamber can comprise a filter through which an extracted
sample flows. For example, if a filter is disposed at the proximal
end of -the lower chamber, an extracted sample then flows through a
filter (e.g., a mesh disk) thereby precluding certain components of
the extraction mixture from passing into the reagent area
compartment comprising a solid reagent bead. Furthermore, a filter
means can also function to restrain the reagent bead during SCD
transportation and storage. As noted herein, the reagent bead can
comprise both the detection and capture probe, or two separate
beads can each contain detection or capture probes.
[0103] The filtering aspect allows an analyte of interest to
migrate through the device in a controlled fashion with few, if
any, interfering substances. The filtering aspect, if present,
often provides for a test having a higher probability of success,
depending on the type of sample being processed, as would be
evident to one of skill in the art (e.g., whole blood sample versus
throat swab). In another embodiment, the SCD may also incorporate
reagents useful to avoid cross-reactivity with non-target analytes
that may exist in a sample and/or to condition the sample;
depending on the particular embodiment, these reagents may include,
but not limited to, non-hCG blockers, anti-RBC reagents, Tris-based
buffers, EDTA, among others. When the use of whole blood is
contemplated, anti-RBC reagents are frequently utilized. In yet
another embodiment, the SCD may incorporate other reagents such as
ancillary specific binding members, fluid sample pretreatment
reagents, and signal producing reagents (e.g., substrates necessary
for reacting with label conjugates).
[0104] In another embodiment, the lower chamber can comprise a
small element of absorbent paper, on which a predetermined
percentage of the extracted sample is retained for archival
purposes. After passing through the collection device and having a
portion restrained for archival purposes, the extracted sample
contacts a reagent solution or solid (e.g., conjugate bead 202),
and the next assay step takes place as the specimen liquid rapidly
dissolves the conjugate bead and allows the reactants to mix and
start the assay.
[0105] In other embodiments, the luer 105 disposed on the distal
end of the SCD can be replaced with or further attached to a valve
structure that also functions to control the flow of liquid out
from the sample collection device. A valve can also be utilized in
practice of the invention, where the valve is disposed in the upper
sealed chamber, thus providing a means to control release of
solutions contained therein. Whether in the upper chamber, lower
chamber, or between any compartments disposed in the SCD, a valve
can be of any type as recognized in the art such as, but not
limited to, a rotary, snap, breakable, stopcock, gate, ball,
needle, butterfly, pinch, bellows, piston, slide, plug, diverter,
or actuator valve. In one embodiment, the valve is a snap valve
which is rendered open where pressure is exerted in the stem
portion of the valve that extends into the upper sealed chamber
(100), thereby breaking open the hollow stem 102 and allowing the
contents of the sealed upper chamber to flow into the hollow tube,
at the end of which is disposed a sampling implement (e.g., swab
107).
[0106] In one embodiment, there is a valve at the distal end of the
SCD. When the valve is in the closed position, a sample or sample
and reagent can be retained in the lower chamber 104. When the
valve is in the open position the contents of a mixing
subchamber--can be released, for example by gravity flow.
Alternatively, the bulb component of the sample collection device
is utilized to release the sample/reagent from the luer, luer-valve
or valve. In a preferred embodiment of the present invention, the
content from the distal or outlet end of the sample collection
device ("SCD") is released from the SCD such that the flow can be
actuated, regulated or modulated.
[0107] In another embodiment, the distal end of the SCD is open,
whereby prior to release of a solution from the upper sealed
chamber, the SCD is engaged (e.g., by friction fit) into the
receiving port of a Test Device. In such an embodiment, the fluid
flow from the distal end of the SCD into the Test Device is not
regulated by a luer or a valve structure.
[0108] In another embodiment, the distal end of the SCD does not
utilize a valve but rather is open. The SCD may be attached to the
test device prior to release of the buffer from the upper chamber.
Upon release of the solution from the upper chamber, the sample is
released and/or extracted from the collection implement by the
solution and mixed with the reagents located in the lower chamber.
The mixture then flows to the test device for analysis of the
presence of one or more analytes. It is possible to include
water-dissolvable membranes within the lower chamber to slow the
flow of the mixture out of the SCD onto the test device. Such
membranes are conventional and can be designed to permit the
retention of the mixture for differing periods of time sufficient
to allow mixing and reaction of the reagents and sample analytes.
For example, such membranes can be prepared from proteins,
polysaccharides or film formers.
[0109] In another embodiment, the distal end of an SCD comprises a
very narrow opening that prevents fluid flow unless and until
pressure is applied to the device (e.g., via the bulb structure of
the SCD, or if the housing is depressible, then by exerting
pressure on the housing itself) to force the fluid out from the
distal end. In other words, there is no valve of any sort disposed
at the distal end of the SCD.
[0110] In another aspect of the present invention the fluid
regulatory means (e.g., luer 105, luer-valve combination and valve)
is closed such that the sample or sample and one or more reagents
can be retained in the lower chamber 104 for any length of time.
The valve structure can then be mechanically, fully or partially,
opened to release the contents through the distal or outlet end of
the SCD into a test device, optionally at a regulated or modulated
rate.
[0111] Reagents utilized in an SCD of the invention can include one
or more salts, chelators, anticoagulants, detergents, stabilizers,
diluents, buffering agents, enzymes, cofactors, specific binding
members, labels, mucolytic and the like. The one or more reagents
can be compounds that facilitate analysis of a sample. Furthermore,
such reagents can readily be adapted for use in a Test Device of
the invention.
[0112] In a one embodiment the SCD is engaged to a second device,
for example the test device of the present invention, such that
opening of the valve structure or removing a cap 106 covering the
opening (no valve) releases the contents into the second device. In
another embodiment, engaging the SCD comprising a male luer to the
test device comprising a female luer results in release of the
contents of the reaction subchamber. In a further embodiment, the
contents from the distal end can be released via the bulb
component. Where a valve structure is utilized in the SCD, the
valve structure can be opened to release the contents by various
means such as, but not limited to, opening a stopcock or by
turning, rotating, twisting or sliding the valve structure such
that the valve can be opened to allow fluid communication into the
test platform or by removing a cap which opens the fluid flow
path.
[0113] Archive Sample. In one aspect of the invention a means for
archiving a portion of a sample is provided. In some embodiments,
an SCD or Test Device, or both, comprise an archival means, which
can comprise an absorbent or adsorbent substrate (e.g., paper or
membrane),--a short capillary tube of defined length, or a small
reservoir/compartment for retaining a portion of the sample in the
lower chamber. In some embodiments, this archival mechanism is
located at a position in the device before the sample encounters
the reaction reagents.
[0114] In one embodiment shown in FIG. 28, a small compartment,
that can provide a small reservoir for an archive sample, 1109 is
positioned in the test device adjacent to the port/aperture 1108
for delivery of sample to the test device. Such an archive
compartment can be configured to be removable or so that a
substrate onto which the archive sample is disposed, is itself
removable from said compartment. For example, a filter/membrane
material sized to fit into the compartment will function to collect
to a predetermined capacity of sample (e.g., cell, cell components,
protein, nucleic acid, etc.). The test device may contain a pouch
1106 of wash buffer with a housing or cover 1107
[0115] In another embodiment, an SCD comprises a means for
retaining an archive sample FIG. 29A. For example, within a SCD
lower compartment, filter paper and hydrophobic membranes can be
provided configured to retain a sample for archiving purposes.
Various combinations of materials are possible. In one embodiment
the means for archiving comprises three disks 1203, 1206, 1207,
that may or may not touch each other. In another embodiment, the
SCD FIG. 29A can be configured with sleeves 1202 which provide a
means to move the sizes of the tube/casing closer to the swab 1205
attached to stem 1201 so that as a fluid exits the swab it will
stay in close proximity to the swab, so as to improve the
efficiency of extracting fluid from the swab. A reagent bead is
depicted as 1208 and the exit port is 1204.
[0116] As shown in FIG. 29B the disks can comprise a grid portion
1210 and a pad portion 1209, wherein the pad portion is designed to
retain an archive sample. The pad portion can be comprised of any
absorptive/adsorptive material and can comprise 5, 10, 15, 20, 25,
30, 35, 40, 45 or 50% of the surface area of a disk. Furthermore,
the grid portion can comprise three dimensional ("3D") substrates
raised relative to the surface of a disk. Such 3D protrusions can
provide a grid into which a reagent bead can be disposed. Such
beads can measure in size from about 0.5, 1, 1.5, 2.0, 2.5, 3.0,
3.5, 4, 4.5, 5.0, 5.5, to about 6.0 mm.
[0117] In one embodiment, the archived material is a cell(s) or
cellular component, including but not limited to a protein,
peptide, protein fragment or nucleic acid molecule. Therefore,
samples can be preserved for further testing depending on the type
of molecule archived (e.g., protein versus nucleic acid).
Furthermore, archive disks 1207 (for example as shown in FIG. 29A)
provide a means of storing samples and maintain stability of said
samples from about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,
21 to 30 days. For example, RNA is stable for about 2, 3, 4, or 5
days.
[0118] In another embodiment, the archival disks are placed in a
preservative solution, which extends storage time for said archive
samples from about 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 weeks. Of course
depending on the in-field setting, samples can be stored
indefinitely (e.g., once the sample is subjected to freezing).
[0119] In another embodiment, a reaction compartment--in the lower
chamber can be removed from the sample receiving tube and placed in
a housing (e.g., plastic tube). In one embodiment, the compartment
(e.g., a cage) retains a small volume of sample mixture to which a
preservative can be added for storage. In another embodiment, the
solutions provided in the upper chamber or a reaction solution in
the lower chamber can also include preservatives necessary to
archive a liquid sample. Such preservatives are known in the art.
See, e.g., U.S. Pat. RE29061; Buccholz et al. Transfusion. 1999
September; 39(9):998-1004; Quiagen specialty reagents, available at
Quiagen.com.
[0120] In one embodiment, an archive sample is retained for later
testing (e.g., by RT-PCR, See Example 8).
[0121] Sample Identification. The SCD also includes anywhere on the
sampling implement or the sample receiving tube, for example,
attached to the outer tube 103, 3 identical identifying labels
(e.g., barcodes allowing at least 10.sup.9 unique values) for
patient identification number. The labels can be peel-off and is
self-adhesive. One label is retained on the SCD 103. The peel-off
copies can be placed on the Test Device FIG. 3 and on any facility
paperwork, or an archival reservoir means. Bar code format will be
to a universal standard such as Codabar 303 or 305. In other
embodiments, the identifying labels can be signal emitting
transponders known in the art, including but not limited to, radio
frequency emitter, light emitter or electromagnetic wave
emitter.
[0122] Compartments in the Upper or Lower Chambers. In some aspects
of the invention, the SCD comprises one or more compartments in the
lower chamber that can include reagents, filters, membranes and
reservoirs. In one embodiment the upper chamber of the SCD may
comprise one, two or more compartments-, each of which can further
contain a solution. In some embodiments, such compartments can
comprise the same or two different solutions, reagents, buffers, or
a combination thereof. Further, multiple compartments can be
arranged in series in a lower chamber (e.g., multiple cages -in
series). In addition, such compartments may be referred to as
"subcompartment" or "subcompartments" in the disclosures
herein.
[0123] In one embodiment, compartment is distal relative to a
sampling implement and comprises a liquid or solid reagent
component that comprises binding agents that are specific to a
particular analyte (or analyte type). For example, the liquid or
solid reagent component includes a specific binding agent (e.g.,
antibody) that is capable of specifically binding an analyte that
may be present in a sample. In some embodiments, a single reaction
or mixing compartment--is utilized in the SCD that is distal to and
in fluid communication with the sampling implement. In other
embodiments, one or more compartments can be utilized where one
compartment functions as a lysis or extraction chamber, while a
second compartment distal to the first compartment functions as a
reagent-sample mixing chamber. In further embodiments, -filtering
means may be disposed on the proximal end of one or more
compartments, which compartment(s) is disposed--distal relative to
the sampling implements. Filter means can be utilized to remove
certain components from the sample, prior to extraction/lysis,
sample-reagent mixing, during processing or before release from the
SCD. Furthermore, the same or different filtering means can be
disposed on multiple compartments if such multiple compartments are
present in the sample receiving tube.
[0124] Samples.
[0125] A sample is any material to be tested for the presence
and/or concentration of an analyte. In general, a biological sample
can be any sample taken from a subject, e.g., non-human animal or
human and utilized in the test devices. For example, a biological
sample can be a sample of any body fluid, cells, or tissue samples
from a biopsy. Body fluid samples can include without any
limitation blood, urine, sputum, semen, feces, saliva, bile,
cerebral fluid, nasal swab, urogenital swab, nasal aspirate, spinal
fluid, etc. Biological samples can also include any sample derived
from a sample taken directly from a subject, e.g., human. For
example, a biological sample can be the plasma fraction of a blood
sample, serum, protein or nucleic acid extraction of the collected
cells or tissues or from a specimen that has been treated in a way
to improve the detectability of the specimen, for example, a lysis
buffer containing a mucolytic agent that breaks down the mucens in
a nasal specimen significantly reducing the viscosity of the
specimen and a detergent to lyse the virus thereby releasing
antigens and making them available for detection by the assay. A
sample can be from any subject animal, including but not limited
to, human, bird, porcine, equine, bovine, murine, cat, dog or
sheep.
[0126] For example, a sample can be derived from any source, such
as a physiological fluid, including blood, serum, plasma, saliva or
oral fluid, sputum, ocular lens fluid, nasal fluid, nasopharyngeal
or nasal pharyngeal swab or aspirate, sweat, urine, milk, ascites
fluid, mucous, synovial fluid, peritoneal fluid, transdermal
exudates, pharyngeal exudates, bronchoalveolar lavage, tracheal
aspirations, cerebrospinal fluid, semen, cervical mucus, vaginal or
urethral secretions, amniotic fluid, and the like. Herein, fluid
homogenates of cellular tissues such as, for example, hair, skin
and nail scrapings and meat extracts are also considered biological
fluids. Pretreatment may involve preparing plasma from blood,
diluting or treating viscous fluids, and the like. Methods of
treatment can involve filtration, distillation, separation,
concentration, inactivation of interfering components, and the
addition of reagents. Besides physiological fluids, other samples
can be used such as water, food products, soil extracts, and the
like for the performance of industrial, environmental, or food
production assays as well as diagnostic assays. In addition, a
solid material suspected of containing the analyte can be used as
the test sample once it is modified to form a liquid medium or to
release the analyte. The selection and pretreatment of biological,
industrial, and environmental samples prior to testing is well
known in the art and need not be described further.
[0127] Other fields of interest include the diagnosis of veterinary
diseases, analysis of meat, poultry, fish for bacterial
contamination, inspection of food plants, restaurants, hospitals
and other public facilities, analysis of environmental samples
including water for beach, ocean, lakes or swimming pool
contamination. Analytes detected by these tests include viral and
bacterial antigens as well as chemicals including, for example,
lead, pesticides, hormones, drugs and their metabolites,
hydrocarbons and all kinds of organic or inorganic compounds.
[0128] Test Device.
[0129] The present disclosure provides a test device, particularly
immunoassay devices, for determining the presence or absence of
multiple analytes in a fluid sample. In general, a Test Device FIG.
3 of the present disclosure includes a matrix defining an axial
flow path. Typically, the matrix further includes a sample
receiving zone, one or more test zones and one or more control
zones. In frequent embodiments, a test region comprises the test
and control zones, which are collectively addressable lines.
[0130] As used herein in the context of the Test Device the terms
"axial flow membrane", "lateral flow membrane", "test membrane",
"test strip" or "matrix" are used interchangeably which employs
capillary action to move or transport the test fluids or employs
the movement of fluid separate from capillary action as where fluid
is pumped by the accumulation of gas pressure, hydraulic pressure
(direct pumping using a piston or rotary, bellows or other type
pump on the assay fluids, electrostatic movement due to an electric
field, gravity, etc.).
[0131] In one aspect of the invention, the Test Device as depicted
in FIG. 3 is comprised of an aperture/port into which the distal
end of a SCD of the invention is engaged either by friction fit,
luer lock, adaptor or valve. An aperture/port 302,--provides an
opening through which a sample from the SCD flows into the Test
Device. A blood separation membrane can be disposed at the port
which provides one way flow. In another embodiment, such a membrane
can also be disposed in the SCD (e.g., immediately distal to the
sample swab implement.
[0132] Upstream of the aperture is a compartment 307 that may be in
fluid communication with the aperture, which aperture is in fluid
communication with a wicking substrate 309. Furthermore, the
compartment can comprise one or more subcompartments 308 that
contain one or more solution(s). Subcompartments in the context of
the Test Device can be made of a pierceable, puncturable, breakable
(e.g., ampule) or depressible bladder like material. In one
embodiment, the collar breaks away, however, the buffer is only
released once additional force is applied to the buffer/wash
compartment 308. As indicated herein, such compartments can be
manipulated by applying pressure so as to puncture, break or
depress the compartment enough so to release it contents. In
addition, such compartments may be pierced by a lance, stab or
appendage that breaks into said compartment upon exertion of force
(e.g., thumb pressing down) onto said compartment.
[0133] In one embodiment, the Test Device comprises two sections,
wherein one section comprises a portion where a sample is applied
302- and a compartment upstream of portion where the sample is
applied comprising a wash or running buffer 308. In another
embodiment, the upstream section can comprise one or more (e.g.,
two) compartments which may contain the same or different buffers,
wherein each compartment can be separately or simultaneously
manipulated to expel its contents.
[0134] In another embodiment, the compartment 307 itself can be
semi-rigid, pliable or depressible, or bladder like, which provides
a means for compacting the compartment to expel any contents
therein. Therefore, in some embodiments, a user can exert pressure
on the compartment 307 that will result in contents therein,
whether self-contained or contained in a subcompartment, to be
released to the wicking substrate 309. Such a solution can function
as a wash or chase buffer, mobilizing or enhancing mobilization of
the processed sample mixture through the wicking pad and into the
test strip 310. Generally, such liquid solutions can comprise wash
buffer, saline or any desired solution. Furthermore, in some
embodiments, such a solution can comprise reagents, enzymes, labels
or chemical compounds. The wash buffer mobilizes any unbound label
causing it to migrate along the strip past the detection zone thus
reducing background.
[0135] Furthermore, downstream of the test strip 310 is disposed an
absorbent substrate 311. The test membrane substrate can overlap or
abut to one or both the wicking substrate and absorptive substrate,
respectively. Furthermore, in some embodiments, the Test Device
upper 306 or lower housing 312 can comprise identity labels 303 and
305, which identify and correspond to an identical identity label
on the SCD and can also identify the lot number of the Test Device
(e.g., for quality assurance and tracking purposes). Window 304
through the upper housing permits visualization and reading of the
results.
[0136] In a related embodiment, the matrix further includes an
absorbent zone disposed downstream of the lateral flow substrate,
membrane or matrix. In one embodiment, a wicking pad can be
disposed upstream of the lateral flow membrane. In another
embodiment, a wicking pad is disposed directly below the sample
entry aperture. Moreover, in preferred embodiments, the test
region, which comprises the test and control zones, is observable
FIG. 8.
[0137] Suitable materials for manufacturing absorbent substrates
311 include, but are not limited to, hydrophilic polyethylene
materials or pads, acrylic fiber, glass fiber, filter paper or
pads, desiccated paper, paper pulp, fabric, and the like. For
example, the lateral flow membrane absorbent zone may be comprised
of a material such as a nonwoven spunlaced acrylic fiber, i.e., New
Merge (available from DuPont) or HDK material (available from HDK
Industries, Inc.), nonwoven polyethylene treated to improve the
hydrophobic property of the material.
[0138] Safety Means. In some embodiments, a safety means 301 is
disposed over the depressible chamber--307 so that the contents of
the chamber cannot be accidentally discharged into the channel in
fluid communication with the lateral flow membrane. A safety means
can be a cover or flange that is lifted or pulled back to expose
the depressible chamber or a push button disposed thereon.
[0139] Furthermore, such a safety means can function as an adaptor
for a specific cognate adaptor, luer or valve present on the distal
end of the SCD. Thus, a safety means can cover an aperture into
which the distal end of the SCD is engaged, for example, prior to
release of a sample into the Test Device. In an additional
embodiment, the reader is designed so that a Test Device can only
be inserted into a receiving port if the safety cover is first
removed. For example, a Test Device with its safety cover removed
indicates that a sample has been introduced into the Test Device
and running buffer has been released from the compartment 307
upstream of the aperture (adapter/safety cover). In one embodiment,
the aperture is disposed above the wicking pad 309.
[0140] Gap Means. In some embodiments, a Test Device comprises a
gap disposed between the lateral flow membrane (e.g., wicking pad)
and the channel in fluid communication with the buffer reservoir.
The gap functions to keep any solution contained in the push button
reservoir and assay sample separate until the appropriate time
according to the assay development FIG. 7. In some embodiments, the
gap can be from about 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 5, 6, 7, 8, 9
or 10 mm. In one embodiment the gap is greater than zero and less
than 3 mm. Thus, where a user exerts pressure on the
compartment--upstream of the sample aperture-, the gap as shown
between 703 and 704 is forced closed and a solution contained in
the compartment--flows in the direction shown by 701 to and through
the wicking pad, thus mobilizing the sample through the test
strip-. As indicated above, the solution can comprise any desired
buffer, reagent, chemical compound, dye, label or bead. It should
be understood that the gap embodiments disclosed herein can be
adapted to any of the Test Device configurations disclosed
herein.
[0141] In one embodiment, the test device is a lateral flow test
strip, preferably, though not necessarily, encased in a housing,
designed to be read by the reader FIGS. 3, 27.
[0142] In one embodiment, shown in FIGS. 27 A, B, and C a
SCD-processed sample is introduced into the Test Device 1001, a
chase or running buffer--is subsequently released and follows the
specimen through the wicking pad and into the test strip-, 1003
where specifically patterned capture agents bind their partner
capture probes. Therefore, if a particular analyte is present, it
will be bound by a detection probe and capture probe (as described
herein above), which capture probe will bind its specific partner
capture moiety immobilized on defined spots or lines on the test
strip--1003. FIG. 27B shows the components of the test device
including the upper housing 1002, lower housing 1004, and aperture
1005.
[0143] In one embodiment, a wash/running buffer solution is
comprised in a foil, sac or buster type packet (e.g., similar to
ketchup/condiment packet) which is disposed in the Test Device
upstream FIG. 27C of the sample entry port 302, 504, 605, 1005,
1108. The sac or packet can be designed so that it is symmetric
about the two orthogonal axes so that it can be loaded into the
Test Device easily. Therefore, in one embodiment, the cover of the
Test Device disposed over the packet when pressed down can cause
the packet to break releasing the contents therein.
[0144] In another embodiment, the button portion can comprise a
piercing appendage that punctures the packet as the button is
depressed thus releasing the contents therein. A leaf spring or
cantilever spring FIG. 27C can rest between the packet and the
button and results in pressure exerted on the packet to ensure all
the contents are released. Further, the geometry of the Test Device
is configured so that the wash buffer is directed toward the
wicking pad 904/905, 1006. In addition the geometry of the button,
spring, and housing also reduces air voids in the packet area
allowing the wash buffer to flow in any direction, even against
gravity (e.g., uphill), as necessary, but not back into the packet
storage area.
[0145] The number and size of the holes created, as well as the
geometry of the hole created can be adjusted relative to one
another in order to allow for predetermined flow of the wash buffer
out of the packet.
[0146] In one embodiment, the piercing appendage (e.g., needle)
will provide a fluid resistance barrier on the top of the packet,
allowing fluid to exit the lower portion of the packet in the
direction of the wicking pad. The piercing needle can also be
tapered in order to achieve or enhance this function.
[0147] In one embodiment, the spring FIG. 27C is an integral part
of the button, top housing or lower housing FIG. 27B or it can be a
separate component altogether that is configured to easily fit and
seal the wash/running buffer chamber.
[0148] In one embodiment, the sides of the button are designed to
minimize pinch points while the button is depressed. Sides can also
be designed to provide a baffle-type function, minimizing the risk
of liquid exiting the Test Device.
[0149] In another embodiment, the geometry of the feature that
supports the end of the wicking strip is designed to allow the
piercing feature (e.g., needle) to pass through the packet and not
allow the packet to form a seal between the packet and the support
feature. The action of the needle pierces both the wicking pad and
the packet. In another embodiment, the piercing is only of the
packet with the wicking pad located directly adjacent to the
pierced hole.
[0150] In one embodiment, the wash/running buffer in the Test
Device is comprised in a breakable/rupturing substrate (e.g., an
ampoule). Pressure exerted on a sealing membrane or button breaks
the ampoule thus releasing its contents. In one embodiment, a
channel, gutter, or trough is designed to direct the buffer to the
wicking pad.
[0151] In one embodiment, the aperture for receiving the SCD distal
end comprises a break-away collar ("Lock Collar") 1104 (FIG. 28A)
which--attaches to the SCD assembly and breaks away from the Test
Device body 1105 as the SCD 1101 is removed, thus releasing wash or
running buffer from a compartment/reservoir 1102 upstream or
immediately upstream of said aperture. In yet another embodiment,
the Lock Collar when twisted into the lock position allows a sample
to be dispensed onto the Test Device while concurrently releasing
buffer or wash buffer from an upstream compartment. For example,
the Lock Collar will comprise a geometry of channels 1103, holes or
openings that line up with openings, channels or holes of the
wash/buffer compartment only when the collar is in the lock
position. Such a Lock Collar can be utilized with any of the one or
more upstream compartments 1102, 1106 that can be utilized to
deliver a buffer/wash or any other liquid.
[0152] In an alternative embodiment, the SCD 1101 can comprise the
Lock Collar 1103 which fits into the Test Device body 1103 and
twists from an unlock position to a lock position 1104.
[0153] In one embodiment as depicted in FIG. 28B, the upstream
wash/buffer compartment comprises a soft membrane (e.g., form fill
seal pack) or ampoule that is easily ruptured/broken upon exertion
of minimal force (e.g., user pressing with finger). Such an onion
skin compartment--1106, can be further covered by a hard removable
cover 1107 which prevents accidental breakage of the onion skin.
The sample enters the test device through a port 1109 and the
device may have a narrow channel 1109 for recovery of an archival
sample.
[0154] As shown in FIG. 8 the sample is delivered to the test strip
by the SCD 802 which includes the stem and swab 806. Upstream of
the test strip is the compartment 801 with wash buffer or other
fluid. The test strip includes test zones 803-, and control zones
A, B, C 804-. The detection probe, via the conjugate label, will
provide a detectable signal. The Test Device is then inserted into
a reader 400 as shown in FIG. 4, where the signal from the label is
measured and/or detected. In another embodiment, the test strip 401
can be inserted into a movable tray 402 in the reader after the
short assay processing period has completed for a very short read
period (.about.20 seconds), this allows for a much higher through
put of tests with one reader. Further, in another embodiment, the
test strip can be inserted into the reader prior to addition of the
sample.
[0155] In any of the embodiments herein directed to a wash/running
buffer release from a chamber upstream of the sample (e.g., sample
entry port), a time delay feature can be configured into the Test
Device, so that a period of time passes between introduction of the
sample and the release of the wash/running buffer. For example, a
dry wicking pad substrate swells when wet (i.e., after wash buffer
release) and due to the swelling connects and otherwise
disconnected wicking strip FIG. 34. For example, a sample is
applied 1405. The ampoule or substrate 1401 comprising the wash
buffer is broken/ruptured to release the liquid into the dry
wicking pad portion 1402, which swells 1403 and provides liquid
communication to the wicking pad portion containing the sample
1405. The sample/buffer run through the test strip via the wicking
pad 1406.
[0156] In another embodiment, a predetermined length/density of
fibrous membrane is placed in between the wash buffer compartment
and the wicking membrane, which fibrous membrane can delay the
contact of the wash buffer to the wicking membrane thus functioning
as a time delay mechanism. Buffer wicks down the fibrous membrane
and accumulates on the end of the membrane fibers until it reaches
the wicking membrane and flows through with the sample disposed on
the wicking membrane. In another embodiment, the buffer accumulates
at the ends of the membrane fibers until there is enough volume to
bridge a gap separating the fibrous membrane from the wicking
membrane.
[0157] In other embodiments, a plunger or spring mechanism is
configured into the Test Device, which functions to reduce the
compartment/ampoule volume, thus ensuring the contents therein are
dispersed onto a wicking pad. A plunger can be moved forward by the
user exerting pressure on the button or a spring loaded plunger can
be driven forwarded in an automated fashion (e.g., when placed in
the reader). The plunger forms a seal as it drives forward so that
the liquid's only means of exit is through to the wicking pad.
[0158] In one embodiment, the liquid transport along the test strip
is based upon capillary action. In a further embodiment, the liquid
transport along the matrix is based on non-bibulous lateral flow,
wherein all of the dissolved or dispersed components of the liquid
sample are carried at substantially equal rates and with relatively
unimpaired flow laterally through the matrix, as opposed to
preferential retention of one or more components as would occur,
e.g., in materials that interact, chemically, physically, ionically
or otherwise with one or more components. See for example, U.S.
Pat. No. 4,943,522, hereby incorporated by reference in its
entirety.
[0159] Any suitable material can be used to make the devices
disclosed herein, such material including a rigid or semi-rigid,
non-water-permeable material, such as glass, ceramics, metals,
plastics, polymers, or copolymers, or any combination thereof. In
some embodiments, either the SCD or Test Device comprise a plastic,
polymer or copolymer such as those that are resistant to breakage,
such as polypropylene, polyallomer, polycarbonate or cycloolefins
or cycloolefin copolymers. Furthermore, devices of the invention
can be made by appropriate manufacturing methods, such as, but not
limited to, injection molding, blow molding, machining or press
molding.
[0160] As used herein, test strip substrate refers to the material
to which a capture moiety is linked using conventional methods in
the art. A variety of materials can be used as the substrate,
including any material that can act as a support for attachment of
the molecules of interest. Such materials are known to those of
skill in this art and include, but are not limited to, organic or
inorganic polymers, natural and synthetic polymers, including, but
not limited to, agarose, cellulose, nitrocellulose, cellulose
acetate, other cellulose derivatives, dextran, dextran-derivatives
and dextran co-polymers, other polysaccharides, glass, silica gels,
gelatin, polyvinyl pyrrolidone (PVP), rayon, nylon, polyethylene,
polypropylene, polybutylene, polycarbonate, polyesters, polyamides,
vinyl polymers, polyvinylalcohols, polystyrene and polystyrene
copolymers, polystyrene cross-linked with divinylbenzene or the
like, acrylic resins, acrylates and acrylic acids, acrylamides,
polyacrylamide, polyacrylamide blends, co-polymers of vinyl and
acrylamide, methacrylates, methacrylate derivatives and
co-polymers, other polymers and co-polymers with various functional
groups, latex, butyl rubber and other synthetic rubbers, silicon,
glass, paper, natural sponges, insoluble protein, surfactants, red
blood cells, metals, metalloids, magnetic materials, or other
commercially available media or a complex material composed of a
solid or semi-solid substrate coated with materials that improve
the hydrophilic property of the strip substrate, for example,
polystyrene, Mylar, polyethylene, polycarbonate, polypropylene,
polybutylene, metals such as aluminum, copper, tin or mixtures of
metals coated with dextran, detergents, salts, PVP and/or treated
with electrostatic or plasma discharge to add charge to the surface
thus imparting a hydrophilic property to the surface.
[0161] In one embodiment, the lateral flow membrane is comprised of
a porous material such as high density polyethylene sheet material
manufactured by Porex Technologies Corp. of Fairburn, Ga., USA. The
sheet material has an open pore structure with a typical density,
at 40% void volume, of 0.57 gm/cc and an average pore diameter of 1
to 250 micrometers, the average generally being from 3 to 100
micrometers. In another embodiment, the label zone is comprised of
a porous material such as a nonwoven spunlaced acrylic fiber
(similar to the sample receiving zone), e.g., New Merge or HDK
material. Often, the porous material may be backed by, or laminated
upon, a generally water impervious layer, e.g., Mylar. When
employed, the backing is generally fastened to the matrix by an
adhesive (e.g., 3M 444 double-sided adhesive tape). Typically, a
water impervious backing is used for membranes of low thickness. A
wide variety of polymers may be used provided that they do not bind
nonspecifically to the assay components and do not interfere with
flow of the fluid sample. Illustrative polymers include
polyethylene, polypropylene, polystyrene and the like. On occasion,
the matrix may be self-supporting. Other membranes amenable to
non-bibulous flow, such as polyvinyl chloride, polyvinyl acetate,
copolymers of vinyl acetate and vinyl chloride, polyamide,
polycarbonate, polystyrene, and the like, can also be used. In yet
another embodiment, the lateral flow membrane is comprised of a
material such as untreated paper, cellulose blends, nitrocellulose,
polyester, an acrylonitrile copolymer, and the like. The label zone
may be constructed to provide either bibulous or non-bibulous flow,
frequently the flow type is similar or identical to that provided
in at least a portion of the sample receiving zone. In a frequent
embodiment, the label zone is comprised of a nonwoven fabric such
as Rayon or glass fiber. Other label zone materials suitable for
use by the present invention include those chromatographic
materials disclosed in U.S. Pat. No. 5,075,078, which is herein
incorporated by reference.
[0162] In a frequent embodiment, the test strip substrate is
treated with a solution that includes material-blocking and
label-stabilizing agents. Blocking agents include bovine serum
albumin (BSA), methylated BSA, casein, acid or base hydrolyzed
casein, nonfat dry milk, fish gelatin, or similar. Stabilizing
agents are readily available and well known in the art, and may be
used, for example, to stabilize labeled reagents. In some
embodiments, the upstream compartment containing a solution 307 can
comprise multiple ampules, which can be selectively punctured or
broken to release their contents. Therefore, in one embodiment,
blocking reagents are contained in one ampule which is utilized to
pre-treat (e.g., "block") the test strip (i.e., lateral flow
membrane), while the additional ampule is reserved for washing the
sample through the test strip.
[0163] In various disclosures herein, the test strip/lateral flow
membrane comprises multiple test zones, one of which is referenced
803 in FIG. 8. Test zones generally contains a pre-selected capture
moieties, where a pre-selected region comprises capture moieties
that are partners for capture moieties conjugate to
analyte-specific binding agents, such as monoclonal antibodies. In
many of the presently contemplated embodiments, multiple types of
labeled reagents are incorporated in SCD such that they may
permeate together with a fluid sample contacted in the device.
These multiple types of labeled reagent can be analyte specific or
control reagents and may have different detectable characteristics
(e.g., different colors) such that one labeled reagent can be
differentiated from another labeled reagent if utilized in the same
device, or in a preferred embodiment, having different capture
moieties. As the labeled reagents are frequently bound to a
specific analyte of interest and are subsequently processed through
to a Test Device comprising a test strip, differential detection of
labeled reagents having different specificities (including analyte
specific and control labeled reagents) may be a desirable
attribute. However, frequently, the ability to differentially
detect the labeled reagents having different specificities based on
the label component alone is not necessarily due to the presence of
defined test and control zones in the device, which allow for the
accumulation of labeled reagent in designated zones.
[0164] In some embodiments, each detection probe is conjugated to a
fluorescent label emitting a different wavelength. Therefore, where
a plurality of specific binding agents are comprised in a SCD,
where the plurality comprises several different groups of specific
binding pairs, each binding pair for a given group comprises labels
different than any other group, where the multiple groups make up
the plurality of specific binding agents. For example, a group of
specific binding antibodies to influenza A can be conjugated to one
type of fluorescent label (i.e., detection probes conjugated to a
first fluorescent label), while a group of specific binding
antibodies (i.e., detection probes conjugated to a second
fluorescent label), and a third, fourth or fifth group can each
comprise detection probes conjugated to different fluorescent
labels. Of course, it should be evident that detection probes can
also comprise a combination of different types of labels (e.g.,
first group comprising a fluorescent label, while second group a
metal, while a control can comprise a chromophore). In one
embodiment, the fluorescent labels emit wavelengths that are
sufficiently distinct so that several test lines can be
differentiated.
[0165] The present description provides for the development and use
of single or multiple control zones in a single immunoassay device
that are positioned in a predetermined manner relative to
individual test zones thereby allowing easy identification of each
of the one or more analytes of interest tested for in the device.
The present description further provides for the making of control
zones of various shapes, physical or chemical identities, and
colors. In part, the use of such control zones allows for
immunoassay devices that are easy to use, and allow for the
identification of multiple analytes during a single assay
procedure. (See, Example 11).
[0166] In one embodiment, the Test Device does not include any
reagents contained therein that are capable of specifically binding
to an analyte (e.g., antibody that is specific for H5N1).
[0167] The test region generally includes one or more control zone
that is useful to verify that the sample flow is as expected. Each
of the control zones comprise a spatially distinct region that
often includes an immobilized member of a specific binding pair
which reacts with a labeled control reagent. In an occasional
embodiment, the procedural control zone contains an authentic
sample of the analyte of interest, or a fragment thereof. In this
embodiment, one type of labeled reagent can be utilized, wherein
fluid sample transports the labeled reagent to the test and control
zones; and the labeled reagent not bound to an analyte of interest
will then bind to the authentic sample of the analyte of interest
positioned in the control zone. In another embodiment, the control
line contains antibody that is specific for, or otherwise provides
for the immobilization of, the labeled reagent. In operation, a
labeled reagent is restrained in each of the one or more control
zones, even when any or all the analytes of interest are absent
from the test sample.
[0168] In some embodiments, a labeled control reagent is introduced
into the fluid sample flow either in the SCD or in the Test Device.
For example, in the Test Device, control reagents can be included
in the upstream solution/buffer reservoir, which are described
herein FIG. 3. In another example, the labeled control reagent may
be added to the fluid sample before the sample is applied to the
Test Device, e.g., present in the mixing subchamber in the SCD.
[0169] Exemplary functions of the labeled control reagents and
zones include, for example, the confirmation that the liquid flow
of the sample effectively solubilized and mobilized the labeled
reagents from the SCD, which are captured in one or more defined
test zones. Furthermore, controls can confirm that a sufficient
amount of liquid traveled correctly through the test strip test and
control zones, such that a sufficient amount of capture moieties
could react with the corresponding specific capture probes
complexed to a specific analyte (i.e., via the antigen specific
binding agent). Further, control reagents confirm that the
immunocomplexes (e.g., analyte-analyte specific binding agent)
migrate onto the test region comprising the test and control zones,
cross the test zone(s) in an amount such that the accumulation of
the labeled analyte would produce a visible or otherwise readable
signal in the case of a positive test result in the test zone(s).
Moreover, an additional function of the control zones may be to act
as reference zones which allow the user to identify the test
results which are displayed as readable zones.
[0170] Since the devices of the present invention may incorporate
one or more control zones, the labeled control reagent and their
corresponding control zones are preferably developed such that each
control zone will become visible with a desired intensity for all
control zones after fluid sample is contacted with the device,
regardless of the presence or absence of one or more analytes of
interest.
[0171] In one embodiment, a single labeled control reagent will be
captured by each of the control zones on the test strip.
Frequently, such a labeled control reagent will be deposited onto
or in the label zone in an amount exceeding the capacity of the
total binding capacity of the combined control zones if multiple
control zones are present. Accordingly, the amount of capture
reagent specific for the control label can be deposited in an
amount that allows for the generation of desired signal intensity
in the one or more control zones, and allows each of the control
zones to restrain a desired amount of labeled control-reagent. At
the completion of an assay, each of the control zones preferably
provide a desired and/or pre-designed signal (in intensity and
form). Examples of contemplated pre-designed signals include
signals of equal intensities in each control zone, or following a
desired pattern of increasing, decreasing or other signal intensity
in the control zones.
[0172] In another embodiment, each control zone will be specific
for a unique control reagent. In this embodiment, the label zone
may include multiple and different labeled control reagents,
equaling the number of control zones in the assay, or a related
variation. Wherein each of the labeled control reagents may become
restrained in one or more pre-determined and specific control
zone(s). These labeled control reagents can provide the same
detectable signal (e.g., be of the same color) or provide
distinguishable detectable signals (e.g., have different colored
labels or other detection systems) upon accumulation in the control
zone(s).
[0173] In yet another embodiment, the control zones may include a
combination of the two types of control zones described in the two
previous embodiments, specifically, one or more control zones are
able to restrain or bind a single type of labeled control reagent,
and other control zones on the same test strip will be capable of
binding one or several other specifically labeled control
reagents.
[0174] In one embodiment, the labeled control reagent comprises a
detectable moiety coupled to a member of a specific binding pair.
Typically, a labeled control reagent is chosen to be different from
the reagent that is recognized by the means which are capable of
restraining an analyte of interest in the test zone. Further, the
labeled control reagent is generally not specific for the analyte.
In a frequent embodiment, the labeled control reagent is capable of
binding the corresponding member of a specific binding pair or
control capture partner that is immobilized on or in the control
zone. Thus the labeled control reagent is directly restrained in
the control zone.
[0175] In another embodiment, the detectable moiety which forms the
label component of the labeled control reagent is the same
detectable moiety as that which is utilized as the label component
of the analyte of interest labeled test reagent. In a frequent
embodiment, the label component of the labeled control reagent is
different from the label component of the labeled test reagent, so
that results of the assay are easily determined. In another
frequent embodiment, the control label and the test label include
colored beads, e.g., colored latex. Also frequently, the control
and test latex beads comprise different colors.
[0176] In a further embodiment, the labeled control reagent
includes streptavidin, avidin or biotin and the control capture
partner includes the corresponding member of such specific binding
pairs, which readily and specifically bind with one another. In one
example, the labeled control reagent includes biotin, and the
control capture partner includes streptavidin. The artisan will
appreciate that other members of specific binding pairs can
alternatively be used, including, for example, antigen/antibody
reactions unrelated to analyte. In yet other embodiment, capture
partners can include any of the binding moieties disclosed
herein.
[0177] The use of a control zone is helpful in that appearance of a
signal in the control zone indicates the time at which the test
result can be read, even for a negative result. Thus, when the
expected signal appears in the control line, the presence or
absence of a signal in a test zone can be noted.
[0178] In still further embodiment, a control zone comprising a
mark that becomes visible in the test region when the test region
is in a moist state is utilized. Control zones of this type are
described in U.S. patent application Ser. No. 09/950,366, filed,
Sep. 10, 2001, currently pending and published as U.S. patent
application Publication No. 20030049167, and Ser. No. 10/241,822,
filed Sep. 10, 2002, currently pending and published as U.S. patent
application Publication No. 20030157699.
[0179] In occasional embodiments, one or more control zones of this
type are utilized. In another embodiment, a combination of control
zones of the type utilizing labeled control reagents and control
zone and of the type that display the control zone when in a moist
state can be used. This allows a simple way to formulate control
zones while allowing to use a reagent-based control zone to
ascertain that the re-solubilization and mobilization of the
reagents in SCD-processed samples has been effective, and that the
specific reactions took place as expected, all along the path
defined Test Device, wick, test strip and absorbent pad. The
present embodiment includes the use of one or more control zones
that become visible when the test region is in the moist state for
each of the control zones of an assay, except the control zone on
the distal or downstream end of the test strip.
[0180] The present description further provides means to build a
rapid, multi-analyte assay, which is needed in many fields of
environmental monitoring, medicine, particularly in the field of
infectious disease. For example, contemplated devices include those
useful for the differential diagnosis of Flu A or Flu B, and
subtypes thereof (e.g., Flu A, H5N1) which may result in different
treatments, or the differential diagnosis of Flu A, Flu B, and/or
RSV in one step. Such devices permit the use of a single sample for
assaying multiple analytes at once, and beneficially allows for a
considerable reduction of the hands-on time and duration of the
diagnostic process for the benefit of the doctor, or user in
general. As such a plurality of immunoreagents can be utilized in
an SCD of the invention, where said plurality comprises populations
of specific binding agents, comprising pairs conjugated
respectively to label and capture moiety, whereby said plurality
comprise multiple populations each specific for a different analyte
as compared to any other population. For example, the plurality of
immunoreagents can be specific for several types of one pathogen or
different pathogens.
[0181] A variety of analytes may be assayed utilizing devices and
methods of the present disclosure. In a particular device useful
for assaying for one or more analytes of interest in a sample, the
collection of analytes of interest may be referred to as a panel.
For example, a panel may comprise any combination (or all of) of
influenza A, influenza B, influenza A subtypes, respiratory
syncytial virus (RSV), adenovirus, and different types of
Parainfluenza viruses (for example Types 1, 2, 3 etc.). Another
panel may comprise testing for a selection of one or more of upper
respiratory infection including, for example, Streptococcus
pneumoniae, Mycoplasma pneumoniae and/or Chlamydia pneumoniae. Yet
another panel can be devised for the diagnosis of sexually
transmitted disease including, for example, Chlamydia, Trichomonas
and/or Gonorrhea. In each case, a particular panel devised to
provide signals on the Test Device for a particular series of
analytes is readily obtained by incorporating a different set of
detection and capture probes in the SCD, which is described herein.
Therefore, a particular SCD will provide all the reagents necessary
to detect a particular panel of analytes which are detected when
using a Test Device employing test strips that have detection
reagents that are not specific for the analytes of interest. In
other embodiments, a broad scope Test Device can comprise
non-specific capture moieties for several series of analytes from
related or distinct pathogens, e.g., detection of HIV and HCV
antigens; HIV and tuberculosis, Influenza A, B, and subtypes of A,
bacterial and viral infections. Thus a single Test Device can be
used with SCDs comprising immunoreagents for a different panel of
analytes, providing enhanced efficiency and cost effectiveness.
[0182] For example, a panel may optionally include a variety of
other analytes of interest, including SARS-associated coronavirus,
influenza A; a hepatitis panel comprising a selection of hepatitis
B surface Ag or Ab, hepatitis B core Ab, hepatitis A virus Ab, and
hepatitis C virus; a phospholipids panel comprising a selection of
Anticardiolipin Abs (IgG, IgA, and IgM Isotypes); an arthritis
panel comprising a selection of rheumatoid factor, antinuclear
antibodies, and Uric Acid; an Epstein Barr panel comprising a
selection of Epstein Barr Nuclear Ag, Epstein Barr Viral Capsid Ag,
and Epstein Barr Virus, Early Antigen; other panels include HIV
panels, Lupus panels, H. Pylori panels, toxoplasma panels, herpes
panels, Borrelia panels, rubella panels, cytomegalovirus panels,
panels testing for recent myocardial infarction with analytes
comprising an isotype of Troponin with myoglobin and/or CKMB and
many others. One of skill in the art would understand that a
variety of panels may be assayed via the immunoassays utilizing the
devices disclosed herein Immunoassay methods are known in the art.
See, e.g., CURRENT PROTOCOLS IN IMMUNOLOGY (Coligan, John E. et.
al., eds. 1999).
[0183] Numerous analytical devices known to those of skill in the
art may be adapted in accordance with the present invention, to
detect multiple analytes. By way of example, dipstick, lateral flow
and flow-through devices, particularly those that are immunoassays,
may be modified in accordance herewith in order to detect and
distinguish multiple analytes. Exemplary lateral flow devices
include those described in U.S. Pat. Nos. 4,818,677, 4,943,522,
5,096,837 (RE 35,306), U.S. Pat. Nos. 5,096,837, 5,118,428,
5,118,630, 5,221,616, 5,223,220, 5,225,328, 5,415,994, 5,434,057,
5,521,102, 5,536,646, 5,541,069, 5,686,315, 5,763,262, 5,766,961,
5,770,460, 5,773,234, 5,786,220, 5,804,452, 5,814,455, 5939,331,
6,306,642. Other lateral flow devices that may be modified for use
in distinguishable detection of multiple analytes in a fluid sample
include U.S. Pat. Nos. 4,703,017, 6,187,598, 6,352,862, 6,485,982,
6,534,320 and 6,767,714. Exemplary dipstick devices include those
described in U.S. Pat. Nos. 4,235,601, 5,559,041, 5,712,172 and
6,790,611. It will be appreciated by those of skill in the art that
the aforementioned patents may and frequently do disclose more than
one assay configuration and are likewise referred to herein for
such additional disclosures. Advantageously, the improvements
described are applicable to various assay, especially immunoassay,
configurations.
[0184] SCDs or Test Devices of the invention can be configured to
be utilized with existing analyte detection systems. For example,
an SCD of the invention can be configured for use with an existing
test device, or an existing test device can be configured/modified
pursuant to disclosures herein for a Test Device. Some exemplary
devices that can be modified in such a fashion include dipstick,
lateral flow, cartridge, multiplexed, microtiter plate,
microfluidic, plate or arrays or high throughput platforms, such as
those disclosed in U.S. Pat. Nos. 4,235,601, 4,632,901, 5,559,041,
5,712,172, and 6,790,611 6,448,001, 4,943,522, 6,485,982,
6,656,744, 6,811,971, 5,073,484, 5,716,778, 5,798,273, 6,565,808,
5,078,968, 5,415,994, 6,235,539, 6,267,722, 6,297,060, 7,098,040,
6,375,896, 4,818,677, 4,943,522, 5,096,837 (RE 35,306), U.S. Pat.
Nos. 5,096,837, 5,118,428, 5,118,630, 5,221,616, 5,223,220,
5,225,328, 5,415,994, 5,434,057, 5,521,102, 5,536,646, 5,541,069,
5,686,315, 5,763,262, 5,766,961, 5,770,460, 5,773,234, 5,786,220,
5,804,452, 5,814,455, 5939,331, and 6,306,642. Other lateral flow
devices that may be modified for use in distinguishable detection
of multiple analytes in a fluid sample include U.S. Pat. Nos.
4,703,017, 6,187,598, 6,352,862, 6,485,982, 6,534,320 and
6,767,714, 7,083,912, 5,225,322, 6,780,582, 5,763,262, 6,306,642,
7,109,042, 5,952,173, and 5,914,241. Exemplary microfluidic devices
include those disclosed in U.S. Pat. Nos. 5,707,799, 5,837,115 and
WO2004/029221. Each of the preceding patent disclosures is
incorporated by reference herein in its entirety.
[0185] Reader.
[0186] The systems and methods include an immunoassay device in
combination with a reader 400, particularly a reader 400 with a
built in computer, such as a reflectance and/or fluorescent based
reader, and data processing software employing data reduction and
curve fitting algorithms, optionally in combination with a trained
neural network for accurately determining the presence or
concentration of analyte in a biological sample. As used herein, a
reader refers to an instrument for detecting and/or quantitating
data, such as on test strips comprised in a Test Device 401. The
data may be visible to the naked eye, but does not need to be
visible. The methods include the steps of performing an immunoassay
on a patient sample, reading the data using a reflectance and/or
fluorescent based reader and processing the resultant data using
data processing software employing data reduction. Preferred
software includes curve fitting algorithms, optionally in
combination with a trained neural network, to determine the
presence or amount of analyte in a given sample. The data obtained
from the reader then can be further processed by the medical
diagnosis system to provide a risk assessment or diagnosis of a
medical condition as output. In alternative embodiments, the output
can be used as input into a subsequent decision support system,
such as a neural network, that is trained to evaluate such
data.
[0187] In various embodiments, the reader can be a reflectance,
transmission, fluorescence, chemo-bioluminescence, magnetic or
amperometry reader (or two or more combinations), depending on the
signal that is to be detected from the Test Device. (e.g., LRE
Medical, USA). In one embodiment, the reader comprises a receiving
port designed to receive a Test Device, but where the Test Device
can only be inserted into the receiving port if a depressible
(e.g., push button) means upstream of the sample entry aperture has
been depressed allowing the Test Device to fit into the receiving
port 402. Thus, in such an embodiment, the Test Device is placed in
a reader only when the contents of the solution reservoir (e.g.,
wash buffer) has been released, ensuring that the sample has been
"run-through" the lateral flow membrane comprised in the Test
Device.
[0188] In one embodiment, the reader is a UV LED reader which
detects a fluorescence signal. The fluorescence signal is excited
by a light emitting diode that emits in the UV region of the optics
spectrum and within the absorbance peak of the fluorescence signal
(e.g., lanthanide label). The emitted fluorescence signal is
detected by a photodiode and the wavelength of the signal detected
may be limited using a long pass filter which blocks stray emitted
light and accepts light with wavelengths at and around the peak
emission wavelength of the fluorescence emitting label. In other
embodiments, the long pass filter may be replaced by a band pass
filter. Furthermore, the excitation light may be limited by a band
pass filter.
[0189] In another embodiment, the diode is a UV laser diode. Any
conventional UV, LED or photodiode may be utilized.
[0190] In any such embodiments, the excitation source and the
detector are mounted in a single machine or molded block. For
simplified reading of the fluorescent signals generated on the test
strip. In a further embodiment, such a machine also comprises hard
standards (e.g., FIG. 33) as described herein.
[0191] In one embodiment, the axis of the excitation light is at 90
degrees to the Test Device or test strip comprised in a Test
Device. Further, the axis of the emitted light is at an angle other
than 90 degrees to the test strip.
[0192] In one embodiment the wavelength of the excitation light is
limited by a short pass filter. In yet another embodiment the
wavelength of the excitation light is limited by a combination of
band pass filter and short pass filter. In yet a further
embodiment, the wavelength of the detected light is limited by a
combination of band pass and long pass filter. The reader can be
configured to detect any of the signal emitters/labels described
herein. In one embodiment, the label is any of the lanthanides
described herein. In a further embodiment, the lanthanide used is
Europium.
[0193] As indicated herein, in one embodiment, the reader is
configured to comprise one or more hard standards. Thus, the reader
can be machined to provide a implement (e.g., a jig) to hold 0.5,
0.75, 1, 1.25, 1.5, 1.75, 2, 2.25, 2.5 or 3 mm standards (e.g.,
encased in acrylic as described herein), which standard is disposed
on about 3, 4, 5, or 6 mm centers. (e.g., See FIG. 33A, 33B,
34).
[0194] In one embodiment, the reader is adapted with a receiving
port for the Test Device FIG. 27, which itself is configured with a
safeguard FIG. 27C. In one embodiment the reader will accept but
not process the Test Device 401 if the push button has not been
depressed, or the reader will accept and read the Test Device, but
will reject the result if the Wash Buffer control does not yield a
positive signal, etc. In this latter embodiment, a wash/running
buffer disposed in a compartment/sac disposed upstream of the
sample FIG. 27C can contain a control signal (e.g., label emitting
at a different wavelength) which the reader is programmed to
detect.
[0195] The signal obtained by the reader is processed using data
processing software employing data reduction and curve fitting
algorithms, optionally in combination with a trained neural
network, to give either a positive or negative result for each test
linie, or a quantitative determination of the concentration of each
analyte in the sample, which is correlated with a result indicative
of a risk or presence of a disease or disorder. This result can
optionally be input into a decision support system, and processed
to provide an enhanced assessment of the risk of a medical
condition as output. In one embodiment, the entire procedure may be
automated and/or computer-controlled.
[0196] Multianalyte Point of Care System.
[0197] Rapid influenza tests have been marketed for years. Most of
these tests are lateral flow immunoassay tests using either gold or
latex as the visualization agent. While most of new rapid
immunoassays are able to differentiate influenza Type A from
influenza Type B, only few of them have both test lines for type A
and type B on the one strip. However, none of these tests are
designed to differentiate subtypes of influenza type A. Therefore
these tests may be able to detect avian influenza, none of them can
tell if a patient is infected by a seasonal flu A virus or a more
severe Type A subtype such as H5N1 termed avian influenza (or
current potential pandemic subtype of influenza A). The present
invention is designed on concepts that when applied are to yield a
highly sensitive assay with improved reproducibility, able to
detect type A, type B and differentiate subtype H5N1 from seasonal
flu (subtypes H1 and H3) and is easy to use. Efforts have been made
to apply multiple new technologies with a new device design, such
as pre-mixing of the sample with the conjugate, use of a chasing or
wash buffer to reduce background, employ a unique generic capture
reagent pRNA which allows multiple analytes detection at high
sensitivity, fluorescent label which is highly sensitive, etc. The
combination of these approaches enables a novel and highly
effective influenza rapid test that is much more sensitive,
provides low cost production, ease of operate and has the ability
to differentiate seasonal flu from pandemic avian flu H5N1.
[0198] In one embodiment, the combination of features described
herein are responsible for the excellent sensitivity and
reproducibility of assays constructed in accordance with the
invention to use the novel system, which serves to concentrate
ligand from a test sample at a test site the test strip, and the
use of a metal sol or other colored particle as a marker system
which permits persistent visual observation of the fluorescence
over a period of one to several hours beyond the minimum time
needed to complete the assay). Background noise is reduced while
maintaining excellent sensitivity by including in the test
implement a controllably released buffer that functions to wash
away excess/unbound label. Furthermore, one or more control sites
whose color is compared with the test site. In some embodiments, a
filtration means is comprised in the sample implement or in the
test implement, which filtration means limits the introduction to
the test site of contaminants from the crude or unprocessed
biological sample. In yet other embodiments, the filtration means
is a membrane and includes detection and capture probes disposed
thereon 604.
[0199] Assay Methods.
[0200] In one embodiment, an assay method comprises the steps of
applying the sampling implement to a subject or subject's
biological sample, to collect a sample (e.g., swabbing inside the
nose, mouth, throat, ear; applying a sampling element to a
biological sample obtained from a subject), inserting the
collection implement into the sample collection device housing
chamber, squeezing the upper chamber to break open the snap-valve
and allowing a buffer to run down to the sampling implement, thus
immersing the biological sample disposed thereon and running the
mixture of buffer and sample into a reaction chamber (e.g., lower
chamber) where a plurality of capture and detection probes bind to
their specific target analyte. Subsequently or concurrently, the
mixture is expelled from the distal end of the SCD into a Test
Device comprising immobilized capture moieties designed to capture
a complex of analyte and detection/capture probe, via the
complementary capture moiety linked to a capture probe. Thus, a
particular capture probe is designed to be complementary to an
immobilized capture moiety for one particular analyte. Furthermore,
as disclosed herein, capture moieties are disposed on a lateral
flow membrane in distinct positions/patterns, where a single line
or spot(s) if detected via the signal emitting label, allows
qualitative and/or quantitative detection of a particular analyte.
Therefore, by patterning particular capture probes on the lateral
flow membrane, an assay method can detect a panel of the same or
related infectious agent or even unrelated infectious agents, as
disclosed herein.
[0201] In some embodiments a sandwich immunoassay format is
utilized but any conventional format, including a competitive
assay, may be used. Typically, an indirect capture of the formed
immunocomplex is utilized in the sandwich format. One or more
analytes in the sample are contacted with one or more pairs of a
detection probe and a capture probe. Each pair contains the
detection probe which is a conjugate comprising a label and a
specific binding agent (SBA) capable of specifically binding to an
analyte and a capture probe which is a conjugate comprising a
detection moiety and another SBA capable of specifically binding
the same analyte. Examples of specific binding agent(s) include
antibodies, aptamers. In one embodiment, each of two SBAs are
specific binding partners in that each bind the same target antigen
or analyte. Examples of SBAs are known in the art and include but
are not limited to antibodies, aptamers or oligonucleotides. In the
sandwich assay, the analyte is simultaneously bound by both the
detection probe and the capture probe. The detection moiety is part
of a specific binding pair and the other partner of the pair is
immobilized on the test device to capture the immunocomplex as it
flows through the test device. The use of different capture moiety
pairs for each different analyte permits the detection of multiple
analyte on one test device from a single sample and reaction
sequence. In most cases, the labels for each analyte are different.
However, by having a specific location for each analyte as a
capture zone with distinct capture moiety pairs for each analyte,
it is possible to utilize the same label for all of the
analyte.
[0202] Examples of sandwich immunoassays performed on test strips
are described in U.S. Pat. Nos. 4,168,146 and 4,366,241, each of
which is incorporated herein by reference. Examples of competitive
immunoassay devices are those disclosed by U.S. Pat. Nos.
4,235,601, 4,442,204 and 5,208,535, each of which is incorporated
herein by reference. Some additional exemplary devices that can be
adapted to incorporate one or more components of the present
invention include dipstick, lateral flow, cartridge, multiplexed,
microtiter plate, microfluidic, plate or arrays or high throughput
platforms, such as those disclosed in U.S. Pat. Nos. 6,448,001,
4,943,522, 6,485,982, 6,656,744,6,811,971, 5,073,484, 5,716,778,
5,798,273, 6,565,808, 5,078,968, 5,415,994, 6,235,539, 6,267,722,
6,297,060, 7,098,040, 6,375,896, 7,083,912, 5,225,322, 6,780,582,
5,763,262, 6,306,642, 7,109,042, 5,952,173, and 5,914,241.
Exemplary microfluidic devices include those disclosed in U.S. Pat.
No. 5,707,799 and WO2004/029221.
[0203] In one such scheme, a test strip contains an enzyme labeled,
mobile binding partner for an analyte which is in a zone downstream
from a sample application zone. If an analyte is present in the
test sample, it will combine with its labeled binding partner to
form a complex which will flow along the strip to a detection zone
which contains a substrate for the enzyme label and capable of
providing a detectable enzymatic response, e.g., color response in
the presence of the enzyme. The strip also contains a zone in which
a predetermined amount of analyte is immobilized, so that any
labeled binding partner which does not combine with the analyte,
due to the absence of the analyte in a sample, will be captured and
thereby inhibited from reaching the detection zone.
[0204] Alternatively the strip can contain a binding partner bears
a moiety having a detectable physical property. For example, the
labeled binding partner can include one or more moieties, e.g.,
chemical groups detectable on the basis of certain physical
properties including without limitation colored species of
fluorescers, phosphorescent molecules, radioisotopes, electroactive
moieties, particles such as gold, etc.
[0205] In another scheme, the detection is based on the competition
between a specific analyte, e.g., ligand, antigen or antibody, the
amount of which is to be determined in a sample, and a known amount
of tracer, which is generally the analyte or appropriate analog
thereof in labeled form, with the analyte and tracer competing for
a limited number of available binding sites on a binder which is
specific towards the analyte and tracer. For example, if the
concentration of tracer and binder is fixed and the only variable
is the level of analyte, then the unknown level of analyte can be
measured by determining the amount of bound and/or free tracer in
the system. The values determined in the assay are compared with
the values given by a range of known amounts of the analyte treated
in the same manner, and by such comparison, one can determine the
amount of analyte in the sample.
[0206] In general, the tracers used in such assays require either
instrumentation and/or treatment of the tracer in order to
determine the tracer in the bound and/or free portion of the assay
as a measure of analyte. For example, in an assay in which an
enzyme is used as the label or marker for the tracer, the enzyme
must be developed with a suitable developer. When the label or
marker is a fluorescent material, the tracer in the bound and/or
free portion is determined by the use of appropriate
instrumentation for determining fluorescence.
[0207] Alternatively the tracer used in the assay is a ligand
labeled with a particulate label which is visible when bound to the
binder on the support or when bound to the analyte bound to the
binder on the support, without further treatment, and wherein the
ligand is bound by either the binder or analyte. See also U.S. Pat.
No. 4,703,017, which is incorporated herein by reference.
[0208] In another particular aspect, the non-nucleic acid based
screening test of the present invention includes any solid phase,
lateral flow, or flow-through tests. In general, solid phase
immunoassay devices incorporate a solid support to which one member
of a ligand-receptor pair, usually an antibody, antigen, or hapten,
is bound. Common early forms of solid supports were plates, tubes,
or beads of polystyrene, which were known from the fields of
radioimmunoassay and enzyme immunoassay. More recently, a number of
porous materials such as nylon, nitrocellulose, cellulose acetate,
glass fibers, and other porous polymers have been employed as solid
supports
[0209] In one embodiment, a sample is collected from a subject via
a sampling implement 102 and placed back into the cylinder housing
of the SCD device 200. The SCD can first be inserted into a Test
Device, or prior to insertion into a Test Device, a solution
contained in the upper sealed chamber of the SCD is released to
effect washing the sample and solution into a mixture downwards
into a reaction chamber. In the reaction chamber is disposed either
liquid or solid reagents comprising detection and capture probes
that target one or more different analytes as disclosed herein,
thereby forming a complex of analyte bound to detection and capture
probe. The sample is then expelled from the SCD into a Test Device
through an aperture that seals the contact between the SCD and the
Test Device from the outside environment (e.g., preventing any
spillage, aerosol or contamination). The sample mixture can flow as
a result of gravity or the force of air pressure produced by
squeezing the SCD (e.g., upper sealed chamber), into a Test Device.
The sample is driven by capillary force and/or by wash buffer
comprised in the Test Device 307 so as to allow any analyte-probe
complex to pass through the lateral flow membrane contained in the
Test Device. Capture probes and complementary immobilized capture
moieties bind or hybridize to each other in predetermined lines or
spots on the lateral flow membrane 803 or 804, whereby detection
probes (via conjugate labels contained thereon) will provide a
detectable signal which can subsequently be read to determine which
analytes were present in the sample processed.
[0210] In one embodiment, Test Devices with samples processed
thereon, can be set aside for time periods of about 1, 2, 3, 4, 5,
6 or 8 hours before reading the results, and yet provide results as
accurately as if read in 15 or 20 minutes after processing. Thus,
the signals produced are stable for long periods of time so that
reading the results may occur at a significantly later time after
the tests are actually performed. This is a great improvement for
point-of-care diagnostics, where in the field conditions often
present limited resources in manpower and time, and where the test
setting can be in remote regions that are not easily or quickly
accessed.
[0211] Binding Reagents.
[0212] One aspect of the invention is directed to binding reagents
disposed in the SCD. For example, in some immunoassays, an antibody
pair is utilized, where each member of the pair can specifically
bind the same target analyte, wherein one antibody is a capture
antibody and the other is a detection antibody. A capture antibody
is linked, directly or indirectly, to a capture moiety which is
"captured" by a cognate immobilized capture moiety disposed on a
solid support (e.g., nitrocellulose membrane). Furthermore, the
detection antibody (i.e., detection probe) is linked to a
detectable label. The detection antibody is preferably labeled by
conjugation to a physically detectable label, and upon contacting
with the sample containing the target analyte forms a complex. The
antibody-analyte complex can then be immobilized on a solid support
via the capture moiety. The resulting complex immobilized on the
solid support, is detectable by virtue of the label.
[0213] In one embodiment, the SCD reagent solution or solid
substrate comprises a plurality of different detection probes, each
detection probe capable of binding to a different target and each
detection probe being labeled with or enabling the formation of a
detection signal so that the presence of each target is indicated
by the formation of a signal at the test zone for that target
(i.e., in the Test Device); wherein the target for at least two of
the capture moieties is an infectious agent or a disease causing
micro-organism or a marker indicating the existence of a disease,
disorder, or condition of the host from which the sample solution
was derived, and wherein at least two of the capture moieties are
capable of binding to different components or markers of the same
infectious agent or disease causing microorganism, or to different
markers for the same disease, disorder, or condition not caused by
an infectious agent or disease causing microorganism, as targets
for those capture moieties. Furthermore, the SCD will also comprise
a plurality of different capture probes, each of which is paired up
with a detection probe, where the pairing is defined by the
capability to bind a particular target analyte.
[0214] As used herein, the term "specifically binds" refers to the
binding specificity of a specific binding pair. "Specific binding
pair member" refers to a member of a specific binding pair ("sbp"),
which means two different molecules wherein one of the molecules
specifically binds with the second molecule through chemical or
physical means. For example, a pair of pRNAs or an aptamer/target
antigen pair, or streptavidin-biotin provide exemplary specific
binding pair members or sbp. The two molecules are related in the
sense that their binding with each other is such that they are
capable of distinguishing their binding partner from other assay
constituents having similar characteristics. The members of the
specific binding pair are referred to as ligand and receptor
(antiligand), sbp member and sbp partner, and the like. A molecule
may also be a sbp member for an aggregation of molecules; for
example an antibody raised against an immune complex of a second
antibody and its corresponding antigen may be considered to be an
sbp member for the immune complex.
[0215] In addition to antigen and antibody specific binding pair
members, other specific binding pairs include, as examples without
limitation, biotin and avidin, carbohydrates and lectins,
complementary nucleotide sequences, complementary peptide
sequences, effector and receptor molecules, enzyme cofactors and
enzymes, enzyme inhibitors and enzymes, a peptide sequence or
chemical moiety (such as digoxin/anti-digoxin) and an antibody
specific for the sequence, chemical moiety or the entire protein,
polymeric acids and bases, dyes and protein binders, peptides and
specific protein binders (e.g., ribonuclease, S-peptide and
ribonuclease S-protein), metals and their chelators, and the like.
Furthermore, specific binding pairs can include members that are
analogs of the original specific binding member, for example an
analyte-analog or a specific binding member made by recombinant
techniques or molecular engineering.
[0216] An sbp member is analogous to another sbp member if they are
both capable of binding to another identical complementary sbp
member. Such an sbp member may, for example, be either a ligand or
a receptor that has been modified by the replacement of at least
one hydrogen atom by a group to provide, for example, a labeled
ligand or labeled receptor. The sbp members can be analogous to or
complementary to the analyte or to an sbp member that is
complementary to the analyte. If the specific binding member is an
immunoreactant it can be, for example, an antibody, antigen,
hapten, or complex thereof. If an antibody is used, it can be a
monoclonal or polyclonal antibody, a recombinant protein or
antibody, a chimeric antibody, a mixture(s) or fragment(s) thereof,
as well as a mixture of an antibody and other specific binding
members. Other examples of binding pairs that can be incorporated
into the detection molecules are disclosed in U.S. Pat. Nos.
6,946,546, 6,967,250, 6,984,491, 7,022,492, 7,026,120, 7,022,529,
7,026,135, 7,033,781, 7,052,854, 7,052,916 and 7,056,679.
[0217] "Antibody" refers to a polypeptide substantially encoded by
an immunoglobulin gene or immunoglobulin genes, or fragments
thereof, and includes any immunoglobulin, including monoclonal
antibodies, polyclonal antibodies, multispecific or bispecific
antibodies, that bind to a specific antigen. A complete antibody
comprises two heavy chains and two light chains. Each heavy chain
consists of a variable region and a first, second, and third
constant region, while each light chain consists of a variable
region and a constant region. The antibody has a "Y" shape, with
the stem of the Y consisting of the second and third constant
regions of two heavy chains bound together via disulfide bonding.
Each arm of the Y consists of the variable region and first
constant region of a single heavy chain bound to the variable and
constant regions of a single light chain. The variable regions of
the light and heavy chains are responsible for antigen binding. The
variable region in both chains generally contains three highly
variable loops called the complementarity determining regions
(CDRs) (light (L) chain CDRs including LCDR1, LCDR2, and LCDR3,
heavy (H) chain CDRs including HCDR1, HCDR2, HCDR3) (as defined by
Kabat, et al., Sequences of Proteins of Immunological Interest,
Fifth Edition (1991), vols. 1-3, NIH Publication 91-3242, Bethesda
Md.). The three CDRs are interposed between flanking stretches
known as framework regions (FRs), which are more highly conserved
than the CDRs and form a scaffold to support the hypervariable
loops. The constant regions of the heavy and light chains are not
involved in antigen binding, but exhibit various effector
functions. The recognized immunoglobulin genes include the kappa,
lambda, alpha, gamma, delta, epsilon, and mu constant regions, as
well as myriad immunoglobulin variable region genes. Light chains
are classified as either kappa or lambda. Heavy chains are
classified as gamma, mu, alpha, delta, or epsilon, which in turn
define the immunoglobulin classes and subclasses include IgG, IgG1,
IgG2, IgG3, IgG4, IgM, IgA, IgA1, or IgA2, IgD, and IgE,
respectively. Typically, an antibody is an immunoglobulin having an
area on its surface or in a cavity that specifically binds to and
is thereby defied as complementary with a particular spatial and
polar organization of another molecule. The antibody can be
polyclonal or monoclonal. Antibodies may include a complete
immunoglobulin or fragments thereof. Fragments thereof may include
Fab, Fv and F(ab')2, Fab', and the like. Antibodies may also
include chimeric antibodies or fragment thereof made by recombinant
methods. The term "antibody" as used herein Antibodies are assigned
to classes based on the amino acid sequence of the constant region
of their heavy chain. The major classes of antibodies are IgA, IgD,
IgE, IgG, and IgM, with several of these classes divided into
subclasses such as.
[0218] In addition to an intact immunoglobulin, the term "antibody"
as used herein further refers to an immunoglobulin fragment thereof
(i.e., at least one immunologically active portion of an
immunoglobulin molecule), such as a Fab, Fab', F(ab').sub.2, Fv
fragment, a single-chain antibody molecule, a multispecific
antibody formed from any fragment of an immunoglobulin molecule
comprising one or more CDRs. In addition, an antibody as used
herein may comprise one or more CDRs from a particular human
immunoglobulin grafted to a framework region from one or more
different human immunoglobulins.
[0219] "Fab" with regards to an antibody refers to that portion of
the antibody consisting of a single light chain (both variable and
constant regions) bound to the variable region and first constant
region of a single heavy chain by a disulfide bond.
[0220] "Fab'" refers to a Fab fragment that includes a portion of
the hinge region.
[0221] "F(ab').sub.2 refers to a dimer of Fab'.
[0222] "Fc" with regards to an antibody refers to that portion of
the antibody consisting of the second and third constant regions of
a first heavy chain bound to the second and third constant regions
of a second heavy chain via disulfide bonding. The Fc portion of
the antibody is responsible for various effector functions but does
not function in antigen binding.
[0223] "Fv" with regards to an antibody refers to the smallest
fragment of the antibody to bear the complete antigen binding site.
An Fv fragment consists of the variable region of a single light
chain bound to the variable region of a single heavy chain.
[0224] "Single-chain Fv antibody" or "scFv" refers to an engineered
antibody consisting of a light chain variable region and a heavy
chain variable region connected to one another directly or via a
peptide linker sequence (Houston 1988).
[0225] "Single-chain Fv-Fc antibody" or "scFv-Fc" refers to an
engineered antibody consisting of a scFv connected to the Fc region
of an antibody.
[0226] The term "epitope" as used herein refers to the group of
atoms and/or amino acids on an antigen molecule to which an
antibody binds.
[0227] The term "monoclonal antibody" as used herein refers to an
antibody or a fragment thereof obtained from a population of
substantially homogeneous antibodies, i.e., the individual
antibodies comprising the population are identical except for
possible naturally occurring mutations that may be present in minor
amounts. Monoclonal antibodies are highly specific, being directed
against a single epitope on the antigen. Monoclonal antibodies are
in contrast to polyclonal antibodies which typically include
different antibodies directed against different epitopes on the
antigens. Although monoclonal antibodies are traditionally derived
from hybridomas, the monoclonal antibodies of the present invention
are not limited by their production method. For example, the
monoclonal antibodies of the present invention may be made by the
hybridoma method first described by Kohler et al., Nature, 256:495
(1975), or may be made by recombinant DNA methods (see, e.g., U.S.
Pat. No. 4,816,567).
[0228] The term "chimeric antibody" as used herein refers to an
antibody in which a portion of the heavy and/or light chain is
identical with or homologous to corresponding sequences in
antibodies derived from a particular species or belonging to a
particular antibody class or subclass, while the remainder of the
heavy and/or light chain is identical with or homologous to
corresponding sequences in antibodies derived from another species
or belonging to another antibody class or subclass, as well as
fragments of such an antibody, so long as such fragments exhibit
the desired antigen-binding activity (U.S. Pat. No. 4,816,567 to
Cabilly et al.; Morrison et al., Proc. Natl. Acad. Sci. USA,
81:6851 6855 (1984)).
[0229] The term "humanized antibody" used herein refers to an
antibody or fragments thereof which are human immunoglobulins
(recipient antibody) in which residues from part or all of a CDR of
the recipient are replaced by residues from a CDR of a non-human
species (donor antibody) such as mouse, rat or rabbit having the
desired specificity, affinity, and capacity. In some instances, FR
residues of the human immunoglobulin are replaced by corresponding
non-human residues. Furthermore, humanized antibodies may comprise
residues which are found neither in the recipient antibody nor in
the imported CDR or framework sequences. These modifications are
made to further refine and optimize antibody performance. In
general, the humanized antibody will comprise substantially all of
at least one, and typically two, variable domains, in which all or
substantially all of the CDR regions correspond to those of a
non-human immunoglobulin and all or substantially all of the FR
regions are those of a human immunoglobulin sequence. The humanized
antibody optimally also will comprise at least a portion of an
immunoglobulin Fc region, typically that of a human immunoglobulin.
For further details, see Jones et al., Nature, 321:522 525 (1986);
Reichmann et al., Nature, 332:323 329 (1988); Presta, Curr. Op.
Struct. Biol., 2:593 596 (1992); and Clark, Immunol. Today 21: 397
402 (2000).
[0230] The present invention provides anti-H5 monoclonal antibodies
that are produced by mice hybridoma cell strains 8H5, 3C8, 10F7,
4D1, 3G4 and 2F2. These monoclonal antibodies are named after the
hybridoma cell strains that produce them. Thus the anti-H5
monoclonal antibodies that are produced by mice hybridoma cell
strains 8H5, 3C8, 10F7, 4D1, 3G4, and 2F2, respectively, are named
monoclonal antibodies 8H5, 3C8, 10F7, 4D1, 3G4, and 2F2,
respectively. Monoclonal antibodies 8H5, 3C8, 10F7, 4D1, 3G4, and
2F2 specifically bind to the hemagglutinin of subtype avian
influenza virus. The mice hybridoma cell strains 8H5, 3C8, 10F7,
4D1, 3G4, and 2F2 were deposited in China Center for Typical
Culture Collection (CCTCC, Wuhan University, Wuhan, China) on Jan.
17, 2006 with deposit numbers of CCTCC-C200607 (hybridoma cell
strain 8H5), CCTCC-C200605 (hybridoma cell strain 3C8),
CCTCC-C200608 (hybridoma cell strain 10F7), CCTCC-C200606
(hybridoma cell strain 4D1), CCTCC-C200604 (hybridoma cell strain
3G4) and CCTCC-C200424 (hybridoma cell strain 2F2).
[0231] In various embodiment, monoclonal antibodies are provided
that block the binding of monoclonal antibodies 8H5, 3C8, 10F7,
4D1, 3G4, or 2F2 to the hemagglutinin of subtype H5 avian influenza
virus. Such blocking monoclonal antibodies may bind to the same
epitopes on the hemagglutinin that are recognized by monoclonal
antibodies 8H5, 3C8, 10F7, 4D1, 3G4, or 2F2. Alternatively, those
blocking monoclonal antibodies may bind to epitopes that overlap
sterically with the epitopes recognized by monoclonal antibodies
8H5, 3C8, 10F7, 4D1, 3G4, or 2F2. These blocking monoclonal
antibodies can reduce the binding of monoclonal antibodies 8H5,
3C8, 10F7, 4D1, 3G4, or 2F2 to the hemagglutinin of subtype H5
avian influenza virus by at least about 50%. Alternatively, they
may reduce binding by at least about 60%, preferably at least about
70%, more preferably at least about 75%, more preferably at least
about 80%, more preferably at least about 85%, even more preferably
at least about 90%, even more preferably at least about 95%, most
preferably at least about 99%.
[0232] The ability of a test monoclonal antibody to reduce the
binding of a known monoclonal antibody to the H5 hemagglutinin may
be measured by a routine competition assay such as that described
in Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory,
Ed Harlow and David Lane (1988). For example, such an assay could
be performed by pre-coating a microtiter plate with antigens,
incubating the pre-coated plates with serial dilutions of the
unlabeled test antibodies admixed with a selected concentration of
the labeled known antibodies, washing the incubation mixture, and
detecting and measuring the amount of the known antibodies bound to
the plates at the various dilutions of the test antibodies. The
stronger the test antibodies compete with the known antibodies for
binding to the antigens, the more the binding of the known
antibodies to the antigens would be reduced. Usually, the antigens
are pre-coated on a 96-well plate, and the ability of unlabeled
antibodies to block the binding of labeled antibodies is measured
using radioactive or enzyme labels.
[0233] Monoclonal antibodies may be generated by the hybridoma
method first described by Kohler et al., Nature, 256: 495 (1975).
In the hybridoma method, a mouse or other appropriate host animal
is immunized by one or more injections of an immunizing agent and,
if desired, an adjuvant. Typically, the immunizing agent and/or
adjuvant will be injected in the host animal by multiple
subcutaneous or intraperitoneal injections. It may be useful to
conjugate the immunizing agent to a protein known to be immunogenic
in the host animal being immunized, such as serum albumin, or
soybean trypsin inhibitor. Examples of adjuvants which may be
employed include Freund's complete adjuvant and MPL-TDM. After
immunization, the host animal makes lymphocytes that produce or are
capable of producing antibodies that will specifically bind to the
antigen used for immunization. Alternatively, lymphocytes may be
immunized in vitro. Desired lymphocytes are collected and fused
with myeloma cells using a suitable fusing agent, such as
polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal
Antibodies: Principles and Practice, pp. 59 103, Academic Press,
1996).
[0234] The hybridoma cells thus prepared are seeded and grown in a
suitable culture medium that preferably contains one or more
substances that inhibit the growth or survival of the unfused,
parental myeloma cells. For example, if the parental myeloma cells
lack the enzyme hypoxanthine guanine phosphoribosyl transferase
(HGPRT or HPRT), the culture medium for the hybridomas typically
will include hypoxanthine, aminopterin, and thymidine (HAT medium),
which substances prevent the growth of HGPRT-deficient cells.
[0235] Preferred myeloma cells are those that fuse efficiently,
support stable high-level production of antibody by the selected
antibody-producing cells, and are sensitive to a medium such as HAT
medium. Among these, preferred myeloma cell lines are murine
myeloma lines, such as those derived from MOP-21 and MC-11 mouse
tumors available from the Salk Institute Cell Distribution Center,
San Diego, Calif. USA, and SP-2 or X63-Ag8-653 cells available from
the American Type Culture Collection, Rockville, Md. USA. Human
myeloma and mouse-human heteromyeloma cell lines also have been
described for the production of human monoclonal antibodies
(Kozbor, J. Immunol., 133: 3001 (1984); Brodeur et al., Monoclonal
Antibody Production Techniques and Applications, pp. 51-63, Marcel
Dekker, Inc., New York, 1987).
[0236] Culture medium in which hybridoma cells are growing is
assayed for production of monoclonal antibodies directed against
the antigen. Preferably, the binding specificity of monoclonal
antibodies produced by hybridoma cells is determined by
immunoprecipitation or by an in vitro binding assay, such as
radioimmunoassay (RIA) or enzyme-linked immunosorbent assay
(ELISA). The binding affinity of the monoclonal antibody can, for
example, be determined by the Scatchard analysis of Munson et al.,
Anal. Biochem., 107: 220 (1980).
[0237] After hybridoma cells are identified that produce antibodies
of the desired specificity, affinity, and/or activity, the cells
may be subcloned by limiting dilution procedures and grown by
standard methods (Goding, Monoclonal Antibodies: Principles and
Practice, pp. 59-103, Academic Press, 1996). Suitable culture media
for this purpose include, for example, DMEM or RPMI-1640 medium. In
addition, the hybridoma cells may be grown in vivo as ascites
tumors in an animal.
[0238] The monoclonal antibodies secreted by the subclones are
suitably separated from the culture medium, ascites fluid, or serum
by conventional immunoglobulin purification procedures such as, for
example, protein A-Sepharose, hydroxylapatite chromatography, gel
electrophoresis, dialysis, or affinity chromatography.
[0239] Monoclonal antibodies of the invention may also be made by
conventional genetic engineering methods. DNA molecules encoding
the heavy and light chains of the monoclonal antibodies may be
isolated from the hybridoma cells, for example through PCR using
oligonucleotide probes that are capable of binding specifically to
genes encoding the heavy and light chains of the monoclonal
antibodies. Then the DNA molecules are inserted into expression
vectors. The expression vectors are transfected into host cells
such as E. coli cells, simian COS cells, Chinese hamster ovary
(CHO) cells, or myeloma cells that do not otherwise produce
immunoglobulin protein. The host cells are cultured under
conditions suitable for the expression of the antibodies.
[0240] The antibodies of the invention can bind to the H5
hemagglutinin with high specificity and affinity. The antibodies
shall have low cross-reactivity with other subtypes of
hemagglutinin, preferably no cross-reactivity with other subtypes
of hemagglutinins. In one aspect, the invention provides antibodies
that bind to H5 hemagglutinin with a K.sub.D value of less than
1.times.10.sup.-5M. Preferably, the K.sub.D value is less than
1.times.10.sup.-6M. More preferably, the K.sub.D value is less than
1.times.10.sup.-7M. Most preferably, the K.sub.D value is less than
1.times.10.sup.-8M.
[0241] The antibodies of the invention may contain the conventional
"Y" shape structure comprised of two heavy chains and two light
chains. In addition, the antibodies may also be the Fab fragment,
the Fab' fragment, the F(ab).sub.2 fragment or the Fv fragment, or
another partial piece of the conventional "Y" shaped structure that
maintains binding affinity to the hemagglutinin. The binding
affinity of the fragments to hemagglutinin may be higher or lower
than that of the conventional "Y" shaped antibodies.
[0242] The antibody fragments may be generated via proteolytic
digestion of intact antibodies (see, e.g., Morimoto et al., J.
Biochem. Biophys. Methods, 24:107-117, (1992) and Brennan et al.,
Science, 229:81 (1985)). Additionally, these fragments can also be
produced directly by recombinant host cells (reviewed in Hudson,
Curr. Opin. Immunol., 11: 548-557 (1999); Little et al., Immunol.
Today, 21: 364-370 (2000)). For example, Fab' fragments can be
directly recovered from E. coli and chemically coupled to form
F(ab).sub.2 fragments (Carter et al., Bio/Technology, 10:163 167
(1992)). In another embodiment, the F(ab').sub.2 is formed using
the leucine zipper GCN4 to promote assembly of the F(ab').sub.2
molecule. According to another approach, Fv, Fab or F(ab').sub.2
fragments can be isolated directly from recombinant host cell
culture. Other techniques for the production of antibody fragments
will be apparent to a person with ordinary skill in the art.
[0243] The present invention provides isolated nucleic acid
molecules encoding antibodies or fragments thereof that
specifically bind to H5 hemagglutinin. Nucleic acid molecules
encoding the antibodies can be isolated from hybridoma cells. The
nucleic acid sequences of the molecules can be determined using
routine techniques known to a person with ordinary skill in the
art. Nucleic acid molecules of the invention can also be prepared
using conventional genetic engineering techniques as well as
chemical synthesis. In one aspect, the present invention provides
an isolated nucleic acid molecule encoding the variable region of
the heavy chain of an anti-H5 (HA) antibody or a portion of the
nucleic acid molecule. In another aspect, the present invention
provides an isolated nucleic acid molecule encoding the variable
region of the light chain of an anti-H5 (HA) antibody or a portion
of the nucleic acid molecule. In another aspect, the present
invention provides an isolated nucleic acid molecule encoding the
CDRs of the antibody heavy chain or light chain variable
regions.
[0244] In one aspect, the present invention provides isolated
nucleic acid molecules encoding the variable regions of the heavy
chain and light chain of monoclonal antibodies 8H5, 3C8, 10F7, 4D1,
3G4, and 2F2. The nucleic acid sequences encoding the heavy chain
variable regions of monoclonal antibodies 8H5, 3C8, 10F7, 4D1, 3G4,
and 2F2 are set forth in SEQ ID NO: 1, SEQ ID NO: 5, SEQ ID NO: 9,
SEQ ID NO:16, SEQ ID NO:20 and SEQ ID NO: 22, respectively. The
nucleic acid sequences encoding the light chain variable regions of
monoclonal antibodies 8H5, 3C8, 10F7, 4D1, and 2F2 are set forth in
SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 11, SEQ ID NO:18, SEQ ID NO:
24, respectively. The present invention also includes degenerative
analogs of the nucleic acid molecules encoding the variable regions
of the heavy chain and light chain of monoclonal antibodies 8H5,
3C8, 10F7, 4D1, 3G4 and 2F2.
[0245] In another aspect, the present invention provides isolated
nucleic acid variants that share sequence identity with the nucleic
acid sequences of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID
NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 16, SEQ ID NO:18,
SEQ ID NO:20, SEQ ID NO:22 or SEQ ID NO:24. In one embodiment, the
nucleic acid variants share at least 70% sequence identity,
preferably at least 75% sequence identity, more preferably at least
80% sequence identity, more preferably at least 85% sequence
identity, more preferably at least 90% sequence identity, most
preferably at least 95% sequence identity, to the sequences of SEQ
ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9,
SEQ ID NO: 11, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID
NO:22 or SEQ ID NO:24.
[0246] The present invention also provides isolated nucleic acid
molecules encoding antibody fragments that are still capable of
specifically binding to subtype H5 of avian influenza virus.
[0247] The present invention further provides isolated nucleic acid
molecules encoding an antibody heavy chain variable region
comprising the amino acid sequence set forth in SEQ ID NOs: 26-28,
SEQ ID NOs: 32-34, SEQ ID NOs: 38-40, SEQ ID NOs: 44-46; SEQ ID
NOs: 50-52, and SEQ ID NOs: 53-55. The present invention provides
isolated nucleic acid molecules encoding an antibody light chain
variable region comprising the amino acid sequence set forth in SEQ
ID NOs: 29-31, SEQ ID NOs: 35-37, SEQ ID NOs: 41-43, SEQ ID NOs:
47-59, and SEQ ID NOs: 56-58.
[0248] The present invention provides recombinant expressing
vectors comprising the isolated nucleic acid molecules of the
invention. It also provides host cells transformed with the nucleic
acid molecules. Furthermore, the present invention provides a
method of producing antibodies of the invention comprising
culturing the host cells under conditions wherein the nucleic acid
molecules are expressed to produce the antibodies and isolating the
antibodies from the host cells.
[0249] Antibody Polypeptide Sequences
[0250] The amino acid sequences of the variable regions of the
heavy chain and light chain of monoclonal antibodies 8H5, 3C8,
10F7, 4D1, 3G4 and 2F2 have been deduced from their respective
nucleic acid sequences. The amino acid sequences of the heavy chain
variable regions of monoclonal antibodies 8H5, 3C8, 10F7, 4D1, 3G4
and 2F2 are set forth in SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO: 10,
SEQ ID NO:17, SEQ ID NO:21, and SEQ ID NO:23, respectively. The
amino acid sequences of the light chain variable regions of
monoclonal antibodies 8H5, 3C8, 10F7, 4D1, and 2F2 are set forth in
SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:19, and SEQ ID
NO:25. In one aspect, the present invention provides anti-H5
antibodies comprising a heavy chain variable region comprising the
amino acid sequences as set forth in SEQ ID NO:2, SEQ ID NO:6, SEQ
ID NO: 10, SEQ ID NO:17, SEQ ID NO:21, and SEQ ID NO:23. In another
aspect, the present invention provides anti-H5 antibodies
comprising a light chain variable region comprising the amino acid
sequences as set forth in SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:12,
SEQ ID NO:19, and SEQ ID NO:25.
[0251] In another aspect, the present invention provides an
antibody heavy chain comprising a variable region having at least
70% sequence identity, preferably at least 75% sequence identity,
more preferably at least 80% sequence identity, more preferably at
least 85% sequence identity, more preferably at least 90% sequence
identity, most preferably at least 95% sequence identity to the
amino acid sequences set forth in SEQ ID NO:2, SEQ ID NO:6, SEQ ID
NO: 10, SEQ ID NO:17, SEQ ID NO:21, and SEQ ID NO:23.
[0252] In another aspect, the present invention provides an
antibody light chain comprising a variable region having at least
70% sequence identity, preferably at least 75% sequence identity,
more preferably at least 80% sequence identity, more preferably at
least 85% sequence identity, more preferably at least 90% sequence
identity, most preferably at least 95% sequence identity to the
amino acid sequences set forth in SEQ ID NO:4, SEQ ID NO:8, SEQ ID
NO:12, SEQ ID NO:19, and SEQ ID NO:25.
[0253] The amino acid sequences of the CDRs of the variable regions
of the heavy chain and light chain of monoclonal antibodies 8H5,
3C8, 10F7, 4D1, 3G4, and 2F2 have also been determined as
follows:
[0254] The amino acid sequences of CDR1, CDR2 and CDR3 of the heavy
chain of monoclonal antibody 8H5 are set forth in SEQ ID Nos:26-28,
respectively. The amino acid sequences of CDR1, CDR2 and CDR3 of
the light chain of monoclonal antibody 8H5 are set forth in SEQ ID
Nos:29-31, respectively.
[0255] The amino acid sequences of CDR1, CDR2 and CDR3 of the heavy
chain of monoclonal antibody 3C8 are set forth in SEQ ID Nos:32-34,
respectively. The amino acid sequences of CDR1, CDR2 and CDR3 of
the light chain of monoclonal antibody 3C8 are set forth in SEQ ID
Nos:35-37, respectively.
[0256] The amino acid sequences of CDR1, CDR2 and CDR3 of the heavy
chain of monoclonal antibody 10F7 are set forth in SEQ ID
Nos:38-40, respectively. The amino acid sequences of CDR1, CDR2 and
CDR3 of the light chain of monoclonal antibody 10F7 are set forth
in SEQ ID Nos:41-43, respectively.
[0257] The amino acid sequences of CDR1, CDR2 and CDR3 of the heavy
chain of monoclonal antibody 4D1 are set forth in SEQ ID Nos:44-46,
respectively. The amino acid sequences of CDR1, CDR2 and CDR3 of
the light chain of monoclonal antibody 4D1 are set forth in SEQ ID
Nos:47-49, respectively.
[0258] The amino acid sequences of CDR1, CDR2 and CDR3 of the heavy
chain of monoclonal antibody 3G4 are set forth in SEQ ID Nos:50-52,
respectively.
[0259] The amino acid sequences of CDR1, CDR2 and CDR3 of the heavy
chain of monoclonal antibody 2F2 are set forth in SEQ ID Nos:53-55,
respectively. The amino acid sequences of CDR1, CDR2 and CDR3 of
the light chain of monoclonal antibody 2F2 are set forth in SEQ ID
Nos:56-58, respectively.
TABLE-US-00001 TABLE 1 Six strains of monoclonal antibody CDRs
amino acid sequence. Mono- clonal anti- Antibody heavy chain CDRs
Antibody light chain CDRs body amino acid sequence amino acid
sequence strains CDR1 CDR2 CDR3 CDR1 CDR2 CDR3 8H5 GYTFSNYW
ILPGSDRT ANRYDGYYFGLDY SSVNF YSS QHFTSSPYT (SEQ ID NO: 26) (SEQ ID
NO: 27) (SEQ ID NO: 28) (SEQ ID NO: 29) (SEQ ID NO: 30) (SEQ ID NO:
31) 3C8 GYSFTNYG INTHTGEP ARWNRDAMDY ESVDSSDNSL RAS QQSIGDPPYT (SEQ
ID NO: 32) (SEQ ID NO: 33) (SEQ ID NO: 34) (SEQ ID NO: 35) (SEQ ID
NO: 36) (SEQ ID NO: 37) 10F7 GYTFTSYW IDPSDSYT ARGGTGDFHYAMDY
QGISSN HGT QYVQFPYT (SEQ ID NO: 38) (SEQ ID NO: 39) (SEQ ID NO: 40)
(SEQ ID NO: 41) (SEQ ID NO: 42) (SEQ ID NO: 43) 4D1 GYTFTSYW
IDPSDSFT ARGGPGDFRYAMDY QGISSN HGT VQYVQFPYT (SEQ ID NO: 44) (SEQ
ID NO: 45) (SEQ ID NO: 46) (SEQ ID NO: 47) (SEQ ID NO: 48) (SEQ ID
NO: 49) 3G4 GYTFTDYA INTDYGDT ARSDYDYYFCGMDY (SEQ ID NO: 50) (SEQ
ID NO: 51) (SEQ ID NO: 52) 2F2 GFSLTGYG IWAEGRT AREVITTEAWYFDV
QSISDY YAS QNGHTFPLT (SEQ ID NO: 53) (SEQ ID NO: 54) (SEQ ID NO:
55) (SEQ ID NO: 56) (SEQ ID NO: 57) (SEQ ID NO: 58)
[0260] In another aspect, the present invention provides an anti-H5
monoclonal antibody heavy chain or a fragment thereof, comprising
the following CDRs: (i) one or more CDRs selected from SEQ ID NOs:
26-28; (ii) one or more CDRs selected from SEQ ID NOs: 32-34; (iii)
one or more CDRs selected from SEQ ID NOs: 38-40; (iv) one or more
CDRs selected from SEQ ID NOs: 44-46; (v) one or more CDRs selected
from SEQ ID NOs: 50-52; or (vi) one or more CDRs selected from SEQ
ID NOs: 53-55. In one embodiment, the anti-H5 monoclonal antibody
heavy chain or a fragment thereof comprises three CDRs having the
amino acid sequences set forth in SEQ ID NOs: 26-28, respectively.
In another embodiment, the anti-H5 monoclonal antibody heavy chain
or a fragment thereof comprises three CDRs having the amino acid
sequences set forth in SEQ ID NOs: 32-34, respectively. In another
embodiment, the anti-H5 monoclonal antibody heavy chain or a
fragment thereof comprises three CDRs having the amino acid
sequences set forth in SEQ ID NOs: 38-40. In another embodiment,
the anti-H5 monoclonal antibody heavy chain or a fragment thereof
comprises three CDRs having the amino acid sequences set forth in
SEQ ID NOs: 44-46. In another embodiment, the anti-H5 monoclonal
antibody heavy chain or a fragment thereof comprises three CDRs
having the amino acid sequences set forth in SEQ ID NOs: 50-52. In
another embodiment, the anti-H5 monoclonal antibody heavy chain or
a fragment thereof comprises three CDRs having the amino acid
sequences set forth in SEQ ID NOs: 53-55.
[0261] In another aspect, the CDRs contained in the anti-H5
monoclonal antibody heavy chains or fragments thereof of the
present invention may include one or more amino acid substitution,
addition and/or deletion from the amino acid sequences set forth in
SEQ ID NOs: 26-28, 32-34, 38-40, 44-46, 50-52, and 53-55.
Preferably, the amino acid substitution, addition and/or deletion
occur at no more than three amino acid positions. More preferably,
the amino acid substitution, addition and/or deletion occur at no
more than two amino acid positions. Most preferably, the amino acid
substitution, addition and/or deletion occur at no more than one
amino acid position.
[0262] In another aspect, the present invention provides an anti-H5
monoclonal antibody light chain or a fragment thereof, comprising
the following CDRs: (i) one or more CDRs selected from SEQ ID NOs:
29-31; (ii) one or more CDRs selected from SEQ ID NOs: 35-37; (iii)
one or more CDRs selected from SEQ ID NOs: 41-43; (iv) one or more
CDRs selected from SEQ ID NOs: 47-49; (v) one or more CDRs selected
from SEQ ID NOs: 56-58. In one embodiment, the anti-H5 monoclonal
antibody light chain or a fragment thereof comprises three CDRs
having the amino acid sequences set forth in SEQ ID NOs: 29-31,
respectively. In another embodiment, the anti-H5 monoclonal
antibody light chain or a fragment thereof comprises three CDRs
having the amino acid sequences set forth in SEQ ID NOs: 35-37,
respectively. In another embodiment, the anti-H5 monoclonal
antibody light chain or a fragment thereof comprises three CDRs
having the amino acid sequences set forth in SEQ ID NOs: 41-43. In
another embodiment, the anti-H5 monoclonal antibody light chain or
a fragment thereof comprises three CDRs having the amino acid
sequences set forth in SEQ ID NOs: 47-49. In another embodiment,
the anti-H5 monoclonal antibody light chain or a fragment thereof
comprises three CDRs having the amino acid sequences set forth in
SEQ ID NOs: 56-58.
[0263] In another aspect, the CDRs contained in the anti-H5
monoclonal antibody light chains or fragments thereof of the
present invention may include one or more amino acid substitution,
addition and/or deletion from the amino acid sequences set forth in
SEQ ID NOs: 29-31, 35-37, 41-43, 47-49, and 56-58. Preferably, the
amino acid substitution, addition and/or deletion occur at no more
than three amino acid positions. More preferably, the amino acid
substitution, addition and/or deletion occur at no more than two
amino acid positions. Most preferably, the amino acid substitution,
addition and/or deletion occur at no more than one amino acid
position.
TABLE-US-00002 TABLE 2 The Amino Acid Sequences of the 7aa peptides
that bind to 8H5 mAb or 3C8 mAb. Monoclonal 7peptide Antibody
sequences Sequence No. 8H5 H G M L P V Y SEQ ID No: 59 P P S N Y G
R SEQ ID No: 60 P P S N F G K SEQ ID No: 61 G D P W F T S SEQ ID
No: 62 N S G P W L T SEQ ID No: 63 3C8 W P P L S K K SEQ ID No: 64
N T F R T P I SEQ ID No: 65 N T F R D P N SEQ ID No: 66 N P I W T K
L SEQ ID No: 67
[0264] The variants generated by amino acid substitution, addition
and/or deletion in the variable regions of the above described
antibodies or the above described CDRs maintain the ability of
specifically binding to subtype H5 of avian influenza virus. The
present inventions also include antigen-binding fragments of such
variants.
[0265] Monoclonal antibody variants of the invention may be made by
conventional genetic engineering methods. Nucleic acid mutations
may be introduced into the DNA molecules using methods known to a
person with ordinary skill in the art. Alternately, the nucleic
acid molecules encoding the heavy and light chain variants may be
made by chemical synthesis.
[0266] In another aspect, the screening method of the invention
comprises the steps of (i) culturing a peptide display library
under conditions suitable for peptide expression; (ii) contacting
the culture solution with monoclonal antibodies of the invention;
(iii) selecting the phage clones that specifically bind to said
monoclonal antibodies. The monoclonal antibodies used for the
screening may include without limitation the monoclonal antibodies
8H5, 3C8, 10F7, 4D1 and 3G4. Example 12 included herein describes
in detail an assay that successfully screened short peptides that
bind to the monoclonal antibodies of the invention using a peptide
phage display libraries.
TABLE-US-00003 TABLE 3 The sequences of the 12aa peptides that bind
to 8H5 mAb. Peptide section Amino Acid No. Sequence Base Sequence
121 MEPVKKYPTRSP ATGGAGCCGGTGAAGAAG (SEQ ID NO: 68)
TATCCGACGCGTTCTCCT (SEQ ID NO: 69) 122 ETQLTTAGLRLL
GAGACTCAGCTGACTACG (SEQ ID NO: 70) GCGGGTCTTCGGCTGCTT (SEQ ID NO:
71) 123 ETPLTETALKWH GAGACGCCTCTTACGGAG (SEQ ID NO: 72)
ACGGCTTTGAAGTGGCAT (SEQ ID NO: 73) 124 QTPLTMAALELF
CAGACGCCGCTGACTATG (SEQ ID NO: 74) GCTGCTCTTGAGCTTTTT (SEQ ID NO:
75) 125 DTPLTTAALRLV GATACTCCGCTGACGACG (SEQ ID NO: 76)
GCGGCTCTTCGGCTGGTT (SEQ ID NO: 77) 126 TPLTLWALSGLR
ACGCCGCTTACGCTTTGG (SEQ ID NO: 78) GCTCTTTCTGGGCTGAGG (SEQ ID NO:
79) 128 QTPLTETALKWH CAGACGCCTCTTACGGAG (SEQ ID NO: 80)
ACGGCTTTGAAGTGGCAT (SEQ ID NO: 81) 129 QTPLTMAALELL
CAGACGCCTCTGACTATG (SEQ ID NO: 82) GCGGCTCTTGAGCTTCTT (SEQ ID NO:
83) 130 HLQDGSPPSSPH CAGACGCCTCTGACTATG (SEQ ID NO: 84)
GCGGCTCTTGAGCTTCTT (SEQ ID NO: 85) 131 GHVTTLSLLSLR
GGGCATGTGACGACTCTT (SEQ ID NO: 86) TCTCTTCTGTCGCTGCGG (SEQ ID NO:
87) 132 FPNFDWPLSPWT TTTCCGAATTTTGATTGG (SEQ ID NO: 88)
CCTCTGTCTCCGTGGACG (SEQ ID NO: 89) 133 ETPLTEPAFKRH
GAGACGCCTCTTACGGAG (SEQ ID NO: 90) CCGGCTTTTAAGCGGCAT (SEQ ID NO:
91)
[0267] As used herein the term "Analyte" refers to the compound or
composition to be detected or measured and which has at least one
epitope or binding site. The analyte can be any substance for which
there exists a naturally occurring analyte specific binding member
or for which an analyte-specific binding member can be prepared.
e.g., carbohydrate and lectin, hormone and receptor, complementary
nucleic acids, and the like. Further, possible analytes include
virtually any compound, composition, aggregation, or other
substance which may be immunologically detected. That is, the
analyte, or portion thereof, will be antigenic or haptenic having
at least one determinant site, or will be a member of a naturally
occurring binding pair.
[0268] Analytes include, but are not limited to, toxins, organic
compounds, proteins, peptides, microorganisms, bacteria, viruses,
amino acids, nucleic acids, carbohydrates, hormones, steroids,
vitamins, drugs (including those administered for therapeutic
purposes as well as those administered for illicit purposes),
pollutants, pesticides, and metabolites of or antibodies to any of
the above substances. The term analyte also includes any antigenic
substances, haptens, antibodies, macromolecules, and combinations
thereof. A non-exhaustive list of exemplary analytes is set forth
in U.S. Pat. No. 4,366,241, at column 19, line 7 through column 26,
line 42, the disclosure of which is incorporated herein by
reference. Further descriptions and listings of representative
analytes are found in U.S. Pat. Nos. 4,299,916; 4,275,149; and
4,806,311, all incorporated herein by reference. In some
embodiments, the SCD or Test Device are configured to detect a
plurality of different analytes (e.g., FIG. 26).
[0269] Labeled Reagents
[0270] "Labeled reagent" refers to a substance comprising a
detectable label attached with a specific binding member (e.g.,
detection probe). The attachment may be covalent or non-covalent
binding, but the method of attachment is not critical to the
present invention. The label allows the label reagent to produce a
detectable signal that is related to the presence of analyte in the
fluid sample. The specific binding member component of the label
reagent is selected to directly bind to the analyte or to
indirectly bind the analyte by means of an ancillary specific
binding member, which is described in greater detail hereinafter.
The label reagent can be incorporated into the Test Device at a
site upstream from the capture zone, it can be combined with the
fluid sample to form a fluid solution, it can be added to the test
device separately from the test sample, or it can be predeposited
or reversibly immobilized at the capture zone. In addition, the
specific binding member may be labeled before or during the
performance of the assay by means of a suitable attachment
method.
[0271] "Label" refers to any substance which is capable of
producing a signal that is detectable by visual or instrumental
means. Various labels suitable for use in the present invention
include labels which produce signals through either chemical or
physical means. Such labels can include enzymes and substrates,
chromogens, catalysts, fluorescent or fluorescent like compounds
and/or particles, magnetic compounds and/or particles
chemiluminescent compounds and or particles, and radioactive
labels. Other suitable labels include particulate labels such as
colloidal metallic particles such as gold, colloidal non-metallic
particles such as selenium or tellurium, dyed or colored particles
such as a dyed plastic or a stained microorganism, organic polymer
latex particles and liposomes, colored beads, polymer
microcapsules, sacs, erythrocytes, erythrocyte ghosts, or other
vesicles containing directly visible substances, and the like.
Typically, a visually detectable label is used as the label
component of the label reagent, thereby providing for the direct
visual or instrumental readout of the presence or amount of the
analyte in the test sample without the need for additional signal
producing components at the detection sites.
[0272] Additional labels that can be utilized in the practice of
the invention include, chromophores, electrochemical moieties,
enzymes, radioactive moieties, phosphorescent groups, fluorescent
moieties, chemiluminescent moieties, or quantum dots, or more
particularly, radiolabels, fluorophore-labels, quantum dot-labels,
chromophore-labels, enzyme-labels, affinity ligand-labels,
electromagnetic spin labels, heavy atom labels, probes labeled with
nanoparticle light scattering labels or other nanoparticles,
fluorescein isothiocyanate (FITC), TRITC, rhodamine,
tetramethylrhodamine, R-phycoerythrin, Cy-3, Cy-5, Cy-7, Texas Red,
Phar-Red, allophycocyanin (APC), epitope tags such as the FLAG or
HA epitope, and enzyme tags such as alkaline phosphatase,
horseradish peroxidase, I.sup.2-galactosidase, alkaline
phosphatase, .beta.-galactosidase, or acetylcholinesterase and
hapten conjugates such as digoxigenin or dinitrophenyl, or members
of a binding pair that are capable of forming complexes such as
streptavidin/biotin, avidin/biotin or an antigen/antibody complex
including, for example, rabbit IgG and anti-rabbit IgG;
fluorophores such as umbelliferone, fluorescein, fluorescein
isothiocyanate, rhodamine, tetramethyl rhodamine, eosin, green
fluorescent protein, erythrosin, coumarin, methyl coumarin, pyrene,
malachite green, stilbene, lucifer yellow, Cascade Blue,
dichlorotriazinylamine fluorescein, dansyl chloride, phycoerythrin,
fluorescent lanthanide complexes such as those including Europium
and Terbium, Cy3, Cy5, molecular beacons and fluorescent
derivatives thereof, a luminescent material such as luminol; light
scattering or plasmon resonant materials such as gold or silver
particles or quantum dots; or radioactive material include
.sup.14C, .sup.123I, .sup.124I, .sup.125I, .sup.131I, Tc99m,
.sup.35S or .sup.3H; or spherical shells, and probes labeled with
any other signal generating label known to those of skill in the
art. For example, detectable molecules include but are not limited
to fluorophores as well as others known in the art as described,
for example, in Principles of Fluorescence Spectroscopy, Joseph R.
Lakowicz (Editor), Plenum Pub Corp, 2nd edition (July 1999) and the
6.sup.th Edition of the Molecular Probes Handbook by Richard P.
Hoagland.
[0273] A number of signal producing systems may be employed to
achieve the objects of the invention. The signal producing system
generates a signal that relates to the presence of an analyte
(i.e., target molecule) in a sample. The signal producing system
may also include all of the reagents required to produce a
measurable signal. Other components of the signal producing system
may be included in a developer solution and can include substrates,
enhancers, activators, chemiluminescent compounds, cofactors,
inhibitors, scavengers, metal ions, specific binding substances
required for binding of signal generating substances, and the like.
Other components of the signal producing system may be coenzymes,
substances that react with enzymic products, other enzymes and
catalysts, and the like. In some embodiments, the signal producing
system provides a signal detectable by external means, by use of
electromagnetic radiation, desirably by visual examination.
Exemplary signal-producing systems are described in U.S. Pat. No.
5,508,178.
[0274] In some embodiments, nucleic acid molecules can be linked to
the detection probe (e.g., antibody-linked oligonucleotides),
whereby the nucleic acid functions as a label by utilizing nucleic
acid labels. For example, a reagent solution or substrate comprised
in a SCD can comprise detection reagents--plurality of detection
and capture specific binding agents ("SBA")--comprising a plurality
of oligonucleotides functioning to provide a detectable signal,
whereby for a given subpopulation of SBAs (specific for a
particular analyte), conjugated oligonucleotides are pre-stained
with a different stain as compared to another subpopulation of
antibodies (specific for a different analyte) are nucleic acid
stains that bind nucleic acid molecules in a sequence independent
manner. Examples include intercalating dyes such as phenanthridines
and acridines (e.g., ethidium bromide, propidium iodide, hexidium
iodide, dihydroethidium, ethidium homodimer-1 and -2, ethidium
monoazide, and ACMA); some minor grove binders such as indoles and
imidazoles (e.g., Hoechst 33258, Hoechst 33342, Hoechst 34580 and
DAPI); and miscellaneous nucleic acid stains such as acridine
orange (also capable of intercalating), 7-AAD, actinomycin D,
LDS751, and hydroxystilbamidine. All of the aforementioned nucleic
acid stains are commercially available from suppliers such as
Molecular Probes, Inc. Still other examples of nucleic acid stains
include the following dyes from Molecular Probes: cyanine dyes such
as SYTOX Blue, SYTOX Green, SYTOX Orange, POPO-1, POPO-3, YOYO-1,
YOYO-3, TOTO-1, TOTO-3, JOJO-1, LOLO-1, BOBO-1, BOBO-3, PO-PRO-1,
PO-PRO-3, BO-PRO-1, BO-PRO-3, TO-PRO-1, TO-PRO-3, TO-PRO-5,
JO-PRO-1, LO-PRO-1, YO-PRO-1, YO-PRO-3, PicoGreen, OliGreen,
RiboGreen, SYBR Gold, SYBR Green I, SYBR Green II, SYBR DX,
SYTO-40, -41, -42, -43, -44, -45 (blue), SYTO-13, -16, -24, -21,
-23, -12, -11, -20, -22, -15, -14, -25 (green), SYTO-81, -80, -82,
-83, -84, -85 (orange), SYTO-64, -17, -59, -61, -62, -60, -63
(red). Other detectable markers include chemiluminescent and
chromogenic molecules, optical or electron density markers,
etc.
[0275] As noted above in certain embodiments, labels comprise
semiconductor nanocrystals such as quantum dots (i.e., Qdots),
described in U.S. Pat. No. 6,207,392. Qdots are commercially
available from Quantum Dot Corporation. The semiconductor
nanocrystals useful in the practice of the invention include
nanocrystals of Group II-VI semiconductors such as MgS, MgSe, MgTe,
CaS, CaSe, CaTe, SrS, SrSe, SrTe, BaS, BaSe, BaTe, ZnS, ZnSe, ZnTe,
CdS, CdSe, CdTe, HgS, HgSe, and HgTe as well as mixed compositions
thereof; as well as nanocrystals of Group III-V semiconductors such
as GaAs, InGaAs, InP, and InAs and mixed compositions thereof. The
use of Group IV semiconductors such as germanium or silicon, or the
use of organic semiconductors, may also be feasible under certain
conditions. The semiconductor nanocrystals may also include alloys
comprising two or more semiconductors selected from the group
consisting of the above Group III-V compounds, Group II-VI
compounds, Group IV elements, and combinations of same.
[0276] In some embodiments, a fluorescent energy acceptor is linked
as a label to a detection probe (i.e., binding moiety conjugated
with a detector molecule). In one embodiment the fluorescent energy
acceptor may be formed as a result of a compound that reacts with
singlet oxygen to form a fluorescent compound or a compound that
can react with an auxiliary compound that is thereupon converted to
a fluorescent compound. Such auxiliary compounds can be comprised
in buffers contained in an SCD and/or Test Device. In other
embodiments, the fluorescent energy acceptor may be incorporated as
part of a compound that also includes the chemiluminescer. For
example, the fluorescent energy acceptor may include a metal
chelate of a rare earth metal such as, e.g., europium, samarium,
tellurium and the like. These materials are particularly attractive
because of their sharp band of luminescence. Furthermore,
lanthanide labels, such as europium (III) provide for effective and
prolonged signal emission and are resistant to photo bleaching,
thereby allowing Test Devices containing processed/reacted sample
to be set aside if necessary for a prolong period of time.
[0277] Long-lifetime fluorescent europium(III) chelate
nanoparticles have been shown to be applicable as labels in various
heterogeneous and homogeneous immunoassays. See, e.g., Huhtinen et
al. Clin. Chem. 2004 October; 50(10): 1935-6. Assay performance can
be improved when these intrinsically labeled nanoparticles are used
in combination with time-resolved fluorescence detection. In
heterogeneous assays, the dynamic range of assays at low
concentrations can be extended. Furthermore, the kinetic
characteristics of assays can be improved by use of detection
antibody-coated high-specific-activity nanoparticle labels instead
of conventionally labeled detection antibodies. In homogeneous
assays, europium(III) nanoparticles have been shown to be efficient
donors in fluorescence resonance energy transfer, enabling simple
and rapid highthroughput screening. Heterogeneous and homogeneous
nanoparticle-label-based assays can be run with various sample
matrixes, e.g., serum, heparin plasma, and mucus.
[0278] In some embodiments, a label (e.g., fluorescent label)
disclosed herein, is comprised as a nanoparticle label conjugated
with biomolecules. In other words, a nanoparticle can be utilized
with a detection or capture probe. For example, a
europium(III)-labeled nanoparticle linked to monoclonal antibodies
or streptavidin (SA) to detect a particular analyte in a sample can
be utilized in practice of the present invention (e.g.,
nanoparticle-based immunoassay). The nanoparticles serve as a
substrate to which are attached the specific binding agents to the
analyte and either the detection (i.e., label) or capture
moiety.
[0279] In various embodiments of the invention, the label utilized
is a lanthanide metal. Lanthanides include but are not limited to
europium, samarium, terbium or dysprosium. Non-specific background
fluorescence has a decay time of only about 10 ns, so that such
background dies away before the sample fluorescence is measured.
Furthermore, Lanthanide-chelates have large Stokes' shifts. For
example, the Stokes' shift for europium is almost 300 nm. This big
difference between excitation and emission peaks means that the
fluorescence measurement is made at a wavelength where the
influence of background is minimal. In addition, the emission peak
is very sharp which means that the detector can be set to very fine
limits and that the emission signals from different lanthanide
chelates can be easily distinguished from each other. Therefore, in
one embodiment, one or more different lanthanides can be utilized
in the same assay.
[0280] Hard Standards. In one embodiment, a fluorescence reader is
configured to comprise an integrated or permanent standard ("hard
standard"). The term "hard standard" as referred to herein means
that the device for reading a test sample in methods of
detecting/quantifying one or more analytes comprises an internal,
integrated or permanent standard, against which samples labeled
with the same label as that used in the hard standard are read. In
one embodiment, the hard standard and the test label comprise a
lanthanide (e.g., Europium III) FIG. 33.
[0281] In one embodiment, the reader is an LED, comprising a lamp
emitting UV A (400 to 315 nm) part of the spectrum. Emission is in
the visible part of the spectrum. Some exemplary or conventional
LEDs or photodiodes are disclosed in U.S. Pat. Nos. 7,175,086,
7,135,342, and 7,106,442, the disclosure of each of which is
incorporated herein in its entirety.
[0282] In another embodiment, a reader comprises at least two hard
standards of different amounts (e.g., low and high concentration of
label), thus providing a two point check of the reader. For
example, two (2) lanthanide hard standards (e.g., Europium) are
mounted permanently on the reader slides and may be read during the
course of each test read. As such, the two hard standards can be
utilized to determine the lower detection limit (i.e., in a analyte
quantification assay or for determining lowest detection threshold
in qualitative assays). Here, fluorescence is read and plotted as
percentage of fluorescence (y axis) against concentration (x axis).
The straight line between the two reads for each of the hard
standards on such a plot allows measuring the intercept of noise
(no label) to give a measurement for the lowest detection
limit.
[0283] In some embodiments, a Test Device comprises a chamber
(compartment or liquid sac) that contains wash or running buffer,
which functions to remove unbound label, to reduce or eliminating
background noise. In various embodiments, devices comprising a hard
standard (s) provide accurate qualitative as well quantitative
measurement of analyte(s) present in a sample and labeled with
label that is the same as that used in the hard standard(s).
[0284] In some embodiments, hard standards are embedded or cast in
a polymer material, including glass, plastic, vinyl, or acrylic
FIG. 33B. Such embedded labels can be cast into appropriate
shapes/sizes. Alternatively, such hard standards can be cut to
appropriate sizes to be integrated into a reader. In one
embodiment, hard standards are cut in rectangular, square, oblong,
circular, or any polygon shape. In one embodiment, hard standards
are cut into rectangular shapes, comprising dimensions for height
of about 0.04, 0.045, 0.05, 0.055, 0.06, 0.065, 0.07, 0.075, 0.08,
0.085, 0.09, 0.095, 0.10, 0.11, 0.12, 0.125, 0.126, 0.127, 0.128,
0.129, 0.130, 0.135, 0.140, 0.150 inch; width of about 0.01, 0.02,
0.03, 0.035, 0.039, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.15,
0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75,
0.8, 0.85, 0.9, 0.95, or 1.0 inch; and lengths of about 0.01, 0.02,
0.03, 0.035, 0.039, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.15,
0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75,
0.8, 0.85, 0.9, 0.95, or 1.0 inch.
[0285] In one embodiment, a reader employing a hard standard as a
reference is utilized for normalizing readers across a population,
e.g., plotting subsequent reader performance against a
pre-determined "Gold Standard" reader as illustrated in the
following table:
TABLE-US-00004 TABLE 4 Gold Std. Test S0 1000 900 S1 5400 5000 S2
10200 11000 S3 19000 20000 S4 22000 23000 S5 50000 50000
[0286] Therefore, where y and x axis are Test reader and Gold
Standard measurements respectively, the lower limit of detection is
the intercept of the plotted line across the noise level (reading
with no label).
[0287] In one embodiment, a Test Device comprises different pRNAs
each patterned based on a specific analyte, a complementary SCD
comprises a plurality of capture antibody linked to cognate pRNAs
to those immobilized on the Test Device, and where said plurality
comprising different subpopulation of antibodies specific for
different analytes). Furthermore, the SCD reagent solution or
substrate (e.g., lyophilized solid substrate) comprise detection
probes, or a plurality of europium(III) labeled antibodies,
consisting of the same subpopulations of antibodies specific for
different analytes. Additional lanthanide labels that can be
practiced in the present invention are known in the art, such as
disclosed in U.S. Pat. No. 7,101,667. See also, e.g., Richardson F.
S., "Terbium(III) and Europium(III) Ions as Luminescent probes and
Stains for Biomolecular Systems," Chem. Rev., 82:541-552
(1982).
[0288] Therefore, depending on choice of labels, in some
embodiments a signal is viewable by the unaided eye, while in other
embodiments, a reader instrument is utilized in the practice of the
present invention.
[0289] Capture Moieties.
[0290] In some embodiments, one member of a pair of complementary
capture moieties will be bound to analyte-specific binding agent
and the other member is immobilized on a line or spot FIG. 8A,
respectively. As referred to herein, the terms "capture moiety"
means a binding moiety that is specific for a partner or
complementary capture moiety (e.g., pRNA specific for complementary
pRNA, or avidin/streptavidin-bioin). In one embodiment, a Test
Device comprises a combination of different capture moieties,
wherein said capture moieties are comprise of different substances
in number and/or type. For example, a test strip disposed in a Test
Device can comprise one or more addressable test lines comprising
pRNA, antibodies and specific binding members (e.g.,
avidin/biotin). Therefore, in various embodiments where two of the
same type of capture moieties are disposed on a test strip, each
will be specific for a different complementary partner molecule.
For example, for two pRNA addressable lines, each line will
comprise pRNA having different sequences which will specifically
bind to pRNA of complementary sequences which are themselves bound
to antibodies targeting a different analyte (e.g., Influenza A
versus Influenza B). Furthermore, in such a configuration,
additional test lines can comprise different capture moieties
(e.g., antibodies or avidin) which themselves bind their cognate
partner molecules. Thus in one embodiment, a test strip comprise 2,
3, 4, 5, 6, 7 or 8 addressable test lines, which can comprise any
combination of 1, 2, 3 or 4 different types of capture
moieties.
[0291] Where multiplexed (i.e., multianalyte) detection is desired,
a plurality of capture moieties is utilized, antibodies specific
for one analyte(s) will comprise a member of one specific pair of
complementary capture moieties and antibodies that specifically
bind a second and different analyte(s) will comprise a member of a
second and different specific pair of complementary capture
moieties, and so on. Thus, in one embodiment, a plurality of
different analyte(s) can be detected, where the cognate member of a
pair of capture moieties is immobilized in a discrete location on a
test membrane comprised in the test implement. For example, a
plurality of antibodies in an SCD is comprised of antibodies
targeting different influenza virus strains and/or subtypes, where
said antibodies are comprised of pairs of detection
antibody-capture antibody and where the capture antibody has a
specific capture moiety. Further, each population of antibodies in
the plurality of antibodies is defined by the particular target
analyte to which the antibody binds. Thus, all capture antibodies
directed to one specific target analyte will have the same capture
moiety, for which cognate/complementary capture moieties are
disposed in the Test Device.
[0292] In various embodiments, capture moieties are comprised of an
oligonucleotide, avidin, streptavidin, pyranosyl RNA (pRNA),
antigen-antibody binding pair selected for high affinity, aptamer
or a combination thereof. In further embodiments, an
oligonucleotide is DNA or RNA. Moreover, in some embodiments a
combination of different capturemoieties are utilized in the same
detection system of the invention. For example, a capture moiety
pair for one specific analyte comprises an oligonucleotide pair,
while a capture pair for a different analyte comprises a capture
moiety pair comprising pRNA, or avidin or streptavidin, etc. In
other embodiments, a combination of different types of capture
moieties is utilized in devices and assays of the invention to
detect multiple analytes (e.g., plurality of capture antibodies
whereby each population of capture antibodies specific for a single
type of target analyte is linked to one type of capture moieties,
and other analyte-specific antibodies are linked to others, such as
aptamers, pRNA or streptavidin, etc.)
[0293] In one embodiment, all capture moieties are pRNAs, with
multiple pairs of pRNA capture moiety and pRNA partner capture
moiety (e.g., one is conjugated to a specific binding agent and the
cognate pRNA is immobilized on the lateral flow membrane).
[0294] pRNA
[0295] In one aspect of the invention, combinations of
complementary pyranosyl RNA (pRNA) sequences are incorporated in
the SCD/Test Devices of the invention enabling simultaneous
specific detection of multiple analytes FIG. 32. In various
embodiments one of a pair of homologous pRNA sequences is
immobilized in a specific stripe or test zone in the Test Device,
while the other of the pair of homologous pRNA sequences is
conjugated to a binding moiety, which specifically binds to a
target analyte. Thus in one embodiment, a target analyte is
captured by the immobilized pRNA through interaction of the
immobilized pRNA sequence with its binding pair pRNA. The analyte
is detected by a detector molecule conjugated to a second binding
moiety 1301 also specific for the same target analyte.
[0296] In some embodiments, pRNA binding partners are selected from
but not limited to the following pRNAs
TABLE-US-00005 Table 5 Name 4'-2' SEQ ID NO: 102a10-3-NH2
TAGAACGAAG 92 102b10-3-NH2 CTTCGTTCTA 93 119a10-1-NH2 TCAGTGGATG 94
119b10-1-NH2 CATCCACTGA 95 3a10-1-NH2 GTATTGCGAG 96 3b10-1-NH2
CTCGCAATAC 97 102a8-2-NH2 AACGATTC 98 102b8-2-NH2 GAATCGTT 99
119a8-1-NH2 AGTGGATG 100 119b8-1-NH2 CATCCACT 101 3a8-1-NH2
GTATTGCG 102 3b8-1-NH2 CGCAATAC 103 4a8 ATGCCTTC 104 4b8 GAAGGCAT
105 5a8 TGATGGAC 106 5b8 GTCCATCA 107 6a8 CAGTAGTG 108 6b8 CACTACTG
109 7a8 TTCCTGAG 110 7b8 CTCAGGAA 111 8a8 GACTCTCT 112 8b8 AGAGAGTC
113 all oligos with 4'-C12 amino and 2'-hexanol groups
[0297] Therefore, where a plurality of capture probes (e.g.,
antibody linked to pRNA), each capture probe is linked to a capture
moiety, for which a cognate capture probe is immobilized in a
predetermined location on a test strip 310, 903, 904, 905, 906
comprised in a Test Device FIGS. 3, 26, 27. See also, FIG. 39-42,
which illustrate the substantial sensitivity of pRNA capture.
[0298] Therefore, a sequence immobilized on a test strip at a
specific addressable line will bind specifically to the
complimentary pRNA conjugated with anti-analyte binding moieties
(e.g., anti-virus antibody). The efficacy of such specificity is
demonstrated in Example 6 (e.g., FIG. 30).
[0299] In some embodiments, a Test Device incorporating one or more
pRNA binding pairs, provides sensitivity of about 0.02, 0.03, 0.04,
0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7,
0.8, 0.9, 1.0, 1.2, 1.5, 1.7, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0,
5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 15, 20, 30, 40
or 50 ng/mL.
[0300] In some embodiments pRNA is attached to a membrane (i.e.,
test strip) utilizing a protein linker For example, pRNA can be
conjugated to a hydrophilic protein. In one embodiment, the linker
protein has a molecular weight of at least from about 500, 600,
700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500,
5000, 5500, 6000, 6500, 7500, 8000, 9000, 10000, 20000, 30000,
40000, 50000, 60000, 70000, 80000, 90000, 100000, 110000, 120000,
130000, 140000, 150000, 160000, 170000, 180000, 190000, 200000,
225000, 250000, 300000, 350000 to about 450000. Such a linker can
range in size from about s about 5 to 10, 6 to 11, 7 to 12, 8 to
13, 9 to 14, 10 to 15, 11 to 16, 12 to 17, 13 to 18, 14 to 19, 15
to 20, 16 to 21, 17 to 22, 18 to 23, 19 to 24, 20 to 25, 21 to 26,
22 to 27, 23 to 28, 24 to 29, 25 to 30, 35, 40, 45 or 50 AA long.
The linker can be a peptide or polypeptide. In one embodiment, the
linker is BSA or IgG.
[0301] In one embodiment, pRNA is coupled to a hydrophilic
protein/peptide via a covalent bond between the pRNA molecule and
the hydrophilic protein. A solution containing the pRNA-protein
complex is applied to defined regions on a test membrane (e.g.,
nitrocellulose), whereby the protein anchor binds to the membrane
in a an irreversible. The pRNA is then available for use in the
assay. In one embodiment, the anchor/linker protein is a
hydrophilic protein and the test membrane is nitrocellulose.
[0302] As indicated previously, a Test Device can comprise
addressable test lines utilizing different types of capture
moieties (e.g., a combination of antibodies, nucleic acids, pRNA,
avidin/streptavidin/biotin). In one embodiment (e.g., FIG. 32), at
least one addressable line or specific capture zone 1305, 1312
comprises a pRNA 1304, 1311 sequence that is bound to a solid
support 1308 (e.g., nitrocellulose, polystyrene, glass, plastic,
metal, etc.) and is specific in binding for a homologous pRNA
sequence conjugated to a second binding moiety 1302, 1310 that is
specific for a particular target analyte 1306. Furthermore, a first
binder molecule is a partner to the first binder molecule and said
second binder molecule is conjugated to a detector molecule 1301,
1309 (e.g., fluorescent label). As demonstrated in Example 6
herein, pRNA is effectively immobilized and can specifically bind a
complementary sequence to allow detection of an analyte. As such,
pRNA provides a novel tool for detecting multiple analytes based on
the great specificity and avidity of pRNA molecules.
[0303] In one embodiment, pRNA molecules are disposed on 1, 2, 3,
4, 5, 6, or 7 distinct addressable lines (i.e., capture zones) in a
Test Device. In other embodiments, pRNA are utilized in combination
with antibodies, nucleic acid binding pairs, and
avidin/streptavidin, digoxin/anti-digoxin. For example, a Test
Device comprise a test strip with 5 addressable test/capture zones,
wherein each test zone is specific for a distinct analyte (e.g.,
influenza type A or B) and/or subtype (e.g., influenza A pandemic
and non-pandemic subtypes). Thus, for example, two test zones
utilized pRNA binding, while one utilizes
strepatvidin/avidin-biotin, while another utilizes fixed antibody,
and yet another utilizes DNA/RNA. In this example, for each type of
binding system, there is a complementary binding partner that is
specific for the target analyte which is "captured" on the
particular test zone. Further, as described herein, the analyte is
also bound by a binder that is conjugated to a detector
molecule/label (e.g., fluorescence label). As such, the once the
analyte-detector- binding partner complex flows through the Test
Device, the complex is captured at the distinct test zone having
the immobilized binding partner.
[0304] As such a central aspect of the present SCD/Test Devices of
the invention is that they can be configured to detect multiple
analytes, including cells, cell components (e.g., cell markers,
cell surface markers), proteins (e.g., enzymes) and the such.
[0305] In one embodiment, SCD/Test Devices of the invention can be
used in a method to assay for any pathogenic conditions for which
particular corresponding analytes are known or are identified in
future. The SCD and Test Device can be configured to provide any
combination of the detection reagents disclosed herein (e.g., pRNA,
nucleic acids, antibodies, specific binding partners, e.g.,
avidin/biotin). For example, multiple analytes corresponding to
myocardial infarction (MI) can be identified in
detecting/diagnosing MI. Markers for various conditions are known
in the art, such as for cardiac markers disclosed in U.S. Pat. Nos.
5,604,105; 5,710,008; 5,747,274, 5,744,358 and 5,290,678, the
disclosures of each of which is incorporated by reference herein in
its entirety.
[0306] In one embodiment, the devices of the invention provide a
three in one assay for protein based cardiac markers of myocardial
infarction would be designed (See Example 10).
[0307] In one embodiment, a sample is applied in a way that enables
mixing with a mixture (in series or in parallel) of a first binder
conjugated to a detector reagent (e.g., combinations of gold
microparticles and fluorescent label) and with a second binder
conjugated to a first member of a pRNA homologous binding pair
which may or may not contain materials to enable an immune binding
reaction and/or specific binding of pRNA homologous binding
pairs.
[0308] If desired a pre-incubation is designed into the system
whereby the first and second binders are allowed to attach to and
bridge the analyte forming complex, and such a step can be during
the flow of reagents to the test or capture zone. Alternatively,
the flow may be stopped to allow for the reaction to come closer to
completion. In yet another embodiment, the reagent mix in an SCD of
the invention prior to be applied to a Test Device.
[0309] A mixture flows via capillary action or hydrostatic pressure
from any of several mechanism or other non-capillary action along
the surface of or within a matrix of a solid material/substrate
(e.g., test strip) allowing the first and second homologous binding
pairs to come into contact as the reaction mixture passes
over/through the test/capture zone. If the first and second binder
have bridged the analyte FIG. 32 the detector reagent (e.g.,
Europium) will accumulate at the test/capture zone yielding a
signal that can be interpreted visually or using an instrument
reader.
[0310] In embodiments where multiple different analytes are sought
to be detected, different detector reagents are used for each
analyte and each different analyte is captured in a single
detection/capture zone. An insturment reader is used to distinguish
between the signal for each analyte. In one embodiment, the
instrument reader can detect a single to provide for qualitative
and quantitative measurement.
[0311] In another embodiment, unique pRNA homologous binding pairs
are utilized for each target analyte.
[0312] In another embodiment a wash buffer or reagent buffer is
released through the flow path (as described herein) where said
buffer can comprise a reagent that yields the release of energy in
any of several forms that can be detected with an instrument,
reader or visually. For example, the reaction can result in release
of light, electrons or can require the input of electrons plus a
substrate yielding the release of light. Such reactions are known
to one of skill in the art.
[0313] Such reactions can result in enablement of a fluorescent
compound to fluoresce after coming into contact with the
running/wash buffer, resulting in release of electrons, protons,
neutrons, which can be detected by an instrument or reader as
described herein.
[0314] Aptamers.
[0315] In some embodiments, capture moieties are aptamer molecules
that are can be interchangeably utilized with a capture probe or as
an immobilized capture moiety included in the Test Device axial
flow membrane. Aptamers include nucleic acids that are identified
from a candidate mixture of nucleic acids. In a preferred
embodiment, aptamers include nucleic acid sequences that are
substantially homologous to the nucleic acid ligands isolated by
the SELEX method. Substantially homologous is meant a degree of
primary sequence homology in excess of 70%, most preferably in
excess of 80%. The "SELEX" methodology, as used herein, involves
the combination of selected nucleic acid ligands, which interact
with a target analyte in a desired action, for example binding to a
protein, with amplification of those selected nucleic acids.
Optional iterative cycling of the selection/amplification steps
allows selection of one or a small number of nucleic acids, which
interact most strongly with the target antigen/biomarker from a
pool, which contains a very large number of nucleic acids. Cycling
of the selection/amplification procedure is continued until a
selected goal is achieved. The SELEX methodology is described in
the following U.S. patents and patent applications: U.S. patent
application Ser. No. 07/536,428 and U.S. Pat. Nos. 5,475,096 and
5,270,163.
[0316] Infectious Agents.
[0317] In various embodiments of the present compositions and
methods, an infectious agent can be any pathogen including without
any limitation bacteria, yeast, fungi, virus, eukaryotic parasites,
etc. In some embodiments, the infectious agent is influenza virus,
parainfluenza virus, adenovirus, rhinovirus, coronavirus, hepatitis
viruses A, B, C, D, E, etc, HIV, enterovirus, papillomavirus,
coxsackievirus, herpes simplex virus, or Epstein-Barr virus. In
other embodiments, the infectious agent is Mycobacterium,
Streptococcus, Salmonella, Shigella, Staphylcococcus, Neisseria,
Clostridium, or E. coli. It will be apparent to one of skill in the
art that the compositions and methods of the invention are readily
adaptable to different infectious agents, by utilizing a different
panel of binding agents (e.g., antibodies) that are specific for
type(s) or subtype(s) of an infectious agent(s).
[0318] Usually the general type of an infectious agent can be the
genus type of an infectious agent or any primary or first instance
typing or identification of an infectious agent. A subtype of an
infectious agent can be the species or strain type of an infectious
agent or any secondary or subsequent typing of an infectious agent.
According to the present invention, identification of the general
type or subtype of an infectious agent can be carried out via
various suitable test set ups. For example, identification of the
general type of an infectious agent can include one or more
screening tests for 1) a specific general type of an infectious
agent, 2) certain desired or selected general types of an
infectious agent, or 3) all or substantially all relevant general
types of an infectious agent, or a combination thereof. Similarly
identification of the subtype of an infectious agent can include
one or more screening tests for 1) one or more specific subtypes of
an infectious agent, 2) one or more specific subtypes of a
particular general type of an infectious agent, 3) one or more
specific subtypes of an infectious agent selected based on
additional information associated with the subject being tested,
e.g., one or more suspected or expected subtypes for a particular
population or geographic location or 4) one or more potentially
pandemic or epidemic subtypes of an infectious agent that is
identical to or associated with the infectious agent tested for the
general type, or a combination thereof.
[0319] According to another aspect of the present invention, the
method provided by the present invention can optionally or
additionally include identification of the general and/or
subtype(s) of a second infectious agent that is closely related to
the first infectious agent, or alternatively the infection of the
second infectious agent is associated or likely coupled with the
infection of the first infectious agent. In one embodiment, the
method provided by the present invention includes identification of
the general and subtype(s) of a virus as well as a bacterium. For
example, HIV infection can be associated with certain bacterial
infection therefore it will be useful to identify the general and
subtyp(s) of HIV as well as Mycobacterium and/or Pneumocystis
carina. Specifically the method provided by the present invention
includes identification of HIV and one or more species of
Mycobacterium and/or Pneumocystis carina.
[0320] In another embodiment, the method provided by the present
invention includes identification of the general and subtype(s) of
a first virus as well as a second virus. For example, the method
provided by the present invention can include identification of the
general and subtype(s) of HIV as well as hepatitis virus or
alternatively the general and subtype(s) of HIV as well as the
general type of hepatitis virus or certain strains of hepatitis
virus. In one embodiment, the methods and compositions of the
invention can be utilized in assays to detect E. coli 0157 (a very
dangerous, often fatal infectious strain) in the presence of other
enteric or infective strains. Another example would be in testing
patients for influenza infection, where mutation or variation of
the strains within subtypes is known to occur and some forms of
influenza are far more pathogenic than others. A further example is
detection of different types of HIV, for example HIV-1 and HIV-2.In
one aspect, identification of the general type of human
immunodeficiency virus (HIV) can include screening for the presence
of HIV whereas identification of the subtype of HIV can include
screening for HIV-1, HIV-2, and/or other subtypes of HIV. Similarly
identification of the general type of herpes virus such as simplex
virus (HSV) can include screening for the presence of HSV whereas
identification of the subtype of HSV can include screening for HSV
type 1 and/or HSV type 2 or for Epstein-Barr virus and subtypes of
EBV.
[0321] In still another particular aspect, identification of the
general type of enterovirus can include screening for the presence
of one or more enteroviruses, e.g., poliovirus, coxsackievirus,
echovirus, designated enterovirus, etc. whereas identification of
the subtype of enterovirus can include screening for poliovirus,
e.g., serotype 1-3, coxsackievirus A, e.g., serotype 1-22 and 24,
coxsackievirus B, e.g., serotype 1-6, echovirus, e.g., serotype
1-9, 11-27, 29-31, and designated enterovirus, e.g., enterovirus
68-71, etc.
[0322] In general, with respect to a bacterial infectious agent
identification of the general and subtype of a bacterial infectious
agent includes screening for the genus and one or more species or
strains of the bacterial infectious agent that are relevant to the
infection and/or the agent's antimicrobial resistance. In one
embodiment, identification of the general and subtype of a
bacterial infectious agent includes screening for Mycobacterium and
one or more species of Mycobacterium including without limitation
tuberculosis, avium, bovis, chelonei, fortuitum, intracellulare,
kansasii, leprae, etc. In another embodiment, identification of the
general and subtype of a bacterial infectious agent includes
screening for Salmonella and one or more species of Salmonella
including without limitation typhi, enteritidis, etc. In yet
another embodiment, identification of the general and subtype of a
bacterial infectious agent includes screening for Shigella and one
or more species of Shigella including without limitation
dysenteriae. In yet another embodiment, identification of the
general and subtype of a bacterial infectious agent includes
screening for Streptococcus and one or more species of
Streptococcus including without limitation pneumonia, pyogenes
(group A), etc. In still yet another embodiment, identification of
the general and subtype of a bacterial infectious agent includes
screening for E coli and one or more strains of E coli including
without limitation enterotoxigenic strains.
[0323] According to the present invention, screening test(s) used
for the identification of the general and subtype(s) of an
infectious agent can be any suitable tests known or later
discovered in the field. For example, the screening tests of the
present invention can be a non-nucleic acid based test including
without any limitation a protein, peptide, amino acid, ligand, or
chemistry based test. In one embodiment, the screening test of the
present invention is a test based on the presence or absence of one
or more structural proteins of an infectious agent, e.g.,
glycoproteins, envelop proteins, polysaccharides, etc. In another
embodiment, the screening test of the present invention is a test
based on the presence or absence of one or more antigens or
epitopes, or antibodies to an infectious agent. In yet another
embodiment, the screening test of the present invention is a test
based on the presence or absence of one or more substances that is
released or metabolized by an infectious agent. In still yet
another embodiment, the screening test of the present invention is
a test based on the presence or absence of one or more substances
derived from a host cell associated with or generated by the
infection of an infectious agent.
[0324] In one particular aspect, the non-nucleic acid based
screening test of the present invention includes the commonly used
ligand binding or immunoassays, e.g., ligand or
immunochromatographic assays. Many of these assays are based on the
highly specific interactions between specific binding pairs.
Examples of such binding pairs include antigen/antibody,
hapten/antibody, lectin/carbohydrate, apoprotein/cofactor and
biotin/(strept)avidin. Furthermore, many of these assays involve
devices (e.g., solid phase, lateral-flow test strips, flowthrough
tests) with one or more of the members of a binding pair attached
to a mobile or immobile solid phase material such as latex beads,
glass fibers, glass beads, cellulose strips or nitrocellulose
membranes (U.S. Pat. Nos. 4,703,017; 4,743,560; 5,073,484).
[0325] In one embodiment, the methods and apparatus of the
invention are utilized to detect or identify an influenza type A
subtype and/or influenza type B and/or influenza type C.
[0326] Influenza virus belongs to the genus orthomyxovirus in the
family of Orthomyxoviridae. ssRNA enveloped viruses with a helical
symmetry. Enveloped particles 80-120nm in diameter. The RNA is
closely associated with the nucleoprotein (NP) to form a helical
structure. The genome is segmented, with 8 RNA fragments (7 for
influenza C). There are 4 principle antigens present, the
hemagglutinin (H), neuraminidase (N), nucleoprotein (NP), and the
matrix (M) proteins. The NP is a type-specific antigen which occurs
in 3 forms, A, B and C, which provides the basis for the
classification of human and non-human influenza viruses. The matrix
protein (M protein) surrounds the nucleocapsid and makes up 35-45%
of the particle mass. Furthermore, 2 surface glycoproteins are seen
on the surface as rod-shaped projections. The haemagglutinin (H) is
made up of 2 subunits, H1 and H2. Haemagglutinin mediates the
attachment of the virus to the cellular receptor. Neuraminidase
molecules are present in lesser quantities in the envelope. The
antigenic differences of the hemagglutinin and the neuraminidase
antigens of influenza A viruses provide the basis of their
classification into subtypes. e.g., A/Hong Kong/1/68 (H3N2)
signifies an influenza A virus isolated from a patient in 1968, and
of subtype H3N2.
[0327] In various embodiments, the methods and apparatus of the
invention are directed to detecting or identifying influenza virus
type A which is defined by HxNy where x is 1-16 and y is 1-9, or
any combination of xy thereof. For example, in one embodiment, the
methods and apparatus of the invention is utilized to detect
influenza A subtype H1N5. Thus, a plurality of detection probes and
capture probes targeting different subtypes of influenza virus are
disposed in an SCD of the invention. In one embodiment, the assay
is utilized to detect Influenza A (with subtypes H1/H3, and a
pandemic subtype H5) and Influenza B.
[0328] In particular, the general type of an influenza virus can be
any type designated based on antigenic characteristics of the
nucleoprotein and matrix protein antigens, e.g., type A, B, or C
influenza virus, whereas the subtype can be one or more subdivided
types of an influenza virus on the basis of an antigen, e.g. one or
more subtypes of influenza type A or type B virus characterized by
a surface antigen such as hemagglutinin (H) or neuraminidase
(N).
[0329] In one embodiment, identification of the general type of
influenza virus includes screening for type A, type B, and/or type
C influenza virus whereas identification of the subtype of
influenza virus, e.g., type A virus includes screening for one or
more expected subtypes of type A, e.g., subtypes expected to be
present in the population at the time of testing, and optionally
one or more suspected subtypes, e.g., subtypes under surveillance
for an outbreak such as epidemic or pandemic outbreak. In another
embodiment, identification of the general type of influenza virus
includes screening for type A and type B influenza virus whereas
identification of the subtype of influenza virus, e.g., type A
virus includes screening for one or more subtypes used for the
production of the influenza vaccine, e.g., current vaccine
subtypes(s) or strain(s) for the testing season including subtypes
and/or strains expected to be in circulation during the next
influenza season. In yet another embodiment, identification of the
general type of influenza virus includes screening for type A and
type B influenza virus whereas identification of the subtype of
influenza virus, e.g., type A includes screening for one or more
subtype(s) or strain(s) used for the production of the influenza
vaccine and one or more subtype(s) or strain(s) suspected for the
cause of a pandemic outbreak, e.g., one or more avian subtype(s) or
strain(s) such as H5N1 or the derivatives thereof.
[0330] In yet another embodiment, identification of general type of
influenza virus includes screening for type A and type B influenza
virus whereas identification of the subtype of an influenza virus,
e.g., type A includes screening for one or more common or expected
subtypes in circulation including, without any limitation, a)
H.sub.1 and H.sub.3, b) H.sub.1, H.sub.3, and H.sub.2, c) H.sub.1,
H.sub.2, H.sub.3, and H.sub.9, d) H.sub.1, H.sub.3, and N.sub.1, e)
H.sub.1, H.sub.3, N.sub.1, and N.sub.2, f) H.sub.1, H.sub.3, and
N.sub.2, g) N.sub.2, and h) N.sub.1 and N.sub.2. For example, a
screening test for the subtype identification of type A influenza
virus can be directed to the identification of the presence of any
one of the subtypes listed in the subtype group of a), b), c), d),
e), f), g), or h) e.g., without necessarily identifying the
presence of a specific subtype in a subtype group. Alternatively
screening test for the subtype identification of type A influenza
virus can be directed to the identification of the presence or
absence of each and everyone of the subtypes listed in a), b), c),
d), e), f), g), or h) e.g., identifying the presence of a specific
subtype in a subtype group.
[0331] In still another embodiment, identification of general type
of influenza virus includes screening for type A and type B
influenza virus whereas identification of the subtype of an
influenza virus, e.g., type A includes screening for one or more
pandemic or un-expected subtypes in circulation including, without
any limitation, a) H.sub.5, b) H.sub.5 and H.sub.7, c) H.sub.5,
H.sub.7, and H.sub.9, d) N.sub.2, N.sub.7, and N.sub.8, e) H.sub.5
and N.sub.2, f) H.sub.5 and N.sub.1, g) H.sub.5 and N.sub.8, h)
H.sub.5, N.sub.8, H.sub.7, and N.sub.7, i) H.sub.5, H.sub.7,
H.sub.9, N.sub.7, and N.sub.8. For example, a screening test for
the subtype identification of type A influenza virus can be
directed to the identification of the presence of any one of the
subtypes listed in the subtype group of a), b), c), d), e), f), g),
h), or i) e.g., without necessarily identifying the presence of a
specific subtype in a subtype group. Alternatively screening test
for the subtype identification of type A influenza virus can be
directed to the identification of the presence or absence of each
and everyone of the subtypes listed in a), b), c), d), e), f), g),
h), or i), e.g., identifying the presence of a specific subtype in
a subtype group.
[0332] In another particular aspect, the general type of hepatitis
virus can be A, B, and C virus with each virus possibly having
several subtypes including mutant strains. In one embodiment,
identification of the general type of hepatitis virus includes
screening for A, B, and/or C hepatitis virus whereas identification
of the subtype of hepatitis virus includes screening for subtypes
or mutant strains of A, B, and C hepatitis viruses, respectively.
In another embodiment, identification of the general type of
hepatitis virus includes screening for hepatitis B virus whereas
identification of the subtype of hepatitis virus includes screening
for one or more subtypes and/or mutant strains of hepatitis B
virus. In yet another embodiment, identification of the general
type of hepatitis virus includes screening for hepatitis C virus
whereas identification of the subtype of hepatitis virus includes
screening for one or more of subtypes 1-9 of type C hepatitis
virus.
[0333] In various embodiments, methods and apparatus of the
invention can detect one or more different infectious agents. For
example, a sampling implement can comprise a plurality of different
antibodies, wherein multiple subgroups of antibodies are present,
whereby each subgroup of antibodies specifically binds a different
infectious agent. For example, a plurality of antibodies can
comprise 2, 3, 4, 5, 6, 7, 8, 9 or 10 subgroups, wherein each
subgroup of antibodies in the plurality of antibodies specifically
binds a different infectious agent. In some embodiments, methods
and apparatus of the invention detect a pandemic and non-pandemic
infectious agent. In one embodiment, the pandemic and non-pandemic
infectious agents are influenza virus.
[0334] The explosive nature of epidemic influenza and the specific
clinical features of this disease have given reliable
epidemiological records of this infection since the beginning of
the nineteenth century. Several epidemics were recorded during the
nineteenth century but the first pandemic was not accurately
recorded until 1889-92. A second pandemic, probably originating in
Europe, occurred in 1918-19, and is known as Spanish Influenza,
which was responsible for 20-25 million deaths, principally in
young adults.
[0335] Pandemics continued to occur regularly after the Spanish
influenza, in 1932-33, 1947-48, 1957 and 1968. The next pandemic is
thought to be overdue. These latter pandemics resembled the
pandemic of 1890, affecting millions of people with a mild URTI and
a small number of deaths. The H1N1 (swine) viruses probably
appeared in 1918 and continued to circulate until 1957, at which
time they were supplanted by the H2N2 (Asian) viruses. The H2N2
viruses were prevalent until 1968, when H3N2 (Hong Kong) strains
appeared. The H1N1 virus reappeared in 1977 and did not replace the
H3N2 subtype and both subtypes continued to cocirculate. Therefore,
it is imperative that subjects are screened in an effective and
accurate manner to determine with what strain and/or subtype an
individual is infected. Furthermore, in some circumstances such
sample collection and processing will necessarily occur in a
point-of-care setting (e.g., in the field, without large numbers of
subjects to sample and process, and with limited man power to
effect such sampling).
[0336] As such, in one embodiment, the methods and apparatus of the
invention are utilized in processing a large number of samples, in
a point-of-care setting, where test results may be visualized
(i.e., read) some period of time after the test is complete. For
example, the period of time can be 30 minutes, 1 hour, 1.5 hour, 2
hours, 2.5 hours, 3 hours, 4 hours or 5 hours. In some embodiments,
methods and apparatus in conjunction with the reagents disclosed
herein provide high sensitivity and specificity where the
fluorescent result can be read with very similar results over a
long period of time. Thus, in some embodiments biological samples
can be collected and processed, but set aside to be read a
significant time later, which is greatly advantageous in
point-of-care settings or where a large number of samples are
collected with limited manpower or time to further process
samples.
[0337] In yet another aspect of the invention, the compositions and
methods of the invention are directed to detecting any one or more
analytes present in a sample. As indicated above, for example, by
utilizing different binding moieties that specifically bind markers
associated with a condition, one or more analytes associated with
MI can be detected. Therefore, an SCD and Test Device can comprise
the necessary reagents to diagnose a disease or pathological
condition, other than infectious diseases.
[0338] In some embodiments, the one or more analytes are markers
associated with a pathological condition or disease. In another
embodiment, the one or more analytes are polypeptides associated
with a nutrional state or condition. In yet other embodiments, the
one or more analytes are cell markers associated with cell cycle
and growth. In another embodiment, the one or more analytes are
associated with cell proliferation and differentiation. In one
embodiment, cell markers are associated with cancer.
EXAMPLES
Example 1
Assay Volume and Chasing Buffer
[0339] To explore the optimal assay sample volume that will give
the best sensitivity and to investigate if a chasing buffer will
also increase the sensitivity and performance of the test
strip.
[0340] Materials: Nitrocellulose Membrane: Millipore HiFlow 135
membrane, 2.5 cm in width. Cat. No. SHF1350425, Lot No. R68N46849,
Code No. RK04414, roll No. 04OLI. Membrane was striped with
monoclonal anti-H5N1 antibody, clone 3C8 at 1.0 mg/ml. Control line
was striped with 1.5 mg/ml rabbit anti-mouse antibody. Wicking pad:
0.05% Tween-20 treated polyester pad, Alhstrom grade 6613. Prepared
Absorbent pad: Ahlstrom Grade 222 paper, 3.5 cm in width, purchased
from Fisher, Cat. #2228-1212, lot #6150502. Anti-H5 3G4 gold
conjugate was prepared by Nanogen POC at Toronto, Canada, OD of 3G4
gold conjugate at maximal absorbent peak was 112.54. Extraction
buffer: 50 mM Tris-Cl, pH 8.15, 0.75 M NaCl, 1% BSA, 0.1% pluronic
F68, 0.05% digested casein, 2 mM TCEP and 0.02% NaN.sub.3.
Recombinant H5 hemagglutinin (0 2 mg/mL) was provided by Nanogen
POC, Toronto, Canada
[0341] Protocol: Preparation of lateral flow test strip.
Nitrocellulose membrane striped with antibodies was laminated on
plastic card with 1-2 mm overlap with wicking pad and absorbent
pad. No cover tape was applied on any components. Strips were cut
in 5 mm in width.
[0342] Test: Extraction buffer ranged from 50-100 .mu.L was mixed
with 1.1 .mu.L 3G4 gold conjugate and with or without 2 .mu.L
recombinant H5 hemagglutinin Test was started by inserting strip
into extraction buffer and result was scanned at 15 min, 20 min, 25
min and 30 min For experiment with chasing buffer, see result for
detail.
[0343] Result: FIG. 9 Test the strips with different volume of
Extraction Buffer, at 15, 20, 25 and 30 minutes. FIG. 9A Test
strips were tested in different volume of Extraction Buffer. At the
15, 20, 25 and 30 min, strips were taken out and scanned with
Bio-Rad GS-800 Calibrated Densitometer. Lane 1, 2: 50 .mu.L
Extraction Buffer; Lane 3, 4: 60 .mu.L Extraction Buffer; Lane 5,
6: 70 .mu.L Extraction Buffer; Lane 7, 8: 80 .mu.L Extraction
Buffer; Lane 9, 10: 100 .mu.L Extraction Buffer; FIG. 9B Compare
the test line intensity of test strip with different volume of
Extraction Buffer at different reading time.
TABLE-US-00006 TABLE 6 Data analysis of FIG. 9 with Quantity One.
Lane Extraction Peak Intensity Number Buffer (.mu.l) 15 min 20 min
25 min 30 min 1 50 55.6 62.3 63.1 62.5 2 51.1 57.3 55.4 62.6 3 60
53.5 65.7 68.5 68.9 4 51.6 63.8 66.2 66.5 5 70 45.6 57.9 60.4 60.7
6 42.1 57.0 61.7 63.9 7 80 39.6 53.7 59.8 61.5 8 42.9 57.9 64.7
65.4 9 100 35.2 49.2 54.6 55.6 10 30.8 44.9 49.3 49.8
[0344] FIG. 10A Test Strip #3 with various amount of extraction
buffer and effect of background clean-up on their negative control
result.
TABLE-US-00007 TABLE 7 Data analysis of FIG. 10 with software
Quantity One Lane Extraction Peak Intensity Number Buffer (.mu.l)
15 min 20 min 1 50 3.8 5.4 2 4.1 4.6 3 60 3.4 4.8 4 3.4 5.5 5 70
3.4 2.9 6 2.7 4.1
To various amount of extraction buffer, 1.1 .mu.L 3G4 gold
conjugate was added. Test was started by inserting strip to the
assay solution and scanned at 15 min (First roll). Then all the
strips were transferred to tubes containing extraction buffer only
and ran for 5 more minutes. The strips were scanned again. (Second
roll).
[0345] FIG. 10B Test Strip #3 in various amount of extraction
buffer and effect of background clean-up on their sensitivity.
[0346] To various amount of Extraction Buffer, 1.1 .mu.L 3G4 gold
conjugate and 2 .mu.L recombinant H5 hemagglutinin was added. Test
was started by inserting strip to the assay solution and scanned at
15 min (First roll). Then all the strips were transferred to tubes
containing extraction buffer only and ran for 5 more minutes. The
strips were scanned again. (Second roll).
TABLE-US-00008 TABLE 8 Data analysis of FIG. 4 with software
Quantity One Lane Extraction Peak Intensity Intensity Number Buffer
(.mu.L) 15 min 20 min change 1 50 56.9 88.7 56% 2 57.8 94.4 63% 3
60 50.2 78.3 56% 4 51.2 77.4 51% 5 70 46.8 67.1 43% 6 47.9 73.4
53%
[0347] Discussion. In the design of most of the rapid lateral flow
tests for point-of-care, antibody conjugate usually is dried on a
conjugate pad which is interfaced with nitrocellulose membrane. The
disadvantage of this design is that dried conjugate may not release
evenly and consistently from strip to strip, and from time to time
due to materials for conjugate pad. Conversely, if antibody
conjugate is premixed with assay solution, as long as the liquid
flow is consistent, which is more likely to occur because once the
wicking pad is wet, the flow speed actually is controlled by the
membrane rather than the wicking material, the flow of antibody
conjugate should be more consistent and even. Therefore in the
above example, antibody gold conjugate was directly mixed with
assay solution.
[0348] Initially, 100 .mu.L Extraction Buffer was used for each
test and the reading time of the assay was set at 15 min It was
found that at the end of 15 min, not all assay solutions were
consumed. There was a waste of both analyte and gold conjugate.
Experiment was designed to investigate what the optimal assay
volume is in a relatively short reading time ranges. FIG. 9B and
table 2 showed that with larger volume, such as 100 .mu.L, of assay
solution, longer time is required to get better sensitivity.
However, with 50 .mu.L of assay buffer, the color intensity of the
test line does not increase much over different reading time after
15 min indicating that most of the analyte and gold conjugate have
passed the capture line at 15 min Although at 30 min the color
intensity of the stripes tested with 100 .mu.L assay solution
almost reach the same level as those tested with low volume assay
solution at 15 min, it is obvious that a 15 min test is more
competitive than a 30 min test in the market.
[0349] However, due to the higher concentration of gold conjugate
in a relatively small volume of assay solution, the background of
test strips showed significant red background which reduced the
contrast between the test line and the background and was
cosmetically unattractive. FIG. 10B showed that running a chasing
buffer after 15 min could significantly clean up the background;
hence increase the sensitivity of the test by 43%-63%. The data
also demonstrated again that a small assay volume, 50 .mu.L, is a
better choice for the test.
Example 2
Europium Bead Versus Prior Art System
[0350] Materials: Inactivated influenza A virus: Texas 1/77 (H3N2)
from Microbix Biosystems, Inc. Cat. #EL-13-02, lot #13037A3;
Inactivated influenza B virus: Hong Kong 5/72 from Microbix
Biosystems, Inc. Cat. #EL-14-03, lot #14057A2; Quidel QuickVue A+B
Test, lot #702391
[0351] Protocol: Virus dilution--Influenza A and influenza B viral
preparations were diluted with saline to 4096 HA/mL and 409.6
HA/mL, respectively, before use. Assay procedure: Procedures
described in the package insert of the Quidel test kit were
followed and briefly reviewed here. Dispense all of the Extraction
Reagent Solution from the reagent tube. Gently swirl the tube to
dissolve its content. Spike virus into the tube. Place the swab
into the Extraction Tube. Roll the swab at least three times while
pressing the head against the bottom and side of the Extraction
Tube. Leave the swab in the tube for 1 min Roll the swab head
against the inside of the tube as you remove it. Place the test
strip into the Extraction Tube. Read result at 10 min
[0352] Result: FIG. 11 Test QuickVue Flu A+B with different amount
of influenza A virus, Texas 1/77. The test line is shown above the
control line.
TABLE-US-00009 TABLE 9 Visual read result and data analysis of FIG.
4 with software Quantity One. Flu A virus (HA/test) Lane No. Peak
intensity Visual read result 0 1 2.5 - 2 1.9 - 1.0 3 3.1 - 4 3.1 -
2.0 5 3.0 - 6 3.9 - 4.1 7 7.2 + 8 8.7 + 8.2 9 18.2 + 10 14.3 + 16.4
11 21.1 + 12 31.9 + 32.8 13 66.2 + 14 48.1 + 65.5 15 56.8 + 16 74.0
+
[0353] Peak intensity was analyzed with software Quantity One.
Values were given by the software. Any values below 4 will be read
as a negative with eyes.
[0354] FIG. 12 Test QuickVue Flu A+B with different amount of
influenza B virus, Hong Kong 5/72.
[0355] The test line for Type B is below the control line.
TABLE-US-00010 TABLE 10 Visual read result and data analysis of
FIG. 4 with software Quantity One. Flu B virus (HA/test) Lane No.
Peak Intensity Visual read result 0 1 2.9 - 2 0.2 - 0.41 3 1.6 - 4
1.9 - 0.82 5 2.3 - 6 3.5 - 1.64 7 4.2 .+-. 8 6.3 + 3.28 9 2.0 - 10
8.1 + 6.55 11 16.3 + 12 14.0 +
[0356] Peak intensity was analyzed with software Quantity One.
Values were given by the software. Any values below 4 will be yield
a negative result with a visual read.
TABLE-US-00011 TABLE 11 Test Quidel QuickVue Flu A + B with
different amount of influenza B virus, Hong Kong 5/72 Virus
(HA/test) 0 0.41 0.82 1.64 No. 1 2 1 2 1 2 1 2 Visual - - .+-. .+-.
.+-. .+-. + + Result
Example 3
Test the Detection Limit of Influenza A and B Test Using Gold
Label
[0357] Materials: Anti-Flu A M4090913 gold conjugate was prepared
by Nanogen POC at Toronto, Canada. OD was 102.69 at maximal
absorbent peak. Anti-Flu B antibody 2/3 gold conjugate was prepared
by Nanogen POC at Toronto, Canada. OD was 94.78 at maximal
absorbent peak. See above for nitrocellulose membrane, wicking pad
and absorbent pad. Membrane striped with anti-Flu B M2110171
antibody at 1.5 mg/ml and rabbit anti-mouse antibody at 1.5 mg/ml
was prepared by Nanogen POC at Toronto, Canada. Membrane striped
with anti-Flu A 7304 antibody at 1.5 mg/ml and rabbit anti-mouse
antibody at 1.5 mg/ml was prepared by Nanogen POC at Toronto,
Canada. Inactivated influenza A H3N2, Texas 1/77 was purchased from
Microbix Biosystems, Inc. lot #13037A3, 40960 HA/mL.
[0358] Inactivated influenza B, Hong Kong 5/72 was purchased from
Microbix, Biosystems, Inc., lot #14057A2, 40960 HA/mL; Molecular
Biology Water, lot #318105; 3.times. extraction buffer containing
0.15 M Tris-Cl, pH 8.0, 2.25 M NaCl, 0.3% Pluronic F68, 3% BSA, 6
mM TCEP, 1.5% digested casein and 0.06% NaN.sub.3; 2.times.
extraction buffer containing 0.1 M Tris-Cl, pH 8.0, 1.5 M NaCl,
0.2% Pluronic F68, 2% BSA, 4 mM TCEP, 1% digested casein and 0.04%
NaN.sub.3; 1.times. extraction buffer, lot #2232-003.
[0359] Protocol: Preparation of lateral flow test strip (See
above); Virus dilution: Inactivated influenza A, Texas 1/77 was
diluted to 409.6 HA/mL with 10 mM PBS with 1% BSA. Inactivated
influenza B, Hong Kong 5/72 was diluted to 40.96 HA/mL with the
same buffer before use. Influenza A and B test with gold conjugate:
For influenza A test, 16.7 .mu.L 3.times. extraction buffer, 1.95
.mu.L M4090913 gold conjugate and different amount of inactivated
influenza A virus were added to a tube. H.sub.2O was added to a
final volume of 50 .mu.L. For influenza B test, 25 .mu.L 2.times.
extraction buffer, 2.12 .mu.L 2/3 gold conjugate and different
amount of virus were added to a tube. H.sub.2O was added to a final
volume of 50 .mu.L. Test was started by inserting the test strip
into the assay solution. At 15 min, 50 .mu.L 1.times. Extraction
Buffer was added to the same tube and test was carried on for 5
more minutes. Results on strips were read with eyes and also
analyzed with software Quantity One after strips were scanned with
Bio-Rad GS-800 Calibrated desitometer.
[0360] Result: FIG. 13. Test the limit of detection of influenza A
test using gold as label.
TABLE-US-00012 TABLE 12 Data analysis of FIG. 7 with software
Quantity One and visual read result Virus Lane Peak Peak Visual
HA/test No. Intensity Intensity(avg) read 0 1 0 0 - 2 0 0.105 3 2.2
2.8 - 4 3.3 0.21 5 4.8 6.7 + 6 8.6 0.41 7 13.2 11.2 + 8 9.1 0.82 9
14.8 14.1 + 10 13.5 1.64 11 26.4 24.5 + 12 22.5
[0361] Values of peak intensity are the analysis result with
Quantity One.
[0362] FIG. 14 Test the limit of detection of influenza B test
using gold as label.
TABLE-US-00013 TABLE 13 Data analysis of FIG. 9 with software
Quantity One and visual read result Virus Lane Peak Peak Visual
HA/test No. Intensity Intensity(avg) read 0 1 2.2 2.18 - 2 2.2
0.021 3 2.8 2.78 - 4 2.7 0.041 5 4.8 4.67 .+-. 6 4.6 0.082 7 7.3
7.76 + 8 8.2 0.164 9 14.4 13.83 + 10 13.3
[0363] Values of peak intensity are the analysis result with
Quantity One.
Example 4
Test the Detection Limit of Influenza A and B Test Using
Fluorescent Europium Conjugate
[0364] Materials: Wicking pad: 0.05% tween-20 treated polyester
pad, 1.4 cm in width provided by Nanogen POC in Toronto, Canada;
Absorbent pad: Ahlstrom Grade 222 paper, 3.5 cm in width, purchased
from Fisher, Cat. #2228-1212, lot #6150502; Nitrocellulose
Membrane: Millipore HiFlow 135 membrane, 2.5 cm in width. Cat. No.
SHF1350425, Lot No. R68N46849, Code No. RK04414, roll No. 04OLI;
Flu A membrane: 1.5 mg/ml anti-Flu A Medix 7304 antibody on test
line and 1.5 mg/ml rabbit anti-mouse IgG on control line striped in
Nanogen POC, Toronto, Canada; Anti-Flu A Fitzgerald
M4090913--Europium conjugate at 1% beads concentration was prepared
in Nanogen POC, Toronto, Canada; Flu B membrane: 1.5 mg/ml anti-Flu
B Medix 9901antibody on test line and 1.5 mg/ml rabbit anti-mouse
IgG on control line striped in Nanogen POC, Toronto, Canada; Flu B
membrane: 1.5 mg/ml anti-Flu B M2110171 antibody on test line and
1.5 mg/ml rabbit anti-mouse IgG on control line striped in Nanogen
POC, Toronto, Canada; Anti-Flu B HyTest 2/3--Europium conjugate at
1% beads concentration was prepared in Nanogen POC, Toronto,
Canada; Inactivated influenza A virus: Texas 1/77 (H3N2) from
Microbix Biosystems, Inc. Cat. #EL-13-02, lot #13037A3; Inactivated
influenza B virus: Hong Kong 5/72 from Microbix Biosystems, Inc.
Cat. #EL-14-03, lot #14057A2; 3.times. extraction buffer containing
0.15 M Tris-Cl, pH 8.0, 2.25 M NaCl, 3% BSA, 0.3% pluronic, 0.06%
NaN3, 1.5% digested casein and 6 mM TCEP, was prepared in Nanogen,
San Diego. Lot #2232-001; Virus dilution buffer: 10 mM PBS with 1%
BSA; Antibody-europium conjugate dilution buffer: 10 mM PBS, 1% BSA
and 0.2% tween-20.
[0365] Protocols. Preparation of the lateral flow test strip:
Nitrocellulose membrane striped with antibodies was laminated on a
plastic card with 1-2 mm overlap between the wicking pad and at the
end of the nitrocellulose strip with the absorbent pad. No cover
tape was applied on any components. Strips were cut in 5 mm widths.
Viruses dilution: Inactivated influenza A and influenza B positive
controls were diluted to 409.6 HA unit/mL and 40.96 HA unit /mL,
respectively for assay with the influenza test.
[0366] Antibody-Europium conjugate pretreatment: 1%
Antibody-Europium conjugate was sonicated in a bath sonicator for 4
min An amount of conjugated beads was then diluted to 0.04% with
antibody-europium dilution buffer and sonicated for another 4
min
[0367] Influenza A and B test procedure with antibody Europium
conjugate: To 16.67 .mu.L, 3.times. Extraction Buffer in a test
tube, add 5 .mu.L 0.04% antibody-europium conjugate and different
amount of virus. Add water to a final volume of 50 .mu.L. Insert a
lateral flow test strip in the assay solution. Wait 15 min at room
temperature. Add 50 .mu.L 1.times. extraction buffer to the same
test tube. Wait for 5 min at room temperature. Place the lateral
flow test strip in a tray and measure the fluorescence intensity.
Each test condition was run with 5 replicates.
[0368] Results: FIG. 15 Analytic sensitivity of Flu A test using
Europium conjugate
TABLE-US-00014 TABLE 14 Statistic analysis of raw data for analytic
sensitivity of Flu A test using Europium conjugate HA Replicates
units 1 2 3 4 5 AVG CV 0 568 677 629 620 650 629 6.4% 0.021 1350
1241 1256 1308 1216 1274 4.2% 0.041 1777 1535 1727 1595 1364 1600
10.2% 0.082 2171 2336 2438 2114 2336 2279 5.8% 0.123 4176 3745 3558
3410 3765 3731 7.7% 0.164 3989 3917 3963 4050 4489 4082 5.7% 0.328
8866 9979 8497 8304 7783 8686 9.5%
[0369] FIG. 16 Test the analytic sensitivity of Flu B test using
Europium conjugate and Medix 9901 on the membrane
TABLE-US-00015 TABLE 15 Statistic analysis of raw data for analytic
sensitivity of Flu B test using Europium conjugate HA Replicates
units 1 2 3 4 5 AVG CV 0 284 196 330 360 331 300 21.4% 0.0021 1419
1361 1572 1600 1496 1490 6.8% 0.0041 1430 1467 1611 1746 1626 1576
8.1% 0.0082 2321 2520 2539 3312 1690 2476 23.4% 0.0123 2580 2791
2661 2737 2781 2710 3.3% 0.0164 5590 5111 5058 5639 5809 5441 6.2%
0.0328 8699 10903 10047 10491 9796 9987 8.4%
[0370] FIG. 17 Test the analytic sensitivity of Flu B test using
Europium conjugate and M2110171 on the membrane
TABLE-US-00016 TABLE 16 Statistic analysis of raw data for analytic
sensitivity of Flu B test using Europium conjugate HA units 1 2 3 4
5 AVG CV 0 511 501 532 481 446 494 6.6% 0.0021 1368 1280 1458 1420
1395 1384 4.9% 0.0041 2697 2535 2708 2677 2711 2666 2.8% 0.0082
3412 3198 3817 3930 3609 3593 8.3% 0.0123 6531 6560 6294 7290 6055
6546 7.1% 0.0164 6707 7328 6679 6853 8098 7133 8.4% 0.0328 12016
13162 12597 12907 13555 12847 4.5%
[0371] Results: Table 5-7 show that the current market leader of
rapid influenza immunoassay, the Quidel QuickVue Flu A+B test has a
detection limit of 4.1 HA units for influenza A Texas 1/77 and 1.6
HA units for influenza B Hong Kong 5/72. When gold label is used,
the present invention's influenza rapid test can detect at least
0.41 HA influenza A Texas 1/77 and 0.082 HA influenza B Hong 5/72
when limited assay volume and chasing buffer were used. This is 10
and 20 times more sensitive than Quidel's QuickVue Flu A+B in
detecting influenza A Texas 1/77 and influenza B Hong Kong 5/72,
respectively. By applying fluorescent Europium conjugate, the
present influenza rapid test is able to detect as low as 0.021 HA
unit of Influenza A Texas 1/77 with a Signal/noise (S/N) ratio of
2.03 and 0.0021 HA unit of Influenza B Hong Kong 5/72 with an S/N
ratio of 4.96. If a S/N ratio of 2 is considered the limit of
detection for the Europium based assay, Flu B would be able to
detect down to 0.001 HA units/test. Or an improvement in
sensitivity over the Quidel assay for Type B of about 1600 fold.
This study clearly demonstrated that the improvements made to
nitrocellulose assay technology does yield significant improvement
in POC assay performance.
Example 5
[0372] Laboratory testing results to demonstrate preliminary proof
of principle that influenza H5N1 can be differentiated from
seasonal human influenza viruses using the proposed product design
and instruments.
[0373] Materials: 3.times. Extraction Buffer containing 0.15 M
Tris-Cl, pH 8.0, 2.25 M NaCl, 0.3% Pluronic F68, 3% BSA, 1.5%
casein and 0.06% NaN.sub.3, lot #2232-001;1.times. Extraction
Buffer, lot #2232-003; 0.2 M Tris(carboxyethyl)phosphine
hydrochloride (TCEP), lot #2232-078; Molecular Biology Water, lot
#318105; Europium dilution buffer, lot #RC 6-8-06; 10 mM PBS, pH
7.2 with 1% BSA, lot #HR 7-19-06; 1% HyTest 2/3 Europium conjugate
with 0.025 mg antibody, lot #E2/3-270706-2; 1% M4090913 Europium
conjugate with 0.025 mg antibody, lot #E13-270706-2; 1% 3C8
Europium conjugate with 0.1 mg antibody, lot #E3C8-180806;
Nitrocellulose membrane striped with anti-Flu A A60010044P,
anti-Flu B 9901, anti-H5 3G4 and rabbit anti-mouse antibody was
prepared by Nanogen POC, Toronto, Canada. The antibody coating
concentration was 2.0 mg/ml for all three test lines. The antibody
coating concentration for the control line was 1.5 mg/ml. The
distance between each test line, and test line and control line is
5 mm.+-.0.5 mm; Wicking pad: 0.05% tween-20 treated polyester pad,
1.4 cm in width provided by Nanogen POC in Toronto, Canada;
Absorbent pad: Ahlstrom Grade 222 paper, 3.5 cm in width, purchased
from Fisher, Cat. #2228-1212, lot #6150502; Inactivated influenza A
H1N1, Beijing/262/95 was purchased from NIBSC, 40 .mu.g HA
activity/ampoule; Inactivated influenza A H3N2, Texas 1/77 was
purchased from Microbix Biosystems, Inc. lot #13037A3, 40960 HA/mL;
Inactivated influenza H5N1, Ck/HK/Yu22/02 from Wantai at China;
Inactivated influenza B, Hong Kong 5/72 was purchased from
Microbix, Biosystems, Inc., lot #14057A2, 40960 HA/mL; Epstein-Barr
virus purchased from ATCC, VR-1491, strain B98-8, lot #3605034
[0374] Procedures: Preparation of influenza test strips with
multiple test line: Nitrocellulose membrane striped with antibodies
was laminated on plastic card with 1-2 mm overlap with wicking pad
and absorbent pad. No cover tape was applied on any components.
Strips were cut in 5 mm in width; Inactivated influenza A H3N2,
Texas 1/77 and influenza B, Hong Kong 5/72 were diluted 10 and 100
times, respectively, with 10 mM PBS/1% BSA; Inactivated H1N1
Beijing 262/95 was reconstituted with 1 mL H.sub.2O; 1% antibody
Europium conjugate was sonicated for 4 min with Branson 1210 bath
sonicator. The individual conjugate was then diluted to 0.04% with
Europium conjugate dilution buffer and sonicated 3 times with 4 min
each time in ice bath. In some experiments, all three Europium
conjugates were diluted together into the same tube. In this case,
each antibody Europium conjugate concentration was still 0.04%
bead; Assay preparation:
[0375] For each assay, following components were added to a tube:
16.67 .mu.L 3.times. Extraction Buffer; 0.5 .mu.L 0.2 M TCEP; 5
.mu.L diluted Europium conjugate if all europium conjugate were
diluted into the same tube or 15 .mu.L Europium conjugate (5 .mu.L
each if individual Europium conjugate was diluted separately);
Virus as indicated in the figures; Adjust the final volume to 50
.mu.L with H.sub.2O.
[0376] Test: Test was started by inserting the test strip into the
assay solution. After 15 min, 50 .mu.L 1.times. Extraction Buffer
was added to the test tube and test was carried on for another 5
min At 20 min, the result of test strip was read with a
fluorescence reader. Each test was repeated 3 times.
[0377] Results: FIG. 18 influenza rapid test strip images under UV
light after strips were tested with buffer, influenza A subtype
H3N2, H1N1 and H5N1, influenza type B and Epstein-Barr virus.
[0378] FIG. 19 Test of influenza rapid test with buffer only
[0379] FIG. 20 Test of influenza rapid test with influenza A
subtype H3N2, Texas 1/77
[0380] FIG. 21 Test of influenza rapid test with influenza type
B
[0381] FIG. 22 Test of influenza rapid test with influenza A
subtype H5N1, Ck/HK/Yu22/02
[0382] FIG. 23 Test of influenza rapid test with influenza A
subtype H1N1 Beijing/262/95
[0383] FIG. 24 Test of influenza rapid test with H5N1 and influenza
type B
[0384] FIG. 25 Test of influenza rapid test with Epstein-Barr
virus
[0385] Results: Based on initial test results, antibodies have been
selected for detecting influenza type A, type B and subtype H5.
Strips with three different test lines that detected type A, type B
and subtype H5, respectively, were prepared. The results in FIGS.
1-7 show that when Flu B is tested, only Flu B peak is observed.
When either H1N1 or H3N2 is tested, only Flu A test peak is seen
and peak corresponding to subtype H5 is not observed. Based one
embodiment herein, another test line corresponding to H1 and H3
(together) will be striped on the membrane once anti-H1 and H3
antibodies are selected. Then another peak corresponding to H1/H3
will be also observed in addition to Flu A peak. When subtype H5N1
was tested, both Flu A peak and H5 peak are observed with the Flu A
peak showing a stronger signal than the H5 peak. This result was
expected as additional optimization of MAb selection and antibody
loading and other factors within the assay are optimized No
cross-reaction of influenza test with EB virus was observed. These
results clearly demonstrate that the approach discovered herein can
yield multiple analyte test results without interference between
the test lines.
[0386] The results shown above have clearly demonstrated that a new
fully integrated POC technology that is highly sensitive and can
independently detect different viral Types and subtypes on the same
test strip has been discovered.
Example 6
Flu B Rapid Assay Using pRNA as Capture
[0387] pRNA was fixed on the membrane to determine efficacy for
capture of complimentary pRNA conjugated with anti-influenza B
nucleoprotein antibody, in order to generate a signal when type B
virus is present in the sample and an antibody-antigen-antibody
complex is formed.
[0388] Materials: Wicking pad: 0.05% tween-20 treated polyester
pad, 1.4 cm in width provided by Nanogen POC in Toronto, Canada;
Absorbent pad: Ahlstrom Grade 222 paper, 3.5 cm in width, purchased
from Fisher, Cat. #2228-1212, lot #6150502 ; Nitrocellulose
Membrane: Millipore HiFlow 135 membrane, 2.5 cm in width. Cat. No.
SHF1350425, Lot No. R68N46849, Code No. RK04414, roll No. 04OLI;
Nitrocellulose membrane striped with 1.5 mg/ml M2110171, lot
#M083106; Nitrocellulose membrane striped with 1.35 mg/ml
102a4-5G4, lot #MZ-42-74247-pRNA135; Anti-Flu B HyTest
2/3--Europium conjugate at 1% beads concentration was prepared in
Nanogen POC, Toronto, Canada, lot #E2/3-270706-3; Inactivated
influenza B virus: Hong Kong 5/72 from Microbix Biosystems, Inc.
Cat. #EL-14-03, lot #14057A2; 3.times. extraction buffer containing
0.15 M Tris-Cl, pH 8.0, 2.25 M NaCl, 3% BSA, 0.3% pluronic, 0.06%
NaN3, 1.5% digested casein and 6 mM TCEP, was prepared in Nanogen,
San Diego. Lot #2232-001; 0.2 M TCEP, lot #2232-078; 1.times.
Extraction Buffer, lot #2232-003; Europium conjugate dilution
buffer, lot #RC 6-8-06; Virus dilution buffer: 10 mM PBS with 1%
BSA; 10 mM PBS, lot #SP09-22-06; Antibody-europium conjugate
dilution buffer: 10 mM PBS, 1% BSA and 0.2% tween-20; Protein 5G4
for conjugation with pRNA that would be fixed on the membrane;
Anti-Flu B nucleoprotein M2110171 was purchased from Fitzgerald,
Bat #578; pRNA 102a4-amn and 102b4-amn (both 13mer) were
synthesized and activated by PDITC.
[0389] Protocol:
[0390] Preparation of pRNA-antibody conjugates
TABLE-US-00017 TABLE 17 Calculation of amounts of oligo and
antibodies required for conjugation. Conjugated with Oligo/antibody
Antibody Oligo Antibody Ratio required 102a4amn 76.3 nmol 5G4 10:1
1.15 mg 102b4amn 54.7 nmol M2110171 5:1 1.64 mg
[0391] Dissolve activated pRNA 102a4amn (total 229 nmol) and
102b4amn (total 164 nmol) in 150 .mu.l of water in two separate
tubes. [0392] a. Aliquot 50 .mu.l into 3 microcentrifuge tubes for
each pRNA and dry them using a spin vac. [0393] b. Place 1.5 mg of
5G4 monoclonal antibody into one spin filter and 2.0 mg M2110171
monoclonal antibody into another spin filter to concentrate both
antibody. Antibodies were rinsed with 0.1 M sodium borate buffer.
The final concentration of 5G4 and M2110171 were determined by the
absorbance at 280 nm and were 33.35 mg/ml and 28.6 mg/ml,
respectively [0394] c. Take 34 .mu.L of concentrated 5G4 (1.15 mg)
and 57 .mu.L of concentrated M2110171. Bring the both volumes to 65
.mu.L with 0.1 M borate buffer. [0395] d. Add each antibody to the
corresponding pRNA and let reaction go for 15-20 hours at room
temperature. [0396] e. pRNA-antibody conjugate was then purified
with a Sephadex G-50 column with bed volume of 7 mL equilibrated
with 0.01 M PBS.
[0397] 1. Preparation of nitrocellulose membrane striped with
102a4-5G4 [0398] Membrane was striped at NPOC at Toronto. See
"Materials".
[0399] 3. Preparation of the lateral flow test strip: [0400]
Nitrocellulose membrane striped with antibody or 102a4-5G4 was
laminated on a plastic card with 1-2 mm overlap with wicking pad at
one end and 1-2 mm overlap with absorbent pad at the other end.
Strips were cut in 5 mm widths.
[0401] 4. Viruses dilution: [0402] Inactivated influenza B virus
was diluted to 4.096 HA unit/mL with 0.01 M PBS and 1% BSA
buffer.
[0403] Antibody Europium conjugate pretreatment [0404] 1% anti-Flu
B 2/3-Europium conjugate was sonicated in a bath sonicator for 4
min. An amount of 10 .mu.L conjugated beads was then diluted to
0.02% with antibody-europium dilution buffer and sonicated for 20
sec. using a probe sonicator (Fisher Model 550) at power setting 2
for 4 pulses, 5 sec. each, with 10 sec. incubation in ice between
each pulse.
[0405] 102b4-M2110171 (0.54 mg/ml after purification) was diluted
to 0.1 mg/ml with 10 mM PBS.
[0406] Assay procedure
[0407] Take 50 .mu.L assay mixture and add it to a test tube.
Insert a test strip into the assay mixture. Wait 15 min at room
temperature. Add 50 .mu.L 1.times. Extraction Buffer to the same
test tube and incubate for another 5 minutes. Read the test strip
with a LRE fluorescence reader
[0408] a. Prepare assay mixture for strips with 102a4-5G4
striped
TABLE-US-00018 TABLE 18 Virus HA units/test Components 0 0.002
0.004 0..008 0.016 0.032 3.times. buffer (.mu.L) 41.7 41.7 41.7
41.7 41.7 41.7 Water (.mu.L) 52.1 50.8 49.6 47.1 42.1 32.1 0.2M
TCEP (.mu.L) 1.3 1.3 1.3 1.3 1.3 1.3 0.1 mg/ml 5.0 5.0 5.0 5.0 5.0
5.0 102b4-M2110171 pRNA conj. (.mu.L) 0.02% 2/3 Europium 25.0 25.0
25.0 25.0 25.0 25.0 conj. (.mu.L) Flu B virus 4.096 HA 0.0 1.3 2.5
5.0 10.0 20.0 units/ml (.mu.L) Total vol. for 2.5 tests (.mu.L)
125.0 125.0 125.0 125.0 125.0 125.0
[0409] b. Prepare assay mixture for strips striped with anti-Flu B
antibody M2110171
TABLE-US-00019 TABLE 19 Virus HA units/test Components 0 0.002
0.004 0..008 0.016 0.032 3.times. buffer (.mu.L) 41.7 41.7 41.7
41.7 41.7 41.7 Water (.mu.L) 57.1 55.8 54.6 52.1 47.1 37.1 0.2M
TCEP (.mu.L) 1.3 1.3 1.3 1.3 1.3 1.3 0.02% 2/3 Europium 25.0 25.0
25.0 25.0 25.0 25.0 conj. (.mu.L) Flu B virus 4.096 HA 0.0 1.3 2.5
5.0 10.0 20.0 units/ml (.mu.L) Total vol. for 2.5 tests (.mu.L)
125.0 125.0 125.0 125.0 125.0 125.0
[0410] Result
[0411] FIG. 30 A: Raw signals of test strips with 102a4-5G4
conjugate on.
TABLE-US-00020 TABLE 20 Net peak height of signals showed in FIG.
30A Table 16. test HA units 1 2 AVG Signal/Noise 0 360.0 331.9
345.9 1.0 0.002 521.1 447.1 484.1 1.4 0.004 682.0 783.3 732.7 2.1
0.008 1048.5 916.2 982.4 2.8 0.016 1799.0 1594.3 1696.7 4.9 0.032
2745.4 3257.2 3001.3 8.7
[0412] FIG. 30B: Raw signals of test strips with antibody M2110171
on.
TABLE-US-00021 TABLE 21 Net peak height of signals showed in FIG.
30B test HA units 1 2 AVG signal/noise 0 155.1 142.6 148.8 1.0
0.002 495.8 538.8 517.3 3.5 0.004 884.2 895.9 890.0 6.0 0.008
1633.7 1698.0 1665.8 11.2 0.016 3329.4 2574.9 2952.2 19.8 0.032
7203.1 6974.3 7088.7 47.6
[0413] FIG. 31 Compare the sensitivity of two Flu B test
strips.
[0414] The data clearly indicates that 1) pRNA conjugated with a
protein, such as 5G4 antibody in this case, can be fixed on the
nitrocellulose membrane; 2) pRNA can hybridize with a complementary
oligo conjugated with an antibody on the nitrocellulose membrane;
3) the analyte specific antibody is still functioning after
conjugated with pRNA. The result has demonstrated that a specific
pair of pRNA can be used as a capture system in lateral flow rapid
assay.
Example 7
Reader Europium Detection
[0415] Six different reference concentrations of Europium were used
to test a reader (note the following numerical references
correspond to references utilized in figures: (1) 5.times.10.sup.-6
M; (5) 5.times.10.sup.-5 M; (3) 5.times.10.sup.-5M; (6)
5.times.10.sup.-4M; (2) 5.times.10.sup.-4M and (4)
5.times.10.sup.-3M. Europium was encased in acrylic and different
sized blocks were cut to provide different concentrations.
Measurements are provided as for counts per meters.times.10.sup.-4
FIG. 33.
[0416] In another example, seven (7) 1 mm wide Europium hard
standards pieces were mounted on 4mm centers FIG. 34.
Example 8
Archive Sample
[0417] Influenza Viruses: Inactived Influenza A/Texas 1/77 (H3N2)
at 40960 HA units/mL from Microbix Biosystems, Inc. of Ontario,
Canada, catalog number EL-13-02; and inactive Influenza B/Hong Kong
5/72 at 40960 HA units/mL from Microbix Biosystems, Inc. of
Ontario, Canada, catalog number EL-14-03.
[0418] Viral RNA Transcript Positive Controls; Influenza A RNA
Control, PN: 606161, LN: RH2174P73 at 50,000 copies/.mu.L; and
Influenza B RNA Control, PN: 606162, LN: RH2174P73 at 50,000
copies/.mu.L
[0419] Extraction Buffer: 3.times. Extraction Buffer
[0420] The table below lists the formulation of the 3x Extraction
Buffer and the corresponding part number, lot number and vendor of
each of the components
TABLE-US-00022 TABLE 22 Concen- Catalog Lot Component tration
Vendor Number Number Trizma Base 150 mM Sigma T-6791 58H5431 BSA
.sup. 3% SeraCare Life AP-4500 013-05-015 Science Pluronic F68 0.3%
Pragmatics Inc. Code 025 WPMS-561B Casein 1.5% Sigma C5890 075K0108
NaN.sub.3 0.06% Sigma S8032 125K2502 NaCl 2.25M Sigma S7653
075K0024
[0421] Tris(2-carboxyethyl)phosphine hydrochloride (TCEP) 200 mM,
made on Jul. 6, 2006 by HR, powder ordered from PIERCE, Product
Number: 20491, Lot Number:GL102548
[0422] Prepare fresh 1.times. Extraction Buffer: Combine 200 .mu.L
of 3.times. Extraction Buffer with 6 .mu.L of 0.2 M TCEP and 394
.mu.L of water to make 600 .mu.L of 1.times. Extraction Buffer. The
formulation of the 1.times. Extraction Buffer is: 50 mM Tris, 1%
BSA, 0.1% Pluronic F68, 0.5% Casein, 0.02% NaN3, 0.75 M NaCl and 2
mM TCEP.
[0423] Sample binding and storage materials: Whatman FTA Elute
Micro Card, Cat. #WB120401, LN: FE6231106; Whatman Harris UNI-CORE
2.00 mm Punch, Cat. #WB640001, LN: 6023; and MicroAmp Reaction
Tubes with Caps 0.2 mL, PN: N801-0540, LN: P34H4QA11, ABI
[0424] Elution materials: Distilled water, DNAse, RNAse free, Cat.
#10977-015, LN: 1317578, Invitrogen
[0425] Heat source: GeneAmp PCR System 9700, ABI, QA2126
[0426] RT and PCR reagents and material:
TABLE-US-00023 TABLE 23 Catalog Lot Component Vendor Number Number
RT Mix Prodesse GLS01 050105RM MgCl.sub.2 1M Ambion 9530G 064R37A
MulV Reverse Transcriptase, ABI N808-0018 H03264 50 u/.mu.L RNase
Inhibitor, 20 u/.mu.L ABI N808-0119 H05526 RVA Primer Mix Nanogen
Not RH2174P100 available AmpliTaq Gold Polymerase, ABI N808-0249
G03670 5 u/.mu.L 96 Well PCR Reaction Plate ABI 4306737
P03F6QA41
[0427] Materials for evaluating RT-PCR reactions: Agilent DNA 1000
Reagents: PN. 5067-1504, LN: 0623; and Agilent DNA Chips: PN
5067-1522, LN: JA21BK01
[0428] Methods
[0429] Preparation of RT Master Mix
TABLE-US-00024 TABLE 24 Component Volume per reaction (.mu.L) RT
Mix 11 1M MgCl.sub.2 0.1 MulV Reverse Transcriptase (50 u/.mu.L) 1
RNase Inhibitor (20 u/.mu.L) 1 DNAse, RNAse Free Water 1.9 Total
15
[0430] Add 15 .mu.L of RT master mix into a well in 96 well PCR
plate
TABLE-US-00025 TABLE 25 Preparation of PCR Master Mix Component
Volume per reaction (.mu.L) RVA Primer Mix 39.5 AmpliTaq Gold
Polymerase (5 u/.mu.L) 0.5 Total 40
[0431] Add 40 .mu.L of the PCR master mix into a well in 96 well
plate
TABLE-US-00026 TABLE 26 Dilution of RNA Positive Controls: Dilute
Influenza A and Influenza B RNA with DNase, RNase free water as
described in the table below Start Conc. End conc. Dilution .mu.L
.mu.L Total Remain (copies/.mu.L) (copies/.mu.L) Factor RNA water
Volume Copies/rxn Volume 50,000 20,000 2.5 8 12 20 N/A 10 20,000
2000 10 10 90 100 10000 90 2000 200 10 10 90 100 1000 90 200 20 10
10 90 100 100 90 20 2 10 10 90 100 10 100
[0432] Add 5 uL of diluted RNA into proper RT reaction
TABLE-US-00027 TABLE 27 Dilution of Influenza A and Influenza B
inactive viruses with 1.times. Extraction Buffer End Start Conc.
Conc. (HA .mu.L of 1.times. HA (HA units/ units/ Dilution .mu.L
Extraction Total units/ Remain .mu.L) .mu.L) Factor virus Buffer
Volume disc Volume 40.96 4.096 10 5 45 50 20.5 45 4.096 0.4096 10 5
45 50 2.05 45 0.4096 0.04096 10 5 45 50 0.2 45 0.04096 0.004096 10
5 45 50 0.02 45 0.004096 0.0004096 10 5 45 50 0.002 50
[0433] Application of Samples to FTA Elute Card:
[0434] Apply 5 .mu.L of following samples onto the sample area of
the Whatman FTA Elute Micro Card; 1.times. Extraction Buffer as a
control; Virus diluted with 1.times. Extraction Buffer; Air dry the
FTA Elute Card for 15 -20 min. at room temperature; punch out a 5
to 6 mm diameter disc with the punch from the area that the sample
was applied to then place in a 0.2 mL MicroAmp reaction tube;
and
[0435] elute RNA from FTA Elute disc: Add 150 .mu.L of water into
the tube containing the disc and vortex 3.times. for 5 seconds;
remove water by using a sterile pipette tip; repeat
addition/removal of water once; centrifuge for 5 seconds, then
pipette off the excess liquid; add 504 of water, heat at 65.degree.
C. for 30 min. then vortex the tubes for 5 seconds; and take out 5
.mu.L of the liquid and add to the appropriate well of the 96 well
RT plate.
[0436] RT-PCR Assay
[0437] Thermal Cycler Program for RT Step
TABLE-US-00028 Temperature (.degree. C.) Time (minutes) Number of
Cycles 22 10 1 42 60 1 95 5 1 4 Hold --
[0438] Transfer 10 .mu.L of the completed RT reaction to the
appropriate PCR well containing PCR master mix. Pipette up and down
to mix.
TABLE-US-00029 TABLE 28 Thermal cycler program for PCR Step
Temperature (.degree. C.) Time Number of Cycles 95 10 minutes 1 95
60 seconds 2 55 30 seconds 72 45 seconds 94 60 seconds 38 60 30
seconds 72 30 seconds 72 7 minutes 1 4 Hold --
[0439] Evaluate PCR Reactions for Amplicon Using Agilent
Bioanalyzer
[0440] RT-PCR Agilent Results: The no template control reactions
did not generate any detectable amplicon.
[0441] RNA Positive Controls
TABLE-US-00030 TABLE 29 Cop- Influenza A Influenza B ies/ band ng/
Pos/ band ng/ Pos/ rxn size .mu.L Rxn size .mu.L Rxn 10K 233 bp
17.33 3/3 242 bp 18.28 2/2 1K 233 bp 11.57 5/5 245 bp 16.96 3/3 100
233 bp 3.84 3/3 246 bp 10.62 2/2 10 [1] 236 bp 0.30 3/3 246 bp 4.08
2/2
[0442] The observed band sizes of Influenza A and Influenza B RNA
Positive Controls are of the correct size.
TABLE-US-00031 TABLE 30 Influenza Viruses HA Influenza A Influenza
B units/disc band size ng/.mu.L Pos/Rxn band size ng/.mu.L Pos/Rxn
20 229 bp 17.39 2/2 246 bp 12.31 2/2 2 229 bp 5.83 2/2 247 bp 3.02
2/2 0.2 230 bp 2.34 2/2 247 bp 7.25 2/2 0.02 230 bp 0.34 1/2 247 bp
3.06 2/2 0.002 Not Not available 0/2 244 bp 1.08 2/2 detected
[0443] Conclusion: Successfully detected RNA from lysed Influenza
types A and B viruses using the Whatman FTA Elute
[0444] Card and RT-PCR Amplification.
[0445] The assay detected down to 0.2 HA units/disc of Influenza
type A virus and 0.002 HA units/disc of Influenza type B virus. The
Whatman FTA Elute Card can be used to archive viral RNA for later
testing in an RT-PCR assay for the detection of Influenza types A
and B.
Example 9
Multi-Analyte Detection
[0446] To demonstrate proof of principle that multianalyte
Influenza diagnostic test can differentiate Influenza H5N1 from
seasonal flu a rapid and high sensitivity assay was developed and
compared with traditional lateral flow tests using colored latex
bead or colloidal gold.
[0447] A lanthanide label was utilized where latex microbeads
filled with Europium chelate, combined with a wash buffer
demonstrated improved sensitivity. Seasonal Influenza (in this
example, Influenza Type B) was clearly differentiated from avian
flu H5N1 using membrane striped directly with anti-Flu A, anti-Flu
B and anti-H5 antibodies at different zones on the test strip.
[0448] The data demonstrates that using pRNA and direct striping of
MAb on to the nitrocellulose as the capture systems on the same
lateral flow test strip, is able to separately detect influenza A
subtype H1N1 and H5N1 as well as Type B and Type A nucleoprotein on
different test zones on a single Nitrocellulose test strip. A table
summarizing the areas of the assay that were exercised in this
study are is shown below.
TABLE-US-00032 TABLE 31 Proof of Concept Table: Assay Component
Description Nitrocellulose membrane blocked with Millipore 135
membrane, same material as currently hydrolyzed casein used by
Operations, the size is different Wicking pad treated with Tween
Standard Operations component Absorbent pad Material same as used
in operations, size is different Backing card Material same as used
in operations, size is different Extraction Buffer Designed to lyse
and inactivate influenza Mucolytic Agent Designed to reduce
viscosity of mucus on the specimen swab Wash Buffer Similar
formulation to extraction buffer, designed to "wash" unbound Eu
latex beads from the test strip and reduce background and "stop"
the assay Type A MAb striped onto Nitrocellulose Showing
feasibility of directly spotting MAb in directly combination with
pRNA Type A MAb conjugated to Europium Latex Conjugate the MAb
directly to the Eu microbeads microbeads Type B MAb Pair striped
onto nitrocellulose Showing feasibility of directly spotting MAb in
directly combination with pRNA Type B MAb conjugated to Eu
Microbeads See comment under Type A above H1N1 MAb conjugated to a
specific pRNA Demonstrating and showing proof of concept that pRNA
oligos can be used as a generic capture agent in the assay. H1N1
MAb conjugated to Biotin Conjugate the MAb to biotin and react the
conjugate with Streptavidin coated Eu beads. H5N1 MAb conjugated to
a specific pRNA Demonstrating and showing proof of concept that
pRNA oligos can be used as a generic capture agent in the assay.
H5N1 MAb conjugated to Eu See comment under Type A above Europium
latex microbeads coated with Developed a procedure to react
Europium latex beads Streptavidin and reacted with H1N1 coated with
Streptavidin with a specific MAb conjugated with biotin. After the
reaction excess free biotin is added to block the reaction sites.
Europium latex microbeads coated with See comment above
Streptavidin and reacted with anti-H1 MAb Europium latex microbeads
coated with See comments above individual MAbs Reader The reader
will have optics better suited to the UV LED light source and with
filters to eliminate background light emission, to improve the
performance of the system. Assay regent, single solution (mixture
of This will enable confirming the design with all assay extraction
reagent, mucolytic agent, reagents mixed together. trehalose,
buffer agents, Europium latex beads with conjugated/bound detection
reagents and capture reagents with detection and capture reagents
Four unique analyte test lines plus a control The assay
demonstrated four separate functioning line analyte test lines
[0449] Materials
[0450] 1. Preactivated pRNAs were provided by Nanogen Bothell
development team. [0451] a. 102a10 (10 mer), lot #825-079A [0452]
b. 102b10 (10 mer, the complimentary pRNA of 102a10), lot
#825-065D, lot #825-065D [0453] c. 3a10 (10 mer), lot #825-079C
[0454] d. 3b10 (10 mer, the complimentary pRNA of 3a10), lot
#825-065F, lot #825-065F
[0455] 2. Antibody [0456] a. Anti-Flu A 7304 from BiosPacific
[0457] b. Anti-Flu A M4090913 from Fitzgerald [0458] c. Anti-Flu B
2/3 from HyTest [0459] d. Anti-Flu B M2110171 from Fitzgerald
[0460] e. Anti-influenza H1 M322210 from Fitzgerald [0461] f.
Anti-influenza H1 8252K from Chemicon [0462] g. Anti-influenza H5
10F7 from Xiamen University [0463] h. Anti-influenza H5 3G4 from
Xiamen University
[0464] 3. OptiLink Carboxylate-Modified Microparticles (Europium
beads) [0465] Europium beads with 0.3 .quadrature.M diameter was
purchased from ThermoFisher Scientific (Seradyn). Cat.
#83470750010250, manufacture lot #CO20512, package lot #201154.
[0466] 4.Virus [0467] Influenza B Hong Kong/5/72 from Microbix,
B/Shanghai/2002 from Dr. Bovian's laboratory, A/Taiwan/1/86 (H1N1)
from NIBSC, A/Beijing/95(H1N1) provided by Dr. Bovian's laboratory,
Bar-headed GS/QH/15/2005 (H5N1) and DK/VNM/568/2005 (H5N1) from Dr.
Guan's laboratory in Hong Kong University.
[0468] 5. Three (3).times. Extraction buffer containing 150 mM
Tris-Cl, pH 8.0, 2.25 M NaCl, 3% BSA, 1.5% digested casein, 0.3%
pluronic F68 and 0.15% ProClin 300, lot #2277-038.
[0469] 6. solution: 0.2 M
Tris(2-carboxyethylphosphine)hydrochloride solution, lot
#2277-067.
[0470] 7. Recombinant Streptavidin.
[0471] 8. Non-specific antibody 5G4 for conjugation with pRNA,
prepared by Nanogen Toronto facility and used to strip onto the
nitrocellulose.
[0472] 9. Strip backing card, 0.1'' super white polystyrene from
G&L
[0473] 10. Absorbent pad, filter paper grade 222 from Alstrom
filtration
[0474] 11. Wicking pad, 0.05% Tween-20 treated polyester pad.
[0475] Procedure
[0476] Preparation of antibody-Europium conjugate
[0477] 1.1 Antibodies M4090913, 2/3, and 3G4 were conjugated with
0.3 .quadrature.M Europium beads by ThermoFisher Scientific
(Seradyn).
[0478] 1.2 Antibody 8252k Europium conjugate was prepared through
an indirect procedure by combining biotinylated 8252K with
streptavidin coated Europium microparticles. Biotinylated 8252K and
streptavidin Europium microbeads were prepared at the Nanogen
Toronto facility. Streptavidin Europium stock at 1% solid was
sonicated with a bath sonicator for 5 min It was then diluted to
0.02% with Europium conjugate dilution buffer and sonicated with a
probe sonicator for 20 s. Biotinylated 8252k was diluted to 10
.mu.g/ml with PBS buffer. Aliquot of 5 .mu.L was added to each
tube. Aliquot of 5 .mu.L 0.02% solid streptavidin Europium
conjugate was then quickly mixed with biotinylated 8252K. The
conjugate was blocked using 2.5 uL of 100 mM Biotin. The 8252k
Europium conjugate was then directly used in the assay.
[0479] 2. Preparation of antibody-pRNA conjugate: All the
antibodies were concentrated to approximately 20 mg/ml using
Microcon 50 from Amicon. The buffer was change to 0.1 M sodium
borate, pH 9.0. Aliquot of 1 mg non-specific antibody 5G4 was added
to tubes containing 33.5 nmol pre-activated pRNA 102b10 or 3b10
(dry powder). Aliquot of 0.5 mg M322210 and 0.5 mg 10F7 were added
to tubes containing 16.7 nmol pre-activated pRNA 102a10 and 3a10,
respectively. The reaction was carried out for 15 hours at room
temperature. The antibody pRNA conjugate was purified with a
Sephadex G-50 column equilibrated with 10 mM PBS buffer, pH 7.0.
Two peaks were resolved, the first peak with conjugate and second
peak with unactivated pRNA(confirmed by HPLC, data not shown).
[0480] 3. Preparation of nitrocellulose membrane striped with four
test lines. For this membrane, anti-Flu A 7304 and anti-Flu B
M2110171 at 1.5 mg/ml were striped directly for test line "A" and
test line "B", respectively. Conjugate 102b10-5G4 and 3b10-5G4,
both at 0.75 mg/ml, were striped for "H1" and "H5" test line. The
control line was striped with 0.5 mg/ml rabbit anti-mouse antibody.
All lines were 5 mm apart. The membrane was then blocked with a
solution containing 0.35% PVP, 0.25% Triton X-100 and 0.5% digested
casein, and cured at 37.degree. C.
[0481] 4. Preparation of test strip Nitrocellulose membrane striped
with 4 test lines and one control line was laminated onto the
backing card and has 1-2 mm overlap with treated polyester pad and
absorbent pad. Strips were cut into 5 mm widths using a Kinematic
2360 strip cutter.
[0482] 5. Test procedure. Assay mixture was prepared as following.
To a test tube, add 16.7 .mu.L 3.times. Extraction Buffer, 0.5
.mu.L 0.2 M TCEP, 0.1 .mu.g 102a10-M322210 conjugate, 0.1 .mu.g
3a10F7 conjugate, antibody Europium conjugate and different
viruses. The final concentration of all antibody Europium
conjugates is 0.002% solid in a final volume of 50 .mu.L. Test was
started by inserting test strip into the assay mixture. After 15
min, another 50 .mu.L of 1.times. Extraction Buffer was added to
wash the strip. Strips were read with a fluorescence reader at 20
min.
[0483] Results.
TABLE-US-00033 TABLE 32 The following table compares the
signal/noise ratio of different test peaks when tested with
different viruses. Signal/noise of each test line Virus Description
H5 H1 B A H5 Bar Headed 116.9 2.0 0.6 245.7 GS/QH/15/2005
DK/VNM/568/2005 122.0 5.7 1.0 606.9 H1 Taiwan/1/86 8.5 63.8 0.4
492.7 Beijing/95 6.4 44.4 0.5 896.5 Flu B Hong Kong 5/72 3.5 1.3
437.1 4.8 Shanghai/2002 2.0 1.2 376.0 3.9
[0484] The data clearly shows that the test system is able to
differentiate multiple analytes. In this example, the analytes
detected included seasonal influenza subtype H1N1, Flu B from avian
flu H5N1 with a good Type A signal. In this test, two pairs of pRNA
were used as the capture system to differentiate H1 test line from
H5 test line. Some low reactivity was observed at the H1 test line
position when H5 viruses were tested indicating small amount of
hybridization of pRNA 3a10 conjugated on anti-H5 antibody with
102b10 striped on test line H1. No false positive reaction effects
were observed on the H5 specific test line when subtype H1N1
viruses were tested.
[0485] In addition, this report clearly shows that the assay, shows
good reactivity, good separation of analyte signals when a single
multi-analyte reaction mix is used for all test specimens. In
addition, the assay can detect all appropriate analytes when
testing a single test specimen at moderate viral loads
simultaneously.
[0486] Conclusion: Results demonstrate that with virus of multiple
subtypes and strains significant separation of the signals for each
analyte for each viral strain or subtype tested is achieved (FIGS.
36-38). To ensure the results had validity two separate strains of
each subtype were tested. Furthermore, the results clearly
demonstrate that a pRNA generic capture system to apply to the H1
and H5 detection systems, thus enabling utilization of the pRNA
capture system for additional panels of different analytes.
[0487] Moreover, the assay can readily be adapted to incorporate
additional analyte-specific binding agents. Further, device/assays
of the invention can be utilized in methods of screening different
analytes. For example, a large number of Anti-H3 expressing clones
are can be screened to determine
[0488] Of course the devices/methods of the invention can be
utilized to screen new viral strains (an important example is the
new Fujan like strain). For example, as new MAb to new strains of
the H5N1 subtype are obtained, they can be incorporated in the SCD
and Test Device of the invention for screening.
[0489] The results shown above have clearly demonstrated that a new
fully integrated assay technology that is highly sensitive and can
independently detect different viral Types and subtypes on the same
test strip has been discovered. Further such assay can be
administered in a Point-of-Care setting.
Example 10
Multianalyte Detection: Marker Proteins
[0490] The device and methods of the invention may also be used to
detect various analytes associated with a disease condition or
nutritional state, or any other associative characteristics. For
example, multiple cardiac markers may be detected and/or
quantified:
[0491] (1) The Troponin marker: Capture Antibody is a polyclonal
rabbit anti-Troponin-I and conjugated to a specific pRNA sequence
(SEQ ID NO: 92). Detection antibody is a mixture of two mouse MAbs
and conjugated to biotin and reacted with streptavidin coated 0.3u
Europium latex microparticle.
[0492] (2) The CK/MB marker. Capture antibody is a Polyclonal Goat
and is conjugated to specific pRNA sequence (SEQ ID NO: 93).
Detection antibody is also a MAb and is conjugated to biotin and
reacted with streptavidin coated 0.3u Europium latex
microparticles.
[0493] (3) The Myoglobin marker: Capture antibody is a Rabbit anti
Myoglobin Polyclonal and is conjugated to specific pRNA sequence
(SEQ ID NO: 94). Detection antibody is also a MAb and is conjugated
to biotin and reacted with streptavidin coated 0.3u Europium latex
microparticle.
[0494] (4) Control line made up of rabbit anti mouse.
[0495] Therefore, the multianlyte "point of care" assay system can
quantitate each of the markers individually in a single assay
format as outlined above and as follows:
[0496] Using the reader with a hard Europium standard (a Europium
standard locked in acrylic and yields a uniform reading under a
uniform set of conditions over a long period of time) which ensures
the reader is able to meet fluorescent signal intensity
requirements reproducibly. Thus each analyte can be quantitated
independently
Example 11
Multianalyte Detection: Viruses
[0497] As disclosed herein above, the devices of the invention can
also be configured to detect multiple different viruses, or
different subtypes of a virus. For example, the devices of the
invention can be configured to provide an influenza multianalyte
plus RSV qualitative assay where multiple analytes can be detected
from a single specimen:
[0498] (1) Influenza Type A: Capture antibody is a MAb and is
conjugated to specific pRNA sequence (SEQ ID NO: 93); Detection
antibody is also a MAb and is conjugated to biotin and reacted with
streptavidin coated 0.3u Europium latex microparticles
[0499] (2) Influenza Type B: Capture antibody is a monoclonal mouse
MAb and is conjugated to a specific pRNA sequence (SEQ ID NO: 94);
Detection antibody is also a MAb and is conjugated to biotin and
reacted with streptavidin coated 0.3u Europium latex
microparticles.
[0500] (3) Influenza H1 and H3: Capture antibodies are MAb specific
for H1 and H3 Hemagglutinin and are all conjugated to specific pRNA
sequence (SEQ ID NO: 95); Detection antibody is also a MAb and is
conjugated to biotin and reacted with streptavidin coated 0.3u
Europium latex microparticles.
[0501] (4) Influenza H5: Capture antibody is a monoclonal mouse MAb
and is conjugated to specific pRNA sequence (SEQ ID NO: 96);
Detection antibody is also a MAb and is conjugated to biotin and
reacted with streptavidin coated 0.3u Europium latex
microparticles.
[0502] (5) RSV: Capture antibody is a monoclonal mouse MAb and is
conjugated to specific pRNA sequence (SEQ ID NO: 97); Detection
antibody is also a MAb and is conjugated to biotin and reacted with
streptavidin coated 0.3u Europium latex microparticles.
[0503] (6) Control Line: Capture antibody is rabbit anti-mouse and
binds to the MAb on the Europium latex microparticle.
Example 12
Expression of Single Chain Antibodies of 10F7 and 4D1 and Test of
Their Activities
[0504] The variable region genes of the heavy and light chains of
each antibody were linked with a nucleic acid encoding a short
peptide (GGGGS (SEQ ID NO:114)) to form the DNA fragment encoding a
single chain antibody. Use 10F7 VHF/10F7 VHR as the primer pair to
amplify the variable region DNA fragment of 10F7 heavy chain. Use
10F7 VKF/10F7 VKR as the primer pair to amplify the variable region
DNA fragment of 10F7 light chain. Use 4D1 VHF/4D1 VHR as the primer
pair to amplify the variable region DNA fragment of 4D1 heavy
chain. Use 4D1 VKF/4D1 VKR as the primer pair to amplify the
variable region DNA fragment of 4D1 light chain.
[0505] Use 10F7 VHF/10F7 VKR as primers to amplify the overlapping
10F7 single chain DNA fragment. Use 4D1 VHF/4D1 VKR as primers to
amplify the overlapping 4D1 heavy chain DNA fragment. The amplified
DNA fragments were recovered, digested with BamH I and Sal I, and
cloned into prokaryotic expression vector pTO-T7 digested with the
same restriction enzymes. Using ER2566 E. coli as host cells, the
single chain antibody proteins were expressed using standard
methods. The expressed proteins were in the form of insoluble
inclusion bodies. The inclusion bodies were broken up by ultrasound
treatment, and the resulting sediments were purified using standard
methods. The purified sediments were dissolved in 8M urea. The urea
solution was dialyzed slowly in 1.times.PBS solution, centrifuged
at 12000 rpm for 10min to remove the remaining sediments. The final
purified single chain antibody solution was tested for
activities.
[0506] Select 26 strains of H5N1 viruses to test the activities of
the above purified 10F7 and 4D1 single chain antibodies using HI
assay as described above. The concentration of 10F7 single chain
antibody is used at 1.06 mg/ml. The concentration of 4D1 single
chain antibody is used at 0.34 mg/ml. The 4D1 single chain antibody
exhibits HA inhibition activity against 23 of the virus strains.
The 10F7 single chain antibody shows HA inhibition activity against
14 of the virus strains (Table 33 below).
TABLE-US-00034 TABLE 33 HA inhibition activities of the three
single chain antibodies against the 25 H5N1 viruses. H5N1 Virus
ScFv Strains 4D1 10F7 8H5 A1 >8 >8 3.5 A2 >8 >8 4.5 A3
>8 >8 3.5 A5 >8 >8 4 A6 7 7.5 3 A7 6 6.5 3 A8 >8 6
2.5 B1 >8 7 4 B2 0 0 0 B3 >8 >8 5 B4 >8 >8 5 B5 0 0
0 B6 >8 7 3.5 B7 >8 7 4 B8 >8 7 3 C2 >8 2 1 C3 >8 1
0 D1 >8 2.5 2.5 D2 7.5 2.5 2 E1 1 0 0 E2 1 0 0 F2 5 0 0 F3 3 0 0
G1 3.5 0 0 H1 6 <1 0 H2 4 0 0 HI titer is diluted by the "n"th
power of 2. "n" is the numbers shown in the table.
[0507] The activity of the above purified 10F7 single chain
antibody was tested using the neutralization method. 7 virus
strains that were isolated from chicken, duck and various wild
birds in Hong Kong, Indonesia, Qinghai and other areas during the
period from 2002 to 2006 were used to test the activity of 10F7
using the HI assay. The antibody showed good neutralization
activity against 5 of the virus strains (Table 34). At 64 times of
dilution, the antibody was still able to inhibit virus infection of
host cells.
TABLE-US-00035 TABLE 34 10F7 single chain antibody neutralization
test results. Virus strain dilution of 10F7 scFv CK/HK/Yu22/02 64
DK/IDN/MS/04 16 CK/IDN/2A/04 32 BhGs/QH/15/05 16 CK/HK/213/03 <1
CP Heron/HK/18/05 8 Oriental Magpie Robin/HK/366/2006 <1
Example 13
Detection of 7aa Peptides Activities
[0508] The three bacteriophages containing the 7aa peptides of
8H5A, 8H5E and 3C8A were amplified in large numbers. They were
dissolved in PBS after being precipitated with PEG. Phage titer was
between 10.sup.11 and 10.sup.12. Microplates were pre-coated with
monoclonal antibodies 8H5, 4A1, 9N7 and 4D11 at 5 .mu.g/ml. The
plates were blocked with PBS containing 5% milk The three
bacteriophages were serially diluted; and added to the plates. The
reaction was carried on for 1 hr. Then the plates were washed for 5
times. 1:5,000 diluted mouse anti-M13/HRP antibody (Amersham
Phamarcia Biotech, UK) was added as the secondary antibody and
incubated for 0.5 hr. The results were read after the reaction was
completed. The results are shown in Table 35 below, which
demonstrated that the specific reactions between the peptide 8H5A
and the monoclonal antibody 8H5 were good, and the specific
reactions between 8H5A and the other three monoclonal antibodies
were weak. The specific reaction between 8H5E and monoclonal
antibody 8H5 was relatively poor.
TABLE-US-00036 TABLE 35 Detection results of the specific binding
activity of 7aa Peptides to monoclonal antibodies Monoclonal
Antibody 8H5A (1:1000) 8H5E (1:1000) 8H5 0.559 0.25 4A1 0.158 0.142
9N7 0.062 0.065 4D11 0.118 0.078
Example 14
MAB Capture System vs. pRNA Capture System Using Nanogen Influenza
A Test
[0509] To compare the sensitivity of Nanogen Influenza A Test using
2 different capture systems and to compare the pRNA capture system
using 2 different test strips
[0510] Procedure for cutting test strips: Prepare the membrane card
by placing absorbent pad (3.5 cm wide) and Tween-20 pre-treated;
precut polyester wicking pad (1.4 cm wide) on the adhesive side
with 1 mm overlap with the membrane, striped with appropriate
antibody. Cut this prepared card into 5 mm wide test strips with a
paper cutter
[0511] Virus Dilution: Dilute Influenza A/Texas/1/77 40960 HA
units/mL virus with 1% BSA in PBS according to the following
dilution scheme.
TABLE-US-00037 TABLE 36 Dilution Scheme for Flu A Virus Start conc.
1% BSA in End conc. (HA dilution Start vol. PBS vol. End vol. (HA
units/mL) factor (.mu.L) (.mu.L) (.mu.L) units/mL) 40960 1:100 1 99
100 409.6 409.6 1:10 50 450 500 40.96 40.96 1:10 20 180 200
4.096
[0512] Europium Dilution: Dilute 17-10-06-1 europium conjugate to
0.02% using the europium dilution buffer; 245 .mu.L of europium
dilution buffer+5 .mu.L of 1% stock . Sonicate the diluted europium
conjugate using a probe sonicator (Fischer Model 550) at power
setting of 3, total time 20 seconds (10 sec on time and 5 sec off
time).
[0513] pRNA Conjugate dilution: Dilute 102a10-7304 (0.39 mg/ml)
conjugate to 0.1 mg/ml with 10 mM PBS, pH 7.2; 37.2 .mu.L of
PBS+12.8 .mu.L of 102a10-7304 conjugate.
[0514] Assay Procedure: Prepare the assay mixtures according to the
following tables
TABLE-US-00038 TABLE 37 Assay Mix for the MAB capture system 4.096
HA 40.96 HA units/ml units/ml HA units/test 0 0.01 0.02 0.04 0.1 1
3.times. extraction 41.75 41.75 41.75 41.75 41.75 41.75 buffer
(.mu.L) 0.2M 1.25 1.25 1.25 1.25 1.25 1.25 TCEP (.mu.L) 0.02% euro-
12.5 12.5 12.5 12.5 12.5 12.5 pium conj. (.mu.L) Flu A virus
(.mu.L) 0 6.25 12.5 25 6.25 62.5 water (.mu.L) 69.5 63.25 57 44.5
63.25 7 Total vol. for 125 125 125 125 125 125 2.5 test (.mu.L)
TABLE-US-00039 TABLE 38 Assay Mix for the pRNA capture system 4.096
HA 40.96 HA units/ml units/ml HA units/test 0 0.01 0.02 0.04 0.1 1
3.times. extraction 83.5 83.5 83.5 83.5 83.5 83.5 buffer (.mu.L)
0.2M TCEP (.mu.L) 2.5 2.5 2.5 2.5 2.5 2.5 0.02% euro- 25.0 25.0
25.0 25.0 25.0 25.0 pium conj. (.mu.L) Flu A virus (.mu.L) 0.0 12.5
25.0 50.0 12.5 125.0 0.1 mg/mL 5.0 5.0 5.0 5.0 5.0 5.0 102a10-7304
conj. (.mu.L) water (.mu.L) 134.0 121.5 109.0 84.0 121.5 9.0 Total
vol. for 250.0 250.0 250.0 250.0 250.0 250.0 5 tests (.mu.L)
TABLE-US-00040 TABLE 39 Materials Material Lot Note 1 Absorbent
Pad: Grade 222, 6150502 cat #2228-1212 12 IN .times. 12 IN 2
Wicking Pad: Tween 20 06PPP0157C Grade 6613 Pre-treated Polyester
Pad 3 Anti-flu A Medix 7304 310806 (1.5 mg/ml) membrane 4
102b10-5G4 striped 131206-2-1 conc. 0.75 mg/ml, membrane w/o
blocking 5 102b10-5G4 striped 131206-2-2 conc. 0.75 membrane
mg/ml/w/0.5% casein (toronto) blocked 6 3x Extraction Buffer
2271-001 DOM Nov. 15, 2006 7 1x Extraction Buffer 2232-003 DOM Nov.
15, 2006 8 0.2M TCEP 2232-180 DOM Nov. 9, 2006 9 Molecular Biology
Water 1347929 CAT #10977-015, GIBCO 10 1% BSA in 0.10M Phosphate
2271-036 DOM Dec. 14, 2006 Buffered Saline 11 Flu A/Texas/1/77
virus @ 13037A3 CAT # EL-13-02 40960 HA units/mL 12 Europium
dilution buffer N/A DOM Jun. 8, 2006 by Roy Chung 13 Europium
conjugate with 17-10-06-1 Prepared by M4090913 Philip Lam 14
102a10-7304 pRNA 2271-028-2 DOM Dec. 8, 2006, conjugate conc. 0.39
mg/ml 15 10 mM PBS pH 7.2 2271-035 DOM Dec. 14, 2006
[0515] Incubate 7304 (1.5 mg/mL) test strips with MAB CAPTURE
SYSTEM assay mix.
[0516] Incubate 102b10-5G4 (0.75 mg/mL) w/o blocking, and w/0.5%
casein blocked test strips with pRNA CAPTURE SYSTEM assay mix.
[0517] Run the experiment in duplicates.
[0518] Let it stand at room temperature for 15 min.
[0519] Add 50 .mu.L of 1.times. extraction buffer to the same tube
to wash the test strips.
[0520] After 5 min, read the test strips using a fluorescence
reader.
Results
TABLE-US-00041 [0521] TABLE 40 MAB capture system (See FIG. 39) HA
units/test 0 0.01 0.02 0.04 0.1 1 1 528.1 793.7 935.7 1421.6 2550.1
21111.3 2 569.6 776.0 1037.5 1440.0 2395.4 19961.8 AVG 548.8 784.9
986.6 1430.8 2472.8 20536.5 S/N 1.4 1.8 2.6 4.5 37.4
TABLE-US-00042 TABLE 41 pRNA capture system with 0.75 mg/ml
membrane blocked with 0.5% casein (See FIG. 40) HA units/test 0
0.01 0.02 0.04 0.1 1 1 114.6 434.1 703.0 1360.9 3222.7 21209.1 2
222.6 628.5 804.6 1385.1 2891.4 19440.1 AVG 168.6 531.3 753.8
1373.0 3057.1 20324.6 S/N 3.2 4.5 8.1 18.1 120.5
TABLE-US-00043 TABLE 42 pRNA capture system with 0.75 mg/ml
membrane without blocking (See FIG. 41) HA units/test 0 0.01 0.02
0.04 0.1 1 1 284.4 737.9 940.0 1880.4 3336.0 22387.5 2 318.1 612.2
814.6 1471.3 3560.8 22383.6 AVG 301.3 675.1 877.3 1675.9 3448.4
22385.6 S/N 2.2 2.9 5.6 11.4 74.3
[0522] Conclusion: As the data indicates the sensitivity of the
test is highest with pRNA capture system when used with test strips
blocked with 0.5% casein from Toronto with a single to noise ration
of 120 with 1 HA unit but using direct spotting of MAb onto the
test strip the S/N ratio was only 37.4 indicating the pRNA capture
system significantly improved the sensitivity of the assay
system.
TABLE-US-00044 8H5 Vh Nucleotide sequence SEQ ID NO: 1 caggttcagc
tgcagcagtc tggagctgag ctgatgaagc ctggggcctc agtgaagata tcctgcaagg
ctactggcta cactttcagt aactactgga tagagtggat aaagcagagg cctggacatg
gccttgagtg gattggagag attttacctg gaagcgatag aacaaactac aatgggaagt
tcaagggcaa ggccacattc actgcagata catcctccaa cacagcccac atgcaactca
gtagcctgac atctgaggac tctgccgtct attactgtgc aaatagatac gacgggtatt
attttggttt ggattactgg ggtcaaggaa cctcagtcgc cgtctcctca gcc SEQ ID
NO: 2 Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Met Lys Pro Gly
Ala 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Ala Thr Gly Tyr Thr Phe
Ser Asn Tyr 20 25 30 Trp Ile Glu Trp Ile Lys Gln Arg Pro Gly His
Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile Leu Pro Gly Ser Asp Arg
Thr Asn Tyr Asn Gly Lys Phe 50 55 60 Lys Gly Lys Ala Thr Phe Thr
Ala Asp Thr Ser Ser Asn Thr Ala His 65 70 75 80 Met Gln Leu Ser Ser
Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Asn Arg
Tyr Asp Gly Tyr Tyr Phe Gly Leu Asp Tyr Trp Gly Gln 100 105 110 Gly
Thr Ser Val Ala Val Ser Ser Ala 8H5 Vk Nucleotide sequence SEQ ID
NO: 3 gaaatcgtgc tcacccagtc tccagcaatc atgtctgcat ctctagggga
gaaggtcacc atgagctgca gggccagctc aagtgtaaat ttcgtttact ggtaccagca
gaggtcagat gcctccccca aactattgat ttactattca tccaacctgg ctcctggagt
cccacctcgc ttcagtggca gtgggtctgg gaactcttat tctctcacaa tcagcggctt
ggagggtgaa gatgctgcca cttattactg ccagcacttt actagttccc cgtacacgtt
cggagggggg accaacctgg aaataaaacg g 8H5 Vk Amino Acid sequence SEQ
ID NO: 4 Glu Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser
Leu Gly 1 5 10 15 Glu Lys Val Thr Met Ser Cys Arg Ala Ser Ser Ser
Val Asn Phe Val 20 25 30 Tyr Trp Tyr Gln Gln Arg Ser Asp Ala Ser
Pro Lys Leu Leu Ile Tyr 35 40 45 Tyr Ser Ser Asn Leu Ala Pro Gly
Val Pro Pro Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Asn Ser Tyr
Ser Leu Thr Ile Ser Gly Leu Glu Gly Glu 65 70 75 80 Asp Ala Ala Thr
Tyr Tyr Cys Gln His Phe Thr Ser Ser Pro Tyr Thr 85 90 95 Phe Gly
Gly Gly Thr Asn Leu Glu Ile Lys Arg 100 105 3C8 Vh Nucleotide
sequence SEQ ID NO: 5 cagatccagt tggtgcagtc tggacctgag ctgaagaagc
ctggagagac agtcaagatc tcctgcaagg cctctgggta cagcttcaca aactatggaa
tgaactgggt gaagcaggct ccaggaaagg gtctaaagtg gatgggctgg ataaacacct
acaccggaga gccagcctat gctgatgact tcaagggacg gtttgccttc tctctggaaa
cctctgccag cactgcctat ttgcagatca acaacctcaa aaatgaggac acggctacat
atttctgtgc aagatggaat agagatgcta tggactactg gggtcaagga acctcggtca
ccgtatctag c 3C8 Vh Amino Acid sequence SEQ ID NO: 6 Gln Ile Gln
Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu 1 5 10 15 Thr
Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Asn Tyr 20 25
30 Gly Met Asn Trp Val Lys Gln Ala Pro Gly Lys Gly Leu Lys Trp Met
35 40 45 Gly Trp Ile Asn Thr Tyr Thr Gly Glu Pro Ala Tyr Ala Asp
Asp Phe 50 55 60 Lys Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala
Ser Thr Ala Tyr 65 70 75 80 Leu Gln Ile Asn Asn Leu Lys Asn Glu Asp
Thr Ala Thr Tyr Phe Cys 85 90 95 Ala Arg Trp Asn Arg Asp Ala Met
Asp Tyr Trp Gly Gln Gly Thr Ser 100 105 110 Val Thr Val Ser Ser 115
3C8 VK Nucleotide sequence SEQ ID NO: 7 gacattgtgc tgacccaatc
tccagcttct ttggctgtgt ctcttgggca gagggccacc atatcctgca gagccagtga
aagtgttgat agttctgaca atagtcttat gcactggtac cagcagaaac caggacagcc
acccaaactc ctcatctatc gtgcatccaa cctagaatct gggatccctg ccaggttcag
tggcagtggg tctaggacag acttcaccct caccattaat cctgtggagg ctgatgatgt
tgcaacctat tactgtcagc aaagtattgg ggatcctccg tacacgttcg gaggggggac
caagctggaa ataaaacgg 3C8 VK Amino Acid sequence SEQ ID NO: 8 Asp
Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly 1 5 10
15 Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Glu Ser Val Asp Ser Ser
20 25 30 Asp Asn Ser Leu Met His Trp Tyr Gln Gln Lys Pro Gly Gln
Pro Pro 35 40 45 Lys Leu Leu Ile Tyr Arg Ala Ser Asn Leu Glu Ser
Gly Ile Pro Ala 50 55 60 Arg Phe Ser Gly Ser Gly Ser Arg Thr Asp
Phe Thr Leu Thr Ile Asn 65 70 75 80 Pro Val Glu Ala Asp Asp Val Ala
Thr Tyr Tyr Cys Gln Gln Ser Ile 85 90 95 Gly Asp Pro Pro Tyr Thr
Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg 100 105 110 10F7 Vh
Nucleotide sequence SEQ ID NO: 9 caggtccaac tgcagcagcc tggggctgaa
cttgtgaagc ctggggcttc agtgaagctg tcctgcaagg cttctggcta caccttcacc
agctactgga tgcactgggt gaagcagagg cctggacagg gccttgagtg gatcggagag
attgatcctt ctgattctta tactaactac aatcagaagt tcaagggcaa ggccacattg
actgtagaca aatcctccag cacagcctac atgcagctca gcagcctgac atctgaggac
tctgcggtct attactgtgc aagggggggt acaggagact ttcactatgc tatggactac
tggggtcaag gcacctcggt caccgtatca tcg 10F7 Vh Amino Acid sequence
SEQ ID NO: 10 Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Lys
Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr
Thr Phe Thr Ser Tyr 20 25 30 Trp Met His Trp Val Lys Gln Arg Pro
Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile Asp Pro Ser Asp
Ser Tyr Thr Asn Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Lys Ala Thr
Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu
Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala
Arg Gly Gly Thr Gly Asp Phe His Tyr Ala Met Asp Tyr Trp Gly 100 105
110 Gln Gly Thr Ser Val Thr Val Ser Ser 115 120 10F7 VK Nucleotide
sequence SEQ ID NO: 11 gacatcctga tgacccaatc tccatcctcc atgtctgtat
ctctgggaga cacagtcagc atcacttgcc atgcaagtca gggcattagc agtaatatag
ggtggttgca gcagaaacca gggaaatcat ttaagggcct gatctatcat ggaaccaact
tggaagatgg agttccatca aggttcagtg gcagtggatc tggagcagat tattctctca
ccatcagcag cctggaatct gaagattttg cagactatta ctgtgtacag tatgttcagt
tcccgtacac gttcggaggg ggcaccaagc tggaaatcaa acgg 10F7 VK Amino Acid
sequence SEQ ID NO: 12 Asp Ile Leu Met Thr Gln Ser Pro Ser Ser Met
Ser Val Ser Leu Gly 1 5 10 15 Asp Thr Val Ser Ile Thr Cys His Ala
Ser Gln Gly Ile Ser Ser Asn 20 25 30 Ile Gly Trp Leu Gln Gln Lys
Pro Gly Lys Ser Phe Lys Gly Leu Ile 35 40 45 Tyr His Gly Thr Asn
Leu Glu Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser
Gly Ala Asp Tyr Ser Leu Thr Ile Ser Ser Leu Glu Ser 65 70 75 80 Glu
Asp Phe Ala Asp Tyr Tyr Cys Val Gln Tyr Val Gln Phe Pro Tyr 85 90
95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg 100 105
Artificial sequence/Unknown Organism SEQ ID NO: 13 catgggatgc
tgccggtgta t. Artificial Sequence/Unknown Organism SEQ ID NO: 14
aattctgggc cttggctgac g. Artificial Sequence/Unknown Organism SEQ
ID NO: 15 tggccgcctc tgtcgaagaa g. 4D1 VH Nucleotide sequence SEQ
ID NO: 16 caggtccaac tgcagcagcc tggggctgag cttgtgaagc ctggggcttc
agtgaacctg tcctgtaagg cttctggcta caccttcacc agctactgga tgcactgggt
gaagcagagg
cctggacaag gccttgagtg gatcggagag attgatcctt ctgatagttt tactacctac
aatcaaaact tcaaagacag ggccacattg actgtagaca aatcatccag cacagcctac
atgcagctca gaagtctgac atctgaggac tctgcggtct attactgtgc cagggggggt
ccaggagact ttcgctatgc tatggattac tggggccaag gcacctcggt caccgtctcc
tca. 4D1 VH Amino Acid sequence SEQ ID NO: 17 Gln Val Gln Leu Gln
Gln Pro Gly Ala Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Asn
Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp
Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40
45 Gly Glu Ile Asp Pro Ser Asp Ser Phe Thr Thr Tyr Asn Gln Asn Phe
50 55 60 Lys Asp Arg Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr
Ala Tyr 65 70 75 80 Met Gln Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala
Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Gly Pro Gly Asp Phe Arg Tyr
Ala Met Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Ser Val Thr Val Ser
Ser 115 120 4Dl VK Nucleotide sequence SEQ ID NO: 18 gacatcctga
tgacccaatc tccatcctcc atgtctgtat ctctgggaga cacagtcagc atcacttgcc
atgcaagtca gggcattagc agtaatatag ggtggttgca gcagaaacca gggaaatcat
ttaagggcct gatctatcat ggaaccaact tggaagatgg agttccatca aggttcagtg
gcagtggatc tggagcagat tattctctca ccatcagcag cctggaatcc gaagactttg
cagactatta ctgtgtacag tatgttcagt ttccctacac gttcggaggg gggaccaagc
tggaaataaa acgggct 4D1 Vk Amino Acid sequence SEQ ID NO: 19 Asp Ile
Leu Met Thr Gln Ser Pro Ser Ser Met Ser Val Ser Leu Gly 1 5 10 15
Asp Thr Val Ser Ile Thr Cys His Ala Ser Gln Gly Ile Ser Ser Asn 20
25 30 Ile Gly Trp Leu Gln Gln Lys Pro Gly Lys Ser Phe Lys Gly Leu
Ile 35 40 45 Tyr His Gly Thr Asn Leu Glu Asp Gly Val Pro Ser Arg
Phe Ser Gly 50 55 60 Ser Gly Ser Gly Ala Asp Tyr Ser Leu Thr Ile
Ser Ser Leu Glu Ser 65 70 75 80 Glu Asp Phe Ala Asp Tyr Tyr Cys Val
Gln Tyr Val Gln Phe Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys
Leu Glu Ile Lys Arg Ala 100 105 3G4 VH Nucleotide sequence SEQ ID
NO: 20 caggtccaac tgcagcagtc tggggctgag ctggtgaggc ctggggtctc
agtgaagatt tcctgcaagg gttctggcta cacattcact gattatgcta tgcattgggt
gaagcagagt catgcaaaga gtctagagtg gattggactt attaatactg actatggtga
tactacttac aaccagaagt tcaagggcaa ggccacaatg actgtagaca aatcctccaa
cacagcctat atggaacttg ccagactgac atctgaggat tctgccatct attactgtgc
aagatcggac tatgattact atttctgtgg tatggactac tggggtcaag gaaccacggt
caccgaatct cta 3G4 VH Amino Acid sequence SEQ ID NO: 21 Gln Val Gln
Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Pro Gly Val 1 5 10 15 Ser
Val Lys Ile Ser Cys Lys Gly Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25
30 Ala Met His Trp Val Lys Gln Ser His Ala Lys Ser Leu Glu Trp Ile
35 40 45 Gly Leu Ile Asn Thr Asp Tyr Gly Asp Thr Thr Tyr Asn Gln
Lys Phe 50 55 60 Lys Gly Lys Ala Thr Met Thr Val Asp Lys Ser Ser
Asn Thr Ala Tyr 65 70 75 80 Met Glu Leu Ala Arg Leu Thr Ser Glu Asp
Ser Ala Ile Tyr Tyr Cys 85 90 95 Ala Arg Ser Asp Tyr Asp Tyr Tyr
Phe Cys Gly Met Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Thr Val Thr
Glu Ser Leu 115 120 2F2 VH Nucleotide sequence SEQ ID NO: 22
caggtgcagc tgaaggagtc aggacctggc ctggtggcgc cctcacagcg cctgtccatc
acatgcaccg tctcagggtt ctcattaacc ggctatggtg tacactggat tcgccagtct
ccaggaaagg gtctggagtg gctgggaatg atatgggctg agggaagaac cgactataat
tcagttctca aatccagact gagcatcaat aaggacaatt ccaggagcca agttttctta
gaaatgaaca gtctgcaaac tgatgacaca gccaggtact actgtgccag agaggtgatt
actacggaag cctggtactt cgatgtctgg ggccaaggaa cctcggtcac cgaatct 2F2
VH Amino Acid sequence SEQ ID NO: 23 Gln Val Gln Leu Lys Glu Ser
Gly Pro Gly Leu Val Ala Pro Ser Gln 1 5 10 15 Arg Leu Ser Ile Thr
Cys Thr Val Ser Gly Phe Ser Leu Thr Gly Tyr 20 25 30 Gly Val His
Trp Ile Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45 Gly
Met Ile Trp Ala Glu Gly Arg Thr Asp Tyr Asn Ser Val Leu Lys 50 55
60 Ser Arg Leu Ser Ile Asn Lys Asp Asn Ser Arg Ser Gln Val Phe Leu
65 70 75 80 Glu Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Arg Tyr Tyr
Cys Ala 85 90 95 Arg Glu Val Ile Thr Thr Glu Ala Trp Tyr Phe Asp
Val Trp Gly Gln 100 105 110 Gly Thr Ser Val Thr Glu Ser 115 2F2 VK
Nucleotide sequence SEQ ID NO: 24 gacattgtga tgactcagtc tccagccacc
ctgtctgtga ctccaggaga tagagtctct ctttcctgca gggccagcca gagtattagc
gactacttat actggtatca acaaaaatca catgagtctc caaggcttct catcaaatat
gcttcccaat ccatctctgg gatcccctcc agattcagtg gcagtggatc agggtcagat
ttcactctca ctatcaacag tgtggaacct gaagatgttg gaatgtatta ctgtcaaaat
ggtcacacct ttccgctcac gttcggtgct ggcaccaagc tggaaatcaa acgg 2F2 VK
Amino Acid sequence SEQ ID NO: 25 Asp Ile Val Met Thr Gln Ser Pro
Ala Thr Leu Ser Val Thr Pro Gly 1 5 10 15 Asp Arg Val Ser Leu Ser
Cys Arg Ala Ser Gln Ser Ile Ser Asp Tyr 20 25 30 Leu Tyr Trp Tyr
Gln Gln Lys Ser His Glu Ser Pro Arg Leu Leu Ile 35 40 45 Lys Tyr
Ala Ser Gln Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly 50 55 60
Ser Gly Ser Gly Ser Asp Phe Thr Leu Thr Ile Asn Ser Val Glu Pro 65
70 75 80 Glu Asp Val Gly Met Tyr Tyr Cys Gln Asn Gly His Thr Phe
Pro Leu 85 90 95 Thr Phe Gly Ala Gly Thr Lys Leu Glu Ile Lys Arg.
Sequence CWU 1
1
1141363DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 1caggttcagc tgcagcagtc tggagctgag
ctgatgaagc ctggggcctc agtgaagata 60tcctgcaagg ctactggcta cactttcagt
aactactgga tagagtggat aaagcagagg 120cctggacatg gccttgagtg
gattggagag attttacctg gaagcgatag aacaaactac 180aatgggaagt
tcaagggcaa ggccacattc actgcagata catcctccaa cacagcccac
240atgcaactca gtagcctgac atctgaggac tctgccgtct attactgtgc
aaatagatac 300gacgggtatt attttggttt ggattactgg ggtcaaggaa
cctcagtcgc cgtctcctca 360gcc 3632121PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
2Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Met Lys Pro Gly Ala1 5
10 15Ser Val Lys Ile Ser Cys Lys Ala Thr Gly Tyr Thr Phe Ser Asn
Tyr 20 25 30Trp Ile Glu Trp Ile Lys Gln Arg Pro Gly His Gly Leu Glu
Trp Ile 35 40 45Gly Glu Ile Leu Pro Gly Ser Asp Arg Thr Asn Tyr Asn
Gly Lys Phe 50 55 60Lys Gly Lys Ala Thr Phe Thr Ala Asp Thr Ser Ser
Asn Thr Ala His65 70 75 80Met Gln Leu Ser Ser Leu Thr Ser Glu Asp
Ser Ala Val Tyr Tyr Cys 85 90 95Ala Asn Arg Tyr Asp Gly Tyr Tyr Phe
Gly Leu Asp Tyr Trp Gly Gln 100 105 110Gly Thr Ser Val Ala Val Ser
Ser Ala 115 1203321DNAArtificial SequenceDescription of Artificial
Sequence Synthetic polynucleotide 3gaaatcgtgc tcacccagtc tccagcaatc
atgtctgcat ctctagggga gaaggtcacc 60atgagctgca gggccagctc aagtgtaaat
ttcgtttact ggtaccagca gaggtcagat 120gcctccccca aactattgat
ttactattca tccaacctgg ctcctggagt cccacctcgc 180ttcagtggca
gtgggtctgg gaactcttat tctctcacaa tcagcggctt ggagggtgaa
240gatgctgcca cttattactg ccagcacttt actagttccc cgtacacgtt
cggagggggg 300accaacctgg aaataaaacg g 3214107PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
4Glu Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Leu Gly1 5
10 15Glu Lys Val Thr Met Ser Cys Arg Ala Ser Ser Ser Val Asn Phe
Val 20 25 30Tyr Trp Tyr Gln Gln Arg Ser Asp Ala Ser Pro Lys Leu Leu
Ile Tyr 35 40 45Tyr Ser Ser Asn Leu Ala Pro Gly Val Pro Pro Arg Phe
Ser Gly Ser 50 55 60Gly Ser Gly Asn Ser Tyr Ser Leu Thr Ile Ser Gly
Leu Glu Gly Glu65 70 75 80Asp Ala Ala Thr Tyr Tyr Cys Gln His Phe
Thr Ser Ser Pro Tyr Thr 85 90 95Phe Gly Gly Gly Thr Asn Leu Glu Ile
Lys Arg 100 1055351DNAArtificial SequenceDescription of Artificial
Sequence Synthetic polynucleotide 5cagatccagt tggtgcagtc tggacctgag
ctgaagaagc ctggagagac agtcaagatc 60tcctgcaagg cctctgggta cagcttcaca
aactatggaa tgaactgggt gaagcaggct 120ccaggaaagg gtctaaagtg
gatgggctgg ataaacacct acaccggaga gccagcctat 180gctgatgact
tcaagggacg gtttgccttc tctctggaaa cctctgccag cactgcctat
240ttgcagatca acaacctcaa aaatgaggac acggctacat atttctgtgc
aagatggaat 300agagatgcta tggactactg gggtcaagga acctcggtca
ccgtatctag c 3516117PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 6Gln Ile Gln Leu Val Gln Ser Gly Pro
Glu Leu Lys Lys Pro Gly Glu1 5 10 15Thr Val Lys Ile Ser Cys Lys Ala
Ser Gly Tyr Ser Phe Thr Asn Tyr 20 25 30Gly Met Asn Trp Val Lys Gln
Ala Pro Gly Lys Gly Leu Lys Trp Met 35 40 45Gly Trp Ile Asn Thr Tyr
Thr Gly Glu Pro Ala Tyr Ala Asp Asp Phe 50 55 60Lys Gly Arg Phe Ala
Phe Ser Leu Glu Thr Ser Ala Ser Thr Ala Tyr65 70 75 80Leu Gln Ile
Asn Asn Leu Lys Asn Glu Asp Thr Ala Thr Tyr Phe Cys 85 90 95Ala Arg
Trp Asn Arg Asp Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser 100 105
110Val Thr Val Ser Ser 1157339DNAArtificial SequenceDescription of
Artificial Sequence Synthetic polynucleotide 7gacattgtgc tgacccaatc
tccagcttct ttggctgtgt ctcttgggca gagggccacc 60atatcctgca gagccagtga
aagtgttgat agttctgaca atagtcttat gcactggtac 120cagcagaaac
caggacagcc acccaaactc ctcatctatc gtgcatccaa cctagaatct
180gggatccctg ccaggttcag tggcagtggg tctaggacag acttcaccct
caccattaat 240cctgtggagg ctgatgatgt tgcaacctat tactgtcagc
aaagtattgg ggatcctccg 300tacacgttcg gaggggggac caagctggaa ataaaacgg
3398113PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 8Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu
Ala Val Ser Leu Gly1 5 10 15Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser
Glu Ser Val Asp Ser Ser 20 25 30Asp Asn Ser Leu Met His Trp Tyr Gln
Gln Lys Pro Gly Gln Pro Pro 35 40 45Lys Leu Leu Ile Tyr Arg Ala Ser
Asn Leu Glu Ser Gly Ile Pro Ala 50 55 60Arg Phe Ser Gly Ser Gly Ser
Arg Thr Asp Phe Thr Leu Thr Ile Asn65 70 75 80Pro Val Glu Ala Asp
Asp Val Ala Thr Tyr Tyr Cys Gln Gln Ser Ile 85 90 95Gly Asp Pro Pro
Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105
110Arg9363DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 9caggtccaac tgcagcagcc tggggctgaa
cttgtgaagc ctggggcttc agtgaagctg 60tcctgcaagg cttctggcta caccttcacc
agctactgga tgcactgggt gaagcagagg 120cctggacagg gccttgagtg
gatcggagag attgatcctt ctgattctta tactaactac 180aatcagaagt
tcaagggcaa ggccacattg actgtagaca aatcctccag cacagcctac
240atgcagctca gcagcctgac atctgaggac tctgcggtct attactgtgc
aagggggggt 300acaggagact ttcactatgc tatggactac tggggtcaag
gcacctcggt caccgtatca 360tcg 36310121PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
10Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Lys Pro Gly Ala1
5 10 15Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser
Tyr 20 25 30Trp Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu
Trp Ile 35 40 45Gly Glu Ile Asp Pro Ser Asp Ser Tyr Thr Asn Tyr Asn
Gln Lys Phe 50 55 60Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser
Ser Thr Ala Tyr65 70 75 80Met Gln Leu Ser Ser Leu Thr Ser Glu Asp
Ser Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly Gly Thr Gly Asp Phe His
Tyr Ala Met Asp Tyr Trp Gly 100 105 110Gln Gly Thr Ser Val Thr Val
Ser Ser 115 12011324DNAArtificial SequenceDescription of Artificial
Sequence Synthetic polynucleotide 11gacatcctga tgacccaatc
tccatcctcc atgtctgtat ctctgggaga cacagtcagc 60atcacttgcc atgcaagtca
gggcattagc agtaatatag ggtggttgca gcagaaacca 120gggaaatcat
ttaagggcct gatctatcat ggaaccaact tggaagatgg agttccatca
180aggttcagtg gcagtggatc tggagcagat tattctctca ccatcagcag
cctggaatct 240gaagattttg cagactatta ctgtgtacag tatgttcagt
tcccgtacac gttcggaggg 300ggcaccaagc tggaaatcaa acgg
32412108PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 12Asp Ile Leu Met Thr Gln Ser Pro Ser Ser Met
Ser Val Ser Leu Gly1 5 10 15Asp Thr Val Ser Ile Thr Cys His Ala Ser
Gln Gly Ile Ser Ser Asn 20 25 30Ile Gly Trp Leu Gln Gln Lys Pro Gly
Lys Ser Phe Lys Gly Leu Ile 35 40 45Tyr His Gly Thr Asn Leu Glu Asp
Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Ala Asp Tyr
Ser Leu Thr Ile Ser Ser Leu Glu Ser65 70 75 80Glu Asp Phe Ala Asp
Tyr Tyr Cys Val Gln Tyr Val Gln Phe Pro Tyr 85 90 95Thr Phe Gly Gly
Gly Thr Lys Leu Glu Ile Lys Arg 100 1051321DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 13catgggatgc tgccggtgta t 211421DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 14aattctgggc cttggctgac g 211521DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 15tggccgcctc tgtcgaagaa g 2116363DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
16caggtccaac tgcagcagcc tggggctgag cttgtgaagc ctggggcttc agtgaacctg
60tcctgtaagg cttctggcta caccttcacc agctactgga tgcactgggt gaagcagagg
120cctggacaag gccttgagtg gatcggagag attgatcctt ctgatagttt
tactacctac 180aatcaaaact tcaaagacag ggccacattg actgtagaca
aatcatccag cacagcctac 240atgcagctca gaagtctgac atctgaggac
tctgcggtct attactgtgc cagggggggt 300ccaggagact ttcgctatgc
tatggattac tggggccaag gcacctcggt caccgtctcc 360tca
36317121PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 17Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu
Val Lys Pro Gly Ala1 5 10 15Ser Val Asn Leu Ser Cys Lys Ala Ser Gly
Tyr Thr Phe Thr Ser Tyr 20 25 30Trp Met His Trp Val Lys Gln Arg Pro
Gly Gln Gly Leu Glu Trp Ile 35 40 45Gly Glu Ile Asp Pro Ser Asp Ser
Phe Thr Thr Tyr Asn Gln Asn Phe 50 55 60Lys Asp Arg Ala Thr Leu Thr
Val Asp Lys Ser Ser Ser Thr Ala Tyr65 70 75 80Met Gln Leu Arg Ser
Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly Gly
Pro Gly Asp Phe Arg Tyr Ala Met Asp Tyr Trp Gly 100 105 110Gln Gly
Thr Ser Val Thr Val Ser Ser 115 12018327DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
18gacatcctga tgacccaatc tccatcctcc atgtctgtat ctctgggaga cacagtcagc
60atcacttgcc atgcaagtca gggcattagc agtaatatag ggtggttgca gcagaaacca
120gggaaatcat ttaagggcct gatctatcat ggaaccaact tggaagatgg
agttccatca 180aggttcagtg gcagtggatc tggagcagat tattctctca
ccatcagcag cctggaatcc 240gaagactttg cagactatta ctgtgtacag
tatgttcagt ttccctacac gttcggaggg 300gggaccaagc tggaaataaa acgggct
32719109PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 19Asp Ile Leu Met Thr Gln Ser Pro Ser Ser Met
Ser Val Ser Leu Gly1 5 10 15Asp Thr Val Ser Ile Thr Cys His Ala Ser
Gln Gly Ile Ser Ser Asn 20 25 30Ile Gly Trp Leu Gln Gln Lys Pro Gly
Lys Ser Phe Lys Gly Leu Ile 35 40 45Tyr His Gly Thr Asn Leu Glu Asp
Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Ala Asp Tyr
Ser Leu Thr Ile Ser Ser Leu Glu Ser65 70 75 80Glu Asp Phe Ala Asp
Tyr Tyr Cys Val Gln Tyr Val Gln Phe Pro Tyr 85 90 95Thr Phe Gly Gly
Gly Thr Lys Leu Glu Ile Lys Arg Ala 100 10520363DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
20caggtccaac tgcagcagtc tggggctgag ctggtgaggc ctggggtctc agtgaagatt
60tcctgcaagg gttctggcta cacattcact gattatgcta tgcattgggt gaagcagagt
120catgcaaaga gtctagagtg gattggactt attaatactg actatggtga
tactacttac 180aaccagaagt tcaagggcaa ggccacaatg actgtagaca
aatcctccaa cacagcctat 240atggaacttg ccagactgac atctgaggat
tctgccatct attactgtgc aagatcggac 300tatgattact atttctgtgg
tatggactac tggggtcaag gaaccacggt caccgaatct 360cta
36321121PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 21Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu
Val Arg Pro Gly Val1 5 10 15Ser Val Lys Ile Ser Cys Lys Gly Ser Gly
Tyr Thr Phe Thr Asp Tyr 20 25 30Ala Met His Trp Val Lys Gln Ser His
Ala Lys Ser Leu Glu Trp Ile 35 40 45Gly Leu Ile Asn Thr Asp Tyr Gly
Asp Thr Thr Tyr Asn Gln Lys Phe 50 55 60Lys Gly Lys Ala Thr Met Thr
Val Asp Lys Ser Ser Asn Thr Ala Tyr65 70 75 80Met Glu Leu Ala Arg
Leu Thr Ser Glu Asp Ser Ala Ile Tyr Tyr Cys 85 90 95Ala Arg Ser Asp
Tyr Asp Tyr Tyr Phe Cys Gly Met Asp Tyr Trp Gly 100 105 110Gln Gly
Thr Thr Val Thr Glu Ser Leu 115 12022357DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
22caggtgcagc tgaaggagtc aggacctggc ctggtggcgc cctcacagcg cctgtccatc
60acatgcaccg tctcagggtt ctcattaacc ggctatggtg tacactggat tcgccagtct
120ccaggaaagg gtctggagtg gctgggaatg atatgggctg agggaagaac
cgactataat 180tcagttctca aatccagact gagcatcaat aaggacaatt
ccaggagcca agttttctta 240gaaatgaaca gtctgcaaac tgatgacaca
gccaggtact actgtgccag agaggtgatt 300actacggaag cctggtactt
cgatgtctgg ggccaaggaa cctcggtcac cgaatct 35723119PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
23Gln Val Gln Leu Lys Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln1
5 10 15Arg Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Gly
Tyr 20 25 30Gly Val His Trp Ile Arg Gln Ser Pro Gly Lys Gly Leu Glu
Trp Leu 35 40 45Gly Met Ile Trp Ala Glu Gly Arg Thr Asp Tyr Asn Ser
Val Leu Lys 50 55 60Ser Arg Leu Ser Ile Asn Lys Asp Asn Ser Arg Ser
Gln Val Phe Leu65 70 75 80Glu Met Asn Ser Leu Gln Thr Asp Asp Thr
Ala Arg Tyr Tyr Cys Ala 85 90 95Arg Glu Val Ile Thr Thr Glu Ala Trp
Tyr Phe Asp Val Trp Gly Gln 100 105 110Gly Thr Ser Val Thr Glu Ser
11524324DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 24gacattgtga tgactcagtc tccagccacc
ctgtctgtga ctccaggaga tagagtctct 60ctttcctgca gggccagcca gagtattagc
gactacttat actggtatca acaaaaatca 120catgagtctc caaggcttct
catcaaatat gcttcccaat ccatctctgg gatcccctcc 180agattcagtg
gcagtggatc agggtcagat ttcactctca ctatcaacag tgtggaacct
240gaagatgttg gaatgtatta ctgtcaaaat ggtcacacct ttccgctcac
gttcggtgct 300ggcaccaagc tggaaatcaa acgg 32425108PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
25Asp Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Thr Pro Gly1
5 10 15Asp Arg Val Ser Leu Ser Cys Arg Ala Ser Gln Ser Ile Ser Asp
Tyr 20 25 30Leu Tyr Trp Tyr Gln Gln Lys Ser His Glu Ser Pro Arg Leu
Leu Ile 35 40 45Lys Tyr Ala Ser Gln Ser Ile Ser Gly Ile Pro Ser Arg
Phe Ser Gly 50 55 60Ser Gly Ser Gly Ser Asp Phe Thr Leu Thr Ile Asn
Ser Val Glu Pro65 70 75 80Glu Asp Val Gly Met Tyr Tyr Cys Gln Asn
Gly His Thr Phe Pro Leu 85 90 95Thr Phe Gly Ala Gly Thr Lys Leu Glu
Ile Lys Arg 100 105268PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 26Gly Tyr Thr Phe Ser Asn Tyr
Trp1 5278PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 27Ile Leu Pro Gly Ser Asp Arg Thr1
52813PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 28Ala Asn Arg Tyr Asp Gly Tyr Tyr Phe Gly Leu Asp
Tyr1 5 10295PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 29Ser Ser Val Asn Phe1 5303PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 30Tyr
Ser Ser1319PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 31Gln His Phe Thr Ser Ser Pro Tyr Thr1
5328PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 32Gly Tyr Ser Phe Thr Asn Tyr Gly1
5338PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 33Ile Asn Thr His Thr Gly Glu Pro1
53410PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 34Ala Arg Trp Asn Arg Asp Ala Met Asp Tyr1 5
103510PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 35Glu Ser Val Asp Ser Ser Asp Asn Ser Leu1 5
10363PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 36Arg Ala Ser13710PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 37Gln
Gln Ser Ile Gly Asp Pro Pro Tyr Thr1 5 10388PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 38Gly
Tyr Thr Phe Thr Ser Tyr Trp1 5398PRTArtificial SequenceDescription
of Artificial Sequence Synthetic peptide 39Ile Asp Pro Ser Asp Ser
Tyr Thr1 54014PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 40Ala Arg Gly Gly Thr Gly Asp Phe His
Tyr Ala Met Asp Tyr1 5 10416PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 41Gln Gly Ile Ser Ser Asn1
5423PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 42His Gly Thr1438PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 43Gln
Tyr Val Gln Phe Pro Tyr Thr1 5448PRTArtificial SequenceDescription
of Artificial Sequence Synthetic peptide 44Gly Tyr Thr Phe Thr Ser
Tyr Trp1 5458PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 45Ile Asp Pro Ser Asp Ser Phe Thr1
54614PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 46Ala Arg Gly Gly Pro Gly Asp Phe Arg Tyr Ala Met
Asp Tyr1 5 10476PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 47Gln Gly Ile Ser Ser Asn1
5483PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 48His Gly Thr1499PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 49Val
Gln Tyr Val Gln Phe Pro Tyr Thr1 5508PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 50Gly
Tyr Thr Phe Thr Asp Tyr Ala1 5518PRTArtificial SequenceDescription
of Artificial Sequence Synthetic peptide 51Ile Asn Thr Asp Tyr Gly
Asp Thr1 55214PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 52Ala Arg Ser Asp Tyr Asp Tyr Tyr Phe
Cys Gly Met Asp Tyr1 5 10538PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 53Gly Phe Ser Leu Thr Gly Tyr
Gly1 5547PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 54Ile Trp Ala Glu Gly Arg Thr1 55514PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 55Ala
Arg Glu Val Ile Thr Thr Glu Ala Trp Tyr Phe Asp Val1 5
10566PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 56Gln Ser Ile Ser Asp Tyr1 5573PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 57Tyr
Ala Ser1589PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 58Gln Asn Gly His Thr Phe Pro Leu Thr1
5597PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 59His Gly Met Leu Pro Val Tyr1 5607PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 60Pro
Pro Ser Asn Tyr Gly Arg1 5617PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 61Pro Pro Ser Asn Phe Gly
Lys1 5627PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 62Gly Asp Pro Trp Phe Thr Ser1 5637PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 63Asn
Ser Gly Pro Trp Leu Thr1 5647PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 64Trp Pro Pro Leu Ser Lys
Lys1 5657PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 65Asn Thr Phe Arg Thr Pro Ile1 5667PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 66Asn
Thr Phe Arg Asp Pro Asn1 5677PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 67Asn Pro Ile Trp Thr Lys
Leu1 56812PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 68Met Glu Pro Val Lys Lys Tyr Pro Thr Arg Ser
Pro1 5 106936DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 69atggagccgg tgaagaagta
tccgacgcgt tctcct 367012PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 70Glu Thr Gln Leu Thr Thr Ala
Gly Leu Arg Leu Leu1 5 107136DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 71gagactcagc
tgactacggc gggtcttcgg ctgctt 367212PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 72Glu
Thr Pro Leu Thr Glu Thr Ala Leu Lys Trp His1 5 107336DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 73gagacgcctc ttacggagac ggctttgaag tggcat
367412PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 74Gln Thr Pro Leu Thr Met Ala Ala Leu Glu Leu
Phe1 5 107536DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 75cagacgccgc tgactatggc
tgctcttgag cttttt 367612PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 76Asp Thr Pro Leu Thr Thr Ala
Ala Leu Arg Leu Val1 5 107736DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 77gatactccgc
tgacgacggc ggctcttcgg ctggtt 367812PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 78Thr
Pro Leu Thr Leu Trp Ala Leu Ser Gly Leu Arg1 5 107936DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 79acgccgctta cgctttgggc tctttctggg ctgagg
368012PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 80Gln Thr Pro Leu Thr Glu Thr Ala Leu Lys Trp
His1 5 108136DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 81cagacgcctc ttacggagac
ggctttgaag tggcat 368212PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 82Gln Thr Pro Leu Thr Met Ala
Ala Leu Glu Leu Leu1 5 108336DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 83cagacgcctc
tgactatggc ggctcttgag cttctt 368412PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 84His
Leu Gln Asp Gly Ser Pro Pro Ser Ser Pro His1 5 108536DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 85cagacgcctc tgactatggc ggctcttgag cttctt
368612PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 86Gly His Val Thr Thr Leu Ser Leu Leu Ser Leu
Arg1 5 108736DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 87gggcatgtga cgactctttc
tcttctgtcg ctgcgg 368812PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 88Phe Pro Asn Phe Asp Trp Pro
Leu Ser Pro Trp Thr1 5 108936DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 89tttccgaatt
ttgattggcc tctgtctccg tggacg 369012PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 90Glu
Thr Pro Leu Thr Glu Pro Ala Phe Lys Arg His1 5 109136DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 91gagacgcctc ttacggagcc ggcttttaag cggcat
369210DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 92tagaacgaag 109310DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 93cttcgttcta 109410DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 94tcagtggatg 109510DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 95catccactga 109610DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 96gtattgcgag 109710DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 97ctcgcaatac 10988DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 98aacgattc
8998DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 99gaatcgtt 81008DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 100agtggatg 81018DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 101catccact
81028DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 102gtattgcg 81038DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 103cgcaatac 81048DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 104atgccttc
81058DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 105gaaggcat 81068DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 106tgatggac 81078DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 107gtccatca
81088DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 108cagtagtg 81098DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 109cactactg 81108DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 110ttcctgag
81118DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 111ctcaggaa 81128DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 112gactctct 81138DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 113agagagtc
81145PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 114Gly Gly Gly Gly Ser1 5
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