U.S. patent application number 15/410424 was filed with the patent office on 2017-07-13 for quantitive analyte assay device and method.
The applicant listed for this patent is Assurance LLC. Invention is credited to Jin Po LEE.
Application Number | 20170199182 15/410424 |
Document ID | / |
Family ID | 45527110 |
Filed Date | 2017-07-13 |
United States Patent
Application |
20170199182 |
Kind Code |
A1 |
LEE; Jin Po |
July 13, 2017 |
QUANTITIVE ANALYTE ASSAY DEVICE AND METHOD
Abstract
The present invention relates to an assay device and a method
for using such for the quantitative determination of an analyte,
based on a test strip, which contains a porous test membrane
allowing for capillary flow of the analyte and complexes of the
analyte, a porous upstream membrane in fluid connection with the
test membrane and a porous downstream membrane in fluid connection
with the test membrane, wherein the test membrane contains a test
site having immobilized thereon a ligand capable of reacting with
the analyte and binding such to the test site, and two standard
band sites having immobilized thereon known high and low
concentrations of a calibrator agent capable of reacting with a
label conjugate and binding such to the standard sites, wherein the
upstream membrane has a site for the application of a sample to be
analyzed, and has a site downstream from the sample application
site for depositing label conjugates capable of reacting with the
analyte and label conjugates capable of reacting with the
immobilized calibrator agents in the standard bands to provide a
known label response in the standards bands, and the downstream
membrane is capable of absorbing said sample and providing the
capillary flow for the sample through the upstream and test
membrane.
Inventors: |
LEE; Jin Po; (Carlsbad,
CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Assurance LLC |
Carlsbad |
CA |
US |
|
|
Family ID: |
45527110 |
Appl. No.: |
15/410424 |
Filed: |
January 19, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14541093 |
Nov 13, 2014 |
9557329 |
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15410424 |
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13199894 |
Sep 12, 2011 |
8900881 |
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14541093 |
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12317814 |
Dec 30, 2008 |
8455263 |
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13199894 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
Y10S 435/97 20130101;
G01N 33/74 20130101; Y10S 435/805 20130101; G01N 2021/7793
20130101; G01N 21/78 20130101; G01N 2021/7786 20130101; Y10S
436/805 20130101; G01N 2021/6439 20130101; G01N 33/78 20130101;
Y10S 435/81 20130101; G01N 33/57434 20130101; Y10S 436/81 20130101;
Y10S 436/811 20130101; Y10S 436/813 20130101; G01N 33/54373
20130101; Y10S 436/814 20130101; G01N 33/57473 20130101; G01N
2021/7796 20130101; G01N 33/54366 20130101; Y10S 436/818 20130101;
G01N 33/558 20130101 |
International
Class: |
G01N 33/543 20060101
G01N033/543; G01N 33/78 20060101 G01N033/78; G01N 33/574 20060101
G01N033/574; G01N 33/74 20060101 G01N033/74 |
Claims
1. An assay device for the quantitative determination of an
analyte, comprising a test strip containing a porous test membrane
allowing for capillary flow of the analyte and complexes of the
analyte, a porous upstream membrane in fluid connection with the
test membrane and a porous downstream membrane in fluid connection
with the test membrane, (a) said test membrane containing a test
site having immobilized thereon a ligand capable of reacting with
the analyte and binding such to the test site, and at least two
standard band sites having immobilized thereon known high and low
concentrations of at least two different calibrator agents capable
of reacting with a label conjugated antibody and binding such to
the standard sites, (b) said upstream membrane having a site for
the application of a sample to be analyzed, and having a site
downstream from the sample application site for removably
depositing label conjugates capable of reacting with the analyte
and at least two label conjugates capable of reacting with the
immobilized calibrator agents in the standard bands to provide a
known label response in the standards bands, and (c) said
downstream membrane capable of absorbing said sample and providing
the capillary flow for the sample through the upstream and test
membrane.
2. The assay device of claim 1 wherein the label conjugates on the
upstream membrane give rise to a color.
3. The label conjugates of claim 2 comprising gold conjugates,
fluorescent dye conjugates, latex conjugates and enzyme
conjugates.
4. The device of claim 1 wherein the test membrane contains two
calibrator agents.
5. The device of claim 4 wherein the calibrator agents are rabbit
IgG and bovine thyroid globulin.
6.
7. The device of claim 1 wherein the test strip is contained in a
cassette allowing for the addition of the sample at the upstream
membrane and viewing the results of color formation at the sites of
the test membrane.
8. The device of claim 1 wherein the label conjugates are different
for the analyte and the calibrator agents.
9. The device of claim 6 wherein the test membrane is microporous
nitrocellulose and the upstream membrane is fiberglass.
10. The device of claim 1 wherein the test membrane contains three
standard bands.
11. The method for the quantitative analysis of an analyte in a
sample comprising (a) immobilizing on a microporous test strip in a
test band an antibody to the analyte to be established in
concentrations adequate to bind to analyte concentrations in the
sample, (b) immobilizing different calibrator agents on the test
strip in at least two standard bands in the amounts necessary which
when treated with excess of labeled antibodies to the immobilized
calibrator agent result in a measurable response corresponding to
different concentration of analyte, (c) removably depositing on an
additional upstream membrane in contact with the test strip labeled
antibodies to the analyte and the calibrator agents (d)
establishing the curve showing the measurable conjugate response to
different analyte concentration, (e) adding a sample suspected of
containing the analyte for which the test strip was designed to the
assay strip and complexing such to an antibody conjugated to a
measurable label and adding measurable labeled antibodies to the
calibrator agents to the sample prior to contact with the standard
and test bands, (f) measuring the label response at the test and
standard bands and comparing such to the standard curve of step (d)
for the label responses in the standard bands as established in
step (b) and (g) converting the measured label responses to
concentration of analyte in the sample.
12. The method of claim 8 wherein the label generates a visual
signal at the binding sites of the assay device.
13. The method of claim 9 wherein the visual signal is generated by
a colloidal gold conjugate, a fluorescent dye conjugate, or a latex
particle.
14. The method of claim 8 wherein the analyte is an antigen
associated with an infectious agent such as a virus, bacterium
fungus or a prion.
15. The method of claim 8 wherein the analyte is a hormone such as
hCG, thyroxin, TSH, glucagons, insulin relaxin, prolactin,
melanotropin, gastrin, somatotropin, gastrin and bradykin.
16. The method of claim 8 wherein the analyte is a cancer or tumor
marker such as prostrate specific antigen (PSA), carcinoembryonic
antigen (CEA), and alpha-fetoprotein.
17. The method of claim, 8 wherein the analyte is a cardiac
marker.
18. The method of claim 8 wherein the calibrator agents are
monoclonal ligands.
19. The method of claim 15 wherein the calibrator agents are BTG
antibodies, rabbit IgG antibodies and the label conjugates are
label bound antigens capable of binding to the calibrator
agents.
20. The method of claim of claim 8 where more than two calibrator
agents are used.
Description
CROSS-REFERENCE TO RELATED APPLICATION(S)
[0001] This application is a continuation of U.S. patent
application Ser. No. 14/541,093, filed Nov. 13, 2014; which is a
continuation of U.S. patent application Ser. No. 13/199,894, filed
Sep. 12, 2011, now U.S. Pat. No. 8,900,881; which is a
continuation-in-part of U.S. patent application Ser. No.
12/317,814, filed Dec. 30, 2008, now U.S. Pat. No. 8,455,263. The
contents of the above-identified applications are incorporated
herein by reference in their entireties.
FIELD OF INVENTION
[0002] The present invention relates to methods and devices for
assaying biological fluid samples and particularly bodily fluid
samples. More particularly the invention relates to methods and
devices for detecting and quantitatively measuring the
concentration of an analyte in a sample.
BACKGROUND
[0003] Quantitative analysis of fluid samples including antigens,
antibodies hormones, and other secreted proteins as well as other
types of proteins frequently provides critical diagnostic data. The
concept of immunoassays is well known in the art and is based on
reagent impregnated capillary membranes in which an immobilized
reagent in the detection zone of the strip binds to analytes
conjugated with visual label such as a latex particle or metal
containing compounds. In addition U.S. Pat. Nos. 3,654,090,
3,791,392 and 3,817,838 disclose enzyme labels and the means to
detect such, and U.S. Pat. No. 4,062,733 discloses radioactive
labels. Preferred labels are derived from metal sols. Labels such
as prepared from a gold sol are bound to a ligand capable of
further binding or conjugating to an analyte or other ligand or
antibody. Test strips have been used in qualitative assays in a
large number of applications involving analytes contained in such
bodily fluids as urine and blood. Probably the earliest and most
common use of assays is in detecting human chorionic gonatropin as
an indicator of pregnancy in humans. These assay devices are based
on test strips where the assay is performed and results read in a
single step, e.g. where the liquid sample is placed onto a porous.
membrane, any analyte of interest therein binds a corresponding
ligand, and the results are indicated visually in a detection zone
separate from the sample loading zone through formation of a
specific complex. The test strips are normally contained in a
dipstick or cassette allowing the user to conduct the test without
any additional equipment. Such devices are disclosed in U.S. Pat.
Nos. 4,235,601, 4,235,601 and 5,384,264. Assay devices that can be
used with whole blood are disclosed in U.S. Pat. No. 5,821,073.
[0004] Known assay devices generally involve a sandwich assay or a
competitive assay. In a sandwich assay, analyte of interest present
in a fluid sample interacts with a ligand bound label or tracer
resulting in a complex of analyte and label. This can occur prior
to adding the analyte to the test strip or the label bound ligand
can be removably deposited on the porous membrane forming the test
strip. The test strip allows the label-bound analyte to move by
capillary action to a sample capture zone where the analyte complex
is retained by virtue of being bound to the immobilized capture
ligand normally an antibody to the analyte. In the case of a gold
label the presence of the analyte is indicated by color formation
at the analyte test zone. Preferably the assay device also contains
a second immobilized ligand band, generally identified as a control
band capable of binding to label ligand even in the absence of an
analyte, to show that the strip is functioning properly.
[0005] In a competitive or inhibition assay both the analyte and
the label ligand are capable of reacting with the immobilized
ligand in the sample capture zone and thus, both the analyte and
the label bound ligand compete for attachment to the immobilized
ligand in the sample capture zone. Any analyte present would
displace the binding of a labeled ligand. The presence of a signal
in the sample capture zone would thus indicate a negative
result.
[0006] Most of the assay devices based on test strips determine the
presence or absence of an analyte but do not provide but a very
rough measure of the quantity of an analyte present in fluid
sample. Even those that give a quantitative measurement by
incorporating a standard in the test device suffer from
inaccuracies resulting from temperature, air moisture, flow time
variations, strip to strip variations and signal strength
variations. These and other deficiencies of the prior art assay
devices are overcome by the chromatographic assay device of the
present invention, which provides a rapid effective, and efficient
quantitative analysis of fluid samples.
SUMMARY OF THE INVENTION
[0007] The present invention provides an assay device for and a
method of measuring the amount of an analyte in a fluid sample
using a solid phase chromatographic assay, such as a sandwich
assay, in which one or more analyte of interest is bound to a label
conjugate and is also bound to a capture reagent in a test band as
part of a specific binding pair. In addition to the test band, the
microporous test strip membranes of the present invention contain
at least two different standard bands of immobilized calibrator
agents capable of binding to their own pair label reagents. The
labeled reagents captured at the standard bands by the calibrator
agents are used to create a template against which to measure the
concentration of an analyte in a sample bound in the test band. The
immobilized calibrator agents in the standard bands are contacted
with the corresponding label reagents as the fluid sample passes
through the assay strip. Any analyte of interest present in the
fluid sample is bound at the sample capture band. Based on the
relative intensity of the label bound in these bands an accurate
concentration of analyte can be obtained. Thus the density or
intensity of a label such as a gold sol conjugate in the various
sample capture bands on the membrane strip reflects the amount of
the analyte present as do the standard bands which then reflect the
density of the calibrator agent bound and which has a known
concentration. It is critical to an determination of the exact
concentration of the analyte in the sample that the correct amount
of calibrator agent be immobilized within a standard deviation of
less than 0.05%. Commercially available optical readers can then
convert the reflected light intensity of the sample into
concentration as measured on the basis of the curve derived from
the reflected intensity of the standard bands of known
concentrations. A competitive assay, in which the analytes of
interest is competitive with a capture reagent in the test band to
a label conjugate as part of a specific binding pair. The assay
device of the present invention can be used to measure the
concentration of analytes contained in any liquid sample including
blood, urine, water and oils.
[0008] The present invention. comprises a lateral flow assay method
and system including a test strip and/or a cassette for holding the
test strip, for determination of the presence and/or quantity of
analytes in samples, including but not limited to biological or
other samples containing materials including antigens, antibodies,
hormones and other secreted proteins, cell surface proteins,
transmembrane proteins, glycoproteins, enzymes, proteins associated
with cells and other proteins, proteins associated with pathogens
such as bacteria, viruses, and fungi, carbohydrates, drugs,
peptides, toxins, nucleic acids, small molecules, and aptamers.
This novel assay or system can detect and/or quantitate analytes in
small volumes of samples. Generally, the sample volume is less than
about 100 .mu.l. Most preferably, the sample volume is about 40
.mu.l. This assay or system can also separate cells from fluid in a
sample, such as red blood cells or white blood cells or other cell
types. The assay or system also provides low background noise and
is highly efficient.
BRIEF DESCRIPTION OF THE DRAWING
[0009] FIG. 1 is an exploded view of the test strip of the
invention.
[0010] FIG. 2 is a perspective view of a test strip of the
invention
[0011] FIG. 3 is a perspective view of a cassette device of the
invention.
[0012] FIG. 4 is an exploded view of a cassette device of the
invention
DETAILED DESCRIPTION OF INVENTION
[0013] A test strip constructed in accord with the invention for
the use with whole blood samples is shown in FIGS. 1-4. A red or
white blood cell layered mesh trap 1 is shown as overlying a sample
introduction membrane 6 and 13 that also contains downstream from
the sample addition point 7 the label ligands required to bind at
the test zone 4 and at the calibrator or standard bands 3 and 5 on
the microporous test membrane 2. Access to cell trap 1 for
application of test sample is obtained through opening 7 in cover 8
in FIGS. 3 and 4. The test strip 2 also includes a terminal sample
flow zone 10 distal to the test and standard zones. Each of the
above mention zones are in fluid communication with each other. All
of the components described with respect to the test strip of
invention may be encased in a fluid tight housing composed of a
solid plastic cover 8 with view window 14 which fits tightly over
solid plastic base 12 as shown in FIG. 3. Opening 9 in the cassette
cover allows for the introduction of a buffer or other solution
aiding in the capillary flow of the sample.
[0014] For ease of understanding, the following definitions will
apply throughout this description:
[0015] 1. The term "antigen" as used herein refers to any analyte,
which is capable of binding antibodies. Antigens may comprise,
without limitation, chemical compounds, polypeptides,
carbohydrates, nucleic acids, lipids, and the like, including viral
particles, viral subunits, bacterial and parasite surface antigens,
and host proteins that may be diagnostic of the subject's
condition.
[0016] 2. A "binder" refers to a ligand for the analyte as in the
format of a sandwich assay, or a ligand for both the analyte and
the tracer or label as in the format of a competitive assay. A
binder can be chosen from a group of molecules or compounds capable
of binding the analyte, such as an antigen to the antibody analyte,
or an antibody to the antigen analyte.
[0017] 3. A "test zone or band" refers to an area in which a binder
or the analyte is attached, movably or immovably, such as to the
analyte test strip portion of an assay device.
[0018] 4. The term "test strip" or "assay strip" refers to a porous
membrane capable of capillary flow of the fluid sample containing
the bound analytes and other antigens involved in the determination
of the analyte concentration. Common porous membranes are
fiberglass, porous nitrocellulose or polyethylene. An "analyte test
strip" of the invention consists of, collectively, all of the zone
supporting membranes and any filters of the assay device.
[0019] 5. A "tracer" refers to a ligand for the analyte or the
binder labeled with a detectable label, preferably a visually
readable particulate label, such as colloidal gold, latex and
liposomes including dye, carbon black, and the like.
[0020] 6. A "sample loading or application zone" refers to an area
of an analyte test strip on which a fluid analyte sample is applied
for migration to the test bands.
[0021] 7. A "fluid analyte sample" can be any fluid suspected of
containing analyte of interest for which a particular assay is
specific. Test sample may represent any body fluid, including
urine, blood, sweat, lymph, intraperitoneal fluid, crude tissue
extract or homogenate, derived from a fetus, neonate, juvenile or
adult subject; a non-biological fluid such as water from some
ecological niche, e.g., a river or a lake; or a solution used in a
laboratory.
[0022] 8. A "label" is a molecule or compound, which directly or
indirectly mediates the formation of a signal (such as a color
change), which is used in assay to indicate the presence, absence
or concentration range of analyte of interest in a test sample.
Labels may include enzymes, fluorescers, liposomes, erythrocyte
ghosts, polymer microcapsules, color polymer particles (latex), and
preferably includes sols of metal-containing compounds. A wide
variety of patents and patent applications provide an extensive
literature of different techniques for producing detectable signals
in immunoassays. The following list of United States patents is
merely illustrative of the type of label which can find application
in this invention: U.S. Pat. No. 3,646,346 discloses radioactive
labels; U.S. Pat. Nos. 3,654,090, 3,791,932, and 3,817,838 disclose
enzyme labels; U.S. Pat. No. 3,996,345 discloses
fluorescer-quencher labels; U.S. Pat. No. 4,062,733 discloses
radioactive label; U.S. Pat. No. 4,067,959 discloses fluorescer or
enzyme label; U.S. Pat. No. 4,104,099 discloses chemiluminescent
label; and U.S. Pat. No. 4,160,645 discloses non-enzymatic catalyst
label. U.S. Pat. No. 3,966,879 discloses an electrophoretic
technique employing an antibody zone and U.S. Pat. No. 4,120,945
discloses a radioimmune assay (RIA) where labeled analyte is
initially bound to a solid support through antibody. U.S. Pat. No.
4,233,402 discloses enzyme pair labels; U.S. Pat. No. 4,720,450
discloses chemically induced fluorescent labels; and U.S. Pat. No.
4,287,300 discloses enzyme anionic charge labels.
[0023] Labels can also be metal-containing sols; i.e., metal or
metal compounds such as metal oxides, metal hydroxides, metal
salts, metals or metal-containing compounds mixed with polymers or
coated onto polymer nuclei. These metal labels may include dry
forms of any of the above-named metal or metal compound sols, and
preferably include colloidal gold in dry form.
[0024] "Label response" refers to the signal strength that is
generated by the label when it becomes attached to the reagents
immobilized in the test or standard bands. With color generating
labels reflective light units are a preferable way of measuring
label response.
[0025] 9. A "complex" means (depending on the context) any
multimolecular complex formed by analyte and one or more ligands,
or by labeled ligand and immobilized ligand. In a sandwich-type
immunoassay, e.g., the following complexes occur: analyte/labeled
ligand duplex first produced in the assay (first complex) and
analyte/labeled ligand/immobilized ligand triplex formed second in
the assay (second complex).
[0026] 10. "Fluid communication" refers to structures which are in
contact with, but not necessarily affixed to one another and which
allow the passage of a fluid from one to the other.
[0027] 11. "Assay" refers to several different types of assay
formats in which an analyte of interest can be detected using an
assay analyte test strip. For example, in a sandwich-type
immunoassay, analytes of interest in the analyte sample, when
present, bind a labeled tracer movably incorporated in the analyte
test strip (consisting of a porous membrane) at the tracer zone to
form a first complex. The tracer is a molecule, which binds the
analyte of interest and is conjugated to a label, preferably a
metal label, and most preferably colloidal gold.
[0028] 12. The term "test band" or "test zone" as used herein
refers to a region or zone on the chromatographic assay strip that
contains at least one analyte binding agent. The analyte binding
agent is usually immobilized in a band or zone such that after
reaction with a detectable agent, the band or zone produces an
observable or measurable result reflecting the presence or amount
of analyte present in the sample. The "test band" may be comprised
of more than one capture zone for capturing more than one analyte
in the sample, in which event, more than one analyte binding agent
may be used. For example, two assay combinations that are
considered to be within the scope of the invention are assay
combinations that simultaneously detect hepatitis C virus. (HCV)
and human immunodeficiency virus (HIV), and assay combinations that
simultaneously detect Hepatitis B surface antigen (HBsAg) and
Treponema pallidum antigen (TP). Still other combinations are
possible and are within the scope of the invention.
[0029] 13. The term "conjugate" and "detectable agent" are used
interchangeably herein to refer to an antibody or an antigen that
is conjugated to a detectable material such as a colored agent, a
fluorescent agent, an enzyme agent or a chemiluminescent agent. In
the practice of the present invention, the "conjugate" or
"detectable agent" specifically binds the analyte to be determined
or the captured analyte immobilized on the capture band.
Optionally, the "conjugate" or "detectable agent" produces a
measurable quantitative reading at the capture band that reflects
the amount of an analyte present at the capture band. As described
further below, the direct measurable quantitative density in the
capture band does not necessarily reflect the amount of an analyte
present at the capture band through binding, but the intensity of
band as measured by reflective light units (RLU) does reflect the
amount of an analyte present at the capture band.
[0030] 14. The term "standard band" or "calibrator band" as used
herein contains calibrator agents immobilized in calibrator binding
zones on the test strip. The calibrator agents bind specifically to
calibrator binding agents to form a calibrator binding pair. The
present invention includes two or more calibrator bands. An
advantage to having calibrator binding pairs is that they act as
internal standards, that is, the calibrator against which the
amount of an analyte present at the capture band may be calculated.
The calibrators may be used to correct for strip to strip
variability. One of the calibrators can be designated a high
calibrator ("HC") and the other of the calibrators can be
designated a low calibrator ("LC"). The reflection density of HC
and of LC, can be used to determine the standard curve. It is also
within the scope of this invention to use more than standard bands
all of different concentrations of the xcalibrator agent. The
standard curve is made for each quantitative assays by using the
RLU values of the calibrator agents to generate a regression
equation to describe the relationship between two variables.
Although, in general, any conventional calibrators can be used
herein, it is generally preferred to use as calibrators compounds
that do not exist in the sample or do not immunologically
cross-react with compounds that exist in the sample; for example,
monoclonal antibody against rabbit immunoglobulin, monoclonal
antibody against bovine thyroid globulin, or antibody against
2,4-dinitrophenylated bovine serum albumin (BSA-DNP), which can be
purchased from Molecular Probes (Eugene, Oreg., cat# A-23018) can
be used as the two points of calibrator reagent. The compound
2,4-dinitrophenol (DNP) is a small molecule which does not exist
within the human body but acts as a hapten; that is, it is
immunogenic when conjugated to a larger molecule such as a protein
carrier and injected into an antibody-producing mammal such as a
mouse, a rat, a cow, a rabbit, a horse, a sheep, or a goat.
[0031] The test devices of the present invention comprise test
strips, which may be enclosed in a cassette for easier use. Such
test strips enclosed in such cassettes are described in U.S. Pat.
Nos. 5,821,073 and 5,384,264, the disclosures of which are hereby
incorporated by this reference. The test strips employed in the
present invention generally comprise a porous test membrane of
sufficient porosity to allow the components of the test including
the analyte and its complexes to flow through the membrane by
capillary action. Preferred membranes are porous nitrocellulose,
porous. polypropylene and paper membranes. Such membranes are well
known in the art. These membranes contain immobilized ligands
capable of binding the analyte in a test zone, preferably in the
form of a band across the width of the test membrane as well as the
HC and LC calibrator agents in separate zones across the width of
the test membrane in the calibrator are standard bands. As
indicated the immobilized ligands used for the test and calibrator
zones are different. Known amounts of calibrator agents are
immobilized in the standard bands to establish the base line for
the quantitative determination of the analyte. The remaining active
sites after the application of the ligands in the various zones on
the membrane are blocked to allow free flow of the analyte, analyte
complexes, labeled ligands and labeled ligand complexes through the
test strip.
[0032] In addition to the porous membrane the test strips of the
present invention contain a second porous membrane upstream from
the first membrane but in fluid communication with the test
membrane containing the immobilized ligand bands. The upstream
membrane is of an inert type in that it does not contain sites,
which would bind ligands flowing through the membrane. Such
membranes are preferably made of unwoven fiberglass or
polypropylene, again of sufficient porosity to allow capillary flow
of analyte, analyte complexes and labeled ligands. These membranes
can also be suitably used to separate the analyte to be tested from
components in the sample, which could interfere in the analysis of
the analyte. Thus in the analysis of blood the red and white blood
cells can be separated from the fluid serum containing the analyte
to be tested. The upstream membrane therefore contains a site for
the application of the sample, and in addition contains the labeled
ligands, downstream from the application site and upstream from the
contact with the test membrane, that react with the analyte in a
sandwich assay or with the bound ligand in the test zone in a
competitive assay, as well labeled ligands that complex with the
bound calibrator ligands in the standard bands. The labeled
reagents are deposited on the upstream membrane in a manner that
they can lift of from the membrane when contacted by the fluid
sample and in the case of the analyte react with the analyte to
form a complex that can then continue to flow from the upstream
membrane into the test membrane.
[0033] The test strip further contains a third membrane, in fluid
connection with the test membrane, at the downstream end of the
test membrane capable of absorbing the fluid sample that has passed
through the test membrane and that acts as the driving force to
cause the fluid sample to flow through the test membrane by
capillary action. Such adsorption pads are preferably made of high
absorbency materials such as paper that are capable of absorbing
the sample and any buffer added to the strip.
[0034] The operation of the assay of the present invention
basically involves the addition of a known quantity of a fluid
sample containing a suspected analyte to the device at the sample
application site using a transfer pipette. Generally the amount of
sample will be in the range of 10 to 100 microliters. Usually when
the test strip is incorporated into a cassette, a sample well is
provided for the addition of the sample to the test strip. The
sample is absorbed by the upstream membrane and drawn by capillary
action through such upstream membrane towards the test membrane. In
the process the sample passes through the region of the upstream
membrane where label bound ligands are removably deposited. In
addition to the label ligand that binds to the analyte if present
the upstream membrane also contains high and low calibrator label
ligands removably deposited on the upstream membrane. In a
preferred embodiment the two calibrator label ligands and the label
ligands used to bind to the analyte to be tested the sample are
mixed and in dry form.
[0035] In the case of the sample being blood the upstream membrane
can also act as a filter to filter out the red and white blood
cells but let the serum or plasma pass through. The flow of the
sample through the test strip is frequently facilitated by the
addition of a buffer. In general the amount of buffer added is at
least equal to but no more than five times the quantity of the
fluid sample. Suitable buffers include any pharmaceutically
acceptable aqueous buffer, which will not react with the test
sample of the other test and control ligands of the test strip. In
general phosphate buffers, based on mono and di sodium hydro
phosphate and commercially available, are preferred although other
buffers such as citrate buffers or Ringer's solution may also be
used.
[0036] The sample stream picks up the label ligands for both the
test and standard bands before contacting the test membrane. The
preferred labels of the present invention are those that give rise
to color complexes in the test and standard bands. Although color
resulting from enzyme bound ligands or latex bound ligands which
have been used in conjunction with qualitative assays using
capillary flow of test strips such as disclosed in the above
mentioned patents and others can be used in the assays of the
present invention, the preferred color forming ligands are gold
conjugates that can bind to the desired analyte to be analyzed and
to the calibrator agents. The quantitative assay of the present
invention is based on the color intensity of an analyte sample and
the color intensity generated by the calibrator agents in the
standard bands. Thus in sandwich assay, the higher the amount of
analyte in the test sample, the more label ligands binds to the
analyte and the higher the amount of label ligand/analyte complex
that will bind to the to the immobilized ligand in the test zone,
increasing the label intensity in the band. Thus label intensity
increases with increasing amount of analyte present in the sample.
In a competitive assay, the higher the amount of analyte in the
test sample, the less label ligand can bind to the immobilized
ligand-BSA (analyte-BSA) in the test zone, decreasing the label
intensity in the test zone. Thus label intensity decreases with
increasing amount of analyte present in the sample. However in
order to accurately establish the actual concentration of the
analyte, factors other than analyte concentration must be excluded
from any quantitative determination. It is for that purpose that
the test strips of the present invention employ the calibrator
agents in the standard bands which then provide the means for an
accurate quantitative determination. Preferably and for higher
accuracy the present invention employs at least two different
calibrator agents in separate standard zones, before and after the
test zone on the test membrane. Since a fixed amount of the
calibrator agent is immobilized in the standard zones and an excess
of the calibrator label conjugate is deposited in the upstream
band, the same intensity of color is generated in the standard
bands at any given time interval time during the test for different
test strips made in the same way. Using the relative light units a
standard curve can be obtained for each high and low calibrator
zone, which then serves as the base for the determination of the
quantitative amount of analyte present. Although, in general, any
conventional calibrators can be used herein, it is generally
preferred to use as calibrators agents that do not exist in the
sample or do not immunologically cross-react with compounds that
exist in the sample; for example, 2,4-dinitrophenylated bovine
serum albumin (BSA-DNP), which can be purchased from Molecular
Probes (Eugene, Oreg., cat# A-23018) can be U.S.ed as the
calibrator reagent. The compound 2,4-dinitrophenol (DNP) is a small
molecule which does not exist within the human body but acts as a
hapten; that is, it is immunogenic when conjugated to a larger
molecule such as a protein carrier and injected into an
antibody-producing mammal such as a mouse, a rat, a cow, a rabbit,
a horse, a sheep, or a goat. Immobilized ligands in the low
calibrator standard zone are for example monoclonal antibody
against bovine thyroid globulin (BTG) and immobilized ligands in
the high calibrator standard zone are for example goat anti-rabbit
IgG proteins. Conjugates used with the immobilized ligands are goat
BTG gold antigens and rabbit IgG gold antigens.
[0037] In order to determine the amount of the analyte present it
is necessary to develop the relationship of color intensity in the
test zone with concentration of analyte in a sample. Such
relationship, plotted as a curve, is obtained by preparing a
solution or dispersion of the analyte to be tested at a known high
concentration and then continuously diluting the test solution or
dispersion and measuring the change in color intensity in exposed
test strips. Obviously this curve will also be different at
different time intervals. However these curves can be correlated
with the standard curves obtained from the calibrator agents for
known concentrations in the case of measurement of a sample
containing an unknown amount of analyte. Thus in each test of an
unknown sample three different color intensities are obtained. The
intensities of the standard band can be correlated to the standard
concentration curves for the analyte and a quantitative value for
the analyte in the sample to be tested calculated. For verification
purposes it is desirable to obtain relative light units for two
exposure times, which can be correlated to the standard values of
concentration vs. label response in reflectance units to obtain a
verification of the concentration of the analyte in the sample. The
reliability of the curve depends in large part on the accuracy and
strip to strip consistency of the binding agent in the standard
bands. Such accuracy and consistency is accomplished by using
linear motion displacement pumps that provide digitally controlled
pulse free linear flows as are available from the IVEK
corporation.
[0038] Although the necessary calibration of the standard strips
and concentration calculations can be carried out manually, they
are preferably embodied in software that can be read by commercial
equipment capable of measuring the color intensity of the test band
and the standard bands. The color intensity data points generated
by the standard bands and known concentrations of analyte in the
test band can be incorporated into a memory device such as an
electro-magnetic card or an RFID card. When such card is inserted
into a commercially available optical camera reader, such as
manufactured by Kaiwood Technology Co. together with a cassette
that has been treated with a sample, the reader can provide the
concentration of any analyte in the sample.
[0039] In general analytes and the corresponding complex forming
antibodies, or antigen if the analyte is an antibody, are known in
the art from the development of qualitative assays for such
analytes, as are the label conjugates that will bind to the
analyte. For example, if the lateral flow assay is intended to
measure hepatitis B virus ("HBV") surface antigen (HBsAg) in a
blood sample, the capture band will contain antibody to HBsAg
immobilized on the test membrane at the test band.
[0040] Suitable analytes include, but are not limited to antigens,
antibodies, hormones, drugs, cell proteins, DNAs, cardiac markers,
tumor or cancer markers, autoimmune disease markers, or any
macromolecule that could raise antibodies. When the analyte is an
antigen, the antigen can be an antigen associated with an
infectious agent. The infectious agent can be a virus, a bacterium,
a fungus, or a prion. When the infectious agent is a virus, the
virus can be selected from the group consisting of HIV, hepatitis
virus A, B, C, and D, herpes simplex virus, cytomegalovirus,
papilloma virus, Ebola vir, SARS virus Rhinovirus, and Vaccinia
virus, but is not limited to those viruses. When the infectious
agent is a bacterium, the bacterium can be a gram-positive
bacterium or a gram-negative bacterium. The bacterium can be
selected from the group consisting of Bacillus anthracis,
Escherichia coli, Helicobacter pylori, Neisseria gonorrheae,
Salmonella species, and Shigella species, but is not limited to
those bacteria. When the infectious agent is a fungus, the fungus
can be a Mycosporum species or an Aspergillus species, but is not
limited to those fungi.
[0041] When the analyte is a hormone, typically it is selected from
the group consisting of hCG, thyroxin, TSH, glucagons, insulin,
relaxin, prolactin, luteinizing hormone, melanotropin,
somatotropin, follicle-stimulating hormone, gastrin, bradykinin,
vasopressin, and other releasing factors; however, other hormones
of physiological or pathological interest can be the analyte.
[0042] When the analyte is a cancer or tumor marker, typically it
is selected from the group consisting of prostate specific antigen
(PSA), carcinoembryonic antigen (CEA), and .alpha.-fetoprotein;
however, other cancer or tumor markers can be the analyte.
[0043] When the analyte is a cardiac marker, the cardiac marker is
typically selected from the group consisting of Troponin-I,
Troponin T, Creatine kinase-MB isoforms (CK-MB), myoglobin,
C-reactive protein (CRP), fatty acid binding protein (FABP),
glycogen phosphorylase isoenzyme BB (GPBB), B-type natriuretic
peptide (BNP) and pro-BNP; however, the analyte can be another
cardiac marker.
[0044] The invention is further illustrated but not limited by the
following examples.
Example 1
[0045] Onto a test strip comprising a porous nitrocellulose strip
(7.5 cm.times.0.7 cm) as the test membrane sufficient monoclonal
antibody against human prostrate specific antigen (PSA) is
immobilized in a test band to give rise to measurable color
intensity when complexed with PSA analyte color conjugates that can
be expected in a sample. In separate experiments the relationship
of color intensity and concentration is established as a curve.
Rabbit IgG and bovine thyroid globulin are immobilized in two
standard bands by using an IVEK ceramic linear pump having an
accuracy of 99.95%, in known amounts sufficient to give rise to a
color intensity corresponding to PSA at concentration of 1 and 10
nanogram per milliliter (ng/ml) respectively when such is complexed
with a gold label monoclonal PSA specific antibody. The test
membrane is dried in a dehumidifying chamber (humidity of 10-15%)
overnight. Excess of the gold label antibody against PSA, gold
label monoclonal antibody against rabbit IgG and gold label
monoclonal antibody against bovine thyroid globulin are also
removable deposited in an upstream fiberglass membrane. The
conjugate fiberglass pad is treated with a buffer to neutralize
interfering sites and then vacuum dried at 2 Torr overnight at room
temperature. The pad was then cut into narrow strips (1.times.0.7
cm). The bands are arranged such that the sample first contacts the
low calibrator band showing the color intensity with the gold
conjugate corresponding to 1 ng/ml of PSA antigen, then the test
band, followed by the high calibrator agent band, capable of
binding the equivalent of 10 ng/ml of PSA. The correlation of the
color intensity in RLU units with analyte concentration, lot #,
test time, slope and intercept for the two standard bands of the
particular test strip is loaded into a RFID memory card. The
antigen concentrations can then be calculated using the equation
Y=AX+B where Y is the concentration of the analyte, X is the color
intensity generated by the sample in RLU units, A is the slope of
the curve obtained from the standard values and B is the intercept
of the test value on the curve. The test membrane is in fluid
connection with an upstream non-woven fiberglass membrane onto
which is deposited the antibody to the PSA antigen conjugated to a
gold label by techniques known in the art. The label conjugate is
deposited down stream from where the sample to be analyzed is added
to the upstream fiberglass membrane. The assay test strip also
contains a sample absorption pad in fluid connection with the
downstream end of the test membrane. The test strip is inserted
into a cassette such as described in U.S. Pat. No. 5,821,073.
[0046] One drop of blood suspected of containing the PSA analyte,
approximately 30 microliters, is added to the sample applicator
site on the upstream fiberglass membrane followed by four drops,
about 200 microliters, of a commercially available phosphate
buffer. The fiberglass membrane is sufficiently dense to filter out
the red and white blood cells but will let the serum pass. The
serum of the blood combined with buffer flows through the upstream
membrane by capillary action to the label conjugate redistributing
the gold antibody conjugate in the serum which then forms a complex
with any analyte present in the sample. The buffer diluted serum
passes from the fiberglass membrane to the porous nitrocellulose
membrane and continues to flow by capillary action through the
nitrocellulose membrane. Analyte gold conjugate complex is captured
by the PSA antibody deposited in the test band and gives rise to a
reddish color the intensity of which corresponds to the amount of
analyte present in the sample. Monoclonal antibody against rabbit
IgG and monoclonal antibody against bovine thyroid globulin gold
conjugate removably deposited on the fiberglass membrane and
contained in the sertum are also captured by the high and low
standard bands containing sufficient calibrator agent to capture
the equivalent of either one or ten ng/ml of analyte. In the
presence of PSA analyte in the sample three bands of differing
color intensity are obtained. After passing the test and standard
bands the remaining buffered sample passes from the nitrocellulose
strip to the absorption pad and is stored there. The cassette with
the color bands is then inserted into a reader together with the
memory card containing the correlation between color intensity as
measured by relative light units (RLU) and concentration of
analyte. The photo-camera reader employed, Kaiwood CHR 100, is
capable of converting the color intensity into concentration using
the data on the memory card. Color intensities were measured 10 and
15 minutes after the addition of the sample and buffer. The
following results are obtained:
10 Minutest Test Result:
TABLE-US-00001 [0047] Analytes PSA Concentration RLU Calibrator H
10 ng/ml 10 Calibrator L 1 ng/ml 1 Sample Y 4.04
[0048] Based on the two point standard curve, the reader showed 4
ng/ml of the PSA antigen (Y) in the sample.
15 Minutes Test Result:
TABLE-US-00002 [0049] Analytes PSA Concentration RLU Calibrator H
10 ng/ml 23.9 Calibrator L 1 ng/ml 10.3 Sample Y 19.49
[0050] Based on the two point standard curve, the reader showed 4
ng/ml of PSA antigen (Y) in the sample.
Example 2
[0051] In a procedure similar to that of Example 1, onto a test
strip comprising a porous nitrocellulose strip (7.5 cm.times.0.7
cm) as the test membrane containing sufficient monoclonal antibody
against human thyroid stimulate hormone antigen (TSH) is
immobilized in the test band to give rise to a measurable color
intensity at TSH concentrations that can be expected in the sample.
In separate prior experiments the relationship of TSH concentration
and color intensity is established. Rabbit IgG and bovine thyroid
globulin are immobilized in two standard bands by using an IVEK
dispensing linear pump, in amounts sufficient to give rise to a
measurable color intensity corresponding to TSH at concentrations
of 5 .mu.IU/ml and 20 .mu.IU/ml respectively when complexed with a
gold label monoclonal TSH. The test membrane is dried in
dehumidifying chamber (humidity: 10-15%) overnight. Excess of the
gold label antibody against TSH, gold label monoclonal antibody
against rabbit IgG and gold label monoclonal antibody against
bovine thyroid globulin are also removably deposited in an upstream
fiberglass membrane. The conjugate fiberglass membrane was then
treated with a conjugate buffer to neutralize any interfering sites
and then vacuum dried at 2 Torr overnight at room temperature. The
pad was then cut into (1.times.0.7 cm) strips. The bands are
arranged such that the sample first contacts the low calibrator
band showing the color intensity with the gold conjugate
corresponding to 5 .mu.IU/ml of TSH antigen, then the test band,
followed by the high calibrator agent band, capable of binding the
equivalent of 20 .mu.IU/ml of TSH. The correlation of the color
density in RLU units with analyte concentration, lot #, test time,
slope and intercept for the two standard bands of the particular
test strip is loaded into a RFID memory card. The antigen
concentrations can then be calculated using the equation Y=AX+B
where Y is the concentration of the analyte, X the color intensity
generated by the sample in RLU units, A is the slope of the curve
obtained from the standard values and B is the intercept of the
test value on the curve.
[0052] The test membrane is in fluid connection with an upstream
non-woven fiberglass membrane onto which is deposited the antibody
to the TSH antigen conjugated to a gold label by techniques known
in the art. The label conjugate is deposited down stream from where
the sample to be analyzed is added to the upstream fiberglass
membrane. The assay test strip also contains a sample absorption
pad in fluid connection with the downstream end of the test
membrane. The test strip is inserted into a cassette such as
described in U.S. Pat. No. 5,821,073.
[0053] One drop of blood suspected of containing the TSH analyte,
approximately 30 microliters, is added to the sample applicator
site on the upstream fiberglass membrane followed by four drops,
about 200 microliters, of a commercially available phosphate
buffer. The fiberglass membrane is sufficiently dense to filter out
the red and white blood cells but will let the serum pass. The
serum of the blood combined with buffer flows through the upstream
membrane by capillary action to the label conjugate redistributing
the gold antibody conjugate in the serum which then forms a complex
with any analyte present in the sample. The buffer diluted serum
passes from the fiberglass membrane to the porous nitrocellulose
membrane and continues to flow by capillary action through the
nitrocellulose membrane. Analyte gold conjugate complex is captured
by the TSH antibody deposited in the test band and gives rise to a
reddish color the intensity of which corresponds to the amount of
analyte present in the sample. Monoclonal antibody against rabbit
IgG and monoclonal antibody against bovine thyroid globulin gold
conjugate removably deposited on the fiberglass membrane and
distributed in the serum are also captured by the high and low
standard bands containing sufficient calibrator agent to capture
the equivalent of either 5 or 20 5 .mu.IU/ml of analyte. In the
presence of TSH analyte in the sample three bands of differing
color intensity are obtained. After passing the test and standard
bands the remaining buffered sample passes from the nitrocellulose
strip to the absorption pad and is stored there. The cassette with
the color bands is then inserted into a reader together with the
memory card containing the correlation between color intensity as
measured by relative light units (RLU) and concentration of
analyte. The photo-camera reader employed, Kaiwood CHR 100, is
capable of converting the color intensity into concentration using
the data on the memory card. Color intensities were measured 15
minutes after the addition of the sample and buffer. The following
results are obtained:
15 Minutest Test Result:
TABLE-US-00003 [0054] Analytes TSH Concentration RLU Calibrator H
20 .mu.IU/ml 102 Calibrator L 5 .mu.IU/ml 47 Sample Y 19.5
[0055] Based on the two point standard curve, the reader showed
0.78 .mu.IU/ml of the TSH antigen (Y) in the sample.
Example 3
[0056] The procedure of Example 1 is repeated to determine the
concentration of human carcinoembryonic antigen (CEA) with gold
label monoclonal specific anti bodies. In separate prior
experiments the relationship of CEA concentration to color
intensity generated by the antibody when combined with the analyte
is established. Rabbit IgG and bovine thyroid globulin are
immobilized in two standard bands by using an IVEK ceramic linear
pump having an accuracy of 99.95%, in amounts sufficient to give
rise to a color intensity when complexed with old label antibodies
corresponding to concentration of 4 and 100 ng/ml respectively CEA
when such is complexed with a gold label monoclonal CEA specific
antibody, an excess of which is removably deposited on the upstream
fiberglass membrane. CEA specific antibody is immobilized in the
test band in sufficient concentration to provide measurable color
intensities when complexed with labeled CEA concentrations expected
to be present in the sample. Excess of the gold label antibody
against CEA gold label monoclonal antibody against rabbit IgG and
gold label monoclonal antibody against bovine thyroid globulin are
also removably deposited in an upstream fiberglass membrane. As in
Example 1 the correlation of color intensities to antigen
concentration is established using the equation Y=AX+B.
[0057] One drop of blood suspected of containing the CEA analyte,
approximately 30 microliters, is added to the sample applicator
site on the upstream fiberglass membrane followed by 4 drops, about
200 microliters, of a testing phosphate buffer. The fiberglass
membrane used is sufficiently dense to filter out the red and white
blood cells but will let the serum pass. The serum of the blood
combined with buffer flows through the upstream membrane by
capillary action to the label conjugate redistributing the gold
antibody conjugate which then forms a complex with any analyte
present in the sample. The buffer diluted serum passes from the
fiberglass membrane to the porous nitrocellulose membrane and
continues to flow by capillary action through the nitrocellulose
membrane. Analyte gold conjugate complex is captured by the CEA
antibody deposited in the test band and gives rise to a reddish
color the intensity of which corresponds to the amount of analyte
present in the sample. Monoclonal antibody against rabbit IgG and
monoclonal antibody against Bovine thyroid globulin gold conjugate
removably deposited on the fiberglass membrane and picked up by the
serum are also captured by the high and low standard bands. In the
presence of CEA analyte in the sample three bands of differing
color intensity are obtained. After passing the test and standard
bands the remaining buffered sample passes from the nitrocellulose
strip to the absorption pad and is stored there. The cassette with
the color bands is then inserted into a RFID reader together with
the memory card containing the correlation between color intensity
as measured by relative light units (RLU) and concentration of
analyte. The reader employed, CHR 100, is capable of converting the
color intensity into concentration using the data on the memory
card. Color intensities were measured at 15 minutes after the
addition of the sample and buffer. The following results are
obtained:
15 Minutest Test Result:
TABLE-US-00004 [0058] Analytes CEA Concentration RLU Calibrator H
100 ng/ml 78 Calibrator L 4 ng/ml 21.4 Sample Y 34.2
[0059] Based on the two point standard curve, the reader showed 25
ng/ml of the CEA antigen (Y) in the sample.
Example 4
[0060] The procedure of Example 1 is repeated to determine the
concentration of human Troponin-I antigen (TNI-I) in a sample using
gold label monoclonal specific TNI-I antibody. The relationship of
TNI-I concentration and the color intensity generated by the
antibody when conjugated to the analyte is established in prior
experiments. Rabbit IgG and bovine thyroid globulin are immobilized
in two standard bands by using an IVEK ceramic linear pump having
an accuracy of 99.95%, in amounts sufficient to give rise to a
measurable color intensity corresponding to TNI-I concentrations of
1 and 50 ng/ml respectively when such is complexed with a gold
label monoclonal TNI-I specific antibody. TN-I specific antibody is
immobilized in the test band. Excess of the gold label antibody
against TNI-I, gold label monoclonal antibody against rabbit IgG
and gold label monoclonal antibody against bovine thyroid globulin
are also removably deposited in an upstream fiberglass membrane. As
in Example 1 the correlation of color intensity to antigen
concentration is established using the equation Y=AX+B.
[0061] One drop of blood suspected of containing the TNI-I analyte,
approximately 30 microliters, is added to the sample applicator
site on the upstream fiberglass membrane followed by 4 drops, about
200 microliters, of a testing phosphate buffer. The fiberglass
membrane used is sufficiently dense to filter out the red and white
blood cells but will let the serum pass. The serum of the blood
combined with buffer flows through the upstream membrane by
capillary action to the label conjugates redistributing the gold
antibody conjugates which can form a complexes with any analyte
present in the sample and with the antigens in the standard band.
The buffer diluted serum passes from the fiberglass membrane to the
porous nitrocellulose membrane and continues to flow by capillary
action through the nitrocellulose membrane. Analyte gold conjugate
complex is captured by the TNI-I antibody deposited in the test
band and gives rise to a reddish color the intensity of which
corresponds to the amount of analyte present in the sample.
Monoclonal antibodies against rabbit IgG and against bovine thyroid
globulin gold conjugates removably deposited on the fiberglass
membrane and picked up by the serum are captured by the high and
low standard bands. After passing the test and standard bands the
remaining buffered sample passes from the nitrocellulose strip to
the absorption pad and is stored there. The cassette with the color
bands is then inserted into a reader together with the memory card
containing the correlation between color intensity as measured by
relative light units (RLU) and concentration of analyte. The reader
employed, CHR 100 is capable of converting the color intensity into
concentration using the data on the memory card. Color intensities
were measured at 15 minutes after the addition of the sample and
buffer. The following results are obtained:
15 Minutest Test Results:
TABLE-US-00005 [0062] Analytes TNI-I Concentration RLU Calibrator H
50 ng/ml 62 Calibrator L 1 ng/ml 12 Sample Y 6
[0063] Based on the two point standard curve, the reader showed 0.5
ng/ml of the CEA antigen (Y) in the sample.
Example 5
[0064] The procedure of example 5 is competitive assay procedure to
determine the concentration of human Thyroxin antigen (T-4) in a
blood or serum sample. In separate experiments the relationship of
T-4 concentration to color intensity of the gold labeled conjugate
is established. In a procedure similar to that of Example 1 rabbit
IgG and bovine thyroid globulin are immobilized in two standard
bands by using an IVEK ceramic linear pump having an accuracy of
99.95%, in amounts sufficient to give rise to a measurable color
intensity corresponding to T-4 concentrations of 5 and 20 ng/ml
respectively is when such is complexed with a gold label monoclonal
(T-4) specific antibody and complexed to the antibody in the test
band. Furthermore T-4 specific monoclonal antibodies are also
deposited in the test band of the nitrocellulose strip. The bands
are arranged such that the sample first contacts the low calibrator
band showing the color intensity with the gold conjugate
corresponding to 5 .mu.g/ml of T-4 antigen, then the test band,
followed by the high calibrator agent band, capable of binding the
equivalent of 20 .mu.g/ml of T-4. The deposited antigen (T-4)
conjugate with bovine serum albumin in the test band are allowed to
react with the gold sol conjugate of T4-antibody on the
nitrocellulose and become permanently immobilized in test bands on
the test membrane after reaction. The assay strip is then exposed
to the gold conjugate monoclonal antibody against rabbit IgG and
monoclonal antibody against bovine thyroglobulin to generate the
standard color intensities from which a standard curved can be
generated. The standard curve showing the correlation of color
intensity with analyte concentration for different lots of assay
strips is loaded into a RFID memory card. The test value may be
calculated by using the equation of Y=AX+B as set forth in Example
1. The test membrane is in fluid connection with an upstream
non-woven fiberglass membrane onto which is deposited the antibody
to the T-4 antigen conjugated to a gold label by techniques known
in the art. The label conjugate is deposited down stream from where
the sample to be analyzed is added to the upstream fiberglass
membrane. The assay test strip also contains a sample absorption
pad in fluid connection with the downstream end of the test
membrane. The test strip is inserted into a cassette such as
described in U.S. Pat. No. 5,821,073.
[0065] One drop of blood suspected of containing the T-4 analyte,
approximately 30 microliters, is added to the sample applicator
site on the upstream fiberglass membrane followed by 4 drops, about
200 microliters, of a testing phosphate buffer.
[0066] The fiberglass membrane used is sufficiently dense to filter
out the red and white blood cells but will let the serum pass. The
serum of the blood combined with buffer flows through the upstream
membrane by capillary action to the label conjugate redistributing
the gold antibody conjugate which then forms a complex with any
analyte present in the sample. The buffer diluted serum passes from
the fiberglass membrane to the porous nitrocellulose membrane and
continues to flow by capillary action through the nitrocellulose
membrane. Analyte gold conjugate complex is captured by the T-4
antibody deposited in the test band and gives rise to a reddish
color the intensity of which corresponds to the amount of analyte
present in the sample. The corresponding standard band gold
conjugate antibodies, removably deposited on the fiberglass and
picked up by the sample, are captured by the antigens in the
standard bands. After passing the test and standard bands the
remaining buffered sample passes from the nitrocellulose strip to
the absorption pad and is stored there. The cassette with the color
bands is then inserted into a reader together with the memory card
containing the correlation between color intensity as measured by
relative light units (RLU) and concentration of analyte. The reader
employed, CHR 100 is capable of converting the color intensity into
concentration using the data on the memory card. Color intensities
were measured at 15 minutes after the addition of the sample and
buffer. The following results are obtained:
15 Minutes Test Result:
TABLE-US-00006 [0067] Analytes T-4 Concentration RLU Calibrator H
20 .mu.g/ml 39 Calibrator L 5 .mu.g/ml 79 79 Sample Y 66
[0068] Based on the two point standard curve, the reader showed 10
.mu.g/ml of the T-4 antigen (Y) in the sample.
Example 6
[0069] Onto a test strip comprising a porous nitrocellulose strip
(7.5.times.0.0.7 cm) as the test membrane sufficient monoclonal
antibody against human prostrate specific antigen (PSA) in a test
band to give rise to measurable color intensity when complexed with
PSA analyte color conjugates that can be expected in a sample.
Rabbit IgG, bovine thyroid globulin and murine hybridoma are
immobilized in three standard bands by using an IVEK ceramic linear
pump having an accuracy of 99.95%, in amounts sufficient to give
rise to a color intensity corresponding to a PSA concentration of
1, 5 and 10 ng/ml respectively when such is complexed with a gold
label monoclonal PSA specific antibody. The test membrane is dried
in a dehumidifying chamber (humidity of 10-15%) overnight. Excess
of the gold label antibody against PSA, gold label monoclonal
antibody against rabbit IgG, anti-mouse antibody capable of
complexing with hybridoma and gold label monoclonal antibody
against bovine thyroid globulin are also removable deposited in an
upstream fiberglass membrane. The conjugate fiberglass pad is
coated with the label conjugates, then treated with a buffer to
neutralize interfering sites and then vacuum dried at 2 Torr
overnight at room temperature. The pad is then cut into narrow
strips (1.times.0.7 cm). The bands are arranged such that the
sample first contacts the low and intermediate calibrator bands
showing the color intensity with the gold conjugate corresponding
to 1 and 5 ng/ml of PSA antigen, then the test band, followed by
the high calibrator agent band, capable of binding the equivalent
of 10 ng/ml of PSA. The correlation of the color density in RLU
units with analyte concentration, lot #, test time, slope and
intercept for the three standard bands of the particular test strip
is loaded into a RFID memory card. The antigen concentrations can
then be calculated using the equation Y=AX+B where Y is the
concentration of the analyte, X is the color intensity generated by
the sample in RLU units, A is the slope of the curve obtained from
the standard values and B is the intercept of the test value on the
curve.
[0070] The test membrane is in fluid connection with an upstream
non-woven fiberglass membrane onto which is deposited the antibody
to the PSA antigen conjugated to a gold label by techniques known
in the art. The label conjugate is deposited down stream from where
the sample to be analyzed is added to the upstream fiberglass
membrane. The assay test strip also contains a sample absorption
pad in fluid connection with the downstream end of the test
membrane. The test strip is inserted into a cassette such as
described in U.S. Pat. No. 5,821,073.
[0071] One drop of blood suspected of containing the PSA analyte,
approximately 30 microliters, is added to the sample applicator
site on the upstream fiberglass membrane followed by four drops,
about 200 microliters, of a commercially available phosphate
buffer. The fiberglass membrane is sufficiently dense to filter out
the red and white blood cells but will let the serum pass. The
serum of the blood combined with buffer flows through the upstream
membrane by capillary action to the label antibody conjugate
location where the analyte present in the sample complexes with the
gold antibody and where the buffer diluted sample picks up the
antibodies of the antigens in the standard bands. The buffer
diluted serum passes from the fiberglass membrane to the porous
nitrocellulose membrane and continues to flow by capillary action
through the nitrocellulose membrane. Analyte gold conjugate complex
is captured by the PSA antibody deposited in the test band and
gives rise to a reddish color the intensity of which corresponds to
the amount of analyte present in the sample. Monoclonal antibody
against rabbit IgG, antimouse antibody and monoclonal antibody
against bovine thyroid globulin gold conjugate deposited on the
fiberglass membrane and picked up by the sample are also captured
by the high, intermediate and low standard bands containing
sufficient calibrator agent to capture the equivalent of either
one, five or ten ng/ml of analyte. In the presence of PSA analyte
in the sample four bands of normally differing color intensity are
obtained. After passing the test and standard bands the remaining
buffered sample passes from the nitrocellulose strip to the
absorption pad and is stored there. The cassette with the color
bands is then inserted into a reader together with the memory card
containing the correlation between color intensity as measured by
relative light units (RLU) and concentration of analyte. The
photo-camera reader employed, Kaiwood CHR 100, is capable of
converting the color intensity into concentration using the data on
the memory card. Color intensities were measured 10 and 15 minutes
after the addition of the sample and buffer. Essentially the same
results as obtained in Example 1 are obtained.
* * * * *