U.S. patent application number 12/643581 was filed with the patent office on 2010-08-19 for dry reagent particle assay and device having multiple test zones and method therefor.
This patent application is currently assigned to BAYER HEALTHCARE LLC. Invention is credited to Michael P. Allen, Joel M. Blatt.
Application Number | 20100210038 12/643581 |
Document ID | / |
Family ID | 34910668 |
Filed Date | 2010-08-19 |
United States Patent
Application |
20100210038 |
Kind Code |
A1 |
Blatt; Joel M. ; et
al. |
August 19, 2010 |
DRY REAGENT PARTICLE ASSAY AND DEVICE HAVING MULTIPLE TEST ZONES
AND METHOD THEREFOR
Abstract
The present invention relates to a dry reagent assay device
having at least one test zone and at least one reference zone,
which provides an internal mechanism for assuring correct and
reliable assay procedures and reagent qualities. In one embodiment,
the present invention relates to an assay device having, at least
one test zone for detecting at least one analyte in a sample by
reacting the sample with a labeled indicator reagent, and a
reference zone for receiving, unreacted labeled indicator
reagent.
Inventors: |
Blatt; Joel M.; (Mountain
View, CA) ; Allen; Michael P.; (Los Altos,
CA) |
Correspondence
Address: |
BAYER;LERNER, DAVID, LITTENBERG, KRUMHOLZ & MENTLIK, LLP
600 South Avenue West
Westfield
NJ
07090
US
|
Assignee: |
BAYER HEALTHCARE LLC
Tarrytown
NY
|
Family ID: |
34910668 |
Appl. No.: |
12/643581 |
Filed: |
December 21, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10826880 |
Apr 19, 2004 |
7635597 |
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12643581 |
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08512844 |
Aug 9, 1995 |
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10826880 |
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Current U.S.
Class: |
436/518 ;
422/69 |
Current CPC
Class: |
Y10S 436/81 20130101;
G01N 33/558 20130101; Y10S 436/808 20130101 |
Class at
Publication: |
436/518 ;
422/69 |
International
Class: |
G01N 33/543 20060101
G01N033/543 |
Claims
1-50. (canceled)
51. A device for determining the presence of an analyte in a
sample, the device comprising: a bibulous member capable of being
traversed by the sample; a first zone on the bibulous member for
receiving and contacting the sample containing a diffusively
immobilized, particle-linked antigen, the particle-linked antigen
reacting in the presence of the analyte to form a mixture; a second
zone on the bibulous member for receiving and contacting the
mixture containing a non-diffusely immobilized antibody capable of
binding the particle-linked antigen and free sample antigen, the
second zone expresses a detectable response inversely related to
the analyte level in the sample; a third zone on the bibulous
member for receiving and contacting the remaining mixture
containing a non-diffusively immobilized first member of a specific
binding pair, capable of specifically binding to its specific
binding partner which is the second member of the specific binding
pair on the surface of the particle-linked antigen, which second
member of the specific binding pair is not antigenically related to
the sample antigen and will not effectively compete with the
antigen to bind to an anti-antigen monoclonal antibody, the third
zone expresses a detectable response related to the analyte level
in the sample; and means for determining the analyte level in the
sample from the detectable responses in the second and third
zones.
52. The device of claim 51 wherein the determining means further
comprises determining the sum of the detectable responses from the
test zones to be within a pre-determined range of response.
53. The device of claim 51 wherein the determining means further
comprises determining the level of analyte in one test zone by
comparison to the total detectable response.
54. The device of claim 51 wherein the determining means further
comprises determining the level of analyte using the detectable
response in one test zone by comparison to a pre-determined
standard.
55. The device of claim 51 wherein the determining means further
comprises determining the level of analyte using the detectable
response in one test zone in comparison to the detectable response
in the other test zone.
56. The device of claim 51 wherein the determining means further
comprises determining the level of analyte using the detectable
response in one test zone in one portion of the pre-determined
range of analyte and the detectable response in the other test zone
in a different portion of the pre-determined range of analyte.
57. A device for determining the presence of an analyte in a
sample, the device comprising: a bibulous member capable of being
traversed by the sample; a first zone on the bibulous member for
receiving and contacting the sample containing a diffusively
immobilized particle-linked antibody, the particle-linked antibody
reacting in the presence of the analyte to form a conjugate
included in a mixture; a second zone on the bibulous member for
receiving and contacting the mixture containing a non-diffusively
immobilized antigen capable of being bound by the particle-linked
antibody, the second zone expresses a detectable response inversely
related to the analyte level in the sample; a third zone on the
bibulous member for receiving and contacting the remaining mixture
containing a non-diffusively immobilized first member of a specific
binding pair capable of specifically binding to its specific
binding partner which is the second member of the specific binding
pair on the surface of the particle-linked antigen, which second
member of the specific binding pair is not antigenically related to
the sample antigen so it will not effectively compete with the
antigen to bind to an anti-antigen monoclonal antibody, the third
zone expresses a detectable response related to the analyte level
in the sample; and means for determining the analyte level in the
sample from the detectable responses in the second and third
zones.
58. The device of claim 57 wherein the determining means further
comprises determining the sum of the detectable responses from the
test zones is within a pre-determined range of response.
59. The device of claim 57 wherein the combining means further
comprises determining the level of analyte in one zone by
comparison to the total detectable response.
60. The device of claim 57 wherein the combining means further
comprises determining the level of analyte using the detectable
response in one test zone by comparison to a pre-determined
standard.
61. The device of claim 57 wherein the combining means further
comprises determining the level of analyte using the detectable
response in one test zone in comparison to the detectable response
in the other test zone.
62. The device of claim 57 wherein the combining means further
comprises determining the level of analyte using the detectable
response in one test zone in one portion of the pre-determined
range of analyte and the detectable response in the other test zone
in a different portion of the pre-determined range of analyte.
63. A method for determining the level of at least one analyte in a
sample, the method comprising the steps of; contacting the sample
with an end portion of a bibulous strip having a plurality of
zones; wicking the sample to a labeled indicator reagent
diffusively immobilized on the bibulous strip; reacting the labeled
indicator reagent in the presence of the analyte to form a mixture;
wicking the mixture to a first reagent non-diffusely immobilized on
the bibulous strip; reacting the first reagent in the presence of
the mixture to form a first reaction product and a detectable
response inversely related to the analyte level in the sample;
wicking the remaining mixture to a second reagent non-diffusely
immobilized on the bibulous strip; reacting the second reagent in
the presence of the remaining mixture to form a second reaction
product and a detectable response related to the analyte level in
the sample; and determining the analyte level in the sample from
the detectable responses in the reacting steps with the first and
second reagents.
64. The method of claim 63 wherein: the reacting step with the
labeled indicator reagent comprises forming a mixture including
particle-linked antigen with the analyte; the reacting step with
the first reagent comprises binding an antibody with the
particle-linked antigen and the analyte; the reacting step with the
second reagent comprises binding a first member of a specific
binding pair to a second member of the specific binding pair on the
particle-linked antigen, the second member of the specific binding
pair is not a specific binding partner to the analyte.
65. The method of claim 63 wherein: the reacting step with the
labeled indicator reagent comprises forming a mixture including a
particle-linked antibody with the analyte; the reacting step with
the first reagent comprises substantially binding the
particle-linked antibody with an immobilized antigen; the reacting
step for the second reagent comprises binding a first member of a
specific binding pair to a second member of the specific binding
pair on the particle-linked antibody, the second member of the
specific binding pair is not a specific binding pair to the
analyte.
Description
RELATED APPLICATION
[0001] The subject matter of this application is related to a
disposable single-use digital electronic, instrument that is
entirely self-contained, including all chemistry reagents, as
disclosed in U.S. application Ser. No. 08/111,347 entitled "Novel
Disposable Electronic Assay Device" filed Aug. 24, 1993 by Michael
P. Allen. The above application has the same assignee as the
present invention and is incorporated herein by reference in its
entirety.
FIELD OF THE INVENTION
[0002] The present invention relates to a dry reagent assay device
having two or more test zones, which provides an internal reference
mechanism for assuring correct and reliable assay procedures and
reagent qualities.
BACKGROUND OF THE INVENTION
[0003] Qualitative and quantitative self-tests have developed
gradually over the last half century. Non-instrumented tests have
become commercially available using immunochemical reagents on a
solid support for diagnostic tests involving HCG, LH, FSH, CKMB,
Staphylococcus, and Rubella. Measurement of the hormone HCG to
detect pregnancy was among the first of these tests to become
commercially successful in the home market. The first home
pregnancy test, the e.p.t..TM., as introduced in 1977 by
Warner-Lambert. The e.p.t..TM. used a solution phase chemical
reaction that formed a brown ring on the surface of the urine
solution in the presence of HCG. The 2 hour long protocol
associated with this test was sensitive to vibration and timing,
causing false results.
[0004] Two additional test systems that appeared in the late 1980s
were the LipoScan.TM. by Home Diagnostics Inc. and the Chemcard.TM.
by Chematics Inc. Bath tests measure cholesterol in whole-blood
using visual color comparison. Since visual color matching is
subjective these tests do not achieve the quantitative performance
necessary for cholesterol testing (Pradella et al, Clin. Chem.
36:1994-1995 (1990)).
[0005] For many analytes such as the markers for pregnancy and
ovulation, qualitative or semi-quantitative tests are appropriate.
There are, however, a variety of analytes that require accurate
quantitation. These include glucose, cholesterol, HDL cholesterol,
triglyceride, a variety of therapeutic drugs such as theophylline,
vitamin levels, and other health indicators. Generally, their
quantitation has been achieved through the use of an instrument.
Although suitable for clinical analysis, these methods are
generally undesirable for point-of-care testing in physicians
offices and in the home due to the expense of the instrument.
[0006] Recently, a number of non-instrumented methods for measuring
analytes use instrument-free quantitation through the use of
migration distance, rather than color matching, as the visual
signal. In migration distance assays, chemical/biochemical
reactions occur as the analyte is wicked through a solid support.
During wicking the analyte reacts with a signal-producing reagent
and forms a visible signal along the support. The migration
distance or the distance of signal border is related to analyte
concentration. The operator reads the height of the color bar much
the same way one reads a thermometer, and finds the concentration
from a calibrated scale.
[0007] There are a few migration-type assays commercially
available. These include Environmental Test Systems' Quantab.TM.,
which measures chloride in swimming pools and during the mixing of
concrete, Syva's AccuLevel.RTM. for the measurement of therapeutic
drugs, and ChemTrak's AccuMeter.RTM. for measurement of cholesterol
in whole blood. Other companies such as Enzymatics and Crystal
Diagnostics have more recently announced the introduction, of their
Q.E.D..TM. and Clinimeter.TM. technologies to measure,
respectively, alcohol in saliva and cholesterol in blood.
ActiMed.TM. discloses a thermometer-type cholesterol assay device
in Ertinghausen, U.S. Pat. No. 5,087,556 (1992).
[0008] Although these single use, thermometer-type,
non-1-instrumented quantitative devices and non-instrumented color
comparison devices for qualitative measurement have shown adequate
performance, they have several problems associated with reliability
and convenience. First, the colors generated on these devices are
not always uniform and sharp in the case of migration type assays
the border is often light in color, unclear and difficult to read.
This translates directly into user errors since the user must make
a judgment related to the position of the color band border. DT the
case of non-instrumented pregnancy tests it is sometimes difficult
to visually interpret the intensity of the colored spot (especially
at HCG concentrations close to the cut-off sensitivity), and
interpretation of the result is sometimes a problem. Anytime a
non-technical operator is required to make a visual judgment or
interpretation, an error is possible, and sometimes, is
unavoidable.
[0009] Second, the assay protocol for these tests is sometimes
difficult and lengthy, taking 15 minutes to 1 hour to obtain a
result. Third, these tests often do not have sufficient procedural
and reagent references to assure adequate test performance. Fourth
and last, non-instrumented devices can only measure single endpoint
type tests since enzyme rates or ratiometric analysis of two
analytes cannot be measured. Therefore, the menu of potential tests
is limited.
[0010] As an example of the significance of the problems, a recent
article in Clinical Chemistry (Daviaud et al, Clin. Chem. 39 53-59
(1993)) evaluated all 27 home use pregnancy tests sold in France.
The authors state, "among the 478 positive, urine samples
distributed, 230 were falsely interpreted as negative".
[0011] In the past, immunoassays were developed for the
quantitative and qualitative, determination of a wide variety of
compounds in a laboratory setting using detailed procedures and
expensive instrumentation. Recent developments in
immuno-diagnostics have resulted in a movement toward more simple
approaches to the rapid analysis of clinical samples. The
development of solid phase bound reagents has eliminated the need
for precipitation in the separation of bound reagents from free
reagents. Further advancements in solid phase immunochemistry have
resulted in non-instrumented dry reagent strip immunoassays. This
configuration allows for the visual qualitative or
semi-quantitative determination of analytes in patient samples
without the use of an instrument.
[0012] There are two basic types of non-instrumented immunoassay
configurations. In the first type, or visual color zone type, a
signal is generated at a specific zone on the strip where the
signal indicates the presence of analyte; and the intensity of the
signal indicates the concentration of the analyte in the sample.
This type of assay requires visual color interpretation either for
the presence of color above a threshold, as in the case of a
qualitative test, or the comparison of the color intensity to a
color chart, as in the case of a semi-quantitative test. In the
second type, the visual signal is produced along the length of a
bibulous assay strip. During wicking, the analyte reacts with a
signal-producing reagent and forms a visible signal along the
support. The migration distance of the signal from the proximal end
of the strip is a direct measure of analyte concentration. This
type of non-instrumented migration height assay can achieve
quantitative results with reasonable performance as disclosed in
Zuk et al, Clin. Chem. 31:1144-1150 (1985).
[0013] The color zone type of strip immunoassay is usually
configured in three ways. First, a one site competitive immunoassay
where labeled reagent and analyte compete for binding sites at a
discrete zone along a strip where one member of the binding pair is
immobilized. Second, a one site inhibition immunoassay where
labeled reagent binds substantially all of the sample analyte prior
to contact the strip zone where the opposite member of the binding
pair is immobilized. Third, a two-site or "sandwich" immunoassay,
where the sample analyte has at least two binding sites.
[0014] The prior art discloses color one immunoassays in lateral
flow and vertical flow configurations limited to the use of
enzymatic signal generating systems. The use of lateral flow
wicking strips has focused in the area of enzyme detection in
one-site competitive or two-site sandwich configurations, and the
use of particle detection has been confined largely to two-site
sandwich immunoassays.
[0015] There are examples of methods developed where chemical or
immunological reactions occurred along the length of a bibulous
assay strip. In U.S. Pat. Nos. 4,094,841, 4,235,801 and 4,363,537
Deutsch and Mead disclose a bibulous strip assay with discrete
immunochemical reagent zones along its length for conducting
specific binding assays. Grubb and Gladd, U.S. Pat. No. 4,168,146,
describe an enzyme immuno-chromatography assay on a bibulous strip
wherein a sample containing antigen is wicked through the assay
strip, and the antigen in the sample binds to the immobilized,
antibody and progressively fills the binding sites as a measure of
analyte concentration. The antigen containing area is visualized by
wetting the strip with an enzyme labeled antibody and developing
color with a chromogenic substrate. David, et al., disclose U.S.
Pat. No. 4,376,110 that monoclonal antibodies with binding
affinities of. 10.sup.8 or greater can be used in forward, reverse
and simultaneous two-site sandwich immunoassays.
[0016] The lateral wicking immunoassays using colored particle
detection for two-site configurations in the prior art are limited
to visually interpretation and usually provide, only qualitative,
or at best, semi-quantitative results. The prior art fails to
disclose colored particle detection in lateral wicking devices
which use competitive or inhibition immunoassay configurations.
Likewise, lateral wicking immunoassay reagent strips designed for
use in a quantitative instrument read format are not disclosed in
the prior art. Furthermore, the multiple test zone reagent strips
of the prior art fail to provide quality reference.
[0017] Thus, a need exists in the field of diagnostics for a
wicking assay which it sufficiently accurate and reliable to permit
point-of-care use by untrained individuals in locations such as the
home, sites of medical emergencies, or locations other than a
clinic.
SUMMARY OF THE INVENTION
[0018] The present invention provides, a device for determining the
presence of at least one of a plurality of analytes in a sample.
The device includes a test zone corresponding to each analyte
selected for determining its presence. Each test zone receives and
contacts the sample and a labeled indicator reagent corresponding
to the selected analyte with a test zone reagent corresponding to
the selected analyte. The test zone reagent corresponds to the
selected analyte reacting in the presence of the sample and the
labeled indicator reagent corresponds to the selected analyte to
form a corresponding test zone reaction product and a corresponding
test zone detectable response inversely related to the selected
analyte level in the sample. The device also includes a reference
zone for receiving from each test zone the labeled indicator
reagent not reacted with its corresponding test zone reagent and
contacting each labeled indicator reagent with a corresponding
reference zone reagent. Each reference zone reagent reacts in the
presence of the corresponding labeled indicator reagent to form a
corresponding reference zone reaction product and a corresponding
reference zone detectable response related to each selected analyte
level in the sample and related to the corresponding test zone
detectable response to establish a substantially constant total
detectable response for a pre-determined range of each selected
analyte. The device also includes cleans for combining the
detectable responses from the test zones to determine the analyte
level in the sample.
[0019] The present invention also includes a device for determining
the presence of an analyte in a sample. The device includes a first
test zone for receiving and contacting the sample and a labeled
indicator reagent with a first reagent. The first reagent reacts in
the presence of the sample and labeled indicator reagent to form a
first reaction product and a detectable response in the first test
zone inversely related to the analyte level in the sample. A second
test zone receives and contacts the labeled indicator reagent hot
reacted with the first reagent with a second, reagent. The second
reagent reacts in the presence of the labeled indicator reagent to
form a second reaction product and a detectable response in the
second test zone related to the analyte level in the sample and
related to the detectable response of the first test zone to
establish a substantially constant total detectable response from
the test zones for a pre-determined range of the analyte. The
device also includes means or combining the detectable responses
from the test zones to determine the analyte level, in the
sample.
[0020] The present invention also provides a device for determining
the presence of an analyte in a sample which includes a porous
member capable of being traversed by the sample. A first zone on
the porous member receives and contacts the sample with labeled
indicator reagent diffusively immobilized on the porous member. The
labeled indicator reagent reacts in the presence of the analyte to
form a mixture. A second zone on the porous member receives and
contacts the mixture with a first reagent non-diffusely immobilized
on the porous material in the second zone. The first reagent reacts
in the presence of the mixture to form a first reaction product and
a detectable response in the second zone inversely, related to the
analyte level in the sample. A third zone, on the porous member
receives and contacts the remaining mixture with a second reagent
non-diffusely immobilized on the porous material in the third zone.
The second reagent reacts in the presence of the remaining mixture
to form a second reaction product and a detectable response in the
third, zone related to the analyte level in the sample. The device
also includes means for determining the analyte level in the sample
from the detectable responses in the second and third zones.
[0021] A preferred embodiment of the present invention provides a
device for determining the presence of an analyte in a sample which
includes a bibulous member capable of being traversed by the
sample. A first zone on the bibulous member receives and contacts
the sample with a particle-linked antigen diffusively immobilized
on the bibulous member. The particle-linked antigen reacts in the
presence of the analyte to form a mixture. A second zone on the
bibulous member receives and contacts the mixture with an antibody
non-diffusely immobilized on the bibulous material in the second
zone. The antibody is a specific binding partner to the
particle-linked antigen and the analyte, the antibody reacts in the
presence of the mixture to bind the particle-linked antigen and
express a detectable response in the second zone inversely related
to the analyte level in the sample. A third zone on the bibulous
member receives and contacts the remaining mixture with an antibody
non-diffusely immobilized on the bibulous material in the third
zone. The antibody is a first member of a specific binding pair
capable of binding to a second member of the specific binding pair
on the particle-linked antigen. The second member of the specific
binding pair is not a specific binding partner to the analyte. The
antibody reacts in the presence of the remaining mixture to bind
with the remaining mixture and express a detectable response in the
third zone related to the analyte level in the sample. The device
also includes means for determining the analyte level in the sample
from the detectable responses in the second and third zones.
[0022] Another preferred embodiment of the present invention
provides a device for determining, the presence of an analyte in a
sample which includes a bibulous member capable of being traversed
by the sample. A first zone on the bibulous member receives and
contacts the sample with a particle-linked antibody diffusively
immobilized on the bibulous member. The particle-linked antibody
reacts in the presence of the analyte to form a mixture. A second
zone on the bibulous member receives and contacts the mixture with
an antigen non-diffusely immobilized on the bibulous material in
the second zone. The antigen is a specific binding partner to the
particle-linked antibody. The antigen reacts in the presence of the
particle-linked antibody to substantially bind the particle-linked
antibody and express a detectable response in the second, zone
inversely related to the analyte level in the sample. A third zone
on the bibulous member receives and contacts the remaining mixture
with an antibody non-diffusely immobilized on the bibulous material
in the third zone. The antibody is a first member of a specific
binding pair capable of binding to a second member of the specific
binding pair on the particle-linked antibody. The second member of
the specific binding pair is not a specific binding partner to the
analyte. The antibody reacts in the presence of the remaining
mixture to bind with the particle-linked antibody and express a
detectable response, in the third zone related to the analyte level
in the sample. The device also includes means for determining the
analyte level in the sample from the detectable responses in the
second and third zones.
[0023] Methods are also provided by the present invention for
determining the presence of an analyte in a test sample. One method
comprising the steps of: contacting the sample with a porous member
having a plurality of zones; transporting the sample sequentially
across the plurality of, zones and contacting the sample to at
least one reagent immobilized, in each zone; detecting a response
from the contact between the sample and the reagent in at least two
zones; and, determining the analyte level in the sample by
combining the response from at least two zones.
[0024] Another method provided by the present invention determines
the level of at least one analyte in a, sample. The method
comprising the steps of contacting the sample with an end portion
of a bibulous, strip having, a plurality of zones; wicking the
sample to a labeled indicator reagent diffusively immobilized on
the bibulous strip; reacting the labeled indicator reagent in the
presence of the analyte to form a mixture; wicking the mixture to a
first reagent non-diffusely immobilized on the bibulous strip;
reacting the first reagent in the presence of the mixture to form a
first reaction product and a detectable response inversely related
to the analyte level in the sample, wicking the remaining mixture
to a second reagent non-diffusely immobilized on the bibulous
strip; reacting the second reagent in the presence of the remaining
mixture to form a second reaction product and a detectable response
related to the analyte level in the sample, and, determining the
analyte level in the sample from the detectable responses in the
reacting steps with the first and second reagents.
[0025] Accordingly, it is an object of the present invention to
provide a wicking device which uses competitive and immunoassay
configurations for test results which are more accurate and
reproducible than in the prior art.
[0026] It is further object of the present invention to provide a
assay method using multiple test zones in a single assay to yield
accurate quantitative results.
[0027] Another object of the present invention is to, provide an
assay which, provides mean for quality reference using the signals
combined from multiple test zones.
[0028] A farther object of the invention is to provide a
quantitative strip immunoassay based on particle detection.
[0029] Other and further advantages, embodiments, variations and
the like will be apparent to those skilled in the art from the
present specification taken with the accompanying drawings and
appended claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0030] In the drawings, which comprise a portion of this
disclosure:
[0031] FIG. 1 shows a top surface view of an embodiment baying
typical structure with three reagent zones that can be used for
quantitative and qualitative immunoassays;
[0032] FIG. 2 shows a top surface view of an embodiment having
typical structure with four reagent zones that can be used for
quantitative and qualitative immunoassays;
[0033] FIG. 3 shows an exploded lengthwise, cross section of the
embodiment of FIG. 1;
[0034] FIG. 4 shows an exploded lengthwise cross section of an
embodiment having a typical structure with a sample
pre-treatment/filtration/separation/blood separation device;
[0035] FIG. 5 shows an exploded lengthwiSe cross section of an
embodiment having a typical structure with a sample
pre-treatment/filtration/separation/blood separation device and a
sample transport;
[0036] FIG. 6 shows the top surface view of one embodiment of the
N-telopeptide (NTx) assay strip;
[0037] FIG. 7 shows the top surface view of a second embodiment of
the N-telopeptide (NTx) assay strip; and
[0038] FIG. 8 is a graphical representation of a dose response to
NTx with quality reference. Plotting reflectance density vs. NTx
concentration.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0039] The present invention concerns a lateral flow immunoassay
strip for use in an instrument system that can produce qualitative
CT quantitative results. A preferred embodiment of the present
invention provides an assay strip including three zones of which
two zones are test zones and one of the test zones is a reference
zone. A first test zone produces a signal with intensity inversely
proportional to analyte concentration and a second test, zone acts
a reference and produces a signal that is directly proportional to
analyte concentration. The sum of the signals from test zones 1 and
2 is substantially equal at all analyte concentrations.
Quantitative or qualitative results are achieved by instrumental
reading of color intensity on test zone 1, test zone 2 or both test
zones 1 and 2. The results expressed by any one test zone can also
be determined as a proportion of the sum of the actual results
expressed by both test zones. Quality reference is achieved by
instrumental reading of both test zones, the sum of which should be
substantially constant within a specified range.
[0040] The present invention represents a substantial improvement
in the art by providing methods, assay devices and assay
instruments which employ (a) a one-step competitive, lateral flow
strip immunoassay, (b) a one-step inhibition lateral flow
immunoassay, (c) a quantitative bibulous strip immunoassay based on
particle detection, (d) two or more detection zones for testing,
and (e) a reference zone for performing detection tests as a
detection zone and perform quality control using the responses
combined from multiple test zones.
[0041] The present invention provides an assay which can use
specific binding members. A specific binding partner or member, as
used herein, is a member of a specific binding pair. That is, two
different molecules where one of the molecules through chemical or
physical means specifically binds to the second molecule.
Therefore, in addition to antigen and antibody specific binding
pairs of common immunoassays, other specific binding pairs can
include biotin and avidin, carbohydrates and lectins, complementary
nucleotide sequences, effector and receptor molecules, cofactors
and enzymes, enzyme inhibitors and enzymes, and the like.
Furthermore, specific binding pairs can include members that are
analogs of the original specific binding members, for example, an
analyte-analog. Immunoreactive specific binding members include
antigens, antigen fragments, antibodies, and antibody fragments,
both monoclonal and polyclonal, and complexes thereof, including
those formed by recombinant DNA molecules. The term hapten, as used
herein, refers to a partial antigen or non-protein binding member
which is capable of binding to an antibody, but which is not
capable of eliciting antibody formation unless coupled to a carrier
protein.
[0042] Analyte, as used herein, is the substance to be detected
which may be present in the test sample. The analyte can be any
substance for which there exists a naturally occurring specific
binding member (such as, an antibody), or for which a specific,
binding member can be prepared. Thus, an analyte is a substance
that can bind to one or more specific binding, members in an assay.
Analyte alto includes any antigenic substances, haptens,
antibodies, macromolecules, and combinations thereof. As a member
of a specific binding pair, the analyte can be detected by means of
naturally occuring specific binding partners (pairs) such as the
use of intrinsic factor protein as a member of a specific binding
pair for the determination of vitamin B12, or the use of lectin as
a member of a specific binding pair for the determination of a
carbohydrate. The analyte can include a protein, a peptide, an
amino acid, a hormone, a steroid, a vitamin, a drug including those
administered for therapeutic purposes as well as those,
administered for illicit purposes, a bacterium, a virus, and
metabolites of or antibodies to any of the above substances. In
particular, such analytes include, but are not intended to
ferritin; creatinine kinase MIB (CK-MB); digoxin; phenyloin;
phenobarbital; carbamazepine; vancoomyin; gentamicin, theophylline;
valproic acid; quinidine; luteinizing hormone (LH); follicole
stimulating hormone (FSH); estradiol, progesterone; IgE antibodies;
vitamin B2 micro-globulin; glycated hemoglobin (Gly. H10);
cortisol; digitoxin; N-acetylprocainamide (NAPA); procainamide;
antibodies to rubella, such as rubella-IgG and rubella-IgM;
antibodies to toxoplasmosis, such as toxoplasmosis IgG (Toxo-IgG)
and toxoplasmosis IgM (Toxo-IM); testosterone; salicylates;
acetaminophen; hepatitis B core antigen, such as anti-hepatitis B
core antigen IgG and IgM (Anti-HBC); human immune deficiency virus
1 and 2 (HIV 1 and 2); human T-cell leukemia virus 1 and 2 (HTV);
hepatitis Be antigen (HBeAg); antibodies to hepatitis Be antigen
(Anti-HBe), thyroid stimulating hormone (TSH), thyroxine (T4),
total triiodothyronine (Total T3); free triiodothyronine (Free T3);
carcinoembryoic antigen (CEA); and alpha fetal protein (AFP). Drugs
of abuse and referenced substances include, but are not intended to
be limited to, amphetamine; methamphetamine; barbiturates such as
amobarbital, secpbarbital, pentobarbital, phenobarbital, and
barbital; benzodiazepines such as librium and valium; cannabinoids
such as hashish and marijuana; cocaine; fentanyl; LSD;
methaqualone; opiates such as heroin, morphine, codeine,
hydromorphone, hydrocodone, methadone, oxycodone, oxymorphone, and
opium; phenyloyalidine; and propoxyhene. The details for the
preparation of such antibodies and the suitability for use as
specific binding members are well known to those skilled in the
art.
[0043] The analyte-analog can be any substance which cross-reacts
with the analyte-specific binding member, although it may do so to
a greater or lesser extent than does the analyte itself. The
analyte-analog can include a modified analyte as well as a
fragmented, or synthetic portion of the analyte molecule, so long
as the analyte-analog has at least one epitope site in common with
the analyte of interest. An example of an analyte-analog is a
synthetic peptide sequence which duplicated at; least one, epitope
of the whole-molecule analyte so that the analyte-analog can bind
to an analyte-specific binding member.
[0044] The test sample can be derived from any biological source,
such as a physiological fluid, including whole blood or whole blood
components including red blood cells, white blood cells, platelets,
serum and plasma; ascites; urine; sweat; milk; synovial raucous;
peritoneal fluid; amniotic fluid; percerebrospinal fluid; and other
constituents of the body which may contain the analyte of interest.
The test sample can be pre-treated prior to use such as preparing
plasma from blood, diluting viscous fluids, or the like; methods of
treatment can involve filtration, distillation, concentration,
inactivation of interfering compounds, and the addition of
reagents. Besides physiological fluids, other liquid samples can be
used such as water, food products and the like for the performance
of environmental or food production assays. In addition, a solid
material suspected of containing the analyte can be used as the
test sample. In some instances it may be beneficial to modify a
solid test sample to form a liquid medium or to release the
analyte. The analyte can be any compound, or composition to be
detected or measured and which, has at least one epitope or binding
site.
[0045] An assay device for the present invention can have many
configurations, several of which are dependent upon the material
chosen as the porous member. By "porous" is meant that the material
is one through which the test sample can easily pass and includes
both bibulous and non-bibulous solid phase materials. In the
present invention, the porous member can include a fiberglass,
cellulose, or nylon pad for use in a pour and flow-through assay
device having multiple layers for multiple assay reagents; a test
strip for wicking or thin layer chromatographic capillary action
(e.g., nitrocellulose) techniques; or other porous or open pore
materials well known to those skilled in the art (e.g.,
polyethylene sheet material).
[0046] In a preferred embodiment, the present dry reagent assay
device uses a lateral flow bibulous material with proximal and
distal ends, containing at least one central zone along its length.
The strip, configuration may be of any dimensions which provide the
desired, number of zones and which permit (a) the desired binding
reactions to be completed in a reproducible manner and (b)
detection of the reaction indicator to occur. Preferably, the
present strip is a total of no more than about 100 mm in length and
about 6 Mt wide, and more preferably, from about 10 mm to about 40
mm in length and about 1 mm to about 5 mm wide.
[0047] The strip is advantageously integrated into any reflectance
based instrument, and more preferably, into a disposable electronic
assay device, such as that described in application Ser. No.
08/111,347, previously incorporated by reference.
[0048] The bibulous strip, can comprise a plurality of zones along
its length. The zones can contain diffusively or non-diffusively
bound reagents. Each zone can, be from about 0.1 mm to about 10 mm
wide, more preferably from about 0.25 mm to about 5 mm, wide. There
will be a minimum of two zones and a maximum of about 10 or more
zones, depending on the number of assays to be conducted on one
bibulous strip.
[0049] In a preferred embodiment, the bibulous strip has three
zones along its length. According to this preferred embodiment
there are two preferred, configurations including a competitive
configuration and an inhibition configuration.
[0050] The present invention provides an assay. Method having a,
competitive type configuration. Referring to FIG. 1, at the
proximal end 11 of a strip 12 is a first zone 14, comprising a
bibulous material containing a diffusively particle-linked antigen.
A second zone. 16 is separate and distinct from the first zone 14,
and is located at some distance toward the distal end 13 of the
bibulous strip 12. The second zone 16 includes a bibulous material
containing a non-diffusively immobilized antibody capable of
binding the particle-linked antigen and free sample antigen. The
bibulous material of the second vine 16 can be the same or
different from the bibulous material of the first zone 14.
[0051] A third one 18 is separate and distinct from the second zone
16, and is located at some distance toward the distal end 13 of the
bibulous strip 12 from the second zone 16. The third zone 18
includes a bibulous material (which may be the same or different
from the bibulous materials of the first and second zones 14 and
16) containing a non-diffusively immobilized first member of a
specific binding pair, capable of specifically binding to its
specific binding partner which is the second member of the specific
binding pair on the surface of the particle-linked antigen. This
second member of the specific binding pair is not antigenically
related to the sample antigen so it will not effectively compete
with the antigen to bind to an anti-antigen monoclonal
antibody.
[0052] The sample is applied to the strip 12 at the application
site or first zone 14 which is preferably at the proximal end 11 of
the assay strip. The particle-linked antigen is located at or near
the application site. The sample containing a sample antigen
reconstitutes the dried particle-antigen conjugate by dissolving or
dispersing the conjugate, and the mixture of conjugated and free
analyte moves, via bibulous wicking action to the second zone 16,
where the free antigen and particle-conjugated antigen compete for
non-diffusively immobilized antibody at this zone. That portion
(e.g., from 0% to 100%) of the particle-conjugated antigen which
binds to the non-diffusively immobilized antibody is retained in
the second zone 16. The antigen-particle conjugate that does not
bind to the second zone 16 and migrates to the third zone la, where
substantially all of the portion of the particle-conjugated antigen
not retained it the tempi zone 16 is bound by the non-diffusively
immobilized first member of the specific binding pair in the third
zone 18.
[0053] The present invention provides an assay method having an
inhibition type configuration. Again referring to FIG. 1, at the
proximal end 11 of the strip 12 is the first zone 14 which includes
a bibulous material containing a diffusively immobilized,
particle-linked antibody capable of binding sample antigen. The
second zone 16 is separate and distinct from the first zone 14, and
is located some distance toward the distal end 13 of the bibulous
strip. The second zone 16 includes a bibulous material containing a
non-diffusively immobilized antigen capable of being bound by the
particle-linked-antibody. The bibulous material of the second one
16 can be the same or different from the bibulous material of the
first zone 14.
[0054] The third zone 18 is separated and distinct from the second
zone 16, and is located some distance toward the distal end 13 of
the bibulous strip. The third zone 18 includes a bibulous material
which may be the same or different from the bibulous materials of
the first and second zones 14 and 16 containing a, non-diffusively
immobilized first member of a specific binding pair capable of
specifically binding to its specific binding partner which is the
second member of the specific binding pair on the surface of the
particle linked antigen. This, second member of the specific
binding, pair is not antigenically related to the sample antigen so
it will not effectively compete with the antigen to bind to an
anti-antigen monoclonal antibody.
[0055] The fluid sample is applied to the strip at the application
site is preferably in the proximal end of the assay strip. The
application site is where the particle-linked antibody is located.
Sample antigen which may be present in the sample reconstitutes the
particle-antibody conjugate and is bound by the conjugate. The
bound antigen:antibody-particle complex, as well as unbound
antibody-particle complex, are transported or migrate via capillary
or wicking action to the second zone 16, where substantially all of
the free antibody-particle conjugate is bound by the
non-diffusively immobilized antigen. The bound sample
antigen:antibody-particle complex migrates through the second zone
16 to the third zone 18, where substantially all of it is bound by
the non-diffusively immobilized first member of the specific
binding pair.
[0056] In the preferred embodiments described above, the amount of
a detectable response or signal present at the second zone 16 is an
inverse measure of the sample analyte concentration, and the amount
of the detectable response or signal at the third zone 18 is a
direct measure of the sample analyte concentration. The detectable
responses or signals combined from second and third zones 16 and 18
are approximately constant across the entire range of sample
analyte concentration. This total detectable response or signal
serves as a reference mechanism for both the assay procedure and
reagent quality. Thus, if the total signal is below a specified
range, the user is notified of an error. Furthermore, the specific
reason for the incorrect assay procedure can be identified. For
example, the error can be identified as operation outside the
specified temperature and/or humidity range, insufficient sample
volume, expired reagents, or the like.
[0057] The assay quantitation can be determined by reading the
second zone 16, the third zone 18, or both second or third zones 16
and 18. The sample concentration output is a result of a
calibration algorithm related to the second zone 16 alone, the
third zone 18 alone or both second and third zones 16 and 18. This
can result in a more reliable quantitative analyte, concentration
result. The summation of the detectable responses or signal from
second and third zones 16 and 18 to produce a substantially
constant total signal regardless of analyte concentration provides
a reference mechanism for accurate assay performance.
[0058] The above strip configurations are advantageously used in
the integrated assay instrument described in U.S. application Ser.
No. 08/111,347. Although the chemistry and configurations of the
present invention may be used in an integrated assay device, the
present invention can be used in any other instrumented reflectance
or transmission meter as a replaceable reagent. Thus, the present
invention also encompasses integrated assay instruments and
analytical assay instruments comprising the present assay
device.
[0059] The present invention preferably uses particle detection for
a detectable response or signal in each test zone related to the
level of analyte in the sample. Other means for providing a
detectable response in the test zones are suitable for use in the
present invention. For example, and not for limitation, the analyte
may be labelled with an indicator to measure electrical conductance
or the reflectance or absorption of a characteristic light
wavelength. As use herein, "indicator" is meant to include all
compounds capable of labelling the analyte or conjugate thereof and
generating a detectable response or signal indicative of the level
of analyte in the sample.
[0060] The present assay device and method represents a substantial
improvement in the reliability of single-use diagnostic devices
utilizing chromatographic strip; binding assays for determining the
presence or amount of an analyte in a sample, e.g. taken from a
medical patient.
[0061] The assay devices include a bibulous substrate to which
members of specific binding pairs, which may be labeled, are
diffusively or non-diffusively immobilized. Non-diffusive
immobilization can be conducted by adsorbing, absorbing,
crosslinking or covalently attaching a reagent such as a labeled
member of a binding pair to the bibulous substrate.
[0062] Diffusive immobilization can be conducted by formulating one
or more assay reagents to be immobilized. Examples of formulating
the reagents include dissolving in a suitable solvent such as
water, a C.sub.1-C.sub.4 alcohol or mixture thereof, along with any
desired additives. The resulting formulation is applied to the
bibulous material of the assay device in one or more desired
locations, and then, the bibulous material is dried. Diffusive
immobilization allows rapid reconstitution and movement of
reagents, whether reacted or unreacted, through the bibulous
substrate.
[0063] The present invention also includes to a one-step lateral
flow assay strip comprising two or more test zones for each analyte
and a particle detection system that is quantitatively read by a
reflectance type instrument. FIGS. 1-7 show various embodiments of
strip configurations suitable for immunoassay devices and
methods.
[0064] The present immunoassay configurations can measure a wide
variety of analytes. The immunoassays can be set up to be either
qualitative (e.g., as in the cases of HCG (pregnancy) assays,
assays for known metalabolites associated with drugs of abuse or
for known antigens associated with infectious diseases) or
quantitative (e.g., in the case of bone collagen N-telopeptide
(NTx; assayed as a marker for bone resorption), theophylline,
digoxin, quantitative HCG (ectopic pregnancy), C-reactive protein,
CKMB and Troponin).
[0065] The present device May be used on-site in the home and in
physician's office, or in remote locations in emergency medicine.
Therefore, the device may advantageously include, sample
pre-treatment as previously defined, as well as a sample withdrawal
device (e.g., a fingerstick) or any combination thereof, sample
pretreatment can also adjust the pH to within a specified range,
reference salt concentration, turbidity and/or viscosity, and/or
reduce or remove immunochemical cross-reactants. Each immunoassay
configuration shown in FIGS. 1-7 can include sample pretreatment,
including one or more chemical, filtration or separation means, or
any combination thereof.
[0066] The present invention provides a device which can be used to
determine the presence of multiple analytes in a test sample. One
test zone corresponds to each analyte selected, for determining its
presence. Each test zone receives and contacts the sample and a
labeled indicator reagent corresponding to the selected analyte
with a test zone reagent corresponding to the selected analyte. The
test zone reagent corresponds to the selected analyte reacting; in
the presence of the sample and the labeled, indicator reagent
corresponding to the selected analyte to form a corresponding test
zone reaction product and a corresponding test zone detectable
response inversely related to the selected analyte level in the
sample.
[0067] One reference zone receives the labeled indicator reagent
not reacted with its corresponding-test zone reagent from all the
test zones. The reference zone contacts each labeled indicator
reagent with a corresponding reference zone reagent. Each reference
zone reagent reacts in the presence of the corresponding labeled
indicator, reagent to form a corresponding reference zone reaction
product and a corresponding reference zone detectable response
related to each selected analyte level in the sample and
proportionately related to the corresponding test zone detectable
response to establish a substantially constant total detectable
response, for a pre-determined range of each selected analyte. The
detectable responses from each test zone are separately combined
with the detectable result from the reference zone to determine
each selected analyte level in the sample.
[0068] Referring again to FIG. 1, a top surface view of an
embodiment is illustrated having a typical structure with three
zones including two test zones for a single analyte, and FIG. 2
shows a top surface view of a similar device with four zones
including three test zones for two analytes. The same reference
numerals are used to identify the same elements between the
Figures.
[0069] In FIG. 1, the first zone 14 of strip 12 is located at or
slightly downstream from the sample application, site at the
proximal end 11 of the strip, and second zone 16, located
downstream from the first zone 14, may be either directly adjacent
to or separated by a bibulous spacer but in fluid communication
with the first zone 14. The third zone 18, located downstream from
zones 14 and 16, may be either directly adjacent to the second zone
16 or separated but in fluid communication with the second zone 16.
The third zone 18 acts as a reference zone for the second zone 16.
As used herein, "fluid communication" refers to a direct or
indirect contact of bibulous material which permits a, fluid sample
to flow from the sample application site or first zone 14 of the
device; through the various zones of the device, to the periphery
of the device.
[0070] FIG. 2 is a similar construction with an additional fourth
tone 20, located downstream from zones 14, 16 and 18, which may be
either directly adjacent td the third zone 18 or separated but in
fluid communication with the third zone 18. The fourth zone 20 acts
as a reference zone for each of the second and third zones 16 and
18. All zones are in fluid communication, both with each other and
with the sample application site.
[0071] The sample application site is preferably in the first zone
14, or alternatively, can be a separate area directly adjacent to
and upstream from the first zone 14 (preferably still positioned at
the proximate end of the strip). Zones 14, 16, 18, and 20 may be of
any dimensions which provide adequate detection of the indicator in
the assay(s), and preferably are from about 0.05 cm to about 1.5 cm
in length (more preferably about 0.1 cm to about 1.0 cm in
length).
[0072] The overall dimensions of the strip may be any dimensions
which provide adequate spacing and resolution for conducting the
assay(s). Preferably, however, the length of the strip is in the
range of about 2 cm to about 10 cm (more preferably about 2 cm to
about 4 cm) and the width can be about 0.1 cm to about 1.5 cm (more
preferably about 0.2 cm to about 0.5 cm). The strips shown in FIG.
1 and FIG. 2 are, for example, about 3 cm long and about 0.3 cm
wide.
[0073] The strip can be one continuous section of bibulous material
or be composed of one, two, three or more sections. Each zone may
be a separate bibulous material where each zone is in fluid
communication with adjacent zones, or two or more adjacent zones
may share a common material, with the other zones being different
materials.
[0074] The assay strip including each of the zones can be composed
of the tame or different bibulous materials. The bibulous material
permits fluid communication, between the various zones, spacers (if
present) and sample application site by wicking or capillary action
upon-application of a fluid sample. Examples of materials that tan
be used include but are not limited to: cellulose papers such as
WHATMAN 1C, 2C, 4C, 31ET, S&S 903C, GB002; membranes such as
S&S nitrocellulose, cellulose acetate, regenerated cellulose at
pore sizes from 1 .mu.m to 20 .mu.m, Pall nylon at, pore, sizes of
1.mu. to 20.mu. including BIODYNE.RTM. A, B, C or IMMUNODYNE.RTM.
ABC, Gelman ULTRABIND.RTM., Millipore IMMOBILON.RTM.; composite
papers or membranes made from mixtures of glass fiber, plastic or
metal fiber or synthetic or natural mesh or fabric made from
cotton, cellulose, polyethylene, polyester or nylon.
[0075] Zones 14, 16, 18 and 20 of FIGS. 1 and 2 can contain,
reagents diffusively or non-diffusively bound including, but not
limited to, antibodies, antigenS, enzymes, substrates, small
molecules, proteins, recombinant proteins, viral, or bacterial
lysate, receptors, sugars, carbohydrates, polymers like PITA and
detergents.
[0076] FIGS. 3-5 shows exploded lengthwise cross sections of the
embodiment of FIG. 1. The backing 24 in FIGS. 3-5, may provide
structural support for the bibulous material. Backing 24 can be of
any convenient material that, provides support for the assay matrix
and is preferably a plastic, such as cellulose acetate, polyester,
vinyl or the like, or a synthetic or natural fabric or mesh.
Backing 24 has a thickness sufficient to support the assay
material, and preferably has a thickness of from about 0.002 inch
to about 0.015 inch (more preferably about 0.005 inch to about
0.010 inch thick). However, if the bibulous material is itself
sufficiently rigid, or is supported by other mechanical means, then
a backing is not necessary.
[0077] An adhesive 22 can, be interposed between backing 24 and the
bibulous material, to promote, adhesion of these layers. Adhesive
22 can be any double stick adhesive, such as 3M 415, 443, 9460 or
the like. Alternatively, a membrane that is cast during
manufacturing to a plastic support such as S&S PB-NC can be
used. In this case, an adhesive is not necessary.
[0078] FIG. 3 shows an exploded lengthwise cross section of the
embodiment of FIG. 1 which does not have sample pretreatment. In
this configuration, the sample is introduced at the proximal end 11
of the strip in the area of the first zone 14.
[0079] FIG. 4 shows an exploded lengthwise cross section of the
embodiment of FIG. 1 with one type of sample pre-treatment. The
sample pre-treatment can include any combination of chemical,
filtration or separation treatments, including blood separation.
The sample treatment zone may be composed of one, two, or several
layers of depth filter material 26 (such as glass fiber, metal
fiber, synthetic fiber, paper, or natural or synthetic fabric) and
a membrane 28 (such as S&S cellulose, acetate, nitrocellulose,
regenerated cellulose having an average pore size of from about 0.2
.mu.m to about 7 .mu.m, and Nucleopore or Poretics polycarbonate at
pore sizes of about 0.2 .mu.m to about 5 .mu.m).
[0080] The layers of materials 26 and 28 can contain any number of
assay reagents including but not limited to buffers, salts,
proteins, enzymes and/or antibodies (either or both of which may be
diffusively or non-diffusively bound to a particle or the bibulous
material), polymers, small molecules, or any combination thereof.
If red blood cells are to be separated, then layers of materials 26
and 28 function to remove substantially all of the red blood cells
from the blood-sample, leaving plasma to operate in the assay.
[0081] As Shown in FIG. 4, sample filtration 26 and 28 is
positioned immediately above and in fluid communication with the
first zone 14. The sample filtration 26, 28 can be of any
dimensions which effectively remove red blood cells from a whole
blood sample to be assayed, and are preferably from about 0.2 to
about 1 cm in length. The sample filtration can be secured with
adhesive or be held in place by the instrument housing. The
adhesive for affixing the sample filtration means in place may be
any adhesive, such as epoxy, hot melt glue, or the like, or an
adhesive tape such as that made by the 3M company.
[0082] FIG. 5 shows an exploded lengthwise dross section of the
embodiment of FIG. 1 with a second type of sample pre-treatment and
transport means. The sample treatment in the device of FIG. 5 can
include any combination of chemical, filtration or separation
means, including blood separation means. The sample treatment and
transport device, of FIG. 5 includes a sample application zone at
filter 32, a membrane 34, a transport mesh 36, a second filter 38
and a membrane 40.
[0083] The filter 32 can be composed of one, two, three or more
layers of any bibulous material, preferably a depth filter such as
glass fiber, metal fiber, synthetic fiber, paper, or natural or
synthetic fabric. Filter 34 can be one or several layers and is
composed of any microporous membrane such as S&S cellulose
acetate, nitrocellulose, regenerated cellulose, at pore sizes from
about 0.2 .mu.m to about 7 .mu.m, Nucleopore or Poretics
polycarbonate at pore sizes of about 0.2 .mu.m to about 7
.mu.m.
[0084] Although filters 32 and 34 are shown in FIG. 5, one or both
of these layers may not be necessary and can be excluded. In the
case where both filter layer 32 an 34 are excluded, the sample will
be applied directly to the transport layer 36.
[0085] The sample transport layer 36 is designed to accept the
sample, either directly or through the filter layers 32 and 34, and
move it horizontally to the area of filter 38. This sample movement
may take from about 2 seconds to about 10 minutes, preferably from
about 2 seconds to about 5 minutes, and more preferably from about
5 seconds to about 2 minutes.
[0086] The sample transport is composed of any bibulous material
including, but not limited to, fabric or mesh that is woven or
cast, synthetic or natural, and made of cotton, nylon, polyester,
polypropylene, polyethylene or the like; paper such as Whatman 31ET
or 3; glass fiber such as Whatman GFA, GFD, S&S 3362 or 32;
plastic fiber, metal fiber and/or any synthetic membrane. The
sample transport area can be untreated, or may have diffusively or
non-diffusively immobilized therein one or more reagents such as
stabilizing proteins, detergents, anticoagulants like heparin or
EDTA, precipitating reagents, salts, proteins, enzymes, antibodies,
enzyme-particle conjugates, antibody-particle conjugates,
antigen-particle conjugates, red cell agglutinating agents like
wheat germ lectin or anti-human RBC, polymers and/or small
molecules.
[0087] The sample transport layer/zone 36 has dimensions sufficient
to permit any desired sample pre-treatment without adversely
affecting assay reactions and indicator measurements, but is
preferably about 0.5 cm to about 5 cm (more preferably about 1 cm
in length) in length and about 0.1 to about 1.5 cm (more preferably
about 0.2 to about 0.5 cm) in width.
[0088] The filter 38 can be composed of one, two, three or more
layers of any bibulous material, preferably a depth filter such as
glass fiber, metal fiber, synthetic fiber, paper, or natural or
synthetic fabric. Filter 40 can be one or several layers and is
composed of any microporous membrane such as S&S cellulose
acetate, nitrocellulose, regenerated-cellulose at pore sizes from
about 0.2 .mu.m to about 7 .mu.m, Nucleopore or Poretics
polycarbonate at pore sizes of about 0.2 .mu.m to about 5 .mu.m.
Although filters 38 and 40 are shown in FIG. 5, one or both of
these layers may not be necessary and can be excluded. In the case
where both filter layers 38 and 40 are excluded then the sample
will be transported directly from the transport layer 36 to the
first zone 14. All of the sample treatment and transport materials
32, 34, 36, 38, and 40 are in fluid communication with each other
and with the first zone 14.
[0089] If the device is used for blood separation then it will
function to remove substantially all of the red cells from the
blood sample, leaving plasma to operate in the assay. The red cells
can be substantially removed by filters 32 and 34 prior to the
sample contacting the transport mesh or the red cells can be
removed by filters 38 and 40 in which case the whale blood will
travel on the transport layer. In a preferred embodiment, filters
32 and 34 are absent and sample blood or urine or any other body
fluid is applied directly to the transport layer and sample
treatment, filtration and/or blood separation occurs at filters 38
and 40.
[0090] Filters 32, 34, 38, and 40 have dimensions sufficient to
permit any desired sample pre-treatment without adversely affecting
assay reactions and indicator-measurements, but preferably are
about 0.2 CM to about 2 cm (more preferably about 0.25 to about
0.75 cm) in length and about 0.1 to about 1.5. cm (more preferably
about 0-2 to about 0.5-cm), in width. The components of the sample
treatment means and transport means of FIG. 5 can be secured with
adhesive or held in place by a rigid housing. The adhesive can be
any convenient adhesive including epoxy, hot melt glue, or the
like, or may be an adhesive tape such as those, made by 3M
company.
[0091] Referring now to FIGS. 6 and 7, each of these immunoassay
formats can have a sample treatment means and/or a transport means
as described for the assay devices in FIGS. 4 and 5. Alternatively,
they may have a sample treatment means as described for the assay
device in FIG. 3.
[0092] FIGS. 6 and 7 show two embodiments of a quantitative assay
to measure the concentration of the crosslinked bone collagen
telopeptide (NTx) in urine, whole blood, plasma or serum. NT is a
product of bone resorption and is known to be present in urine and
blood. The concentration of NTx is a direct measure of the rate of
bone resorption and is a useful marker for (a) the onset of
osteoporosis and (b) monitoring the progress of therapy for
osteoporosis. Although NTx is shown as an example assay according
to the present invention, it is understood that any analyte can be
quantitatively or qualitatively measured.
[0093] The assay strips of FIGS. 6 and 7 each have two test zones.
The two-test zone design provides improved performance in
quantitative assays and improved reliability, since the sum of the
signals from both test zones is substantially constant regardless
of the analyte/antigen concentration, thus providing a robust
quality reference and assuring accurate assay operation.
[0094] FIG. 6 is a top surface view of an inhibition type
immunoassay configuration, a preferred embodiment of the present
invention. In the assay strip 50, zone 52 contains a diffusively
bound anti-NTx antibody (of any other antibody), conjugated to
colloidal gold, colored latex beads or an enzyme. The diffusively
immobilized anti-NTx-particle conjugate can also be located on
filters 26 or 28 of the device of FIG. 4 or on filters 32, 34, 38
or 40 or transport layer 36 of the device of FIG. 5.
[0095] The antibody can be monoclonal (e.g., derived from fusion of
spleen cells from an immunized mouse with a suitable immortal cell
line in accordance with known methods; see Kohlstein and Milner,
1975) or polyclonal prepared from any suitably immunized animal
species in accordance with known methods).
[0096] A preferred embodiment uses conjugates of anti-NTx antibody
to particles of colloidal gold, or to blue or black latex beads.
Particles can be from about 5 nm to about 2000 nM in diameter (Mote
preferably from about 5 nm to about 500 nm in diameter).
[0097] Diffusive immobilization can be conducted by formulating the
assay reagent(s) to be immobilized (e.g., by dissolving in a
suitable solvent, such as water, a C.sub.1-C.sub.4 alcohol or
mixture thereof, along with any desired, additives), applying the
resulting formulation to the bibulous material of the membrane,
filter or transport layer in the desired location(s), and drying
the material. Suitable additives may include, detergents, proteins,
blocking agents, polymers, sugars or the like. Alternatively the
additive(s) and assay reagent(s); may be applied to the membrane,
filter or transport layer by precoating with a "blocking agent",
water soluble polymer, sugar or detergent, followed by depositing
the conjugate or conjugate formulation and drying the material.
[0098] Zone 54 is the first test zone of strip 50. Zone 54 contains
non-diffusively bound NTX, NTx-macromolecule conjugate or
NTx-particle conjugate. NTx is conjugated to a macromolecule or
particle to help in the immobilization of the NTx peptide to the
membrane (bibulous material) surface. Suitable macromolecules which
can be used for NTx conjugation include any large molecule capable
of adsorption or covalent binding to the membrane, including but
not limited to: bovine serum albumin (BSA), keyhole limpet
hemocyanin (KLH), immunoglobulin G (IgG), mouse IgG, bovine gamma
globulin (BGG), lactalbumin, polylysine, polyglutamate,
polyethylenimine, or aminodextran. Suitable particles which can be
used for NTx conjugation can include particles of about 1-20 .mu.m
in diameter, including, but not 0.39 limited to, latex particles,
microcapsules, liposomes or metal sol particles.
[0099] Non-diffusive immobilization can be accomplished by
covalently attaching, adsorbing or absorbing the NTX, NTX-protein
conjugate or NTX particle conjugate to the membrane. Suitable
membranes for adsorption or absorption include, but are not limited
to, S&S nitrocellulose and cellulose acetate at pore sizes from
0.45 .mu.m to. 12 .mu.m, and Pall nylon at pore sizes of 0.45 .mu.m
to 20 .mu.m (such as BIODYNE A, B, and C). Suitable membranes for
covalent attachment include, but are not limited to, membranes such
as Millipore IMMOBILON.RTM., Gelman ULTRABIND.RTM. and Pall
IMMUNODYNE.RTM. ABC. Alternatively, the antigen, antigen-protein
conjugate or antigen-particle conjugate can be covalently attached
to the membrane by chemically activating the membrane or paper
prior to applying a solution or formulation of. antigen/conjugate.
Covalent attachment of the NTx peptide to the membrane occurs
through a linkage to the primary amine on the NTX molecule.
[0100] Zone 56 is the second test zone of strip 50. Zone 56
contains a non-diffusively bound member of a specific binding pair
capable of binding to a complementary member of the specific
binding pair which is not related, to the sample analyte/antigen on
the surface of the particle-linked antibody.
[0101] For example, if the particle-linked antibody is a mouse
monoclonal antibody, then the non-diffusively complementary binding
partner in zone 56 can be any anti-mouse polyclonal or monoclonal
antibody, including but not limited to: goat-anti-mouse,
sheep-anti-mouse, cow anti-mouse, rabbit-anti-mouse, monoclonal rat
anti-mouse or any other anti-mouse species antibody.
[0102] Alternatively, a generic binding partner such as Protein A,
Protein G or Protein A/G (e.g., obtained from Pierce) can be
non-diffusively immobilized at zone 56, as long as it binds the
particle-antibody conjugate. Lectins can also be immobilized at
zone 56, provided that the particle-antibody conjugate can be bound
at this zone. Biotin, avidin or streptavidin can be linked to
particle or to the particle-linked antibody, and the complementary
binding partner may then be non-diffusively immobilized at zone
56.
[0103] For example, if biotin is conjugated to the particle along,
with the antibody, thus producing an anti-NTx-particle-biotin
conjugate, then avidin or streptavidin can be non-diffusively
immobilized, at zone 56 and used to capture particles not bound in
zone 54. Any non-human antigen, including proteins or small
molecules such as dinitrophenol, known dinitrophenyl
group-containing molecules or fluorescein can be co-conjugated with
anti-NTx to the particle. The complementary antibody can then be
immobilized to zone. 56, the requirement being that the particle
conjugate hot bound in zone 54 is substantially all captured
(bound) in zone 56 in the assay.
[0104] In the assay operation of FIG. 6, the sample is introduced
to the proximal end of the assay strip in the area of the
particle-linked antibody conjugate zone 52. The sample can be
applied directly, or can be pre-treated, filtered, and/or separated
as described above. The fluid sample (sample antigen, in this case
NTx) then reconstitutes the particle-antibody (particle-anti-NTX)
conjugate, and any antigen in the fluid sample is bound by the
conjugate in zone 52. The particle-antibody conjugate is applied in
excess, such that most of the antigen is bound by the
conjugate.
[0105] The bound antigen:antibody-particle complex
(NTx:anti-NTX-particle), as well as unbound antibody-particle
(anti-NTx-particle) conjugate, migrate from zone 52 via capillary
action to zone 54, where substantially all of the free
antibody-particle conjugate is bound by the non-diffusively
immobilized antigen (NTx) at this site. The
antigen:antibody-particle complex cannot bind to the
non-diffusively immobilized antigen at zone 54 since the binding
sites are occupied by sample antigen. Consequently, the antigen
antibody-particle complex migrates via capillary action to zone 56
and is substantially all bound by the non-diffusively immobilized
complementary member of the specific binding pair immobilized at
this site.
[0106] At zero sample antigen concentration, the binding sites on
the particle-antibody conjugate are free, and the particles are
mostly bound at zone 54, where a dark color is produced. At very
high sample antigen concentrations, the binding sites on the
particle-linked antibody are mostly occupied, and the particles
move past zone 54 and are substantially all bound at zone 56.
Intermediate concentrations of sample antigen result in a
predictable response relative to the bound particle signals at
zones 54 and 56. In general, low sample concentrations result in
high signal in one 54 and low signal in zone 56. As analyte/antigen
concentration increases, the signal in zone 54 becomes
progressively lower, and the signal in zone 56 becomes.
correspondingly higher. The total signal, which is the sum of
signal from zones 54 and 56, remains substantially constant across
the entire concentration range. This provides a reliable quality
reference for the assay result, since the sum of the signals must
stay within a specified range. Otherwise, an assay failure is
indicated.
[0107] Assay calibration and sample quantitative measurement can
be, achieved using zone 54 alone, zone 56 alone, or both zones 54
and 56. Under certain conditions, one test zone may produce better
performance in a particular analyte/antigen concentration range,
and the other test zone may produce better performance in a
different analyte/antigen concentration range. In this case, a
hybrid calibration can be done that uses the optimal calibration
range of each zone. Thus, the present two-test zone measurement
provides substantial improvements over previously described
methods.
[0108] FIG. 7 is a top surface view of a competitive-type
immunoassay configuration, another preferred embodiment of the
present invention. In the assay strip 60, zone 62 contains
diffusively bound NTx (or other sample antigen) conjugated to
colloidal gold, colored latex beads or an enzyme. The NTx can be
coupled directly to the particle. Alternatively, NTx can be coupled
indirectly to the particle through the macromolecule moiety of a
macromolecule-NTX conjugate. The macromolecule used for NTx
conjugation can be any large molecule capable of adsorption or
covalent binding to the particle, including but not limited, to:
bovine serum albumin (BSA), keyhole limpet hemocyanin (KLH),
immunoglobulin G (IgG), mouse. IgG, bovine gamma globulin (BGG),
lactalbumin, polylysine, polyglutamate, polyethylenimine, or
aminodextran.
[0109] A preferred embodiment uses conjugates of NTx to particles
of colloidal gold, or to, blue or black latex beads. Particles can
be from about 5 nm to about 2000 nm in diameter (more preferably
from about 5 nm to about 500 nm in diameter).
[0110] The NTx-particle conjugate can also be diffusively
immobilized on filters 26 or 28 of the device of FIG. 4 or on
filters 32, 34, 38 or 40 or transport layer 36 of the device of
FIG. 5. Diffusive immobilization can be accomplished as described
above.
[0111] Zone 64 is the first test zone of strip 60. Zone 64 contains
non-diffusively bound anti-NTx. Non-diffusive immobilization can be
accomplished by covalent attachment or adsorption of the anti-MIX
to the membrane as described, above. Alternatively, anti-NTx can be
conjugated to another protein, and this conjugate is then adsorbed
to the membrane. Adsorption can be accomplished using membranes
including, but not limited to, S&S nitrocellulose and cellulose
acetate at pore sites from 0.45 .mu.m to 12 .mu.m, and Pall nylon
at pore sizes of 0.4.5 .mu.m to 20 .mu.m (such as BIODYNE A, B, and
C). Covalent attachment can be accomplished using membranes such as
Millipore IMMOBILON.RTM., Gelman ULTRABIND.RTM. or Pall
IMMUNODYNE.RTM. ABC, or by chemically activating the membrane or
paper prior to contacting the antibody with the membrane or
paper.
[0112] Zone 66 is the second test one of strip 60. Zone 66 contains
a non-diffusively bound member of a specific binding pair such as
an antibody or an antigen which is not immunologically related to
the sample analyte/antigen, avidin, biotin, Protein A or G, lectin
or the like) which binds to a complementary member of the specific
binding pair on the surface of the particle-linked antigen. For
example, if the particle is linked to both antigen and protein
(e.g., an antigen-macromolecule-particle conjugate), then an
antibody to that protein can be non-diffusively immobilized in zone
66.
[0113] Furthermore, for example, if NTx is conjugated to mouse IgG,
and the particle is linked to this, conjugate (NTx-mouse
IgG-particle), then any anti-mouse antibody can be non-diffusively
immobilized at zone 66. Any protein carrier can be used to
conjugate to NTx, and the corresponding antibody (to the protein
carrier) it then non-diffusively immobilized to zone 66.
[0114] Alternatively, any generic binding partner such as Protein
A, Protein G or Protein A/C (e.g., obtained from Pierce) can be
non-diffusively immobilized at zone 66 as long as it binds the
particle-antigen conjugate. Lectins can also be immobilized at zone
66, provided that the particle-antigen conjugate can be bound at
this zone.
[0115] Biotin, avidin or streptavidin can be conjugated to the
particle-linked antigen, and the complementary binding partner can
then be non-diffusively immobilized in zone 66. For example, if
biotin is conjugated to the particle along with the antigen (in
this case NTx), thus producing a biotin-particle-NTx conjugate,
then avidin or streptavidin can be non-diffusively immobilized in
zone 66.
[0116] Any non-human antigen, including proteins or small
molecules, such as dinitrophenoi, known dinitrophenyl
group-containing molecules or fluorescein, can be co-conjugated
with NTx to the particle, and the complementary antibody can be
immobilized in zone 66, the requirement being that the particle
conjugate, that is not bound in zone 64 is substantially all
captured (bound) in zone 66 in the assay.
[0117] in the assay operation, the sample is introduced to the
proximal end of the assay strip in the area of the particle-linked
antigen conjugate zone 62. The sample can be directly applied, or
alternatively, it can be pre-treated, filtered, and/or separated as
described above. The fluid sample (which may contain antigen, in
this case NTx) then reconstitutes the particle-antigen
(particle-protein-NTx) conjugate, and the mixture of
particle-protein-NTx and free analyte (NTx) moves via capillary
migration or bibulous wicking action from zone 62 to zone 64, where
the free antigen and particle-conjugated antigen compete for
bon-diffusively immobilized antibody. The antigen-particle
conjugate that does not bind to one 64 migrates to zone 66 and is
substantially all bound by the non-diffusively immobilized member
of the specific binding pair immobilized at this site.
[0118] At zero sample analyte/antigen concentration, the
particle-antigen conjugate is mostly bound in zone 64, resulting in
a dark color being produced in this zone. At very high sample
analyte/antigen concentrations, the analyte/antigen occupies most
of the binding sites of zone 64, causing the particle-linked
conjugate to move past zone 64 to zone 66, where it is
substantially all bound. Intermediate concentrations of sample
analyte/antigen result in a predictable response relative to the
bound particle signals. in zones 64 and 66.
[0119] In general, low analyte/antigen concentrations result high
signal in zone 64 and low, signal in zone 66. As sample
analyte/antigen concentration increases, the signal in zone 64
becomes progressively smaller, and the signal in zone 66 becomes.
correspondingly higher. The total signal or detectable response
(i.e., the sum of the signals from zones 64 and 66), remains
substantially constant regardless of the analyte/antigen
concentration (e.g., across the entire concentration range of from
0 to about 100 mM). This provides a reliable assay result and
quality, reference since the sum must stay within a specified
range, otherwise an assay failure is indicated.
[0120] Assay calibration and sample quantitative measurement can be
achieved using zone 64, alone, zone 66 alone- or both zones 64 and
66. Under certain conditions, one test zone may produce better
performance in a particular analyte/antigen concentration range,
and another test zone may produce better performance in a different
analyte/antigen concentration range. In this case, a hybrid
calibration can be done that uses the optimal calibration range of
each zone. Thus, the present two-test zone measurement provides
substantial improvements over previously described methods.
[0121] The present test strip, may be advantageously used in an
instrument which reads the signals in zones 64 and 66. Thus, the
indicator signals need not be visually detectable.
[0122] Having generally described the present invention, a further
understanding can be obtained by reference to the following
specific examples, which are provided herein for purposes of
illustration only and are not intended to be limiting of the
present invention. Unless otherwise specified, temperatures are in
degrees Centigrade and percents are weight percents.
Example 1
[0123] The devices of the embodiments shown in FIGS. 1-7 are
quantitative or qualitative immunoassay strips. The assay strips
shown in these embodiments can be configured by one of two assembly
methods.
[0124] In a first strip assembly method, the strip is composed of
several Separate bibulous membrane sections in fluid communication
by lamination to a plastic strip. The reagents can be diffusively
or non-diffusively immobilized to the membrane prior to lamination.
Alternatively, the reagents can be immobilized after
lamination.
[0125] For convenience, the assay strips are constructed in bulk in
a card form with the discrete assay zones forming lines along the
length of the card. Each card can be of any convenient size,
depending only on the length of the assay strip and the number of
assay strips desired. For example, if the assay strip (as shown in
FIG. 1) is 6 cm long and 0.5 cm wide, then the card can be 6 cm by
10 cm (20.times.0.5 cm), thus providing 20 strips. Two sizes of
strips were used in the examples below. For a strip size of 6 cm
long by 0.5 cm wide, the card was 6 cm by 10 cm (yielding 20
strips); for a strip size of 3 cm long by 0.3 cm wide, the cards
were 13 cm by 6 cm (allowing 20 strips).
[0126] In a second strip assembly method, the strip is one
continuous material that may optionally be laminated or cast to a
plastic support. The reagents are diffusively or non-diffusively
immobilized to a continuous assay strip, and are applied to the
strip using a process that "prints" the reagents in discrete zones
along the length of the strip. The assay strips may be constructed
in bulk in a card form in accordance with the first strip assembly
method described above.
[0127] The following strip assembly and reagent immobilization
methods were used in the construction of the present invention.
[0128] One method of constructing a strip assembly for the present
invention laminated the membrane or paper to a plastic backing by
joining the membrane or paper assay matrix to a sheet of polyvinyl
acetate (0.01'' thick) using a double-stick adhesive or a transfer
adhesive. This is illustrated in FIGS. 3-5.
[0129] A card of polyvinyl acetate sheet (0.01'' thick) was cut to
about 6 cm by 10 cm (or 3 cm by 6 cm). The size of the card varied,
depending on the desired assay card site. The polyvinyl acetate
backing was marked with pencil lines along the length at
appropriate positions indicating the location of the various assay
strip zones. Double stick adhesive, such as 3M 415 to the polyvinyl
acetate, was applied so as to, cover the surface with the pencil
lines, and firm pressure was applied with a roller assembly making
sure to eliminate the formation of bubbles. The release liner of
the double-stick adhesive was removed and the membrane or paper
assay sections applied to the correct location, guided by the
pencil lines, and firm pressure was applied with, a. roller
assembly making sure to eliminate bubbles. Care was taken to ensure
that each section of the bibulous assay matrix was in fluid
communication with its neighbor. Finally, individual assay strips
were cut, each 0.5 cm wide (or 0.3 cm) along the length of the
card, resulting in assay strips 0.6 cm long by 0.5 cm wide (or 3 cm
long by 0.3 cm wide). This was accomplished using a die cutter or a
standard paper cutter.
[0130] A non-diffusive immobilization was accomplished using a
variety of methods. In a preferred method, nitrocellulose or nylon
membrane (pore sizes of 0.45 .mu.m to 12 .mu.m) was incubated with
a protein, protein-hapten conjugate, peptide; small molecule or the
like (immobilization compound) to be non-diffusively immobilized,
in 50 mM sodium-phosphate, pH 7, for 60 minutes. The membranes were
then washed twice in 50 mM. sodium phosphate pH 7, 0.1 M NaCl (PBS)
for 15 minutes and preserved in 5 Mg/mL BSA, 1% sucrose solution
for 10 minutes. Drying was done at 50.degree. C. for 15 minutes or
until dry.
[0131] In a second non-diffusive immobilization method, an
applicator (e.g., a. fountain pen, a pad printer, pipette, air
brush, inkjet print head or the like) was used to accurately
measure the reagents onto appropriate zones of the as matrix. In
this case, the immobilization compound was diluted to between 0.01
mg/mL and 10 mg/mL with 50 mM phosphate, pH 7, and introduced into
the application device. The application device was then positioned.
above the appropriate assay zone and the immobilization material
was coated onto the assay matrix. The strips were washed using PBS
and preserved with BSA/sucrose prior to drying, or they may not be
washed or preserved. The assay membrane was then-dried at
50.degree. C. for 15 minutes or until dry. This method provides
flexibility in "printing" reagents in a referenceled manner at any
location along the assay strip.
[0132] In a third method, the immobilization compound can be
covalently coupled to latex microparticles of about 1-20 .mu.m and
these part idles are drawn into the membrane matrix using suction
or pressure. The microparticle method is accomplished by first
covalently immobilizing the desired protein to microspheres with
carboxyl functional groups as follows: To a suspension of 10 .mu.m
microspheres-COOH (e.g., Bangs. Laboratories stock #PO100000CN) add
1.1 molar equivalents (relative to the COOH groups on the bead
surface) of 1-ethyl-3-(dimethylaminopropyl)carbodiimide (EDAC,
Sigma E 0388) and 1.1 molar equivalents of N-hydroxysuccinimide
(NHS, Pierce 24500) in 0.1 M sodium phosphate, pH 7.0, at room
temperature with stirring for 30 minutes. Add this mixture to a
stirring solution of the desired protein in 0.1 M sodium phosphate,
pH 7.0, (the protein is at a 10 fold molar excess over the COOH
functional groups on the bead surface). Allow to react for 2 hours
at room temperature and then purify by centrifuging, followed by
washing and dialysis. The microparticles now have the desired
protein covalently immobilized. The protein-particle suspension is
then mixed and 2-10 .mu.L is picked up using a pipette. The
membrane or paper assay strip is placed on a sintered glass
filtration platform with vacuum and the bead-protein suspension is
applied from the pipette across the assay strip in the correct
location. The vacuum pressure draws the conjugated beads into the
matrix of the membrane or paper where they are mechanically
non-diffusively immobilized. Alternately these beads can be applied
to the membrane using an air brush or inkjet type print head.
[0133] A fourth type of non-diffusive immobilization, involved
covalent attachment of the immobilization compound to the assay
matrix. This was accomplished by contacting a solution or
formulation of the compound(s). to be immobilized with a
commercially available activated membrane or paper, such as Pall
IMMUNODYNE.RTM. ABC, Gelman ULTRABIND.RTM. or Millipore
IMMOBILON.RTM., using procedures recommended by the
manufacturer.
[0134] Alternatively, chemical activation, of the
hydroxyl-group-containing assay matrix (cellulose paper or
membrane) can be performed by incubating a 20.times.25 cm sheet of
membrane or paper in a covered baking dish 23.times.28 cm for 2;
bouts at room temperature in 500 mL of 0.2 M
1,1'-carbonyldiimidazole (CDI, Aldrich product no 11, 553-31.
Following this incubation, the activated membrane is washed
extensively in several (4-8) 250 mL volutes of methylene chloride
and dried under nitrogen. This procedure results in activated
membrane to which proteins or small molecules with primary amine
functional groups can be covalently immobilized (non-diffusive
binding). Non-diffusive-immobilization of the protein to the
activated membrane, either prepared at outlined above or using ore
of the commercial activated membranes, is accomplished by
incubating the activated membrane in 100 m. of a 0.01 mg/mL to 10
mg/mL solution of the desired immobilization compound in 0.1 M
sodium phosphate, pH 7, at room temperature for two hours. The
paper is then washed by incubation for 20 Minutes in 500 mL of 0.1
M sodium phosphate pH 7. The washing step is repeated 4 times, then
the paper is soaked in 150 mL of 0.5% polyvinyl alcohol (PVA,
Aldrich 18, 965-0) for 10 minutes, gently blotted and dried in a
convection oven at 45.degree. C. for 10 to 30 minutes or until
dry.
[0135] Colloidal gold conjugates of mouse IgG, 1H11 (monoclonal
anti-NTx), and NTx are commercially available from EY Laboratories,
San Mateo, Calif. Methods for the preparation, of colloidal gold
conjugates are disclosed in Muller, C., et al., J. Imm. Methods,
37, 185-190 (1980); Roth, J., "Techniques in Immunocytochemistry,"
Academic Press, pp. 219-284. Conjugates include: Gold-1H11 (15 nm
particle, monoclonal anti-NTx); Gold-Mouse IgG1 Kappa (15 nM
particle); Gold-NTx (15 nm particles).
[0136] Preparation of Latex Particle Conjugates Accomplished by
immobilizing NTx, NTx-protein conjugate, MAb-1H11 or Mouse IgG to
0.356 .mu.m microspheres (Bangs stock #D0003561CB) to produce.
NTx-Latex, NTx-protein-latex and MAb 1H11-Latex conjugates. To a
suspension of 0.356 .mu.m carboxylated microspheres add 10 molar
equivalents (relative to the COOH groups on the bead surface) of
1-ethyl-3-(dimethylaminopropyl) carbodiimide (EDAC, Sigma E 0388)
and 10 molar equivalents of N-Hydroxysuccinimide (NHS, Pierce
24500) in 0.1 M sodium phosphate, pH 7.0, and 0.5% Tween 20 at room
temperature with stirring for 3.0 minutes. Purify by centrifugation
at 13,000 RPM, for 15 minutes. followed by washing with 10 mM
sodium phosphate pH 7, 0.5% Tween 20. Add either the NTx,
NTx-protein conjugate, or MAb-1H11 at a ten fold molar excess to a
stirring solution of the activated microspheres in 0.1 M sodium
phosphate, pH 7.0, 0.5% Tween 20, and allow to react for 2 hours at
room temperature; then purify by centrifugation with washing and
dialysis. The microparticles now have the desired immobilization
compound non-diffusively immobilized.
[0137] Assay strips and reagents were prepared as discussed in the
first strip preparation method and the first non-diffusive
immobilization method. above. The strips were either 0.5 cm wide by
6 cm long or 0.3 cm wide, by 3 cm long, depending on the
experiment. The following assay protocol was used: [0138] 1. Sample
was added (25-100 .mu.L) to the bottom, of. a 12.times.7.5 mm. test
tube. [0139] 2. An assay strip (reagents already applied) was
inserted into the test tube. Sufficient time, was allowed for
wicking to completely saturate the strip. This required about 5-8
minutes for the 0.5 cm.times.6 cm strips and about 1-3 minutes for
the 0.3 cm by 3 cm strips. [0140] 3. The test zone on the assay
strips were read with a Gretag model D182 reflectance
densitometer.
Example 2
[0141] This example demonstrates a single-step, quantitative,
lateral flow, inhibition type immunoassay for a small molecule
using colored particles as the detection method. The assay
summarized below in Table 1 was conducted using reagents and
methods as described in Example 1.
[0142] The assay strips of this example were 6 cm wide and 0.5 cm
long and were similar to those shown in FIG. 1 and FIG. 3, with the
exception that the third zone 18 (second test zone) was not
included. The immunoassay strip configuration was as follows:
[0143] (1) a lower 8 .mu.m pore size nitrocellulose section
containing a reagent zone 14 having diffusively applied
MAb-1H11-colloidal gold, prepared as described above; [0144] (2) a
0.5 cm test zone 16 containing non-diffusively immobilized NTx
covalently linked via the native primary amine group to Pall
IMMUNODYNE.RTM. ABC, prepared as described above; and [0145] (3) an
upper wick area of 8 .mu.m pore size nitrocellulase that extended
from the upper edge of zone 16 to the top of the strip.
[0146] All strip zones were laminated in physical communication
with adjacent zone (s), permitting fluid to flow through the entire
strip by wicking action, and were supported on a plastic backing as
outlined above.
[0147] NTx was diluted in PBS to the concentrations indicated in
Table 1 below, and the general assay protocol of Example 1 was used
to generate the data shown in Table 1. These data indicate a
dose-response demonstrating good sensitivity and quantitative
performance for the present invention. The assay results in Table 1
demonstrate that the present assay strip and method can distinguish
a 1 nM concentration of the peptide marker NTx from the background
(0 concentration) using colloidal gold as the signal reagent.
TABLE-US-00001 TABLE 1 NTx Dose Response Conjugate: Colloidal
Gold-1H11 Reflectance Density NTx (nM) (Gretag) 0 0.60 1 0.57 30
0.55 100 0.52 300 0.41 1000 0.21 3000 0.18
Example 3
[0148] This example demonstrates a quantitative, lateral flow,
inhibition typo, immunoassay which shows excellent performance
using colored particles at the indicator. The assay summarized
below in Table 2 was conducted using strips, reagents and methods
as described in Example 2.
[0149] The assay protocol as indicated above in Example 1 was used
to generate data in the non-amplified data. column of Table 2. For
the silver amplified assays, the protocol was, followed as in
Example 1, with the exception, that a silver enhancement reagent
was added to the test zone after the colloidal gold binding. The
results in Table 2 demonstrate a dose-response showing excellent.
sensitivity and quantitative performance, for the present
invention.
TABLE-US-00002 TABLE 2 NTx Dose Response Silver Amplification
Conjugate: Colloidal-Gold-1H11 Reflectance Density Gretag NTx Non-
Silver- (nM) Amplified Amplified 1 0.30 1.07 30 0.25 0.91 100 0.19
0.84 300 0.15 0.59 1000 0.09 0.23 3000 0.08 0.02
Example 4
[0150] This example, demonstrates a single-step, quantitative,
lateral flow, competitive type immunoassay for a protein using
colored particles as the indicator. The assay summarized below. in
Table 3 was conducted using reagents and methods as described in
Example 1.
[0151] The assay strips of thia example were 6 cm wide and 0.5 cm
long and were similar to those shown in FIG. 1 and FIG. 31, with
the exception that the first test zone (second. zone 16) was not
included. The immunoassay strip configuration was as follows:
[0152] (1) a lower 8 .mu.m pore size nitrocellulose section
containing continuous reagent zones 14 and 16, zone 14 having
diffusively applied mouse IgG-colloidal gold, prepared as described
above; [0153] (2) a 0.5 cm test zone 18 containing non-diffusively
immobilized goat anti-mouse adsorbed to 8 .mu.m pore size
nitrocellulose, prepared as described above; and [0154] (3) an
upper wick area of 8 .mu.m, pore size nitrocellulose that extended
from the upper edge of zone 18 to the top of the strip.
[0155] All strip zones were laminated in physical communication
with adjacent zone(s), permitting fluid to flow through the entire
strip by wicking action, and were supported on a plastic. backing
as outlined above.
[0156] Mouse IgG was diluted in PBS to the concentrations indicated
in Table 3 below, and the assay protocol of Example 1 was used to
generate the data shown in Table 3. These data. demonstrate a
dose-response showing good sensitivity and quantitative performance
for the present invention.
TABLE-US-00003 TABLE 3 IgG Dose Response Conjugate: Colloidal
Gold-IgG1 Reflectance Mouse IgG Density (.mu.g/mL) (Gretag) 0 0.13
8 0.10 33 0.09 50 0.08 100 0.06 200 0.05
Example 5
[0157] This example demonstrates a single-step, quantitative,
lateral flow, inhibition type immunoassay for a small molecule
using colored particles as the indicator and an assay reference
that is directly related to the assay function. The assay
summarized below in Table 4 was conducted using reagents and
methods described, in Example 1
[0158] The assay strips of this example were 6 cm wide and 0.5 cm
long and axe shown in FIG. 1 and FIG. 3. The immunoassay strip
configuration was as follows: [0159] (1) a lower 8 .mu.m pore size
nitrocellulose section containing a reagent zone 14 having
diffusively applied MAb-1H11-colloidal gold, prepared as described
above; [0160] (2) a (1.5 cm test zone 16 containing non-diffusively
immobilized NTx covalently linked via the native primary amine
group to Pall IMMUNODYNE.RTM. ABC, prepared as described above;
[0161] (3) a 0.5 cm spacer 22 of 8 .mu.m pore size nitrocellulose;
[0162] (4) a 0.5 cm long test zone 18 of 8 .mu.m pore size
mitrocellulose containing non-diffusively immobilized, adsorbed
goat-anti-mouse; and [0163] (5) an upper wick area of 8 .mu.m pore
size nitrocellulose that extended from the upper edge of zone 18 to
the top of the strip.
[0164] All strip zones were laminated in physical communication
with adjacent zone(s), permitting fluid to flow through, the entire
strip by wicking action, and were supported on a plastic lacking as
outlined above.
[0165] NTx was diluted in PBS to the concentrations indicated in
Table 4 below, and the general assay protocol of Example 1 was used
to generate the data shown in Table 4. These data demonstrate a
dose-response in both test zone 16 and test zone 18 showing good
sensitivity and quantitative performance for the present invention.
The sum of the two test zones remained substantially constant, this
indicating, a correct functioning of the internal reference feature
of the present invention, wherein the sum of the signal from test
zone one and test one two provides a reliable quality reference to
assure correct assay performance.
TABLE-US-00004 TABLE 4 NTx Dose Response Two Test Zones Conjugate:
Colloidal Gold-1H11 Reflectance Density (Gretag) NTx (nM) Test Zone
1 Test Zone 2 Sum 1 0.38 0.10 0.48 30 0.32 0.16 0.48 100 0.21 0.25
0.46 300 0.14 0.35 0.49 1000 0.09 0.40 0.49 3000 0.07 0.47 0.54
Example 6
[0166] This example demonstrates a quantitative, lateral flow,
inhibition type immunoassay for a small molecule using an enzyme
label as the detection system. The assay summarized, below in Table
5 was conducted using strips, reagents and methods as described in
Example 2, with the exception that the lower reagent zone 14
contained, diffusively applied HRP-1H11. Five pi, of 0.1 .mu.g/mL
HRP-1H11 was applied to zone 14. As indicated in table 5, an
approximately 1.5 ing/mL solution of NT-IgG conjugate was diluted
1:10, 1:20 and 1:40, the adsorbed as described in Example 1 to the
8 .mu.m pore size nitrocellulose of test zone 16.
[0167] NTx was diluted in PBS to the concentrations indicated in
Table 5 below, and the assay protocol of Example 1 was used to
generate the data shown in Table 5. These data illustrate
dose-responses at each of three different NTx immobilization
conditions which show good sensitivity and quantitative
performance. for the present invention when an enzyme indicator is
used.
TABLE-US-00005 TABLE 5 NTx Dose Response Conjugate HRP-1H11 at 0.1
.mu.g/mL Various NTx-protein immobilization concentrations
NTx-Protein Dilution NTx 1:10 1:20 1:40 (nM) Reflectance Density
(Gretag) 24 0.47 0.49 0.52 80 0.41 0.40 0.42 240 0.32 0.38 0.30 800
0.27 0.27 0.20 2400 0.16 0.19 0.17
Example 7
[0168] This example demonstrates a single-step, quantitative,
lateral flow; competition-type immunoassay for a small molecule
using colored particles as the indicator. The assay summarized
below in Table 6 was conducted using reagents and methods as
described in Example 1.
[0169] The assay strips of this example were 3 cm wide and 0.3 cm
long, and were similar to those shown in FIG. 1 and FIG. 3, with
the exception that the third zone 18 (second test zone) was not
included. The immunoassay strip configuration was as follows:
[0170] (1) a lower 8 .mu.m pore size nitrocellulose section
containing continuous reagent zone 14 having diffusively
immobilized. NTx-latex beads (blue), prepared as described above;
[0171] (2) a 0.3 cm test zone 18 containing non-diffusively
immobilized 1H11 monoclonal anti-NTx, adsorbed to 8 .mu.m pore size
nitrocellulose, prepared as described above; and [0172] (3) an
upper wick area of 8 .mu.m pore size nitrocellulose that extended
from the upper edge of zone 16 to the top of the strip.
[0173] All strip zones were laminated in physical communication
with adjacent zone(s), permitting fluid to flow through the entire
strip by wicking action, and were supported on a plastic backing as
outlined above.
[0174] NTx was diluted in PBS to the concentrations indicated in
Table 6 below, and the general assay protocol of Example 1 was used
to generate the data shown in Table 6. These data illustrate a
dose-response demonstrating good sensitivity and quantitative
performance for the present invention using blue latex particles as
the signal reagent.
TABLE-US-00006 TABLE 6 NTx Dose Response Conjugate: Latex Bead-NTx
1H11 Immobilized NTx Reflectance (nM BCE) Density 30 0.79 100 0.64
300 0.46 1000 0.33 3000 0.20
Example 8
[0175] This example demonstrates a single-step, quantitative,
lateral flow, competition-type immunoassay for a proteins using
colored particles as the indicator. The assay summarized below in
Table 7 was conducted using reagents and methods as described In
Example 1. The assay strips of this example were 3 cm wide and 0.3
cm long, and were similar to those shown in FIG. 1 and FIG. 3, with
the exception that the second zone 16 (first test zone) was not
included. The immunoassay strip configuration was as follows:
[0176] (1), a lower 8 .mu.m pore size nitrocellulose section,
containing reagent zone 14 having diffusively applied mouse
IgG-latex beads (blue); [0177] (2) a 0.3 cm intermediate section,
of 8 .mu.m pore size nitrocellulose, containing test zone 18 having
non-diffusively immobilized goat anti-mouse adsorbed thereto; and
[0178] (3) an upper wick area of 8 .mu.m pore size nitrocellulose
that extended from the upper edge of zone 18 to the top of the
strip.
[0179] All strip zones were laminated in physical communication
with adjacent zone(s), permitting fluid to flow through the entire
strip by wicking action, and were supported on a plastic backing as
outlined above.
[0180] Mouse IgG was diluted in PBS to the concentrations
indicated. in Table 7 below, and the general assay protocol of
Example 1 was used to generate the data shown in Table 7. These
data illustrate a dose-response demonstrating good sensitivity and
quantitative performance for the present invention using blue latex
particle as the signal reagent.
TABLE-US-00007 TABLE 7 IgG Dose Response Conjugate: NTx-IgG-Latex
Beads Goat anti-mouse immobilized Mouse IgG Reflectance (.mu.g/mL)
Density 0 0.62 4 0.52 8 0.44 33 0.37 50 0.33 100 0.26 200 0.23
Example 9
[0181] This example demonstrates a single-step, quantitative,
lateral flow, competitive-type immunoassay for a small molecule and
a large molecule, using, colored particles as the indicator and an
assay reference that is directly related to the assay function. The
assay summarized below in Table was conducted. using reagents and
methods as described in Example 1. The assay strips of this example
were 3 cm wide and 0.3 cm long, and are shown in FIG. 1 and FIG. 3.
The immunoassay strip configuration was as follows: [0182] (1) a
lower 8 .mu.m pore size nitrocellulose section, containing reagent
zone 14 having diffusively immobilized NTx-latex beads 10.412
.mu.m, blue); [0183] (2) a 0.3 cm intermediate section of 8 .mu.m
pore size nitrocellulose, containing test zone 18 having
non-diffusively immobilized 1H11 monoclonal anti-NTx adsorbed
thereto; [0184] (3) a 0.5 cm spacer of 8 .mu.m pore size.
nitrocellulose; [0185] (4) a 0.3 cm long section of 8 .mu.m pore
size nitrocellulose, containing, test zone 18 having
non-diffusively immobilized goat-anti mouse adsorbed thereto; and
[0186] (5) an upper wick area of 8 .mu.m pore size nitrocellulose
that extended from the upper edge. of zone. 18 to the top of the
strip.
[0187] All strip zones were laminated in fluid communication with
adjacent zone(s), permitting fluid. to flow through the entire
strip by wicking action, and were supported on a plastic backing as
outlined above.
[0188] NTx was diluted in PBS to the concentrations indicated in
Table 8, and the general, assay protocol of Example 1 was used to
generate the data shown in Table 8. These data illustrate a
dose-response in both test zone 16 and test zone 18 demonstrating
good sensitivity and quantitative performance for the present
invention. The sum of the signals from the two test zones remains
substantially constant throughout the NTX concentration, range,
indicating a correct functioning, of the assay.
[0189] According to the present invention, the sum of the signals
from test zones 16 and 18 provides an internal quality reference to
assure reliable and, correct. assay performance. In addition,
better assay sensitivity is seen in test zone 18 at the lower end
of the curve (0 to 30 nM NTx), while better separation
(Sensitivity) is seen. in test zone. 16 at the upper end of the
curve (100 to 300 nM NTx). These results suggest a that hybrid
calibration algorithm using both test zones can provide improved
performance, relative to previously de-Scribed single-test-zone
methods. According to the present invention, assay calibration and
instrumental reading can use a single test zone alone, or both test
zones dither separately or combined, to provide maximum sensitivity
and reliability.
TABLE-US-00008 TABLE 8 NTx Dose Response Two Test Zones Conjugate:
Latex Bead-NTx Immobilized Zone 1-1H11, Zone 2-Goat Anti-Mouse
Reflectance Density (Gretag) NTx (nM) Zone 1 Zone 2 Sum 0 1.01 0.23
1.24 1 1.06 0.21 1.27 30 1.05 0.40 1.45 100 0.96 0.50 1.46 300 0.68
0.57 1.25
Example 10
[0190] The assay summarized below in Table 9 was conducted using
strips, reagents. and methods as described in Example 9, with the
exception that monoclonal rat anti-mouse was immobilized in zone
18.
[0191] The conclusions of Example 9 are supported by these
data.
TABLE-US-00009 TABLE 9 NTx Dose Response Two Test Zones Conjugate:
Latex Bead-NTx Immobilized Zone 11H11, Zone 2 Monoclonal
Rat-Anti-Mouse Reflectance Density (Gretag) NTx (nM) Zone 1 Zone 2
Sum 0 0.99 0.37 1.36 1 0.86 0.40 1.26 30 0.84 0.44 1.28 100 0.79
0.56 1.35 300 0.38 0.62 1.00
Example 11
[0192] The test results graphically represented in FIG. 8 were from
an assay conducted using strips, reagents and methods as described
in Example 9, except as otherwise noted. FIG. 8 illustrates the NTx
dose response using IgG-C-peptide in the first test zone on
nitrocellulose membrane, S&S AE 98, with a pore size of 5
.mu.m. The second-test zone immobilized monoclonal rat anti-mouse
on nitrocellulose.
[0193] Numerous modifications and variations of the present
invention are possible in light of the above teachings. It is
therefore to be understood that within the scope of the appended
claims, the invention may be practiced otherwise than as
specifically described herein.
* * * * *