Competitive immunoassay device

A chromatographic assay device for detection and/or determination of an analyte in a competitive immunoassay gives a semiquantitative or quantitative indication of analyte concentration in a single assay device while also giving a positive indication that flow has occurred properly through the device. In one form, the device comprises: (1) a first opposable component including a sample preparation zone and an absorber; and (2) a second opposable component including a first chromatographic medium with capture and detection zones, a second chromatographic medium with a comparison zone, and a comparison label. In another form, the second opposable component includes one chromatographic medium with capture, detection, and control zones. Test kits incorporating the devices and methods for their use are also disclosed.

BACKGROUND OF THE INVENTION 
This invention is directed to test strips or assay devices for 
determination of characteristics of samples, unitized housings, and kits 
incorporating test strips and housings, and methods of determining the 
characteristics of samples using the test strips and housings, 
particularly for the performance of competitive assays. 
Among the many analytical systems used for detection and/or determination 
of analytes, particularly analytes of biological interest, are 
chromatographic assay systems. Among the analytes frequently assayed with 
such systems are: 
(1) hormones, such as human chorionic gonadotropin (hCG), frequently 
assayed as a marker of human pregnancy; 
(2) antigens, particularly antigens specific to bacterial, viral, and 
protozoan pathogens, such as Streptococcus, hepatitis virus, and Giardia; 
(3) antibodies, particularly antibodies induced as a result of infections 
with pathogens, such as antibodies to the bacterium Helicobacter pylori 
and to human immunodeficiency virus (HIV); 
(4) other proteins, such as hemoglobin, frequently assayed in 
determinations of fecal occult blood, an early indicator of 
gastrointestinal disorders such as cancer; 
(5) enzymes, such as aspartate aminotransferase, lactate dehydrogenase, 
alkaline phosphatase, and glutamate dehydrogenase, frequently assayed as 
indicators of physiological function and tissue damage; 
(6) drugs, both therapeutic drugs, such as antibiotics, tranquilizers, and 
anticonvulsants, and illegal drugs of abuse, such as cocaine, heroin, and 
marijuana; 
(7) environmental pollutants such as pesticides and aromatic hydrocarbons; 
and 
(8) vitamins. 
Such chromatographic systems are frequently used by physicians and medical 
technicians for rapid in-office diagnosis and therapeutic monitoring of a 
variety of conditions and disorders. They are also increasingly used by 
patients themselves for at-home monitoring of such conditions and 
disorders. 
Among the most important of such systems are the "thin layer" systems in 
which a solvent moves across a thin, flat absorbent medium. 
Among the most important of tests that can be performed with such thin 
layer systems are immunoassays, which depend on the specific interaction 
between an antigen or hapten and a corresponding antibody. The use of 
immunoassays as a means of testing for the presence and/or mount of 
clinically important molecules has been known for some time. As early as 
1956, J. M. Singer reported the use of an immune-based latex agglutination 
test for detecting a factor associated with rheumatoid arthritis (J. M. 
Singer et al., Am. J. Med. 22:888-892 (1956)). 
Among the chromatographic techniques used in conjunction with immunoassays 
is a procedure known as immunochromatography. In general, this technique 
uses a disclosing reagent or particle that has been linked to an antibody 
to the molecule to be assayed, forming a conjugate. This conjugate is then 
mixed with a specimen and, if the molecule to be assayed is present in the 
specimen, the disclosing reagent-linked antibodies bind to the molecule to 
be assayed, thereby giving an indication that the molecule to be assayed 
is present. The disclosing reagent or particle can be identifiable by 
color, magnetic properties, radioactivity, specific reactivity with 
another molecule, or another physical or chemical property. The specific 
reactions that are employed vary with the nature of the molecule being 
assayed and the sample to be tested. 
Immunochromatographic assays fall into two principal categories: "sandwich" 
and "competitive," according to the nature of the antigen-antibody complex 
to be detected and the sequence of reactions required to produce that 
complex. The antigen to be detected can itself be an antibody, such as 
serological assays for H. pylori-specific antibody. In such cases, the 
antibody to be detected can be bound to a specific antigen. Alternatively, 
the antigen to be detected can be detected indirectly by using a labeled 
second antibody that binds to the first antibody to the analyte to be 
detected. 
In competitive immunoassays, the label is typically a labeled analyte or 
analyte analog which competes for binding of an antibody with any 
unlabeled analyte present in the sample. Competitive immunoassays are 
typically used for detection of analytes such as haptens, each hapten 
being monovalent and capable of binding only one antibody molecule. 
Examples of competitive immunoassay devices are those disclosed by U.S. 
Pat. No. 4,235,601 to Deutsch et al., U.S. Pat. No. 4,442,204 to Liotta, 
and U.S. Pat. No. 5,208,535 to Buechler et al., all which are incorporated 
herein by this reference. 
Although useful, currently available chromatographic techniques using test 
strips have a number of drawbacks. Many samples, such as fecal samples, 
contain particulate material that can clog the pores of the 
chromatographic medium, greatly hindering the immunochromatographic 
process. Other samples, such as blood, contain cells and colored 
components that make it difficult to read the test. Still other samples, 
such as milk, contain fat globules or other components that can create 
interference. Even if the sample does not create interference, it is 
frequently difficult with existing chromatographic test devices to apply 
the sample to the chromatographic medium so that the sample front moves 
uniformly through the chromatographic medium to insure that the sample 
reaches the area where binding is to occur in a uniform, straight-line 
manner. 
Other problems exist with currently-available test strips because of the 
nature of the sample to be assayed or the assay to be carried out. With 
such devices, it is impractical to perform washing steps which are 
frequently desirable to improve sensitivity and to reduce to background. 
Also, it is difficult, and in many cases impossible, to carry out 
preincubation steps within the device. Additionally, there is a need for 
an immunochromatographic assay device that can carry out a broad range of 
separations, such as a separation of fat from milk or the separation of 
organic chemicals such as the separation of benzene from toluene. 
Sample preparation and waste generation are responsible for other problems 
with currently available devices and techniques for immunochromatography. 
The increased prevalence of diseases spread by infected blood and blood 
fractions, as well as other bodily secretions, such as AIDS and hepatitis, 
has exacerbated these problems. It is rarely possible to apply a sample 
(such as feces) or a sampling device (such as a throat swab) directly to 
the chromatographic medium. Several extraction and pretreatment reactions 
are usually required before the sample can be applied to the 
chromatographic medium. These reactions are typically carried out by the 
physician or technician performing the test in several small vessels, such 
as test tubes, or microfuge tubes, requiring the use of transfer devices, 
such as pipettes. Each of these devices is then contaminated and must be 
disposed of using special precautions so that workers or people who may 
inadvertently come into contact with the waste do not become contaminated. 
Additionally, currently available test devices, although useful, are 
generally not suited to give a semi-quantitative or quantitative 
indication of the concentration of an analyte present in a test sample, 
particularly for competitive immunoassays. Typically, the obtaining of 
such a quantitative or semi-quantitative indication requires the use of 
more than one test device, such as for calibration. The use of more than 
one test device requires a greater expenditure of time and material and 
also increases the possibility that a mistake may be made during the 
performance of the assay. In particular, with possibly contaminated 
samples, there may be concern that, inadvertently, a device thought to 
contain only a control actually contains a sample that may contain an 
infectious agent. Thus, it would be preferable to be able to determine a 
quantitative or semi-quantitative indication of analyte concentration in 
one assay device without the use of a separate assay device as a control 
or for calibration. 
There is further need for an assay device that gives a positive indication 
that the test has been performed correctly and that flow has occurred 
properly within the chromatographic assay device. 
Accordingly, there is a need for an improved assay device capable of 
handling a broad range of chromatographic assays. Such a device should be 
able to handle all types of immunoassays, particularly competitive 
immunoassays, as well as other types of assays using chromatography. Such 
a device should be capable of receiving a possibly contaminated sample or 
a sample preparation device directly so as to eliminate the need for 
extraction vessels and transfer devices. Such a device, particularly in 
the form of a test strip, should also be capable of performing 
immunochromatographic assays on colored samples or samples containing 
particulates without interference and should be able to deliver the sample 
to the chromatographic medium uniformly and evenly to improve accuracy and 
precision of the tests. Additionally, such an improved test strip should 
be capable of carrying out semi-quantitative or quantitative indications 
of analyte concentration in a single assay device without the need for 
additional control assay devices, and should give a positive indication 
that flow within the device has occurred properly and that the assay has 
operated correctly. 
SUMMARY 
I have developed an assay device that meets these needs, and performs 
semiquantitative or quantitative determinations of analyte concentration 
in a single assay device without requiring additional control assay 
devices. The assay device of the present invention also provides a 
positive indication of proper performance of the assay. The device can 
perform competitive immunoassays for a wide variety of analytes, 
particularly haptens. 
One aspect of the present invention is a competitive immunoassay device 
comprising: 
(1) a first opposable component including: 
(a) a sample preparation zone including a labeled specific binding partner 
for an analyte conjugated to a first member of an auxiliary specific 
binding pair in resolubilizable form; and 
(b) an absorber for absorbing fluid therein separated from the sample 
preparation zone on the first opposable component; and 
(2) a second opposable component hingedly attachable to the first opposable 
component including: 
(a) a first chromatographic medium having first and second ends and 
including thereon: 
(i) a first zone of immobilized analyte or analog thereof bound to the 
first chromatographic medium; and 
(ii) a second zone of an immobilized molecule that is a second member of 
the auxiliary specific binding pair with specific affinity for the first 
member bound to the first chromatographic medium; 
(b) a second chromatographic medium having a first end and a second end and 
including thereon a comparison zone containing a known quantity of the 
analyte or analog thereof immobilized to the comparison zone; and 
(c) a comparison label zone including therein a labeled specific binding 
partner to the analyte or analog thereof in resolubilizable form in 
operable contact with the second chromatographic medium. 
When the first and second opposable components are brought into opposition 
from a position in which they are not in opposition, the sample 
preparation zone comes into operable contact with the first end of the 
first chromatographic medium to apply the sample and the labeled specific 
binding partner for the analyte conjugated to biotin to the first 
chromatographic medium. The absorber also comes into operable contact with 
the second end of the first chromatographic medium and the second end of 
the second chromatographic medium to draw fluid through the first and 
second chromatographic medium from their first end to their second end so 
that the device gives a detectable indication of the presence of an 
analyte at a quantity greater than a predetermined amount by a comparison 
of the intensity of the label bound at the detection zone of the first 
chromatographic medium and at the comparison zone of the second 
chromatographic medium. 
Preferably, the sample preparation zone on the first opposable component 
further includes a second labeled specific binding partner that binds a 
molecule not substantially cross-reactive with the analyte in 
resolubilizable form, and the first chromatographic medium further 
includes a flow control indicator including a molecule binding the second 
labeled specific binding partner so that the flow control indicator gives 
a positive indication that flow has occurred through the first 
chromatographic medium. 
Preferably, the label of both the labeled specific binding partner for the 
analyte conjugated to the first member of the auxiliary specific binding 
pair and the labeled specific binding partner to the analyte in the 
comparison label zone are visibly detectable labels. Preferably, the 
labeled specific binding partner to the analyte conjugated to the first 
member of the auxiliary specific binding pair at the sample preparation 
zone and the labeled specific binding partner to the analyte at the 
comparison label zone are the same label. 
Preferably, the first member of the auxiliary specific binding pair is 
biotin. When the first member of the auxiliary specific binding pair is 
biotin, the second member of the auxiliary specific binding pair is 
preferably streptavidin. 
In one typical embodiment of this device, the analyte is a .beta.-lactam 
antibiotic, the first zone of immobilized analyte or analog thereof is 
7-aminocephalosporanic acid conjugated to immunoglobulin, and the labeled 
specific binding partner for the analyte conjugated to the first member of 
the auxiliary specific binding pair is biotinylated penicillin binding 
protein. 
In another typical embodiment of this device, the analyte is an antibiotic 
selected from the group consisting of gentamycin, sulfamethazine, and 
tetracycline, the first zone of immobilized analyte or analog thereof is 
analyte covalently conjugated to immunoglobulin, and the labeled specific 
binding partner for the analyte conjugated to the fist member of the 
auxiliary specific binding pair is a biotinylated anti-analyte antibody. 
Another aspect of the present invention is a test kit for detection and/or 
determination of an analyte comprising, packaged in separate containers: 
(1) the immunoassay device described above; and 
(2) a liquid for resolubilizing the labeled specific binding partner to the 
analyte or analog thereof in the comparison label zone. 
Another aspect of the present invention is a method for detecting and/or 
determining an analyte in a test sample by a competitive immunoassay 
comprising the steps of: 
(1) adding a test sample to the sample preparation zone of the competitive 
immunoassay device described above; 
(2) allowing the test sample to resolubilize the labeled specific binding 
partner for the analyte conjugated to the first member of the auxiliary 
specific binding pair; 
(3) adding a liquid to the comparison label zone to resolubilize the 
labeled specific binding partner to the analyte or analog thereof; 
(4) bringing the first and second opposable components into opposition from 
a position in which they are not in opposition to bring the absorber on 
the first opposable component into operable contact with the second end of 
the first chromatographic medium and the second end of the second 
chromatographic medium and to bring the sample preparation zone into 
operable contact with the first end of the first chromatographic medium; 
(5) allowing the sample and the labeled specific binding partner for the 
analyte conjugated to the first member of the auxiliary specific binding 
pair to flow through the first chromatographic medium and the labeled 
specific binding partner to the analyte or analog thereof to flow through 
the second chromatographic medium so that, if analyte is present in the 
test sample, the labeled specific binding partner to the analyte 
conjugated to the first member of the auxiliary specific binding pair 
binds to the detection zone on the first chromatographic medium and so 
that the labeled specific binding partner to the analyte or analog thereof 
binds to the comparison zone on the second chromatographic medium; and 
(6) comparing the intensity of label at the detection zone on the first 
chromatographic medium and at the comparison zone on the second 
chromatographic medium to detect and/or determine the analyte in the test 
sample. 
Yet another aspect of the present invention is another version of a 
competitive immunoassay device that uses a single chromatographic medium 
to provide both detection and control functions. This version comprises: 
(1) a first opposable component including: 
(a) a sample preparation zone including: 
(i) a labeled specific binding partner for an analyte conjugated to a first 
member of an auxiliary specific binding pair in resolubilizable form; and 
(ii) a predetermined quantity of the analyte or an analog thereof 
covalently bound to a labeled specific binding partner for a molecule that 
does not substantially cross-react with the analyte in resolubilizable 
form; and 
(b) an absorber separated from the sample preparation zone; and 
(2) a second opposable component including a chromatographic medium with 
first and second ends, the chromatographic medium including thereon in 
discrete, nonoverlapping zones: 
(a) a first, capture, zone of immobilized analyte or analog thereof bound 
to the chromatographic medium; 
(b) a second, detection, zone including an immobilized molecule that is a 
second member of the auxiliary specific binding pair with specific 
affinity for the first member bound to the chromatographic medium; and 
(c) a third, control, zone including an immobilized molecule capable of 
being specifically bound to the labeled specific binding partner 
covalently conjugated to the analyte or analog thereof bound to the 
chromatographic medium. 
In this embodiment of an assay device according to the present invention, 
the first and second opposable components are configured so that bringing 
the first and second opposable components into opposition from a position 
in which they are not in opposition causes the absorber to come into 
operable contact with the second end of the chromatographic medium and 
causes the sample preparation zone to come into contact with the first end 
of the chromatographic medium so that the test sample, the resolubilized 
labeled specific binding partner for the analyte conjugated to the first 
member of the auxiliary specific binding pair, and the predetermined 
quantity of the analyte or analog thereof covalently bound to a labeled 
specific binding partner flow through the chromatographic medium from the 
first end to the second end. As a result, the device gives a detectable 
indication of the presence of an analyte at a quantity greater than a 
predetermined amount by a comparison of the intensity of the label bound 
at the detection zone and at the control zone of the second 
chromatographic medium. 
Typically, the label of the labeled specific binding partner for the 
analyte conjugated to the first member of the auxiliary specific binding 
pair and the label of the labeled specific binding partner for the 
molecule that does not substantially cross-react with the analyte are both 
visually detectable labels. 
In one typical embodiment of this device, in which the first member of the 
auxiliary specific binding pair is biotin and the second member of the 
auxiliary specific binding pair is streptavidin, the analyte is a 
.beta.-lactam antibiotic, the labeled specific binding partner for the 
analyte conjugated to the first member of the auxiliary specific binding 
pair is a penicillin binding protein, the immobilized analyte or analog 
thereof bound at the capture zone on the chromatographic medium is 
7-aminocephalosporanic acid covalently conjugated to immunoglobulin G, the 
labeled specific binding partner for a molecule that does not 
substantially cross-react with the analyte is an anti-rabbit 
immunoglobulin G antibody, and the immobilized molecule bound at the 
control zone on the chromatographic medium is rabbit immunoglobulin G. 
Alternatively, the analyte can be an antibiotic selected from the group 
consisting of gentamycin, sulfamethazine, and tetracycline, the labeled 
specific binding partner for the analyte conjugated to the first member of 
the auxiliary specific binding pair can be an anti-analyte antibody, the 
labeled specific binding partner for a molecule that does not 
substantially cross-react with the analyte can be anti-rabbit 
immunoglobulin G, the immobilized analyte or analog thereof bound at the 
capture zone on the chromatographic medium can be the analyte covalently 
bound to immunoglobulin G of a species other than the species other than 
rabbit, and the immobilized molecule bound at the control zone can be 
rabbit immunoglobulin G. 
Another aspect of the present invention is a method for detecting and/or 
determining an analyte in a test sample by a competitive immunoassay 
comprising the steps of: 
(1) applying the sample to the sample preparation zone of the 
single-chromatographic medium competitive immunoassay device described 
above; 
(2) allowing the sample applied to the sample preparation zone to 
resolubilize the labeled specific binding partner for the analyte 
conjugated to the first member of the auxiliary specific binding pair and 
the predetermined quantity of the analyte or analog thereof covalently 
bound to the labeled specific binding partner for the molecule that does 
not substantially cross-react with the analyte; 
(3) bringing the first and second opposable components into opposition from 
a position in which they are not in opposition so that the absorber is 
brought into operable contact with the second end of the chromatographic 
medium and so that the sample preparation zone is brought into operable 
contact with the first end of the chromatographic medium; 
(4) allowing the sample, the labeled specific binding partner for the 
sample, and the analyte or analog thereof covalently bound to the labeled 
specific binding partner for the molecule that does not substantially 
cross-react with the analyte to migrate through at least a portion of the 
chromatographic medium including the detection zone and the control zone; 
and 
(5) detecting and/or determining the concentration of analyte in the test 
sample by comparing the intensities of label at the detection zone and the 
control zone of the chromatographic medium. 
Yet another aspect of the present invention is multiplex assay devices that 
can perform more than one assay simultaneously. One version of such a 
multiplex assay device comprises: 
(1) a first opposable component including: 
(a) a plurality of sample preparation zones, each sample preparation zone 
including a labeled specific binding partner for an analyte conjugated to 
a first member of an auxiliary specific binding pair in resolubilizable 
form; and 
(b) an absorber for absorbing fluid therein separated from the sample 
preparation zones on the first opposable component; and 
(2) a second opposable component hingedly attachable to the first opposable 
component including: 
(a) a plurality of first chromatographic media, one for each sample 
preparation zone, each first chromatographic medium having first and 
second ends and including thereon: 
(i) a first zone of an immobilized analyte or analog thereof bound to the 
first chromatographic medium; and 
(ii) a second zone of an immobilized molecule that is a second member of 
the auxiliary specific binding pair with specific affinity for the first 
member bound to the first chromatographic medium; 
(b) a plurality of second chromatographic media, one for each sample 
preparation zone, each second chromatographic medium having a first end 
and a second end and including thereon a comparison zone containing a 
known quantity of an analyte or analog thereof immobilized to the 
comparison zone; and 
(c) a plurality of comparison label zones, one for each second 
chromatographic medium, each comparison label zone including therein a 
labeled specific binding partner to the analyte or analog thereof in 
resolubilizable form in operable contact with the second chromatographic 
medium. 
In this version of a multiplex assay device according to the present 
invention, when the first and second opposable components are brought into 
opposition from a position in which they are not in opposition, the sample 
preparation zones come into operable contact with the first end of each 
first chromatographic medium to apply the sample and the labeled specific 
binding partner for the analyte conjugated to the first member of the 
auxiliary specific binding pair to each first chromatographic medium, and 
the absorber comes into operable contact with the second end of each first 
chromatographic medium and the second end of each second chromatographic 
medium to draw fluid through the first and second chromatographic media 
from their first ends to their second ends. As a result, the device gives 
a detectable indication of the presence of an analyte in each first and 
second chromatographic medium at a quantity greater than a predetermined 
amount by a comparison of the intensity of the label bound at the 
detection zone of each first chromatographic medium and at the comparison 
zone of each second chromatographic medium. 
A test kit incorporating this version of a multiplex assay device according 
to the present invention comprises: 
(1) the immunoassay device; and 
(2) a liquid for resolubilizing the labeled specific binding partner to the 
analyte or analog thereof in each comparison label zone. 
A method for using this multiplex assay device comprises: 
(1) adding a test sample to at least one of the sample preparation zones of 
the multiplex assay device; 
(2) allowing the at least one test sample to resolubilize the labeled 
specific binding partners for the analyte conjugated to the first member 
of the auxiliary specific binding partner; 
(3) adding a liquid to at least one of the comparison label zones to 
resolubilize the labeled specific binding partner to the analyte or analog 
thereof; 
(4) bringing the first and second opposable components into opposition from 
a position in which they are not in opposition to bring the absorber on 
the first opposable component into operable contact with the second end of 
the first chromatographic media and the second end of the second 
chromatographic media and to bring the sample preparation zones into 
operable contact with the first ends of the first chromatographic media; 
(5) allowing the at least one sample and the labeled specific binding 
partner for the analyte conjugated to the first member of the auxiliary 
specific binding pair to flow through the first chromatographic media and 
the labeled specific binding partner to the analyte or analog thereof to 
flow through the second chromatographic media so that, if analyte is 
present in any test sample, the labeled specific binding partner to the 
analyte conjugated to biotin binds to the detection zone on the 
corresponding first chromatographic medium and so that the labeled 
specific binding partner binds to the comparison zone on the corresponding 
second chromatographic medium; and 
(6) comparing the intensity of label at the detection zone on each first 
chromatographic medium and at the comparison zone on each second 
chromatographic medium to detect and/or determine the analyte in each test 
sample. 
Another version of a multiplex assay device according to the present 
invention is based on the version of the device using a single 
chromatographic medium and comprises: 
(1) a first opposable component including: 
(a) a plurality of sample preparation zones, each sample preparation zone 
including: 
(i) a labeled specific binding partner for an analyte conjugated to a first 
member of an auxiliary specific binding pair in resolubilizable form; and 
(ii) a predetermined quantity of an analyte or an analog thereof covalently 
bound to a labeled specific binding partner for a molecule that does not 
substantially cross-react with the analyte in resolubilizable form; and 
(b) an absorber separated from the sample preparation zones; and 
(2) a second opposable component including a plurality of chromatographic 
media with first and second ends, each chromatographic medium including 
thereon in discrete, nonoverlapping zones: 
(a) a first, capture, zone of immobilized analyte or analog thereof bound 
to the chromatographic medium; 
(b) a second, detection, zone including an immobilized molecule that is a 
second member of the auxiliary specific binding pair with specific 
affinity for the first member bound to the chromatographic medium; and 
(c) a third, control, zone including an immobilized molecule capable of 
being specifically bound to the labeled specific binding partner 
covalently conjugated to the analyte or analog thereof bound to the 
chromatographic medium. 
In this version of a multiplex assay device according to the present 
invention, the first and second opposable components are configured so 
that bringing the first and second opposable components into opposition 
from a position in which they are not in opposition causes the absorber to 
come into operable contact with the second end of each chromatographic 
medium and causes the sample preparation zones to come into contact with 
the first end of each chromatographic medium so that the test samples, the 
resolubilized labeled specific binding partners for the analyte conjugated 
to the first member of the auxiliary specific binding pair and the 
predetermined quantities of the analyte or analog thereof covalently bound 
to a labeled specific binding partner flow through each chromatographic 
medium from the first end to the second end. 
A method for using this version of the multiplex assay device comprises: 
(1) applying a sample to at least one of the sample preparation zones of 
the multiplex assay device; 
(2) allowing the at least one sample applied to the sample preparation 
zones to resolubilize the labeled specific binding partner for the analyte 
conjugated to the first member of the auxiliary specific binding pair and 
the predetermined quantity of the analyte or analog thereof covalently 
bound to the labeled specific binding partner for the molecule that does 
not substantially cross-react with the analyte; 
(3) bringing the first and second opposable components into opposition from 
a position in which they are not in opposition so that the absorber is 
brought into operable contact with the second end of each chromatographic 
medium and so that each sample preparation zone is brought into operable 
contact with the first end of each chromatographic medium; 
(4) allowing the at least one sample, the labeled specific binding partner 
for the sample, and the analyte or analog thereof covalently bound to the 
labeled specific binding partner for the molecule that does not 
substantially cross-react with the analyte to migrate through at least a 
portion of each chromatographic medium including the detection zone and 
the control zone; and 
(5) detecting and/or determining the concentration of analyte in each test 
sample by comparing the intensities of label at the detection zone and the 
control zone of each chromatographic medium.

DESCRIPTION 
Definitions 
In the context of this disclosure, the following terms are defined as 
follows unless otherwise indicated: 
Specific Binding Partner: A member of a pair of molecules that interact by 
means of specific non-covalent interactions that depend on the 
three-dimensional structures of the molecules involved. Typical pairs of 
specific binding partners include antigen-antibody, hapten-antibody, 
hormone-receptor, nucleic acid strand-complementary nucleic acid strand, 
substrate-enzyme, .beta.-lactam antibiotic-penicillin binding protein, 
inhibitor-enzyme, carbohydrate-lectin, biotin-avidin, and virus-cellular 
receptor. 
Immunoassay: As used herein, the term "immunoassay" includes assays 
involving at least one specific binding partner as defined above and is 
not necessarily limited to assays in which the specific binding partner is 
an antibody unless such a limitation is specified. 
Operable Contact: Two solid components are in operable contact when they 
are in contact, either directly or indirectly, in such a manner that a 
liquid, typically an aqueous liquid, can flow from one of the two 
components to the other Substantially uninterruptedly, by capillarity or 
otherwise. "Direct contact" means that the two elements are in physical 
contact, such as edge-to-edge or front-to-back. Typically, when two 
components are in direct contact, they are overlapped with an overlap of 
about 0.5 to about 3 mm. However, the components can be placed with 
abutting edges. "Indirect contact" means that the two elements are not in 
physical contact, but are bridged by one or more conductors. 
Analyte: The term "analyte" includes both the actual molecule to be assayed 
and analogues and derivatives thereof when such analogues and derivatives 
bind another molecule used in the assay in a manner substantially 
equivalent to that of the analyte itself in terms of affinity and 
cross-reactivity. 
Antibody: The term "antibody" includes both intact antibody molecules of 
the appropriate specificity and antibody fragments (including Fab, F(ab'), 
and F(ab').sub.2 fragments) as well as chemically modified intact antibody 
molecules and antibody fragments, including hybrid antibodies assembled by 
in vitro reassociation of subunits. 
Auxiliary Specific Binding Pair: The term "auxiliary specific binding pair" 
refers to a pair of molecules that are specific binding partners of each 
other, as that term is defined above, with neither member of the pair 
having specific binding affinity for any other molecule participating in 
the assay performed by assay devices according to the present invention. 
An example of an auxiliary specific binding pair is biotin-avidin. 
I. CHROMATOGRAPHIC ASSAY DEVICES 
One aspect of the present invention comprises chromatographic assay devices 
particularly useful for the assay of analytes and biological samples, 
particularly by competitive immunoassays. These devices are suitable for 
the direct application of biological samples, without preliminary 
extraction steps, and are constructed so as to minimize interference with 
assay results caused by particulates or colored samples. 
The device has at least two substantially planar opposable components. One 
of the substantially planar components has on its surface a 
chromatographic medium. 
The device also has means for opposing the opposable components and 
applying pressure thereto. Typically, bringing the opposable components 
into opposition is performed by direct manual closure. The pressure 
applied is sufficient to transfer fluid from one opposable component to 
another opposable component in a direction substantially normal to the 
opposable components so that the sample is applied to the chromatographic 
medium for detection and/or determination of the analyte thereon. The 
pressure also drives fluid through the chromatographic medium to 
accelerate the process of chromatography, giving a detectable result in 
less time. Additionally, the pressure makes possible the performance of 
steps, such as extraction steps, in the device, and can be used to remove 
excess fluid from the chromatographic medium by absorbers to reduce the 
background of the assays. The pressure is generated by placing the 
opposable components into opposition and maintained by holding the 
components in opposition by engagers such as locks or clasps. The pressure 
is calibrated to be optimum for a particular assay, and can vary depending 
upon the construction of the device, the material used in the 
chromatographic medium, the volume and nature of the sample, the nature of 
the specific binding partner and label, and other factors. 
Assay devices according to the present invention perform immunoassays 
without requiring the application of additional liquid to the 
chromatographic medium of the devices once the sample is applied. This 
avoids dilution of the sample or the specific binding partners and 
preserves maximum sensitivity of the assays. 
Typically, devices according to the present invention are constructed for 
the performance of a competitive immunoassay. However, devices can be 
constructed by similar principles for the performance of other types of 
immunoassays and are within the scope of the invention. 
In a device suitable for the performance of a competitive immunoassay, the 
chromatographic medium has incorporated thereon a detection zone of a 
specific binding partner for a labeled component. The labeled component is 
a first member of an auxiliary specific binding pair, and the detection 
zone includes a second member of the auxiliary specific binding pair. 
In one particularly preferable alternative employing an auxiliary specific 
binding pair, the detection zone is an immobilized molecule with specific 
affinity for biotin, and the assay includes a labeled specific binding 
partner for an analyte conjugated to biotin. The immobilized molecule with 
specific affinity for biotin is typically streptavidin, but can also be 
avidin or an anti-biotin antibody. 
The term "biotin", as used herein, includes not only biotin itself, but 
also derivatives of biotin in which the binding between the derivative of 
biotin and its specific binding partner is substantially equivalent to 
that between biotin itself and the specific binding partner. These 
derivatives include iminobiotin and biotin covalently conjugated to a 
spacer, such as .epsilon.-caproamidobiotin. Other derivatives of biotin, 
such as those incorporating spacers of varying lengths, can also be used. 
The detection zone is substantially smaller than the chromatographic 
medium. It typically extends the entire width of the chromatographic 
medium, but can be confined to a spot that is smaller than the entire 
width of the chromatographic medium. 
Typically, detection and/or determination of the analyte after the 
performance of the assay occurs by use of a visually detectable labeled 
component. The labeled component is preferably either the analyte or an 
analog thereof linked to a visually detectable label, or a specific 
binding partner for the analyte linked to a visually detectable label. In 
the arrangements described below for competitive immunoassays, the labeled 
component is typically a labeled specific binding partner for an analyte 
conjugated to a first member of an auxiliary specific binding pair. 
Preferably, the first member of the auxiliary specific binding pair is 
biotin. When the first member of the auxiliary specific binding pair is 
biotin, the second member of the auxiliary specific binding pair is 
preferably streptavidin. 
Ligand analogs, including analyte analogs, are well known in the art and 
are described, for example, in U.S. Pat. No. 3,817,837 to Rubenstein et 
al., incorporated herein by this reference. Analyte analogs can be 
conjugated to a larger protein, such as immunoglobulin that is unreactive 
in the immunoassay, through a linker. The length of the linker can be 
selected for optimal performance in the assay performed by the device. 
Various linking reactions can be used to synthesize the analyte analogs, 
depending on the functional groups available. Such linking reactions are 
described, for example, in U.S. Pat. No. 3,817,837 to Rubenstein et al. 
Assay devices according to the present invention can be constructed for the 
performance of more than one assay simultaneously. In such devices, one of 
the substantially planar components has at least two separate and 
non-contacting chromatographic media thereon. These are described further 
below. 
A. Elements Common to Devices According to the Present Invention 
A number of elements are common to assay devices according to the present 
invention and are discussed here for convenience. 
1. The Chromatographic Medium 
The chromatographic medium is a strip. Typically, the strip is 
substantially planar, although this is not required in all applications. 
It is typically rectangular, having first and second ends and first and 
second surfaces. Throughout this description, the term "first end" refers 
to the end at which liquid is first applied to the chromatographic medium 
and the term "second end" applies to the opposite end of the 
chromatographic medium. The liquid applied at or near the first end of the 
chromatographic medium can be, but is not necessarily, a sample or a 
treated sample. The chromatographic medium is composed of material 
suitable as a medium for thin layer chromatography of analyte and 
analyte-antibody conjugates, such as nitrocellulose, nylon, rayon, 
cellulose, paper, or silica. The chromatographic medium can be pretreated 
or modified as needed. 
The reagent located in the detection zone is immobilized on the 
chromatographic medium in such a way that it is stabilized against 
diffusive movement. The reagent can be bound to the chromatographic medium 
by either covalent or non-covalent means; both are well-known in the art 
and need not be described further here in detail. If the chromatographic 
medium is nitrocellulose, the reagent in the detection zone can be bound 
by a hydrophobic interaction. 
The binding of reagents to solid phases is described, for example, in P. 
Tijssen, "Practice and Theory of Enzyme Immunoassays" (Elsevier, 
Amsterdam, 1985), pp. 297-328. 
Typically, the chromatographic medium is translucent, so that colored zones 
appearing on it can be viewed from either side. 
2. Absorbers 
In a number of devices according to the present invention, absorbers are in 
operable contact with one or both ends of the chromatographic medium. The 
absorbers can be made of any bibulous material that holds a liquid, 
typically an aqueous liquid, sufficiently so that liquid can be drawn 
through the chromatographic medium and accumulated in the absorber. 
Typical materials include, but are not limited to, filter paper. 
3. Other Fluid-Carrying Elements 
As described below, in particular devices according to the present 
invention, other fluid-carrying elements can be employed as sample 
preparation zones, applicators, and/or conductors. These elements are 
prepared of hydrophilic media that pass liquids, typically aqueous 
liquids, without substantially absorbing them. Such materials are 
well-known in the art. 
In some cases, these elements can have incorporated therein a component in 
dry form that can be resolubilized by addition of a liquid, typically an 
aqueous liquid, to the element. This component can be a labeled component 
or another component of the reaction. 
4. Opposable Components 
Many of the embodiments of the assay device according to the present 
invention comprise two opposable components. The bodies of the opposable 
components are preferably made of laminated cardboard that is sufficiently 
impervious to moisture to contain the liquids involved in the performance 
of the assay carried out by the device for the period of time during which 
the assay is carried out. 
Other cellulose-based materials, such as paperboard or solid bleached 
sulfite (SBS), can also be used. Alternatively, the bodies of the 
opposable components can be made of plastic that is impermeable to 
moisture. A suitable plastic is a polycarbonate plastic such as Lexan.TM.. 
The opposable components are typically joined by a hinge, preferably made 
of a material impermeable to liquids, including aqueous liquids, such as a 
plastic that can be compatibly joined with or is the same as the material 
used for the first and second opposable components. 
5. Labeled Components 
For assay devices according to the present invention intended to perform a 
competitive immunoassay, the labeled component is typically a labeled 
specific binding partner to the analyte that is also conjugated to biotin. 
However, other labelling arrangements can be used. 
The label is preferably a visually detectable label, such as a colloidal 
metal label. Preferably, the colloidal metal label is gold, silver, 
bronze, iron, or tin; most preferably, it is gold. The preparation of 
gold-labeled antibodies and antigens is described in J. DeMey, "The 
Preparation and Use of Gold Probes", in Immunocytochemistry: Modern 
Methods and Applications (J. M. Polak & S. Van Noorden, eds., Wright, 
Bristol, England, 1986), Ch. 8, pp. 115-145; other references to the 
preparation of such labeled antibodies and antigens are known in the art. 
Antibodies labeled with colloidal gold are commercially available, such as 
from Sigma Chemical Company, St. Louis, Mo. Alternatively, other colloidal 
labels, such as a colloidal sulfur label or a dye-silica label, can also 
be used. In a less preferred alternative, the visually detectable label 
can be a colloidal latex label. It is also possible to use other labels, 
such as a radioactive label, a fluorescent label, a chemiluminescent 
label, a bioluminescent label, or an enzyme label. 
B. Assay Devices for Competitive Immunoassays 
1. Assay Device for Competitive Immunoassays with Two Chromatographic Media 
One embodiment of the present invention suitable for performing competitive 
immunoassays uses two separate chromatographic media on a single opposable 
component. One of these chromatographic media is used to perform the 
immunoassay, while the other provides a comparison using a known quantity 
of analyte in order to determine whether or not the analyte is present in 
the test sample at a concentration higher than a predetermined 
concentration. 
In general, this immunoassay device comprises: 
(1) a first opposable component including: 
(a) a sample preparation zone including a labeled specific binding partner 
for an analyte conjugated to a first member of an auxiliary specific 
binding pair in resolubilizable form; and 
(b) an absorber for absorbing fluid therein separated from the sample 
preparation zone on the first opposable component; and 
(2) a second opposable component hingedly attachable to the first opposable 
component including: 
(a) a first chromatographic medium having first and second ends and 
including thereon: 
(i) a first zone of immobilized analyte or analog thereof bound to the 
first chromatographic medium; and 
(ii) a second zone of an immobilized molecule that is a second member of 
the auxiliary specific binding pair with specific affinity for the first 
member bound to the first chromatographic medium; 
(b) a second chromatographic medium having a first end and a second end and 
including thereon a comparison zone containing a known quantity of the 
analyte or analog thereof immobilized to the comparison zone; and 
(c) a comparison label zone including therein a labeled specific binding 
partner to the analyte or analog thereof in resolubilizable form in 
operable contact with the second chromatographic medium. 
When the first and second opposable components are brought into opposition 
from a position in which they are not in opposition, the sample 
preparation zone comes into operable contact with the first end of the 
first chromatographic medium to apply the sample and the labeled specific 
binding partner for the analyte conjugated to biotin to the first 
chromatographic medium, and the absorber comes into operable contact with 
the second end of the first chromatographic medium and the second end of 
the second chromatographic medium to draw fluid through the first and 
second chromatographic medium from their first end to their second end. 
The device gives a detectable indication of the presence of an analyte at 
a quantity greater than a predetermined amount by a comparison of the 
intensity of the label bound at the detection zone of the first 
chromatographic medium and at the comparison zone of the second 
chromatographic medium. 
This device is shown in FIG. 1. 
The device 10 includes a first opposable component 12 and a second 
opposable component 14, attached by a hinge 16. The first opposable 
component 12 comprises a sample preparation zone 18. The sample 
preparation zone 18 includes a labeled specific binding partner for an 
analyte conjugated to a first member of an auxiliary specific binding pair 
in a form that can be resolubilized by the addition of a liquid to the 
sample preparation zone 18. The first opposable component 12 further 
includes an absorber 20 for absorbing fluid. The absorber 20 is separated 
from the sample preparation zone 18 on the first opposable component 12. 
The second opposable component 14 includes a first chromatographic medium 
22. The first chromatographic medium 22 has a first end 24 and a second 
end 26. The first chromatographic medium 22 includes thereon: (i) a first 
zone 28 of immobilized analyte or analog thereof bound to the first 
chromatographic medium 22 and (ii) a second zone 30 of an immobilized 
molecule that is a second member of the auxiliary specific binding pair 
bound to the first chromatographic medium 22. When the first member of the 
auxiliary specific binding pair is biotin, the immobilized molecule with 
specific affinity for biotin is typically streptavidin but can 
alternatively be avidin or an anti-biotin antibody. 
The second opposable component 14 also includes a second chromatographic 
medium 32. The second chromatographic medium 32 has a first end 34 and a 
second end 36 and includes thereon a comparison zone 38 containing a known 
quantity of the analyte or an analog thereof immobilized to the comparison 
zone 38. The second opposable component 14 further includes a comparison 
label zone 40 including therein a labeled specific binding partner to the 
analyte or analog thereof in a form that can be resolubilized by the 
addition of a liquid, typically an aqueous liquid, to the comparison label 
zone 40. The comparison label zone 40 is in operable contact with the 
second chromatographic medium 32 so that, when the labeled specific 
binding partner to the analyte or analog is resolubilized, it diffuses 
into the second chromatographic medium 32. The first and second 
chromatographic media 22 and 32 can be backed by a backing 42, which can 
be a plastic such as polycarbonate (Lexan) or other impermeable plastic. 
When the first and second opposable components 12 and 14 are brought into 
opposition from a position in which they are not in opposition, the sample 
preparation zone 18 comes into operable contact with the first end 24 of 
the first chromatographic medium 22 to apply the sample and the labeled 
specific binding partner for the analyte conjugated to the first member of 
the auxiliary specific binding pair to the first chromatographic medium 
22. The absorber 20 on the first opposable component 12 comes into 
operable contact with both the second end 26 of the first chromatographic 
medium 22 and the second end 36 of the second chromatographic medium 32 to 
draw fluid through the first and second chromatographic media 22 and 32 
from their first ends 24 and 34 to their second ends 26 and 36. The 
absorber 20 is large enough so that it can absorb the fluid from both the 
first chromatographic medium 22 and the second chromatographic medium 32. 
The first opposable component 12 can have an aperture 44 therein to view 
the second zone 30 on the first chromatographic medium 24 and the 
comparison zone 38 on the second chromatographic medium 32. The device 10 
can also include engagers such as locks 46 and 48 to hold the first and 
second opposable components 12 and 14 in opposition and a gasket 50 to 
prevent the escape of fluids from the device. 
Preferably, the first chromatographic medium 22 on the second opposable 
component 14 further includes a flow control indicator 52. The flow 
control indicator 52 is located near the second end 26 of the first 
chromatographic medium 22. When a flow control indicator 52 is used, the 
sample preparation zone 18 on the first opposable component 12 further 
includes a second labeled specific binding partner that binds a molecule 
not substantially cross-reactive with the analyte in a form that can be 
resolubilized by the addition of a liquid, typically an aqueous liquid, to 
the sample preparation zone. The flow control indicator 52 includes a 
molecule binding the second labeled specific binding partner so that the 
flow control indicator 52 gives a positive indication the flow has 
occurred through the first chromatographic medium 22. 
In carrying out the assay with the assay device 10, a sample is added to 
the sample preparation zone 18 on the first opposable component 12. The 
sample resolubilizes the labeled specific binding partner for the analyte 
conjugated to the first member of the auxiliary specific binding pair, and 
also the second labeled specific binding partner that binds the molecule 
not substantially cross-reactive with the analyte, to give the indication 
of flow control. The assay device 10 is then allowed to incubate for a 
predetermined period of time, typically 1 minute to 10 minutes. This 
incubation can be performed at room temperature; alternatively, the assay 
device 10, or the first opposable component 12 of the assay device 10, can 
be placed in an incubator that raises the temperature to a temperature 
higher than room temperature, such as 30.degree. C. or 37.degree. C., for 
a faster assay. Even higher temperatures can be used if such temperatures 
would not result in inactivation or denaturation of the antibodies or 
other specific binding partners. 
After the incubation, a buffer or other aqueous reagent is applied to the 
comparison label zone 40 on the second opposable component 14. The 
resolubilized labeled specific binding partner to the analyte or analog 
thereof is then allowed to migrate through the second chromatographic 
medium 32 from its first end 34 toward its second end 36. 
The assay device 10 is then closed, bringing the first and second opposable 
components 12 and 14 into opposition, and applying the resolubilized 
labeled specific binding partner for the analyte conjugated to the first 
member of the auxiliary specific binding pair and, if present, the second 
labeled specific binding partner that binds a molecule not cross-reactive 
with the analyte to the first end 24 of the first chromatographic medium 
22. If analyte is present in the sample, the labeled specific binding 
partner to the analyte conjugated to the first member of the auxiliary 
specific binding pair binds to the first zone (capture zone) of 
immobilized analyte or analog 28 on the first chromatographic medium 22. 
In this case, no signal is detectable at the second zone 30 (detection 
zone) including the immobilized second member of the auxiliary specific 
binding pair bound to the first chromatographic medium 22. 
In either case, whether analyte is present in the sample or not, however, 
the second labeled specific binding partner that binds a molecule not 
substantially cross-reactive with the analyte binds to the flow control 
zone 52 near the second end 26 of the first chromatographic medium 22 and 
gives a positive indication that proper flow through the first 
chromatographic medium has occurred. 
Simultaneously, the resolubilized labeled specific binding partner to the 
analyte or analog thereof originally present in the comparison label zone 
40 is migrating through the second chromatographic medium 32 from the 
first end 34 to the second end 36, and the resolubilized labeled specific 
binding partner to the analyte or analog thereof then binds to the 
immobilized analyte or analog thereof present in a predetermined quantity 
at the comparison zone 38 on the second chromatographic medium 32. 
After a period for migration, typically 1 to 10 minutes, the user then 
views the second zone of immobilized analyte 30 on the first 
chromatographic medium 22 and the comparison zone 38 on the second 
chromatographic medium 32 through the aperture 42 to compare the relative 
intensities of the label at the zones in order to obtain a 
semi-quantitative estimate of the concentration of analyte in the test 
sample. For example, if the comparison zone 38 contains 5 ng of analyte, 
if the intensity present at the second zone 30 on the first 
chromatographic medium 22 is greater than the intensity at the comparison 
zone 38 on the second chromatographic medium 32, there is more than 5 ng 
of analyte present in the volume of the test sample applied to the assay 
device 10. A range of concentrations can be used. 
2. Assay Device Employing Control Line Combined With Flow Indicator 
Another embodiment of an assay device according to the present invention 
employs a combined control line and flow indicator that provide a 
semi-quantitative indication of the analyte in the test sample. This 
device also operates by a competitive immunoassay procedure. 
In general, this embodiment comprises: 
(1) a first opposable component including: 
(a) a sample preparation zone including: 
(i) a labeled specific binding partner for an analyte conjugated to a first 
member of an auxiliary specific binding pair in resolubilizable form; and 
(ii) a predetermined quantity of the analyte or an analog thereof 
covalently bound to a labeled specific binding partner for a molecule that 
does not substantially cross-react with the analyte in resolubilizable 
form; and 
(b) an absorber separated from the sample preparation zone; and 
(2) a second opposable component including a chromatographic medium with 
first and second ends, the chromatographic medium including thereon in 
discrete, nonoverlapping zones: 
(a) a first, capture, zone of immobilized analyte or analog thereof bound 
to the chromatographic medium; 
(b) a second, detection, zone including an immobilized molecule that is a 
second member of the auxiliary specific binding pair with specific 
affinity for the first member bound to the chromatographic medium; and 
(c) a third, control, zone including an immobilized molecule capable of 
being specifically bound to the labeled specific binding partner 
covalently conjugated to the analyte or analog thereof bound to the 
chromatographic medium. 
This embodiment of the assay device is shown in FIG. 2. The assay device 
100 has a first opposable component 102 and a second opposable component 
104. The first and second opposable components 102 and 104 are joined by a 
hinge 106. The first opposable component 102 has a sample preparation zone 
108. The sample preparation zone 108 includes: (1) a labeled specific 
binding partner for an analyte conjugated to a first member of an 
auxiliary specific binding pair in a form that can be resolubilized by a 
liquid; and (2) a predetermined quantity of the analyte or an analog 
thereof covalently bound to a labeled specific binding partner for a 
molecule that does not substantially cross-react with the analyte in a 
form that can be resolubilized by a liquid, typically an aqueous liquid. 
Typically, as described above, the first member of the auxiliary specific 
binding pair is biotin and the second member of the auxiliary specific 
binding pair is an immobilized molecule having specific binding affinity 
for biotin, such as avidin, streptavidin, or an anti-biotin antibody. More 
typically, the second member of the auxiliary specific binding pair is 
streptavidin. The first opposable component 102 also has an absorber 110 
separated from the sample preparation zone 108 on the first opposable 
component 102. 
The second opposable component 104 includes a chromatographic medium 112 
that has a first end 114 and a second end 116. The chromatographic medium 
112 includes thereon: (1) a first, capture zone 118 of immobilized analyte 
or analog thereof bound to the chromatographic medium 112; (2) a second, 
detection zone 120 including an immobilized molecule that is a second 
member of the auxiliary specific binding pair with specific affinity for 
the first member bound to the chromatographic medium 112; and (3) a third, 
control zone 122 including an immobilized molecule capable of being 
specifically bound to the labeled specific binding partner covalently 
conjugated to the analyte or analog thereof bound to the chromatographic 
medium 112. These zones are arranged so that the first, capture zone 118 
is closest to the first end 114 of the chromatographic medium 112 and the 
third, control zone 122 is closest to the second end 116 of the 
chromatographic medium 112, with the second, detection zone 120 being 
between the first, capture zone 118 and the third, control zone 122. The 
chromatographic medium 112 can be backed by a plastic backing 124 as 
described above. 
The second opposable component 104 has an aperture 126 to allow viewing of 
a portion of the chromatographic medium 112, the portion including the 
detection zone 120 and control zone 122. The first and second opposable 
components 102 and 104 can be held together by engagers such as locks 128 
and 130. The first and second opposable components 102 and 104 can be 
surrounded by a gasket 132 to prevent the escape of fluids from the 
device. 
The first and second opposable components 102 and 104 are configured so 
that bringing the first and second opposable components 102 and 104 into 
opposition from a position in which they are not in opposition causes the 
absorber 110 to come into operable contact with the second end 116 of the 
chromatographic medium 112. It also causes the sample preparation zone 108 
on the first opposable component 102 to come into contact with the first 
end 114 of the chromatographic medium 112 so that the test sample, the 
resolubilized labeled specific binding partner for the analyte conjugated 
to the first member of the auxiliary specific binding pair, and the 
predetermined quantity of the analyte or analog thereof covalently bound 
to the labeled specific binding partner flow through the chromatographic 
medium 112 from the first end 114 to the second end 116. 
In use, the sample is added to the sample preparation zone 108 on the first 
opposable component 102, to resolubilize the labeled reagents. The assay 
device 100 or the first opposable component 102 can be inserted into an 
incubator, as described above for the first embodiment. The first and 
second opposable components 102 and 104 are then brought into opposition, 
and the sample and resolubilized labeled components are allowed to migrate 
through the chromatographic medium 112 from the first end 114 to the 
second end 116. 
In the absence of analyte, the labeled specific binding partner for the 
analyte conjugated to the first member of the auxiliary specific binding 
pair binds to the first, capture zone 118 of immobilized analyte or analog 
thereof and does not reach the second, detection zone 120 including the 
immobilized second member of the auxiliary specific binding pair. If, 
however, analyte is present in the sample, it competes for binding to the 
labeled specific binding partner for the analyte conjugated to biotin with 
the immobilized analyte or analog thereof present in the first, capture 
zone 118, and at least some of the labeled specific binding partner for 
the analyte then reaches the second, detection zone 120 including the 
immobilized second member of the auxiliary specific binding pair. The 
labeled specific binding partner for the analyte conjugated to the first 
member of the auxiliary specific binding pair is then bound at the second, 
detection zone 120, creating a detectable signal at that point. The 
intensity of the detectable signal is proportional to the concentration of 
analyte in the original test sample. 
However, the control zone 122 produces a detectable signal, whether or not 
analyte is present in the sample, because the predetermined quantity of 
the analyte or analog thereof covalently bound to the labeled specific 
binding partner for the molecule that does not substantially cross-react 
with the analyte present in the sample preparation zone 108 binds to the 
immobilized molecule capable of being specifically bound to the labeled 
specific binding partner at the control zone 122. This gives a constant 
signal which serves both as a control zone for proper flow through the 
device and a comparison zone so that a quantitative determination of the 
analyte concentration can be obtained. 
C. Multiplex Assay Devices 
Although the devices described above are described in terms of devices that 
perform a single assay, the same principles can be used to construct 
multiplex assay devices capable of carrying out more than one assay at the 
same time. These assay devices can be constructed so that the assays that 
can be performed simultaneously are for the same analyte or different 
analytes. For example, a multiplex device according to the present 
invention can be used to assay a number of different analytes and 
different aliquots of the same sample, such as different antibiotics in 
different aliquots of a sample of milk, or can be used to assay the same 
analyte in a number of different samples. This latter mode is particularly 
useful in assaying for a condition for which samples taken at different 
times from the same patient are to be assayed for the analyte of interest. 
Alternatively, one or more of the assays can be used for controls or 
reference standards. 
Multiplex devices according to the present invention can contain from 2 to 
12 or more sample preparation zones and chromatographic media, depending 
upon the assay for which the device is to be employed. Typically, the 
device contains from two to five separate sample preparation zones and 
chromatographic media. 
The principles of operation for the multiplex assay devices are exactly the 
same as those for the assay devices shown above in FIGS. 1 and 2, except 
for the performance of multiple assays on the same device. 
One embodiment of a multiplex assay device, employing the same principle as 
that shown in FIG. 1, is shown in FIG. 3. 
In general, this device comprises: 
(1) a first opposable component including: 
(a) a plurality of sample preparation zones, each sample preparation zone 
including a labeled specific binding partner for an analyte conjugated to 
a first member of an auxiliary specific binding pair in resolubilizable 
form; and 
(b) an absorber for absorbing fluid therein separated from the sample 
preparation zones on the first opposable component; and 
(2) a second opposable component hingedly attachable to the first opposable 
component including: 
(a) a plurality of first chromatographic media, one for each sample 
preparation zone, each first chromatographic medium having first and 
second ends and including thereon: 
(i) a first zone of an immobilized analyte or analog thereof bound to the 
first chromatographic medium; and 
(ii) a second zone of an immobilized molecule that is a second member of 
the auxiliary specific binding pair with specific affinity for the first 
member bound to the first chromatographic medium; 
(b) a plurality of second chromatographic media, one for each sample 
preparation zone, each second chromatographic medium having a first end 
and a second end and including thereon a comparison zone containing a 
known quantity of an analyte or analog thereof immobilized to the 
comparison zone; and 
(c) a plurality of comparison label zones, one for each second 
chromatographic medium, each comparison label zone including therein a 
labeled specific binding partner to the analyte or analog thereof in 
resolubilizable form in operable contact with the second chromatographic 
medium. 
In this version of a multiplex assay device according to the present 
invention, when the first and second opposable components are brought into 
opposition from a position in which they are not in opposition, the sample 
preparation zones come into operable contact with the first end of each 
first chromatographic medium to apply the sample and the labeled specific 
binding partner for the analyte conjugated to the first member of the 
auxiliary specific binding pair to each first chromatographic medium, and 
the absorber comes into operable contact with the second end of each first 
chromatographic medium and the second end of each second chromatographic 
medium to draw fluid through the first and second chromatographic media 
from their first ends to their second ends. As a result, the device gives 
a detectable indication of the presence of an analyte in each first and 
second chromatographic medium at a quantity greater than a predetermined 
amount by a comparison of the intensity of the label bound at the 
detection zone of each first chromatographic medium and at the comparison 
zone of each second chromatographic medium. 
The device 200 has a first opposable component 202 and a second opposable 
component 204, joined by a hinge 206. The first opposable component 202 
has a plurality of sample preparation zones 208. The first opposable 
component 202 has a plurality of absorbers 210, separated on the first 
opposable component 202 from each of the sample preparation zones 208. 
Alternatively, a single absorber 210 can be used, as long as there is no 
cross-contamination between samples. 
The second opposable component 204 has a plurality of first chromatographic 
media 212, each with a first end 214 and a second end 216. Each first 
chromatographic medium 212 has a first, capture zone of immobilized 
analyte 218, and a second, detection zone of an immobilized molecule that 
is a second member of the auxiliary specific binding pair with specific 
affinity for the first member 220. Each second opposable component 204 
also has a plurality of second chromatographic media 222, each with a 
first end 224 and a second end 226. Each of the second chromatographic 
media 222 has thereon a comparison zone 228. The second opposable 
component 204 has a plurality of comparison label zones 230. Each of the 
first and second chromatographic media 212 and 222 can be backed with a 
plastic backing 232. The second opposable component has one or more 
apertures 234 to allow viewing of the second, capture zones 220 and the 
comparison zones 228. The chromatographic device also has locks 236 and 
238 and a gasket 240. Preferably, each first chromatographic medium 212 
has a flow control indicator 242. 
A multiplex assay device operating by the same principles as the assay 
device of FIG. 2 is shown in FIG. 4. In general, this device comprises: 
(1) a first opposable component including: 
(a) a plurality of sample preparation zones, each sample preparation zone 
including: 
(i) a labeled specific binding partner for an analyte conjugated to a first 
member of an auxiliary specific binding pair in resolubilizable form; and 
(ii) a predetermined quantity of an analyte or an analog thereof covalently 
bound to a labeled specific binding partner for a molecule that does not 
substantially cross-react with the analyte in resolubilizable form; and 
(b) an absorber separated from the sample preparation zones; and 
(2) a second opposable component including a plurality of chromatographic 
media with first and second ends, each chromatographic medium including 
thereon in discrete, nonoverlapping zones: 
(a) a first, capture, zone of immobilized analyte or analog thereof bound 
to the chromatographic medium; 
(b) a second, detection, zone including an immobilized molecule that is a 
second member of the auxiliary specific binding pair with specific 
affinity for the first member bound to the chromatographic medium; and 
(c) a third, control, zone including an immobilized molecule capable of 
being specifically bound to the labeled specific binding partner 
covalently conjugated to the analyte or analog thereof bound to the 
chromatographic medium. 
In this version of a multiplex assay device according to the present 
invention, the first and second opposable components are configured so 
that bringing the first and second opposable components into opposition 
from a position in which they are not in opposition causes the absorber to 
come into operable contact with the second end of each chromatographic 
medium. This also causes the sample preparation zones to come into contact 
with the first end of each chromatographic medium so that the test 
samples, the resolubilized labeled specific binding partners for the 
analyte conjugated to the first member of the auxiliary specific binding 
partner, and the predetermined quantities of the analyte or analog thereof 
covalently bound to a labeled specific binding partner flow through each 
chromatographic medium from the first end to the second end. 
The assay device 300 has a first opposable component 302 and a second 
opposable component 304, joined by a hinge 306. The first opposable 
component 302 has a plurality of sample preparation zones 308 and 
absorbers 310, separated from the sample preparation zones 308. 
The second opposable component 304 has a plurality of chromatographic media 
312, each with a first end 314 and a second end 316. Each of the 
chromatographic media 312 has a first, capture zone 318, a second, 
detection zone 320, and a third, control zone 322. Each of the 
chromatographic media 312 can be backed by a plastic backing 324. The 
second opposable component 304 has an aperture or plurality of apertures 
326 to allow viewing of the detection zones 320 and control zones 322. The 
first and second opposable components 302 and 304 are held together by 
engagers such as locks 328 and 330, and are surrounded by a gasket 332 to 
prevent the escape of fluids. 
In use, the multiplex assay devices are used exactly as the assay devices 
performing single assays as shown above in FIGS. 1 and 2, allowing 
sufficient time to allow resolubilization of the reagents on the device 
and migration through the chromatographic media. 
II. ANALYTES AND SPECIFIC BINDING TNERS 
A. Analytes 
Typically, competitive assays such as those carried out by assay devices 
according to the present invention are used for monovalent analytes. The 
monovalent analytes are typically haptens, but the same principles can be 
used to assay any analyte that is monovalent, such as a normally 
multivalent antigen on which the additional antibody-binding sites are 
blocked or modified. 
Assayable analytes include the following: theophylline; digoxin; 
disopyramide; lidocaine; procainamide; propranolol; quinidine; amikacin; 
penicillin and other .beta.-lactam antibiotics including ampicillin, 
ampicillin derivatives, synthetic and semi-synthetic penicillins, and 
cephalosporins; gentamycin; kanamycin; netilmycin; tobramycin; 
tetracycline; sulfonamides such as phthalylsulfathiazole, sulfamethizole, 
sulfisoxazole, sulfamethazine, sulfisomidine, sulfacetamide, 
sulfanilamide, sulfaphenazole, sulfamethoxazole, sulfadiazine, 
sulfamethoxydiazine, sulfamethoxypyridazine, sulfadimethoxine, 
sulfamethoxypyrazine, sulfadoxine, and 4,4'-diaminodiphenylsulfone; 
tricylic antidepressants; ethosuximide; phenobarbital; diazepam; 
phenytoin; primidone; valproic acid; acetaminophen; acetylsalicylic acid; 
ibuprofen; methotrexate; drugs of abuse such as morphine, codeine, 
cocaine, fentanyl, 3-methylfentanyl, amphetamines, lysergic acid 
diethylamide, phencyclidine, 
N,.alpha.-dimethyl-1,3-benzodioxyl-5-ethanamine ("Ecstasy"), and heroin 
and their metabolites; DNP; 1-substituted-4-hydroxy-2-nitrobenzenes; 
4-substituted 2-nitro-trialkylanilinium salts; and environmental 
contaminants such as benzene, toluene, xylene, ethylbenzene, chlordane, 
DDT and its metabolites, 2,4-D, 2,4,5-T, and atrazine. 
Of these analytes, particularly important analytes are .beta.-lactam 
antibiotics, sulfamethazine, gentamycin, and tetracycline. These 
antibiotics often occur in milk, and it is desirable to have a rapid 
method of detecting their presence in milk. 
B. Specific Binding Partners 
Specific binding partners suitable for performance of assays using devices 
according to the present invention include, but are not limited to, 
antibodies and specific binding proteins. An example of the latter is the 
penicillin binding protein (PBP) isolated from Bacillus 
stearothermophilus. Other examples of specific binding proteins are 
protein receptors for hormones. 
Typically, antibodies against haptens are produced by immunization of an 
antibody-producing animal, generally a mammal such as a goat, rabbit, cow, 
horse, or sheep, with a hapten conjugated covalently to a carrier protein. 
The conjugation of a hapten to a carrier protein for antibody production 
is generally preferred and in some cases may be required. This is because 
most haptens are at most weakly immunogenic if injected into an 
antibody-producing animal without conjugation. 
A variety of carrier proteins are well-known in the art, and can include 
serum albumins of various species, keyhole limpet hemocyanin, 
thyroglobulin, ovalbumin, fibrinogen, polylysine, and purified protein 
derivative of tuberculin (PPD). 
A large number of coupling reagents are known in the art. Among the types 
of reactions used for conjugation of haptens to proteins are acylating 
reagents, alkylating reagents, redox reagents, and electrophilic reagents. 
The choice of reaction typically depends on the groups available in the 
hapten for reaction. If a hapten contains a free carboxyl group or a group 
that can readily be carboxylated, widely used methods include the mixed 
anhydride method, the carbodiimide method, using carbodiimides such as 
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, 
N,N'-dicyclohexylcarbodiimide, or 
1-cyclohexyl-3(2-morpholinoethyl)carbodiimide metho-p-toluenesulfonate, 
and the N-hydroxysuccinimide ester procedure, in which a carbodiimide is 
esterified with N-hydroxysuccinimide. 
Haptens with amino groups or nitro groups reducible to amino groups can be 
coupled to proteins through a number of reactions. If the amines are 
aromatic, they can be converted to diazonium salts by the slow addition of 
nitric acid and reacted with proteins at a moderately alkaline pH. Haptens 
with aliphatic amines can be conjugated to proteins by various methods, 
such as the use of carbodiimides, tolylene-2,4-diisocyanate, the use of 
maleimide compounds, or reaction with sodium periodate. Another approach 
is to convert aliphatic amines to aromatic amines by reacting with 
p-nitrobenzoylchloride and subsequently reducing to a p-aminobenzoylamide, 
which then can be coupled to proteins with diazotization. 
Bifunctional imidate esters, which react with amino groups to form 
amidines, can be used to conjugate haptens to proteins. Such imidate 
esters include dimethyladipimidate, dimethylpimelimidate, 
dimethylsuberimidate, and other similar analogs. 
Haptens with sulfhydryl groups can be conjugated to proteins with 
maleimides. Other reactions are possible, such as activation of the 
protein with bromoacetamide groups, or the formation of disulfide bonds 
between the carrier and hapten in acetate buffer, pH 4.0. 
For haptens with hydroxyl groups, it is generally preferred to convert the 
hydroxyl to another group. For example, an alcohol can be converted to the 
half ester of succinic acid, which introduces a carboxyl group available 
for conjugation. The bifunctional reagent sebacoyldichloride converts an 
alcohol to an acyl chloride, which reacts readily with proteins at 
slightly alkaline pH. Phenols can be activated with diazotized 
p-aminobenzoic acid, which introduces a carboxyl group, and then can be 
reacted with the carrier by a mixed anhydride reaction as described above. 
Sugars can be activated by the formation of a p-nitrophenyl glycoside 
followed by the reduction of the nitro group to an amino group and 
conjugation after diazotization, as for aromatic amines. Another method is 
based on the cleavage of vicinal glycols of sugars to aldehydes, which are 
then coupled to amines by reductive alkylation. Also, haptens can be 
conjugated through chlorocarbonates, prepared with an equimolar amount of 
phosgene. 
For haptens with aldehyde or ketone groups, carboxyl groups can be readily 
introduced through the formation of O-(carboxymethyl)oximes. Ketone groups 
can also be derivatized with p-hydrazinobenzoic acid to produce carboxyl 
groups which could be conjugated to the carrier as described above for 
carboxyl groups. Haptens containing aldehydes can be directly conjugated 
through the formation of Schiff bases which are stabilized by reduction 
with sodium borohydride. 
Conjugation methods are described generally in P. Tijssen, "Practice and 
Theory of Enzyme Immunoassays", (Elsevier, Amsterdam, 1985), ch. 12, pp. 
279-296. Other conjugation methods are known in the art and can be used 
for particular haptens. 
III. TEST KITS 
Another aspect of the present invention is test kits for performance of 
assays using assay devices according to the present invention. These test 
kits incorporate an assay device of the embodiment described above in 
Section I(B)(1), with two chromatographic media. 
Test kits according to the present invention can comprise: 
(1) the immunoassay device; and 
(b) a liquid for resolubilizing the labeled specific binding partner to the 
analyte or analog thereof in the comparison label zone. 
The liquid is typically an aqueous liquid. The aqueous liquid is typically 
water, saline, or a buffered solution, but other aqueous liquids can be 
used. The immunoassay device in the test kit can be either an assay device 
that performs a single assay or a multiplex assay device. If the 
immunoassay device is a multiplex assay device, the kit typically includes 
enough liquid for resolubilizing the labeled specific binding partner to 
the analyte or analog thereof in each comparison label zone. 
Test kits according to the present invention can also contain other 
reagents, such as reagents to extract or treat the sample. 
EXAMPLES 
The invention is illustrated by the following Examples. These Examples are 
for illustrative purposes only and are not to be construed as limiting the 
scope of the invention in any manner. 
Example 1 
Assay Device for Detection of .beta.-Lactam Antibiotics in Milk 
(Prospective Example) 
The assay device described in this example is intended for the detection of 
.beta.-lactam antibiotics (penicillins, ampicillin, or cephalosporins) in 
milk. It can be used for other assays as well. 
This device is constructed according to FIG. 1. The first opposable 
component 10 and the second opposable component 12 are solid bleached 
sulfite. The first opposable component 12 comprises a sample preparation 
zone 18 made of Cytosep (Ahlstrom Filtration, Holly Springs, Pa.). The 
sample preparation zone includes penicillin binding protein conjugated to 
biotin and labeled with colloidal gold. The sample preparation zone 18 
further includes 10% anti-rabbit immunoglobulin G from an animal such as 
sheep or goat. The biotin-labeled penicillin binding protein and 
anti-rabbit immunoglobulin G were separately mixed with equal volumes of 
conjugate diluent (5 mM borate, 0.1% Triton X-100, 1% bovine serum 
albumin, 5% sucrose, pH 8.0) and are dried at 37.degree. C. for 30 
minutes. 
The second opposable component 14 includes a first chromatographic medium 
22 of 20 .mu.M nitrocellulose (Schleicher & Schuell (Keene, N.H.)). The 
first chromatographic medium 22 has bound thereon at a first zone 28 
7-aminocephalosporanic acid conjugated to goat immunoglobulin G and dried 
on the first chromatographic medium from conjugate diluent as described 
above. Typically, the chromatographic medium has dimensions of about 0.5 
inch to about 1 inch for its length and of about 0.125 inch to about 0.375 
inch for its width. The first chromatographic medium also has a second 
zone 30 of streptavidin bound to the first chromatographic medium 22. The 
streptavidin is dissolved in conjugate diluent and dried down as above. 
The first chromatographic medium 22 further includes a flow control 
indicator 52 which is rabbit immunoglobulin G dissolved in conjugate 
buffer and dried down. The first chromatographic medium 22 is backed by a 
polycarbonate test strip backing (Lexan) 42. 
The first chromatographic medium 22 also includes a flow control indicator 
52, which is rabbit immunoglobulin G dried down from conjugate diluent as 
described above. The second chromatographic medium 32 is made of 20 micron 
nitrocellulose (Schleicher & Schuell). The dimensions of the second 
chromatographic medium are typically the same as those of the first 
chromatographic medium. The second chromatographic medium 32 includes 
thereon 5 ng penicillin G immobilized on the chromatographic medium 
through a protein carrier, such as keyhole limpet hemocyanin (KLH). The 
second chromatographic medium 32 further includes gold-labeled penicillin 
binding protein as the comparison label zone 40. 
In use, a raw milk sample is added to the sample preparation zone 18. The 
first opposable component 12 can be slid into an incubator. After the 
incubation step, 2 drops of reagent A (0.005M phosphate buffered saline, 
0.4% Tween 20, 1.25 mM HEPES, 0.0025% Triton X-100, 0.0015% EDTA, 0.25% 
sodium azide, pH 7.5) is added to the comparison label zone 40 on the 
second opposable component 32. The device is then closed and the results 
read. If the test line is less intense than the control line, the milk 
sample contains less than 5 ng of penicillin G or equivalent. Conversely, 
if the test line is more intense than the control line, the sample 
contains more than 5 ng of penicillin G. Color always appears at the flow 
control area provided proper flow has occurred. 
Example 2 
Test Device for Detection of Gentamycin, Sulfamethazine or Tetracycline in 
Milk 
(Prospective Example) 
A device for the detection of one of the antibiotics gentamycin, 
sulfamethazine, or tetracycline in milk is constructed according to 
Example 1, except that the gold-labeled penicillin binding protein 
conjugated to biotin on the sample preparation zone 18 of the first 
opposable component 12 is replaced with a monoclonal antibody for the 
antibiotic conjugated to biotin and labeled with colloidal gold. 
Similarly, the comparison label zone is the same monoclonal antibody 
labeled with colloidal gold. The first zone 28 of immobilized antigen or 
analog thereof bound to the first chromatographic medium 22 is the antigen 
covalently bound to an immunoglobulin G species that is not specifically 
bound by anti-rabbit immunoglobulin G antibody. The control line is 5 ng 
of the antigen or an equivalent immobilized on the second chromatographic 
medium 32. 
Other details of construction and performance of the assay are as in 
Example 1. 
Example 3 
Test Device for Detection of .beta.-Lactam Antibiotics in Milk (Alternative 
Format) 
(Prospective Example) 
A test device for the detection of .beta.-lactam antibiotics in milk can be 
constructed in the alternative format of FIG. 2. This device is 
constructed according to FIG. 2. The first and second opposable components 
102 and 104 are solid bleached sulfite. The sample preparation zone 108 
includes 5 ng/ml penicillin G (or equivalent) bound to colloidal gold via 
anti-rabbit immunoglobulin G. The sample preparation zone 108 also 
includes biotin-labeled penicillin binding protein labeled with colloidal 
gold. These reagents are dried down on the sample preparation zone, which 
is Cytosep (Ahlstrom), as above. The absorber 110 is Ahlstrom 270. 
The second opposable component 104 has a chromatographic medium 112 that is 
20-micron nitrocellulose (Schleicher & Schuell). The chromatographic 
medium 112 is backed by a Lexan backing. The chromatographic medium 112 
includes thereon: (1) the first capture zone 118 of 7-aminocephalosporanic 
acid conjugated to goat immunoglobulin G and immobilized on the 
chromatographic medium; (2) a detection zone 120 of streptavidin as in 
Example 1; and (3) a control zone 122, which also serves as a flow 
indicator, which is immobilized rabbit immunoglobulin G. In use, a raw 
milk sample is added to the sample preparation zone 108, and the test card 
is slid into an incubator as for Example 1. The device is then closed and 
the results are read. If the test line is less intense than the control 
line, the milk sample contains less than 5 ng of penicillin G or 
equivalent. Conversely, if the test line is more intense than the control 
line, the sample contains more than 5 ng of penicillin G. In each case, 
the control line, which also serves as the flow indicator, should give a 
detectable signal. 
Example 4 
Assay Device for Detection of Gentamycin, Sulfamethazine, or Tetracycline 
in Milk (Alternative Format) 
(Prospective Example) 
A device for the detection of gentamycin, sulfamethazine, or tetracycline 
in milk, in the alternative format, is constructed according to Example 3, 
except that the sample preparation zone 108 on the first opposable 
component 102 has 5 ng of antibiotic (or equivalent) covalently bound to 
anti-rabbit immunoglobulin G bound to colloidal gold, as well as a 
monoclonal antibody to the antigen (see Example 2) labeled with colloidal 
gold and bound to biotin. 
The capture zone 118 on the chromatographic medium 112 is the antigen 
covalently bound to goat immunoglobulin G. Other details of construction 
and performance of the assay are as in Example 3. 
ADVANTAGES OF THE INVENTION 
Chromatographic assay devices according to the present invention can 
perform semiquantitative assays for a wide variety of antigens and haptens 
while providing a positive indication of correct performance of the assay. 
Chromatographic assay devices according to the present invention also 
provide an advantage in being constructed of opposable elements. The use 
of opposable elements provides great versatility, as it permits the 
performance of reactions in a number of different sequences. This is 
possible because the use of such opposable elements allows the delivery of 
reagents to precisely defined regions of a test strip or other reaction 
component. The use of opposable elements also provides optimum performance 
with minimum consumption of reagents by ensuring that reagents are not 
wasted by being sequestered in dead volumes of apparatus. Finally, the use 
of opposable components provides optimum containment of possibly 
contaminated blood samples, each as those containing HIV or hepatitis 
virus. 
The use of opposable elements that can be brought into opposition by direct 
manual closure allows the operator to perform assays according to the 
present invention without the use of additional liquids or breakable 
capsules. This allows the performance of assays without the addition of 
additional liquid once the sample or samples are applied to the 
chromatographic medium, avoiding dilution and preserving the sensitivity 
of assays performed with assay devices according to the present invention. 
Additionally, chromatographic assay devices according to the present 
invention allow the rapid and accurate detection of clinically important 
analytes. The construction of the devices allows more even application of 
the samples to the chromatographic medium, and reduces interference that 
might otherwise be introduced by particulates or colored samples. The use 
of colloidal metal labels in a resolubilizable form provides extremely 
rapid kinetics of labeling and improves the performance of the assay. 
Test methods using devices according to the present invention have a wide 
dynamic range and are substantially free from false negatives that may 
occur in other test methods at high concentrations of analyte. 
Although the present invention has been described with considerable detail, 
with reference to certain preferred versions thereof, other versions and 
embodiments are possible. These versions include other arrangements of 
two-component devices that operate by the basic principles described 
herein and perform competitive immunoassays. Therefore, the scope of the 
invention is determined by the following claims.