"Immunochromatographic assay with support having bound ""MIP"" and second enzyme"

Chromatographic immunoassay employing a specific binding pair member and a label conjugate which delineates a border whose distance from one end of the chromatograph relates to the amount of analyte present. By combining the label conjugate and sample in a solution and immunochromatographing the solution, or employing a combination of enzymes, one enzyme being the label and the other enzyme affixed to the chromatographic support, the position of the border defined by the label can be related to the amount of analyte in the sample solution. Preferably, an immunochromatograph is employed having both a specific binding pair member and an enzyme affixed to the support. A sample is chromatographed and the amount of analyte is determined by (1) contacting the chromatograph with a second enzyme conjugated with a specific binding pair member which binds to the chromatograph in proportion to the amount of analyte bound to the chromatograph, or (2) including the second enzyme conjugate with said sample, resulting in a defined border related to the amount of analyte in the sample. The two enzymes are related in that the substrate of one is the product of the other, so that upon contact of the chromatograph with appropriate reagents, a detectable signal develops which permits detection of the border to which the analyte traveled. This distance can be related to the amount of analyte present in the sample.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
The field of competitive protein binding assays or specific binding assays 
has greatly expanded, as its importance in the diagnostic field has become 
recognized. The ability to be able to detect a specific compound and 
measure the compound quantitatively has permitted the monitoring of the 
administration of a wide variety of drugs, the determination of an 
imbalance in a wide variety of hormones, the quantitation of 
physiologicaly active proteins, and the diagnosis of the presence of a 
pathogen. The different techniques have been distinguished in requiring or 
not requiring separation steps, the nature of the signal developed by a 
label, the development of the signal in a solution or on a surface and the 
manner of measurement for a quantitative determination. 
In developing an assay, there are a number of considerations in devising 
the reagents and protocol. One consideration is the degree of 
sophistication of the individual performing the assay. There are many 
situations where it is desirable to have a relatively untrained individual 
to be able to carry out an assay and obtain reasonably quantitative 
results. In these situations, it is desirable that the assay be reasonably 
free from interference by materials in the sample, be relatively free of 
fluctuations with changes in environmental conditions and provide for easy 
measurement. Also, washings can be a source of error, either because of 
inadequate washing, leaving non-specific binding material, or by reversing 
specific binding. 
2. Description of the Prior Art 
U.S. Pat. No. 4,168,146 describes an immunoassay employing 
immunochromatography with antigens followed by contacting the 
immunochromatograph with an aqueous solution containing labeled 
antibodies. U.S. Pat. No. 4,233,402 describes a homogeneous assay method 
employing a combination of enzymes, where the substrate of one enzyme is 
the product of another. Enhanced production of the product is related to 
the amount of analyte in the assay medium. U.S. Pat. No. 4,275,149 
describes the use of particles where combinations of enzymes may be 
employed, where the presence of the particles enhances the interaction 
between two enzymes, where the product of one enzyme is the substrate of 
the other. Enhanced production of the final product due to the presence of 
the two enzymes bound to the particle as a result of binding of specific 
binding pair members is related to the amount of analyte in the assay 
medium. 
SUMMARY OF THE INVENTION 
Novel immunochromatographic methods are provided for detecting an analyte 
where a quantitative determination may be readily made without special 
equipment. The analyte is immunochromatographed on a bibulous carrier in 
the presence or absence of a labeled conjugate where the label is a member 
of an enzymatic signal producing system, which includes one or more 
enzymes. After chromatographing the analyte, if the enzyme conjugate was 
not included in the sample, the chromatograph is contacted with a labeled 
specific binding pair member which binds to the chromatograph in relation 
to the distance travelled by the analyte. By providing appropriate 
reagents, in the case of two enzymes where the substrate of one enzyme is 
the product of the other enzyme, a final product is produced which 
provides for a detectable signal, where the distance traveled by the 
analyte may be defined, which distance is related to the amount of analyte 
in the sample. 
DESCRIPTION OF THE SPECIFIC EMBODIMENTS 
Methods and compositions are provided for use in determining the presence 
of an analyte in a sample. The method involves an immunochromatograph 
where a border is developed intermediate the ends of the 
immunochromatograph, where the distance of the border from one end of the 
immunochromatograph is related to the amount of analyte in a sample. 
While various protocols may be employed, invariably, the 
immunochromatograph will be contacted at one end with a sample solution 
and the solution will travel up the immunochromatograph. Various 
techniques may then be used by combining one or more reagents which 
provide a clear delineation between the region to which the analyte is 
bound and the region free of analyte. The techniques may involve a single 
reagent or a combination of reagents, in order to enhance the clear 
delineation between the region where analyte is bound from the region 
where it is absent. This may involve, in the analyte bound or the analyte 
free region, providing a detectable signal throughout one or the other 
region, resulting in two distinct regions, or developing a distinctive 
border between the two regions. By appropriate choice of the reagents and 
materials employed, one can provide for production of signal in either of 
the regions, analyte bound or analyte free, or primarily at the border 
between the two regions. By employing the subject protocols and reagents, 
wash steps are minimized and adventitious interference with a quantitative 
result is substantially avoided. 
In performing the subject assay, an analyte is measured which is a member 
of a specific binding pair consisting of ligand and receptor. The ligand 
and receptor are related in that the receptor specifically binds to the 
polar and spatial organization of the ligand, being able to distinguish 
the ligand from other compounds having similar characteristics. An 
immunochromatograph is employed which is characterized by having one of 
the members of the specific binding pair non-diffusely bound to a bibulous 
support which allows for movement of a liquid by capillarity through the 
support. In addition to the specific binding pair member, there will also 
be an enzyme member of a signal producing system. The signal producing 
system involves reagents which allow for an accurate determination of the 
region in which the analyte is bound or absent, while minimizing washing 
or other steps to reduce non-specific binding. 
In carrying out the assay, the immunochromatograph is contacted with the 
sample containing solution. The sample containing solution may also 
include a member of the signal producing system bound to a specific 
binding pair member. Alternatively, or in addition, the signal producing 
members other than any signal producing members initially part of the 
immunochromatograph may be added in one or more successive solutions. The 
sample will traverse a region of the immunochromatograph by elution or 
solvent transport, and, in some protocols, a conjugate of a specific 
binding pair member and a signal producing system member will also 
traverse the immunochromatograph. 
To further explain the subject invention, in the subsequent description of 
the subject invention, the following definitions will be used. 
DEFINITIONS 
Analyte--The compound or composition to be measured, which may be a ligand, 
which is mono- or polyepitopic, antigenic or haptenic, a single or 
plurality of compounds which share at least one common epitopic site or a 
receptor. 
Specific Binding Pair ("Mip")--Two different molecules, where one of the 
molecules has an area on the surface or in a cavity which specifically 
binds to a particular spatial and polar organization of the other 
molecule. The members of the specific binding pair are referred to as 
ligand and receptor ("anti-ligand"). For the most part, the receptor will 
be an antibody and the ligand will serve as an antigen or hapten and to 
that extent are members of an immunological pair. Therefore, each of the 
members may be referred to as a mip, it being understood that "mip" is 
intended to include all ligands and all receptors. 
Ligand--Any organic compound for which a receptor naturally exists or can 
be prepared. 
Receptor ("Anti-Ligand")--Any compound or composition capable of 
recognizing a particular spatial and polar organization of a molecule, 
i.e. epitopic site. Illustrative receptors include naturally occuring 
receptors, e.g. thyroxin binding globulin, antibodies, enzymes, FAB 
fragments, lectins and the like. 
Label--The label may be any molecule conjugated to another molecule or 
support and where two molecules are involved is arbitrarily chosen as to 
which molecule is the label. In the subject invention, the labels will be 
a mip which is conjugated to a support or a member of the signal producing 
system that is conjugated to a support or a mip. 
Signal Producing System--The signal producing system may have one or more 
components, at least one component being conjugated to a mip. The signal 
producing system produces a measureable signal which is detectable by 
external means, normally by measurement of the electromagnetic radiation, 
desirably by visual examination. For the most part, the signal producing 
system involves chromophores and enzymes, where chromophores include dyes 
which absorb light in the ultraviolet or visible region, phosphors, 
fluorescers and chemiluminescers. 
Immunochromatograph--The immunochromatograph has a plurality of mips, 
either ligand or receptor, bound in an region to a bibulous support which 
allows for the movement of a liquid across the region with transport of 
the analyte and, as appropriate, any members of the signal producing 
system. The mips are non-diffusively bound to the support, either 
covalently or non-covalently. In addition, one or more members of the 
signal producing system may be non-diffusively bound to the bibulous 
support, either covalently or non-covalently. 
METHOD 
The subject method is carried out on a bibulous support involving a 
stationary solid phase and a moving liquid phase. The stationary solid 
phase may be contacted with a plurality of reagents in sequence in a 
number of different solutions, normally omitting wash steps between 
contacting with subsequent reagent compositions. 
The region in which the mip is non-diffusively bound to the bibulous 
support is referred to as the immunosorbing zone. The analyte from the 
sample will traverse this zone being carried along with a solvent whose 
front crosses the zone. The analyte, which is the homologous or reciprocal 
mip to the mip bound to the support, becomes bound to the support through 
the intermediacy of mip complex formation. The signal producing system 
provides the manner in which the area in the immunosorbing zone to which 
the analyte is bound may be distinguished from the area in which it is 
absent, so that the distance from a predetermined point on the 
immunochromatograph is a measure of the amount of analyte in the sample. 
The incremental movement of the sample through the immunosorbing zone 
results from dissolving the sample in an appropriate solvent and the 
transport of the solution through the immunosorbing zone due to 
capillarity. 
The solvent will normally be an aqueous medium, which may be up to about 40 
weight percent of other polar solvents, particularly oxygenated solvents 
of from 1 to 6, more usually of from 1 to 4 carbon atoms, including 
alcohols, ethers and the like. Usually, the cosolvents will be present in 
less than about 20 weight percent. 
The pH for the medium will usually be in the range of 4-11, more usually 
5-10 and preferably in the range of about 6.5-9.5. The pH is chosen to 
maintain a significant level of binding affinity of the mips. Various 
buffers may be used to achieve the desired pH and maintain the pH during 
the elution. Illustrative buffers include borate, phosphate, carbonate, 
tris, barbital and the like. The particular buffer employed is not 
critical to this invention, but in individual assays, one buffer may be 
preferred over another. 
Desirably, from about 0.05 to 0.5 wt. % of a non-ionic detergent is 
included with the sample. Various polyoxyalkylene compounds may be 
employed of from about 200 to 20,000 daltons. 
Moderate, and desirably substantially constant, temperatures are normally 
employed for carrying out the assay. The temperatures for the elution and 
production of a detectable signal will generally be in the range of about 
10.degree.-50.degree. C., more usually in the range of about 
15.degree.-50.degree. C., and frequently will be ambient temperatures, 
that is, about 15.degree.-25.degree. C. 
The concentration of analyte which may be assayed will generally vary from 
about 10.sup.-4 to about 10.sup.-15 M, more usually from about 10.sup.-6 
to 10.sup.-14 M. Considerations, such as whether the assay is qualitative, 
semi-qualitative, or quantitative, the particular detection technique; the 
concentration of the analyte of interest; and the protocol will normally 
determine the concentration of the other reagents. 
While the concentrations of many of the various reagents in the sample and 
reagent solutions will generally be determined by the concentration range 
of interest of the analyte, the final concentration of each of the 
reagents will normally be determined empirically to optimize the 
sensitivity of the assay over the range of interest. However, with certain 
protocols, individual reagents may be used in substantial excess without 
detrimentally affecting the sensitivity of the assay. 
The size of the immunosorbing zone need have no upper limit, except for 
practical considerations. Since, for the most part, low concentrations are 
being assayed, the width of the immunoabsorbing zone will tend to be 
relatively narrow, so that the analyte may traverse a reasonable distance 
and provide for reasonable differentiation over the concentration of 
interest. Generally, the width of the strip will not be less than about 
0.2 mm and not more than about 2 cm, generally ranging from about 5 mm to 
20 mm, preferably from about 5 mm to 15 mm. A plurality of individual 
strips may be used. Instead of strips, cylinders may be employed, e.g. a 
rod. 
The length of the immunoabsorbing zone will be desirably at least about 2 
to 10 times the width, usually at least about 2 mm, more usually at least 
about 10 mm, preferably at least about 23 mm, and not more than about 12 
cm, usually not more than about 10 cm, preferably from about 5 to 10 cm. 
The distance traversed is a factor in the time required for the assay, 
which will be taken into account with the other factors affecting the time 
required for the assay. 
Other reagents which are members of the signal producing system may vary 
widely in concentration depending upon the particular protocol and their 
role in signal production. In a "true" competitive situation between a 
labeled mip and the analyte, usually the labeled mip will not exceed 10 
times the maximum concentration of interest of the analyte and will not be 
less than about 0.5 times the minimum concentration of interest. In most 
other situations, the amount of the other reagents involved in mip complex 
formation may be present in an amount substantially less than the binding 
equivalent of analyte or in substantial excess to the binding equivalent 
of analyte. Therefore, no simple relationship can be provided. 
In carrying out the assay, the protocol will normally involve dissolving 
the sample into the eluting solvent. The sample may be derived from a wide 
variety of sources, such as physiologic fluids, illustrated by blood, 
serum, plasma, urine, ocular lens fluid, spinal fluid, etc., chemical 
processing streams, food, pesticides, pollutants, etc. 
One end of the chromatograph will then be contacted with the eluting sample 
containing solvent, which will normally be a buffered aqueous medium which 
may contain one or more members of the signal producing system. Where a 
member of the signal producing system is present, at least one member will 
be conjugated to a mip to provide a mip-label conjugate. 
Sufficient time will be allowed for the solvent front to completely 
traverse the immunosorbing zone. The zone has sufficient mip to insure 
that all of the analyte becomes bound in said zone without exhausting the 
mip bound in the zone. 
The labeled mip may be employed in three different ways, two of the ways 
involving the labeled mip being present in the eluting solvent and the 
third way involving the labeled mip being present in a reagent solution 
used after elution of the sample. The two ways involving the eluting 
solvent are either where the mip-label traverses the immunosorbing zone 
concurrently with the analyte, so as to actually compete with the analyte 
for available binding sites; or, where the mip-label does not have an 
apparent competition and primarily binds in a zone immediately beyond the 
zone in which the analyte has bound. In one instance, one obtains a zone 
extending from the initial line of contact of the sample containing 
solvent with the immunochromatograph, while in the other situation, a 
border results distinguishing between the zone in which the analyte is 
bound and the zone which is analyte free. 
In the third way, where one has antigenic analytes, the method can involve 
an initial contact with the sample, where the sample traverses the 
immunosorbing zone, followed by immersing the immunosorbing zone in a 
sample containing labeled mip, which binds to the antigen. This assay is 
conventionally referred to as a sandwich assay. Of course, where a hapten 
is involved, one can provide for a fixed amount of a polyligand, that is, 
the ligand can be polymerized or conjugated to a hub nucleus, so as to 
provide for a plurality of determinant sites common to both the haptenic 
analyte and the polyligand. In effect, one produces an antigen where the 
extent of travel of the synthetic antigen will be related to the amount of 
analyte in the sample. When the immunochromatograph is contacted 
substantially uniformly, e.g., immersing, spraying, etc., with a solution 
containing labeled mip, the labeled mip will bind to the available 
determinant sites of the antigen, resulting in a detectable signal 
defining a region related to the amount of analyte in the sample. 
Rather than employ an antigen, which acts as a bridge between two 
antibodies, one can employ one mip in the solution with the reciprocal mip 
on the immunochromatograph. After allowing the analyte to traverse the 
immunosorbing zone, the immunosorbing zone is contacted, substantially 
uniformly, e.g., immersing, spraying, etc., with a solution of labeled 
mip. The labeled mip is complementary to the mip bound to the 
immunoabsorbing zone, so that the labeled mip will bind to available 
binding sites defining the region in the immunoabsorbing zone free of 
analyte. In this way, the distance the analyte has traversed is indicated 
by the absence of an observable signal in the region containing the 
analyte, and the border is defined by the presence of the signal in the 
region free of the analyte. 
Depending upon the particular protocols, washings may be useful or 
desirable or may be avoided all together. The subject invention permits 
the elimination of washing steps. Preferred protocols are those which 
provide for a minimal number of steps with minimal possibility of operator 
error. Therefore, the devised protocols should minimize measurement steps 
where the results are responsive to errors in measurement. 
Where the immunochromatograph is not standardized to the extent that 
variations in conditions may change the distance the analyte traverses, a 
standard sample can be provided having a known amount of analyte. The 
analyte sample and the standard can be run at the same time, and a 
quantitative comparison can be made between the standard sample and the 
analyte sample. If necessary, more than one standard can be employed, so 
that the distance traversed can be graphed for the different 
concentrations and used to quantitate a particular sample. 
For the most part, relatively short times are involved for the 
immunochromatograph. Usually, the traverse of the sample through the 
immunosorbing zone will take at least 30 sec and not more than 1 hour, 
more usually from about 1 min to 30 min. The development of the signal 
will generally range from 30 sec to 30 min, more usually from about 30 
sec. to 5 min. 
The signal producing system has at least one enzyme and may have two or 
more other components of the signal producing system or one or more 
substrates, and may also include coenzymes. Any member of the signal 
producing system may be employed as a label, where the presence of the 
label on the immunochromatograph provides for a substantial change in 
signal in the area of the label. Therefore, labels may include enzymes or 
coenzymes, but not substrates. Usually, the label will be an enzyme. 
The label provides for a multiplicity of events in its vicinity by 
providing for enzyme turnover of a substrate. Thus, the member of the 
signal producing system which is used as the label will be referred to as 
the enzymatic signal amplifier and is limited to those members indicated 
above. 
The individual or combination of enzyme labels may be varied widely. The 
product producing the detectable signal may be a dye, fluorescer or 
chemiluminescer, with the signal detected by visual observation, due to 
absorption, fluorescence, or chemiluminescence, or a spectrophotometric 
measurement, employing measuring absorption, reflectance, fluorescence or 
chemiluminescence. 
For the most part the enzymes of interest will be oxidoreductases and 
hydrolases. A large number of enzymes of interest are set forth in U.S. 
Pat. No. 4,275,149. For combinations of enzymes one enzyme is 
non-diffusively bound to the immunochromatograph, while the other enzyme 
is conjugated to a mip. 
After the sample has traversed the immunosorbing zone, if the label-mip 
conjugate was not combined with the sample, the immunosorbing zone is 
contacted substantially uniformly with a solution having labeled-mip 
conjugate and depending on the label and protocol one or more other 
members of the signal producing system. 
In the case of an enzyme-mip conjugate the immunosorbing zone is contacted 
with a solution of enzyme-mip conjugate and substrate, optionally with a 
scavenger. In this situation, an enzyme is bound to the 
immunochromatograph in the immunosorbing zone, which is related to the 
enzyme bound to the mip, by the substrate of one being the product of the 
other. The enzyme-mip conjugate will normally be in an aqueous buffered 
solution and may be present in substantial excess of available binding 
sites. The pH range and buffers have been previously considered. After a 
sufficient time for the enzyme-mip conjugate to bind either to ligand or 
receptor, and for color to form, the immunochromatograph is removed from 
the solution. 
By having the two enzymes, a step in the protocol is eliminated since the 
enzyme-mip conjugate and substrate may be combined in the same solution 
without reaction prior to contacting the immunosorbing zone. 
After the enzyme-mip conjugate is bound to the immunochromatograph, by 
being present in the sample, the immunochromatograph is developed by 
immersion in a substrate solution. In this case an enzyme may or may not 
be bound to the immunochromatograph. 
With the coenzyme label, the developer solution will usually contain one or 
more enzymes to provide for regeneration of the coenzyme and substrate. 
Since the enzymatic reaction requires the coenzyme, the enzyme and 
substrate may be combined as a single developer reagent without any 
reaction prior to contact with the immunosorbing zone. 
The substrates will vary with the enzymes and are normally in substantial 
excess, so as not to be rate limiting (greater concentration than Km). The 
aqueous solution will usually be appropriately buffered for the enzyme 
system and may include a scavenger for the product of the enzyme which is 
the substrate of the other enzyme e.g. catalase for hydrogen peroxide from 
uricase. 
The immunochromatograph is contacted with the developer solution for a 
sufficient time to produce sufficient detectable signal producing compound 
so as to define the region of the immunosorbing zone in which the analyte 
is bound. Once the detectable signal has been produced, the distance from 
one end of the chromatograph may be measured as a quantitative measure of 
the amount of analyte in the sample. 
While some distortion may be observed at the border, in most situations the 
border is reasonably well defined, so that changes in concentration of 
factors of two or less in the .mu.g to pg range can be detected with a 
wide variety of analytes. Thus, by employing an appropriate dye precursor 
as a substrate, the amount of an analyte can be quantitatively determined 
by visual observation with a single measurement (the sample) by the user 
and a two-step protocol which is relatively insensitive to interference. 
MATERIALS 
The components employed in the subject immunochromatography are: the 
bibulous support, the mip conjugates, (which include the mip and the 
label), the mip bound to the bibulous support in the immunosorbing zone, 
remaining members of the signal producing system, analyte, and, as 
appropriate, polyligand or polyvalent receptor. 
ANALYTE 
The ligand analytes of this invention are characterized by being 
monoepitopic or polyepitopic, while the receptor analytes may have a 
single or plurality of binding sites. The polyepitopic analytes will 
normally be poly (amino acids), i.e. polypeptides and proteins, 
polysaccharides, nucleic acids, and combinations thereof. Such 
combinations or assemblages include bacteria, viruses, chromosomes, genes, 
mitochondria, nuclei, cell membranes and the like. 
For the most part, the polyepitopic ligand analytes employed in the subject 
invention will have a molecular weight of at least about 5,000, or usually 
at least about 10,000. In the poly(amino acid) category, the poly (amino 
acids) of interest will generally be from about 5,000 to 5,000,000 
molecular weight, more usually from about 20,000 to 1,000,000 molecular 
weight, and among hormones of interest, about 5,000 to 60,000 molecular 
weight. 
An extensive listing of useful ligands may be found in U.S. Pat. No. 
4,275,149, the disclosure bridging columns 12 to 17, which disclosure is 
incorporated herein by reference. 
The monoepitopic ligand analytes will generally be from about 100 to 2,000 
molecular weight, more usually from about 125 to 1,000 molecular weight. 
The analytes of interest include drugs, metabolites, pesticides, 
pollutants, and the like. 
A large number of analytes of interest are listed in U.S. Pat. No. 
4,275,149, columns 17 and 18, which disclosure is incorporated herein by 
reference. 
For receptor analytes, the molecular weights will generally range from 
about 10.sup.4 to 2.times.10.sup.8, more usually from about 
3.times.10.sup.4 to 2.times.10.sup.6. For immunoglobulins, IgA, IgD, IgE, 
IgG and IgM, the molecular weights will generally vary from about 160,000 
to about 10.sup.6. Enzymes will normally vary from about 10,000 to 600,000 
daltons. Natural receptors vary widely, being generally at least about 
25,000 molecular weight and may be 10.sup.6 and higher, including such 
materials as avidin, thyroxine binding globulin, thyroxine binding 
prealbumin, transcortin, membrane surface proteins, etc. 
Where a ligand is conjugated to another molecule or support, frequently the 
ligand will be modified to provide for a particular functional group at a 
particular site. This modification produces a product referred to as a 
ligand analog. U.S. Pat. No. 4,275,149 also has an extensive description 
of ligand analogs, bridging columns 18 and 19, which description is 
incorporated herein by reference. 
Immunochromatograph 
The immunochromatograph involves a bibulous support providing liquid travel 
through capillarity, a nondiffusively bound mip, and may also include one 
or more members of the signal producing system. 
A wide variety of supports may be used of different dimensions, 
particularly thicknesses, different materials and different shapes. For 
the most part, the shape will be elongated, conveniently a rectangular 
strip. At least a portion of the strip will have a mip uniformly bound to 
the strip. The size of the strip will be governed to some degree by 
convenience in handling. Also, the immunosorbing zone must be of 
sufficient size to be able to accommodate all of the analyte which may be 
present in the concentration range of interest of the analyte. Where the 
protocol involves binding of both analyte and labeled mip, then the 
immunosorbing zone must include capacity for both the analyte and labeled 
mip. 
A wide variety of bibulous supports may be used, which include both natural 
and synthetic polymeric materials, particular cellulosic materials, such 
as fiber containing papers, e.g. filter paper, chromatographic paper, 
etc., synthetic or modified natural occurring polymers, such as poly(vinyl 
chloride), cross-linked dextran, acrylates, etc., either used by 
themselves or in conjunction with a ceramic material, such as silica. 
The thickness of the immunochromatograph bibulous support will generally 
vary from about 0.05 mm to about 2 mm, more usually being about 0.1 mm to 
0.5 mm, preferably from about 0.2 mm to about 0.4 mm. The structure of the 
paper may be varied widely and includes fine, medium fine, medium, medium 
coarse and coarse. The surface may be varied widely with varying 
combinations of smoothness and roughness combined with hardness and 
softness. 
The immunochromatograph may be supported by a variety of inert supports, 
such as Mylar, polystyrene, polyethylene, or the like. The supports can be 
used as a backing spaced from the immunochromatograph, edging, or other 
structure to enhance the mechanical integrity of the immunochromatograph. 
The immunochromatograph may be coated with a wide variety of materials to 
provide for enhanced properties. Coatings may include protein coatings, 
polysaccharide coatings, sugars or the like, which are used particularly 
to enhance the stability of the materials conjugated to the support. These 
compounds may also be used for improved binding of the materials, such as 
the mip or signal producing system member bound to the 
immunochromatograph. 
The immunochromatograph may be activated with reactive functionalities to 
provide for covalent bonding of the organic materials to be conjugated to 
the support. Various techniques which may be used to activate the 
immunochromatograph's bibulous support, including functionalization with 
an acyl group e.g. carbonyldiimidazole, treatment with cyanogen bromide or 
difunctional agents such as glutaraldehyde, succinic acid, etc. Methods 
for binding of a wide variety of materials to a bibulous support may be 
found in the literature. See for example, U.S. Pat. No. 4,168,146. 
The amount of mip which is bound to the support will vary depending upon 
the size of the support and the amount required to bind all of the analyte 
and, as required, labeled mip. Generally, the amount of mip will range 
from about 10.sup.-5 to 10.sup.-14 moles/cm.sup.2, more usually from about 
10.sup.-7 to 10.sup.-12 moles/cm.sup.2. The number of moles per unit area 
will be varied in order to insure that there is sufficient discrimination 
in the concentration range of interest for the distance traversed by the 
analyte. 
In a preferred embodiment, a signal producing system member is 
non-diffusively bound to the bibulous support. Particularly, an enzyme is 
bound to the support which will interact with the labeled mip, where the 
label is another enzyme. The relationship of the enzymes will be discussed 
in the description of the signal producing system. 
Both the mip and the signal producing system member may be bound to a 
variety of supports by adsorption, rather than covalent bonding. This will 
involve contacting the bibulous support with the solution containing the 
mip and/or signal producing member, removing the immunochromatograph from 
the solution, and allowing the immunochromatograph to dry. Alternatively, 
the solution may be applied by spraying, painting, or other technique 
which will provide uniformity. 
Generally, relatively large sheets will be used which may then be cut to 
the appropriate dimensions. 
Signal Producing System 
The signal producing system will, for the most part, involve the production 
of a detectable signal involving the absorption or emission of 
electromagnetic radiation, particularly light in the ultraviolet and 
visible region, more particularly radiation having a wavelength in the 
range of about 400 to 800 nm. Because of the nature of the 
immunochromatograph, in order to have a detectable signal, it is necessary 
that there be a sufficient concentration of the label over a unit area. 
Therefore, for the most part, individual labels will not be sufficient to 
provide the desired sensitivity. To that extent, means must be provided 
for the generation of a plurality of detectable molecules associated with 
a single labeled mip, where the label which provides the means for such 
generation does not interfere with the traversing of the labeled mip, when 
the labeled mip traverses the immunosorbing zone. Therefore, one employs a 
label which produces a large number of molecules which can be detected, 
such as an enzyme or coenzyme. Amplification is then obtained by the 
presence of a single label. 
An enzyme or coenzyme is employed which provides the desired amplification 
by producing a product, which absorbs light, e.g. a dye, or emits light 
upon irradiation or chemical reaction, a fluorescer, or chemiluminescer. A 
large number of enzymes and coenzymes for providing such products are 
indicated in U.S. Pat. No. 4,275,149 bridging columns 19 to 23, and U.S. 
Pat. No. 4,318,980, columns 10 to 14, which disclosures are incorporated 
herein by reference. 
Of particular interest is the use of a combination of enzymes, where the 
enzymes are related by the product of one enzyme being the substrate of 
the other enzyme. In this manner, non specific interference is 
substantially reduced and the border between the zones containing the 
bound analyte and free of analyte is more effectively defined. 
A number of enzyme combinations are set forth in U.S. Pat. No. 4,275,149, 
bridging columns 23 to 28, which combinations can find use in the subject 
invention. This disclosure is incorporated herein by reference. 
Of particular interest are enzymes which involve the production of hydrogen 
peroxide and the use of the hydrogen peroxide to oxidize a dye precursor 
to a dye. Particular combinations include saccharide oxidases e.g. glucose 
and galactose oxidase, or heterocyclic oxidases, such as uricase and 
xanthine oxidase, coupled with an enzyme which employs the hydrogen 
peroxide to oxidize a dye precursor, e.g. peroxidase, microperoxidase, and 
cytochrome C oxidase. Additional enzyme combinations may be found in the 
subject matter incorporated by reference. While the above oxidoreductase 
combination is preferred, other enzymes may also find use such as 
hydrolases, transferases, and oxidoreductases other than the ones 
indicated above. 
Illustrative coenzymes which find use include NAD[H]; NADP[H], pyndixal 
phosphate; FAD[H]; FMN[H], etc., usually coenzymes which combine with 
oxidoreductases. For a number of coenzymes involving cycling reactions see 
particularly U.S. Pat. No. 4,318,980. 
The product of the enzyme reaction will usually be a dye or fluorescer. A 
large number of illustrative fluorescers are indicated in U.S. Pat. No. 
4,275,149, columns 30 and 31, which disclosure is incorporated herein by 
reference. 
By appropriate manipulation or choice of the label-mip conjugate, the 
receptors, the bibulous support and the conditions employed in performing 
the assay, two different embodiments of the subject invention can be 
achieved where the analyte and enzyme-mip are applied to the 
immunochromatograph in the same solution. In one embodiment, the region of 
the immunosorbing zone traversed by the analyte is observable due to 
production of the detectable signal substantially uniformly throughout the 
region in which the analyte is present. In the other embodiment, the 
detectable signal is primarily observable at a border related to the 
region in the immunosorbing zone occupied by the analyte. 
The different results may be related to different binding constants, rates 
of travel, adsorption or the like, of the label-mip conjugate as compared 
to the analyte. The variations can be achieved by varying the number of 
mips, particularly haptenic analytes, bound to the labels, varying the 
binding specificity of receptors bound to the bibulous support e.g. by 
preparing antibodies to an immunogen having one linking group between the 
hapten analyte and antigen and employing a different linking group with 
the label-hapten analyte conjugate, varying the solvent and/or support to 
vary the Rf factors, or other techniques. 
As a result of the use of two enzymes in the signal producing system with 
one enzyme as a label, a simplified protocol can be employed, also a 
strong detectable signal is obtained providing for accurate delineation of 
the front to which the analyte progressed. By having the product of the 
enzyme bound to the bibulous support be the substrate of the enzyme 
conjugated to the mip, a sharp, rapid and uniform development of the 
detectable signal is observed on the immunochromatograph. Furthermore, one 
establishes a high localized concentration of substrate for the enzyme 
bound to the immunochromatograph, so as to encourage the rapid deposit of 
the detectable signal producing compound at the surface. 
Kits 
As a matter of convenience, the immunochromatograph can be provided in 
combination with other reagents for use in assaying for an analyte. Where 
two enzymes are involved, the other reagents will include enzyme labeled 
mip, substrate for the enzyme bound to the support, any additional 
substrates and cofactors required by the enzymes, and the dye precursor, 
which provides the detectable chromophore or fluorophore. With the 
coenzyme label the coenzyme labeled mgs, appropriate enzyme(s) including 
the dye precursor will be included. In addition other additives may be 
included, such as stabilizers, buffers, and the like. The relative amounts 
of the various reagents may be varied widely, to provide for 
concentrations in solution of the reagents which substantially optimize 
the sensitivity of the assay. Particularly, the reagents may be provided 
as dry powders, usually lyophilized, including excipients, which on 
dissolution will provide for a reagent solution having the appropriate 
concentrations for combining with the sample.

EXPERIMENTAL 
The following examples are offered by way of illustration and not by way of 
limitation. 
The following abbreviations are used hereafter: HRP--horse radish 
peroxidase; NHS--N-hydroxy succinimide; EDCA--ethyl dimethylaminopropyl 
carbodiimide; DMF--dimethyl formamide; BSA--bovine serum albumin. 
Temperatures not otherwise indicated are Celsius, while parts are by 
weight except for mixtures of liquids which are by volume. 
EXAMPLE 1 
Preparation of Immunochromatograph 
Antibodies to theophylline (antitheophylline) and glucose oxidase are the 
materials to be conjugated. A sheet of Whatman 31ET of about 550 cm.sup.2 
is immersed in 1.8 L pyridine, 0.2 M in carbonyldiimidazole and the 
mixture gently stirred for one hour at room temperature. Additional sheets 
may be activated in the same activating solution. Each sheet is then 
washed with 300 ml tetrahydrofuran and air dried with an air gun over 
about 20 sec. The sheet is then immersed in a solution of 500 .mu.l of a 
49 mg/ml solution of antitheophylline, 790.5 .mu.l of a 16 mg/ml solution 
of glucose oxidase amine and 200 ml of buffer 0.1 M sodium phosphate, pH 
7.0, 0.2 M NaCl and the mixture mildly shaken for 4 hours at room 
temperature. After washing with the phosphate buffer, the solution is then 
immersed in a 4% aqueous Dextran T10 solution to serve as a preservative, 
followed by blotting the sheet, freeze drying and lyophilizing. 
EXAMPLE 2 
Conjugation of Theophylline and HRP 
Into a reaction flask was introduced 8.1 mg of 
1-methyl-3-(3'-carboxypropyl)xanthine, 3.8 mg of NHS, 6.7 mg EDAC and 125 
.mu.l DMF and the mixture allowed to stand overnight at room temperature. 
To four 1.3 ml samples of HRP-oxyamine (1 mg) in 0.1 M sodium carbonate, pH 
9.0 was added varying amounts of the ester prepared above to provide for 
preparations having mole ratios of theophylline to HRP of 400; 200, and 
two of 100 each. Into the first reaction mixture (400 mole ratio) was 
added 0.217 ml of DMF and 66 .mu.l of the above ester in 8.25 .mu.l 
increments over a period of about 2 hrs. Into the second reaction mixture 
(200 mole ratio), 0.238 ml of DMF was added and 33 .mu.l of the ester 
added incremently in 8.25 .mu.l increments. Into the third reaction 
mixture (100 mole ratio), 0.24 ml of DMF was added and 16.5 .mu.l of the 
ester added in 8.2 .mu.l increments, while in the final reaction mixture 
(100 mole ratio), no DMF was added, and 8.25 .mu.l of the ester was added 
in 2.1 .parallel.l increments. During the addition, the temperature was 
maintained at 4.degree., and the mixture then allowed to stand overnight 
at 4.degree.. 
The reaction mixtures were then worked up by chromatography on G-25 
Sephadex with standard buffer. Folin and UV spectroscopic analysis 
indicated theophylline/HRP ratios of 6.9, 4.0, 1.6 and 2.1, respectively. 
EXAMPLE 3 
Preparation of Glucose Oxidase Amine 
Glucose oxidase (Sigma, E.C. 1.1.3.4) was concentrated from 360 ml to 60 ml 
with Amicon PM10 membrane at a pressure below 30 psi. The concentrate of 
glucose oxidase was dialyzed twice against 4 L of water at 4.degree., 
filtered and shown spectrophotometrically to have a concentration of 32 
mg/ml. To 51.5 ml of the glucose oxidase solution was added dropwise 5.15 
ml of 0.2 M sodium periodate, the reaction occurring over 25 min. The 
product was chromatographed on a 2.5.times.60 cm column of Sephadex G-50 
using 2 mM sodium acetate pH 4.5, and the major glucose oxidase peaks 
pooled to yield 91.5 ml of a solution containing the aldehyde derivative. 
To the solution was added dropwise 6 ml of 3 M ethylene diamine in 0.2 M 
sodium carbonate, pH 9.5, and the reaction allowed to proceed for 3 hr. To 
the mix was then added about 3.9 ml of 10 mg/ml sodium borohydride, the 
mixture incubated overnight and then chromatographed to remove the sodium 
borohydride. 
EXAMPLE 4 
Preparation of HRP-Oxyamine 
To 5 ml of 10 mg/ml horse radish peroxidase in 5 mM sodium acetate, pH 4.5 
buffer, was added 50 ml 0.2 M sodium periodate and the mixture stirred for 
30 min, followed by chromatography on a G-50 Sephadex column, eluting with 
2 mM sodium acetate buffer, pH 4.5. The protein fractions were pooled to 
29 ml, the mixture cooled to 4.degree. C. and 2.9 ml of 0.2 M 
2,2'-oxy-bis-ethylamine in 0.5 M carbonate buffer, pH 9.5 at 4.degree. C. 
added. The pH of the mixture was adjusted to 9.5 with 1 N sodium 
hydroxide, stirred for 2 hrs and 3.52 ml of a 4 mg/ml sodium 
borohydride-water solution added and the mixture allowed to react for 3 
hr, followed by chromotography through a Sephadex G-50 column. 
The above procedure was repeated using 400 mg of HRP and 3.5 g of 
2,2'-oxy-bis-ethylamine. No significant change in enzyme activity was 
observed between the native amine and the modified amine, which has about 
four additional amino groups. 
In carrying out the assay, 90.times.8 mm strips were prepared from the 
sheet prepared in Example 1 and the end of the strip dipped into 1 ml of 
sample in 0.1 M NaPO.sub.4, 0.2 M NaCl, pH7.0 and 1 mg/ml BSA, with a 
number of samples prepared containing different amounts of theophylline 
and containing 0.2% Triton DN-65. After 12 min, the strip was then dipped 
into 5 ml of 0.2 .mu.g/ml HRP-theophylline conjugate, so as to be immersed 
in the solution and allowed to stand for 10 min. The strip was then 
removed from the enzyme solution and dipped in a development solution 
comprising 5 ml of 50 mM glucose and 200 .mu.g/ml 4-chloro-1-naphthol and 
allowed to stand for 20 min. The distance of the border from the top of 
the wick was graphed against the samples having differing concentrations 
of theophylline to provide the following results. 
TABLE I 
______________________________________ 
Theophylline Distance of Border 
ng/ml From Strip Top, mm 
______________________________________ 
50 66 
100 55 
200 47 
500 42 
______________________________________ 
In the next study, the procedure of Example 1 was repeated. The strips 
employed were 65.times.8 mm in size. The protocol employed was to dip the 
end of the strip into 0.5 ml of the solution containing 0.4 .mu.g/ml of 
the HRP-theophylline conjugate with different samples having varying 
concentrations of theophylline, each sample containing 0.2% Triton DN-65 
and the strip allowed to stand in the sample solution for 6 min. At the 
end of this time, the strip was immersed in the 
glucose-(4-chloro-1-naphthol) developer solution to provide the following 
results. 
TABLE II 
______________________________________ 
Theophylline Distance of Border 
ng/ml From Strip Top, mm* 
______________________________________ 
0 53 
50 46 
100 38 
200 31 
500 26 
______________________________________ 
*average of 2 values 
In the next example, the paper employed was S.S.589WH, employing a 4% 
dextran T10 solution as a preservative. The sample solution was 0.5 ml 
containing varying amounts of theophylline, 0.4 .mu.g/ml of a 
theophylline-HRP conjugate, having an average of about three theophyllines 
per enzyme, 0.1% Triton X-100, in the buffer indicated previously. After 
the sample solution had traversed the immunochromatograph, the 
immunochromatograph was developed for 10 min with the development solution 
previously described. It was noted that most of the color which developed 
was in a narrow band at the border. The following table indicates the 
results. 
TABLE III 
______________________________________ 
Theophylline Distance of Border 
ng/ml From Strip Top, mm* 
______________________________________ 
0 26, 25 
50 21, 23 
100 18, 18 
200 15, 12 
500 11, 9 
______________________________________ 
*two different determinations 
It is evident from the above results that a sensitive simple method is 
provided for quantitatively determining a wide variety of analytes. The 
protocols are particularly free of nonspecific interference, avoid a 
plurality of measurements of reagents which introduce errors and are 
capable of giving a visual result, so as to avoid the need for expensive 
measuring equipment. In addition, the assay is rapid, so that the result 
can be carried out in the time a patient stays in a doctor's office. Also, 
the protocols do not require intermediate washing steps, which is 
particularly important where relatively untrained personnel are to carry 
out the determination. Washing steps, where required have been a 
continuous source of substantial error. 
Although the foregoing invention has been described in some detail by way 
of illustration and example for purposes of clarity of understanding, it 
will be obvious that certain changes and modifications may be practices 
within the scope of the appended claims.