CA2110920A1 - Assay for the detection of specific ligands - Google Patents

Abstract

The invention disclosed herein relates to the field of one-step assays and particle agglutination tests. In one embodiment of the present invention, there is disclosed an analytical device for the detection or determination of an analyte antibody in a bodily fluid comprising a layer of a plurality of substantially planar zones adjacent one another and in absorbent contact with one another, the layer including a sample application zone, a conjugate zone containing antigen bound to mobile particles, and a detection zone containing immobilized antigen, wherein the antigen is the same in both the conjugate and detection zones and is an antigen that binds with the analyte antibody, the liquid sample is capable of moving from the sample application zone through the conjugate zone and on to the detection zone, and if the analyte antibody is present in the sample it is detected in the detection zone. The present invention also discloses methods of detecting various analytes, particularly analyte antibodies, including specific immunoglobulins. The invention further provides improved methods of preparing and using one-step assays, as well as improved methods of preparing coated particles for use in diagnostic assays.

Description

W;~ 92/22797 PCI'/US92/03680 A88AY FOR TB DETBCT~ON OF 8PECIFIC LIGA~

RELATION TO RELATED APPLICATION
This application is a continuation-in-part of the U.S.
Patent Application, serial no. unknown, filed June 13, 1991, naming Tzenq, et al., as inventors. Priority of subject matter in this spplication common with subject matter in that application is hereby claimed.

~CKGRQUND OF THE INVENTION
Recent technological advances have made it po~sible to tailor assays for a wide variety of analytes, especially tho~e molecules exhibiting antigenic characteristics, such as polypeptides, nucleotides, whole cells, and cellular fragments, to name but a few. In general, moct assays currently in use tend to use antibodies to "capture~ antigenic materials in a liquid-p~a~e or a ~olid-phase format.
Neverthelec~, a~says with an improved sensitivity are needed, as many conditions and di~ea~e~ do not lend themselves to early diagno~i~ via antigenic detection. In many instances, it would be preferable to be able to detect an increase in a specific population of cells or molecules in an ~ organism, which population is produced in response to an - "invasion" of the organism by a specific antigenic material.
For example, in the case of certain viral diseases, by the time a sufficiently detectable titer of ~iral particles i5 present in an organism's blood, the time for effective therapy may well have passed. In addition, diseases that trigger highly specific, but virtually undetectable, responses by an organism's immune system, do not lend themselves to accurate or easy detection via currently-available a6says. Moreover, agents which provoke a response by a specific population or subpopulation of immunoglobulins are often not detectable until the resultinq disease is full-blown, thus limiting diagnostic, as well as therapeutic, options. For example, an assay claiming to facilitate the differential detection of W092/22797 PCT/USs2/036~D
211092~ ~

various immunoglob~lin species from each other is described in U.S. Patent No. 4,292,403 to Duermeyex. Specifically, this assay purports to detect an antigen-specific immunoglobulin of a particular immunoglobulin class, which classes include IgM, IgG, IgA, IgD, or IgE, by using anti-antibodies against the specific immunoglobulin class. Like many other assays disclosed in the art which use anti-antibodies (e.g., U.S.
Patent No. 4,818,688 to Adamich, et al., No. 4,828,981 to Maggio, et al., and No. 4,962,023 to Todd, et al.), the assay described by Duermeyer is complex and involves a multiplicity of specific reagents. Furthermore, none of the~e as6ays can easily distinguish an antigen-specific subpopulation of an immunoglobulin cla~s from other, non-antigen-specific members of its class. That is, prior to the advent of the present invention, it has not been a simple matter to detect subpopulations of specific IgG or IgM molecules, for example, which are precisely sensitized to a single antigenic substance.
An illustrative example of the need for as6ays with increased sensitivity i8 provided by consideration of available assays for the detection of antibodies, and in particular, for the detection of immunoglobulins directed against specific antigens. Lyme Di~ease provides such an illustrative example.
Lyme Borreliosis was first identified in Lyme, Connecticut in 1i75. It has now been reported across several continents, including North America, Europe, Russia, Asia, Africa and Australia. The disease is caused by the tick-transmitted spirochete Bome~a b~gdo~en. This infection can produce a wide spectrum of clinical symptoms which can be confusèd with other entities. Therefore, precise d~agnosis is critical.
~yme disease is the most commonly reported tick-borne illness in the United States. The disease is most pre~alent WOs2/22797 PCT/US92/03680 2~ 2 0 in the northeast, upper midwest, and west coast states. The seasonal onset of the disease is synchronous with the ny~phal stage of the ticks. Thus, the disease is more common in summer and early fall.
Antibody titers specific to Bomelia bu~do~en are typically negative during early illness. Patients with only erythema migrans also rarely have elevated antibody titers. The specific IgM levels begin to rise two week~ after the onRet of the disea~e and peak at three to 6iX week~. The specific IgG
levels tend to lag behind the ~pecific IgM titers by approximately two weeks, but ~re often positive during the latter part of the clinical stage of erythema migran~ and usually remain positive during the second and third clinical stages with manifestations of carditis, neurological disea~e or arthriti~.
Generally, the ~natural history" or progre~sion of Ly~e disease may be divided into three clinical stages. The first ~ta~e is cbaracterized by the development of an eKpanding annular red rash-like ~kin lesion, erythema migrans, which occurs at the ~ite of the tick bite and typically la~ts two to four week~. The erythema migrans may be followed by cardiac, joint and neurological abnormalities in the second stage, which occurs one to four months after the disease onset. The last stage is characterized by arthritiE involving a few large joint~ which may begin as early as three months after onset.
This stage can last several years or may become chronic.
Early diagnosis permits prompt treatment with appropriate antibiotics that can halt the progression of the disease.
Since the spirochete is often difficult to culture from affected skin or body fluids of patients, serological detection of antibodies is considered the best àvailable diagnostic means for Lyme diseaeQ. Thè specific IgM against Bome~a bugdo~en is often not detectable during the first two weeks, but it usually peaks three to six weeks after the initial infection, and then persists or declines. The W092/227s7 PCT/US92/036~0 21 iû92() ~ 4-response of the specific IgG to the spirochete is frequently not detectable for four to six weeks, but peaks in the arthritic stage and often remains elevated for years.
Therefore, in response to an express need for an assay procedure with diverse applicability, which is capable of detecting specific ligands, including various polypeptides, polynucleotides, and immunoglobulins, with great sensitivity, Applicants disclose the present invention. In addition, the presently-described assays avoid the agglomeration problems of other assays, which promotes the goals of improved accuracy and greater resolution. The present invention, which i~
elegant in its simplicity, is hereby disclosed by Applicants, including its equivalents thereof.

SUMMARY OF THE INVENTION
In one embodiment of the present invention, Applicants disclose an analytical device for the detection or determination of an analyte antibody in a bodily fluid comprising a layer of a plurality of substantially planar zones adjacent one another and in absorbent contact with one another, the layer including a sample application zone, a conjugate zone containing antigen bound to mobile particles, and a detection zone containing immobilized antigen, wherein the antigen is the same in both the conjugate and detection zones and is an antigen that binds with the analyte antibody, the liquid 6ample is capable of moving from the 6ample application zone through the conjugate zone and on to the detection zone, and if the analyte antibody is present in the sample it is detected in the detection zone.
In another embodiment, the mobile particles are colored plastic particles or a metal 801- Another variation di6closes a device wherein the antigen will bind to antibodies which in turn bind to epitopes of the Bome~abu~do~en microorganism. In another variation, the mobile particles are colored 3S polystyrene microparticles. In yet another embodiment, the layer is made from nitrocellulose.

W~92/22797 2 11 0 9 2 D PC~r/US92/03680 The present invention also discloses a method employing the disclosed device~, which compri~es adding sample su~pected of containing the analyte antibody to the ~ample application zone and waiting for sufficient time for the sample to traverse the layer through the detection zone, and reading the recults in the detection zone. In one variation, the mobile particles are colored plastic particleE; or al-etal 601.
In another variation, the antigen used will bind to antibod1es which in turn bind to epitopes of the ~on~ba b~do~on microorganism. Yet another aspect discloses that the mobile particles are colored poly~tyrene microparticles. In another embodiment, the layer is made from nitrocellulo~e.
The present invention further discloses a proce~s for the determination of the presence or concentration of an analyte antibody in a sample fluid which comprises contacting a sample of the fluid with a fir~t antigen for the analyte antibody, wherein the first antigen is labelled, in order to form a soluble complex between the fir~t antigen and the analyte antibody; contacting the ~oluble complex with a 6econd antigen, wherein the second antigen is bound to a solid phase insoluble in the fluid, in order to form an in~oluble complex of thæ fir6t antigen, the analyte antibody, and the ~econd antigen; ~eparating the solid phase from the fluid ~ample and the unreacted, first antigen; measuring either the fir~t, labelled antigen associated with the solid pha~e of the unreacted amount of the first, labelled antigen; relating the amount of first, labelled antigen measured for a control sample prepared in accordance with the first four steps, the control sample being free of the analyte antibody, to determine the presence of the analyte antibody in the fluid sample, or relating the amount of first, labelled antigen measured for the fluid sample with the amount of làbelled antigen measured for samples containing known amounts of analyte antibody prepared in accordance with the first four steps in order to determine the concentration of the analyte antibody in the fluid sample; wherein both the fir6t and w092/22797 PCT/US92/036~0 2~ g20, second antigen are the same before they are labelled or attached to the solid phase; respectively. Those ~killed in the art will realize that, in the case where the label is a visible particle, such as a gold sol or colored or colorable microparticle, the complex fo~med by the labelled antigen and the analyte antibody may be insoluble.
In yet another embodiment, the label is an enzyme or a radioisotope. In another alternative embodiment, the antigen reacts with antibodies that in turn react with epitopes of lo Bomel~ bu~do~en. Other variations disclose that the label is alkaline phosphatase and the solid phase i8 a bead, the inner walls of a test tube or the wells of a microtitre plate;
alternatively, the label is alkaline phosphatase and the solid phase is a non-chromatographic device.
~5 The present invention further discloses an a ~ay kit comprising a first antigen bound to a solid phase insoluble in the fluid to be tested and a reagent containing a second antigen bound to a label, wherein the ~olid phase and the reagent are present in sufficient amount to perform at lea~t one assay for analyte antibody in the fluid, and wherein the first and second antigen are the same before they are bound to the solid phase or labelled, respectively. In another variation, the label is an enzyme or a radioisotope. In yet another embodiment, the antigen is an antigen that bind~ to antibodies which in turn bind to epitopes of Bome~a bu~do~on.
Another variation discloses that the solid pha~e is~a bead, the inner walls of a test tube or a non-chromatographic apparatus, and the label is alkaline phosphata6e. In another embodiment, the label is alkaline pho~phata~e and the solid phase is a non-chromatographic device.
In another embodiment, an a88ay kit i8 disclosed, which comprises a first antigen bound to a solid phase insoluble in the fluid to be tested and a reagent containing a second antigen bound to a label, wherein the solid phase and the reagent are present in sufficient amounts to perform at least ` W O 92/22797 PC~r/US92/03680 ~li332~ ;
one assay for analyte antibody in the fluid, and wherein the first and second antigens are the Fame before they are bound to the solid phase or are labelled, re~pectively.
In another embodiment, a process is disclosed, whereby S the process is for the determination of the pre~ence or concentration of an analyte antibody in a fluid comprising the following steps: a) simultaneously contacting a sample of the fluid with a first and second antigen, wherein the fir~t antigen is bound to a solid phase insoluble in the fluid and the second antigen is labelled and provided in a measured in order to form a insoluble complex between the fir~t and ~econd antigens and the analyte antibody; b) separating the ~olid carrier from the fluid sample containing unreacted ~econd, labelled antigen; c) measuring the amount of the second, labelled antigen associated with the solid phase or the amount of unreacted second, labelled antigen; d) relating the amount of second labelled antigen with the amount of labelied antigen measured for a control sample prepared in accordance with steps (a) through (c), the control sample known to be free of analyte antibody, to determine the prQsence of analyte antibody in the fluid sample, or relating the amount of labelled antigen mea6ured for the fluid sample with the amount ~f labelled antigen measured for 6amples containing known amount of analyte antibody prepared in accordance with ~teps a) through c) to determine the concentration of the an~lyte antibody in the fluid sample; wherein the first antigen and second antigen are the same before they are bound to the solid phase or labelled, respectively.
In alternative embodiments, the label is an enzyme or raaioisotope; further, the first and second antigen may bind to antibodies that in turn bind to epitope~ of Bo~el~b~do~en.
In another variation, the label i8 alkaline phosphatase and the solid phase is a plastic bead, the inner walls of a test tube, the wells of a microtitre plate. In ye~ another embodiment, the label is alkaline phosphata6e and the solid phase is a non-chromatographic device.

w092/22797 211~ PCT/US92/036gO
, . ` . .

Another embodiment discloses a proces~ for the determination of the presence or amount of an analyte antibody in a fluid sample comprising the steps of: a) contacting a sample of the fluid with a first antigen, wherein the first antigen is bound to a solid phase insoluble in the fluid, in order to form an insoluble complex between the first antigen and the analyte antibody; b) separating the fluid 6ample containing the unreacted analyte antibody from the insoluble complex of the analyte antibody and first antigen; c) reacting a measured amount of a second, labelled antigen with the insoluble complex of the first antigen and the analyte antibody to form an insoluble complex composed of the first and second antigens and the analyte antibody; d) separating the solid phase from the unreactQd second, labelled antigen;
e) mea~uring either the amount of second, labelled antigen a6sociated with the solid phase or the amount of unreacted ~econd, labelled antigen;
f) relating the amount of ~econd, labelled antigen`measured with the amount of labelled antigen mea6urQd for a control sample prepar~d in accordance with 6teps a) through e) being known to be free of the analyte antibody to determine the presence of analyte antibody, or relating the amount of labelled ~ntigen measured in the fluid sample with the amount of labelled antigen measured for 6amples containing known amounts of analyte antibody prepared in accordance with stQps a) through e) to determine the concentration of analyte antibody in the fluid sample; wherein the first and second antigens, before they are bound to the solid phase or labelled, respectively, are the same.
In various alternative ~mbodiment6, the label is an enzyme, a radioisotope, or alkaline phosphata~e. In another variation, the first and second antigens are both antigen6 that complex with epitopes of the Bon~l~ b~do~en microorganism. Yet another embodiment discloses a process W ~ 92/22797 2 1 ~ O 9 2 0 PC~r/US92/03680 wherein the solid phase is a bead, the inner walls of a test tube, or the wells of a microtitre plate; in another variation, the solid phase is a non-chromatographic device.

~E~rAILED DESC~RIPTION
The present invention is directed to devices and methodR
for simply and rapidly performing assays that are capable of differentiating specific molecules, such as antibodie6. While any convenient solid material may be u6ed as a solid support, it is preferable to use any one (or more) of several bibulous or absorbent solid materials, which may be employed to allow for capillary transport of a liquid away from the interface Detween the sir and liquid. Various bibulous material~
include paper, cellulose particles, silica gel, cellulosic beads, glass fiber, filter paper, and the like. The surface of the solid support should be relatively smooth, 80 ~8 to allow for the formstion of a concentrated particle 6ite, for example, in the form of a sharp band or point. The Qize and shape of the bibulous material may be varied widely depending upon the purpose of the as~ay. For example, the bibulou~
material may be ~haped as a narrow strip of from about one to about five millimeters in width, or it may be in any other convenient geometric or non-gesmetric shape. In each ca~e, the bibulous material will usually have a support which provides structural strength. The non-bibulous material may be a water impermeable layer or coating.
One embodiment of the present invention has the antigens as capture antigens bound to a porous membrane. A porous membrane may be comprised of a flexible or rigid matrix made from any of a variety of filtration or chromatographic materials including glass fibers, micro-fibers, and natural or synthetic materials. Fluids should be able to flow into and pass easily through the porous membrane. The membrane should also preferably have pore sizes of at least 0.1~ and preferably no more than 20~. The porous membrane can be used alone or as part of a more elaborate device. Such devices W O 92/22797 2 1 ~ O 9 2 0 PC~r/US92~0368~ .
. ~, , ., ~ ~ .

include the non-chromatographic ICON0 and like devices described in Valkirs, et al., U.S. Pat. Nos. 4,632,901 ~nd 4,727,01~, issued December 20, 1986 and February 23, 1988, respectively, herein incorporated by reference. ICON~ is a s trademark of Hybritech Incorporated (San Diego, CA) for the devices described in the Valkirs, et al. patents listed above.
More specifically, Valkirs, et al. describe an apparatus for the detection of a target antigen in a liquid sample, comprising: (a) a first member which is a porous membrane or filter and to which is bound an antibody against the target antigen, which member has upper and lower surfaces, the sample being applied to the upper surface, and wherein the antibody is bound within an area smaller than the area of the me~ber to which the sample is applied; and (b) a ~econd member, which is a body of absorbent material having a ~urface over which the first member is placed and having capillaries therethrough in a direction generally transverse to the surface over which the first member is placed, which capillaries are in communication with the pores on the lower surface of the fir6t ~1~ ber BO aB
to draw liquid added to the upper ~urface which has perme~ted the first member into the capillaries of the me~ber, the capillary communication between said first and ~econd ~e~ber~
having been established prior to, and maintained during, the addition of liquids to the apparatus in the immunoassay process.
Another such device is the TestPack~ device of Abbott Laboratories (North Chicago, IL), described in European Patent Application No. 217,403, published April 8, 1987. Still other devices containing porous membranes useful in the present invention include the devices of Bauer, et al., U.S. Pat. No.

3,811,8`40, issuèd May 21, 1974; Brown, III, et al., U.S. Pat.
No. 4,916,056, issued April 10, 1990; Cole, et al., U.S. Pat.
No. 4,407,943, issued Oct. 4, 1983; Cole, eg al., U.S. Pat.
No. 4,246,339, is~ued Jan. 20, 1981; Intengan, U.S. Pat. No.

4,440,301, issued April 3, 1984; ~olley, U.S. Pat. No.
4,704,255, issued Nov. 3, 1987; Katz, et al., U.S. Pat. No.
4,496,654, issued Jan. 29, 1985; and Tom, et al., U.S. Pat.

W~s2/22797 PCT/US92/03680 21i~920 No. 4,366,241, issued Dec. 28, 1982, all of which are incorporated herein by reference.
The present methods also may be accomplished by chromatoqraphic methods such as, for example, those de6cribed in Weng, et al., U.S. Pat. No. 4,740,468, issued April 26, 1988, incorporated herein by reference, and published European Application No. 186,100 to Yue, et al., published July 2, 1986.
The porous membrane of the present invention may also be used in chromatographic assays de6cribed, for exaJple, in U.S.
Pat. No. 4,861,711, issued August 29, 1989 to Friesen, et al.;
U.S. Pat. No. 4,855,453, issued August 8, 1989 to Rosenstein, et al.; U.S. Pat. No. 4,857,453, i~sued August 15, 1989 to Ullman, et al., all of which are incorporated herein by reference, and May, et al., EP0 Publication No. 291,194, published Nov. 17, 1988; Ching, et al., EP0 Publication No.
299,428, published Jan. 18, 1989, and Devereaux, et al., EP0 publication No. 323,605, published July 12, 1989.
The capture antigens may be directly or indirectly bound to the membrane. The direct binding may be covalent or non-covalent and may be accompli6hed by any method known in the art 6uch as, for example, the use of glutaraldehyde and aminosilanes as well a~ other methods described in ~Immobilized Enzymes", Ichiro Chibata, Halstead Pre~s, NY
(1978); Cuatreca6as, J. Bio. Chem. 245: 3059 (1970); and March, et al., Anal. Biochem. 60: 149, et seq. (1974). The non-covalent binding takes advantage of the natural adhe~ion of antibodies to the non-synthetic and especially the 6ynthetic fiber~. Thus, appropriately buffered 601ution6 can be mixed with the membrane then evaporated, leaving a coating of the desired ligand on the membrane.
The non-direct method f or applying the ligand to the membrane employs microparticles that may be bound to or entrapped by the membrane, such that the microparticles are within the matrix of the membrane, on the ~urface of the membrane, or bound to other particles which are in turn bound to the membrane. The microparticles may be any 6hape, ~110~2~ -12-preferably spherical. The size of the particles should be such that they do not migrate through the membrane to any significant degree. The size of the particles may vary, but in general, they may be slightly larger than the minimum pore size of the membrane and smaller-than the Daximum pore size, and in addition or in the alternative, may be larger than the maximum pore size. Thus, the particle may be bound within the matrix of the membrane, on the surface of the membrane, or to other particles which are in turn bound to the membrane. The particles may be made of a variety of naturally occurring or synthetic materials. Exemplary of such partic~es are those made from polyethylene, polyacrylates, polyacrylamide, and preferably poly~tyrene or naturally occurring materials such as cross-linked polysaccharides like agaro~e, dextran, cellulose, starch, or the like. The primary requirement is that the particles do not contribute a signal, usually light absorption, that would cause the zone in which the p rticles were located to have a different gignal than the re~t of the membrane.
The ligand may be covalently or non-covalently bound to the microparticle. The binding of ligand to the particle may be by methods similar to tho~e discus~ed above for binding the ligand directly to the membrane or other ~ethod~ known to those skilled in the art. The preferred method for coating ligands to the microparticles i~ de6cribed in the Ex~mple~
herein.
The particles may be applied (or "spotted"~ to the membrane in a zone within the surface area of the membrane.
Thus, spotting localizes the antigen-coated microparticles to a discrete area on the membrane to localize the antigen-coated microparticles on or within the membrane. Any of the methods known in the art may be employed. One such method employs various mechanical means such as, for example, the Sandy Springs spotting Machine (Germantown, MD) to apply a suspension, frequently aqueous (nlatexn), to the membrane.
The ~ethods of preparing and using such microparticles for the instant invention are further discussed in Weng, et - W ~ 92/22797 PC~r/US92~03680 211032~ :

al., U.S. Pat. No. 4,740,468, is~ued April 26, 1988 (~ee especially columns 13-15) ineorporated herein by referenee;
Brown, III, et al., European Patent App. No. 217,403, published April 8, 1987, ; and A.S. Rubenstein, European Patent App. No. 200,381, published Nov. 5, 1986.
The separation steps for the various assay formats (e.g.,, forward, simultaneou~, and reverse) may be performed by any of the methods known in the art. For membranes and . .
filters, additional washing with buffer may often be suffieient, preferably drawing the liquid through the membrane or filter by applying vacuum to the opposite side of the membrane or filter or eontacting the oppo~ite side of the filter or membrane with a liguid absorbing member that draw6 the liquid through, for example, via eap~llary aetion. The ICON deviee (Hybritech, Incorporated, San Diego, CA), which i~ preferred for use in one e~bodi~ent of the pre~ent invention, uses th~ latter method.
In addition, non-ehromatographie devices and assay~ are appropriate for use aceording to the present invention. Bead as~ay~, for example, provide an alternative to ehromatographie as~ays. See, e.g., Geigel, et al., U.S. Pat. No. 4,517,288, issued May 14, 1985, whieh is ineorporated herein by reference.
Moderate temperatùres are normally employed for earrying out the assay. Constant temperature~ during the period of the mea~urement are generally required only if the assay is performed without eomparison with a eontrol sample. The temperatures for the determination will generally range from about 15--45-C.
The term "labeled antigen" refers to any antigen baving speeifie reaetivity with the partieular antibody of intere~t.
Sueh an antigen may be labeled by eonventional methods to form all or part of a signal generating sy~tem. For example, the antigen may be labeled with radioaetive isotopes, enzymes, biotin, avidin, ehromogenie or fluorogenie ~ubstances, W092/22797 PCT/USg2/036~0 2 1 1 ~ 9 2 0 -14-chemiluminescent labels, colloidal metal particles, colored microparticles, colorable particles, and colorable latex particles.
Thus, the labelled antigen may be covalently bound to s radioisotopes such as tritium, carbon 14, phocphorus 32, iodine 125, and iodine 131 by methods well known in the art.
For example, Il~ can be introduced by procedures such as the chloramine-T procedure, enzymatically via the lactoperoxidase procedure, or by the prelabeled Bolton-Hunter technique.
These techniques plus others are discus~ed in h. Van Vunakis and J.J. Langone, eds., Methods in Enzymolo~y 70: Part A
(1980). See also U.S. Pat. No~. 3,646,346, issued Feb. 29, 1972, and 4,062,733, issued Dec. 13, 1979, incorporated herein by reference, for further examples of radioactive labels. Chromogenic labels are those compounds that absorb light in the visible ultraviolet wavelengths. Such compound~
are usually dyestuffs and include quinoline dye~, triarylmethane dyes, phthalein~, insect dyQs, azo dyes, anthraquimoid dyes, cyanine dyes, and phenazoxonium dyes.
Fluorogenic compounds include tho~e which emit light in the ultraviolet or visible wavelength sub~equent to irradiation by light. The fluorogens can be employed by themselves or with quencher molecules. The primary fluorogens are those of the rhodamine, fluorescein and umbelliferone families. The method of conjugation and use for these and other fluorogens can be found in the art. See, for example, Langone and Van Vunakis, Methods in Enzymolooy 74: apart C
~1981), especially at pages 3-105. For a representative listing of other suitable fluorogens, see Tom, et al., U.S.
Pat. No. 4,366,241, issued Dec. 28, 1982, especially at columns 28 and 29; and U.S. Pat. No. 3,996,345, both of which are incorporated herein by reference.
Chemiluminescent labels may also be used in t~e present invention. For example, the labels listed in Maier, et al., U.S. Pat. No. 4,104,029, issued August 1, 1978, herein incorporated by reference, may be used as detection signals in the present invention.

W O 92/22797 2 1 1 0 ~ 2 0 PC~r/US92/03680 ~

Alternate methods of detection include the u~e of colored microparticles, colorable particle~, including colorable latex particles and colloidal metal particles. Colored microparticle~ and their use in assays known in the art include, for example,- those described in U.S. Pat. No.
4,703,017, issued Oct. 27, 1987 to Campbell, et al., incorporated herein by reference. The use of colloidal metal particles in assay6 is also well known in the art. See, for example, U.S. Pat. No. 4,313,734, i~sued Feb. 2, 1982 to Leuvering; U.S. Pat. No. 4,775,636, issued Oct. 4, 1988 to Moerman~, et al.; both of which are incorporated herein by reference, and by Yost, et al., EP0 Pub. No. 298,368, published Jan. 11, 1989. Colorable particles and colorable latex particles are also known in the art to be useful as marker~ and are described, for example, in U.S. Pat. No.
4,373,932, is6ued Feb. 15, 1983 to Gribnau, et al., and U.S.
Pat. No. 4,837,168, issued June 6, 1989 to deJaeger, et al., re~pectively, both of which are incorpor~ted herein by reference.
The6e non-enzymatic signal sy6tems are adequate for the pre~ent invention. However, tho6e skilled in the art will recognize that an enzyme-catalyzed ~ignal systQm i~ in general more ~ensitive than a non-enzymatic sy~tem and is, therefore, preferred. Catalytic label~ are well known in the art and include ~ingle and dual (nchanneledn) enzymes ~uch as alkaline phosphatase, horseradi6~ peroxidase, lucifera~e, ~-galactosidase, glucose oxidase, lysozyme, malate dehydrogenase, glucose-6-phosphate dehydrogenase, and the like. Dual catalytic systems include, for example, alkaline phosphatase and glucose oxidase using gluco~e-6-pho~phate as the initial substrate. A ~econd example of a dual catalytic sy6tem is illustrated by the oxidation of glucose to hydrogen peroxide by glucose oxidase, which hydrogen peroxide would react with a leuco dye to produce a signal generator. A more detailed discussion of catalytic systems can be found, for example, in U.S. Pat. No. 4,366,241 to Tom, et al., W O 92/22797 P ~ /US92/0368~
2ll0.,s~n,., particularly columns 27-40; U.S. Pat. No. 4,843,000, issued June 27, 1989 to Litman, et al., and U.S. Pat. No. 4,849,338, issued July 18, 1989 to Litman, et al., all of which are incorporated herein by reference. A1BO~ ~;ee Weng, et al., U.S. Pat. No. 4,740,468, which is also incorporated herein by reference, especially at columns 2 and 6-8.
The procedures for coupling enzymes to the antigen~ are well known in the art and are describ~d, for example, in Kennedy, et al., Clin. Chim. ACTA 70: 1 (1976). Reagents that may be u6ed for this procedure include, for example, glutaraldehyde, p-toluene diisocyanate, various carbodiimide reagents, p-benzoquinone m-periodate, N,N'-o-phenylenedimaleimide and the like.
- The substrates for the catalytic systems include simple chromogens and fluorogens such a8 para-nitrophenyl phosphate (PNPP), ~-D-glucose (plus pos~ibly a suitable redox dye), homovanillic acid, o-dianisidine, bxomocre601 purpie powder, 4-alkyl-umbelliferone, luminol, para-dimethylaminolophine, paramethyloxylophine, and the like, with indoxyl phosphate being the preferred substrate.
Depending on the nature of the label and catalytic signal producing system, a signal can be detected by irradiating with light and observing the level of fluorescence; by providing for a catalyst 6ystem to produce a dye, fluorescence, or chemiluminescence, where the dye can be observed visually or in a spectrophotometer and the fluorescence could be observed visually or in a fluorometer; or in the case of chemiluminescence or a radio~ctive label, by employing a radiation counter. Where the appropriate equipment is not available, it will normally be desirable to have a chromophore produced which results in a visible color. Where sophisticated equipment is involved, any of the technigues is applicable. For example, when the preferred combination of alkaline phosphatase is used as the enzyme and indoxyl phosphate ~s the substrate, a color change may be detected visually for a qualitative positive reaction. For a 2110921) quantitative analysis, the ICON~ reader and accompanying software (Hybritech Incorporated, San Diego, CA) may be used according to the manufacturer's in6truction~ and are preferred for use in the present invention.
While latex particles tend to be preferred for use in conjunction with the presently-di~closed invention, it 6hould be appreciated that other particles may be used aucceFsfully.
For example, the particles which are involved in the assay may be present in the sample, may be added a~ reagents or formed ~ s~u. The n~ture of the particle may vary widely, being naturally occurring or synthetic, being a single material, a few materials, or a combination of a wide variety of materials. Naturally-occurring particles include nuclei, mycoplasma, plasmids, pl~stids, mammalian cells, unicellular microorganisms (e.g., bacteria). Synthetic particles may be prepared from synthetic or natur~lly occurring materials, such as metal colloids or latex particles made from polystyrene polyacrylates or naturally-occurring material~, such as polysaccharides, e.g., agarose, or the like. Non-naturally-occurring particles may be varied depending upon the particular as~ay, the protocol for the assay, or other considerations. (See, e.g., Gould, et al., U.S. Pat. No.
4,837,168, which describes the use of a variety of particles.) Uniform latex particles (nULPs~) are, in general, extremely uniform sphere~ of 6mall diameter. Typical diameters range from le6s than about 0.1~m to about 100 ~.
Particles smaller than 5 ~m are usually prepared by emulsion polymerization. The result of this process is a ~eries o$
particles with extremely uniform size distribution~.
The principal use for ULPs is in the medical diagnostic area, wherein the particles are utilized for latex agglutination test6. Other varieties of particles, 6uch as amide-modified latex ( HAML~ ) and carboxylate-modified latex (nCML~) have amide and carboxylic acid groups, respectively, W O 92/22797 PC~r/US92/03680 ~ 1~0920 on their surfaces. These functional groups permit covalent bindin~ of ligands -- for example, antigens or antibodies --to the surface of the ULPs for improved agglutination te~t~.
For example, if one is attempting to measure a particular antibody ("~b"), an appropriate antigen (nAgN) is coated onto the latex particles. Since the Ab is divalent, it may bind to identical sites on two adjacent particle6 and link them together. Thus, if Ab present in an individual's sample is mixed with the Ag-coated particles, it will cause lo agglutination or coagulation of the particles; the~e aggregates are generally visible to the naked eye. This phenomenon is, essentially, the basis for latex agglutination te8ts ( nL~.Tn ) one major difficulty with mobile particles is the fact that the coated particles tend to spontaneously agglutinate.
Latex suspensions, in particular, are colloidal suspen~ions of - hydrophobic particles. The stability of the ~uspension is dependent upon the surface active charges; addition of small amounts of protein (approximately 10 ~g per mg of latex) can cause agglomeration, whereas continued addition of larger amount~ of protein tends to increase particle stability. This type of agglutination is also a problem in the chromat~graphic assays using colored or visible particles.
Various methods of addressing this problem and the related problem of nonspecific agglutination have been suggested, including the u~e of linkers and spacer~. However, few of these suggestions prove entirely satisfactory, as they tend to interfere with the assay, many doing 80 in a manner that inhibits agglutination. Thi6 is, of course, a completely unacceptable result for most assays.
The means for detecting a detectable ~ignal at or away from the concentration site may or may not be an intrinsic property of the particles. The particleF may be labeled with a wide variety of materials which allow for detection, such as radionuclides, dyes, fluorescer~, enzymes, or other convenient label providing for a detectable signal, either vi~ually ~ .

W092/22797 2 1 1 0 9 ~ O PCT/US92/03680 --19-- , observable or detectable by instrumentation. The variou6 labels would normally be covaiently bonded to the particle, using linking arms as appropriate. The labels may be bound to the surface or, when feasible, extend throughout the particle.
The size of the particles may vary widely, generally ranging from about 0.05 to 100 micrometers (~m), more u6ually from about 0.1 to 75~m. The particles may be charged, either positively or negatively, may be amphoteric ~r lack any charge, being neutral. The presence or absence of charge may affect other parameters involved in the a6say.
A large number of patents have been issued which de6cribe a wide variety of labels which have found u~e in diagno6tic assays. Various protocols can be developed where the~e label6 may be used with advantage. Illustrative of 6uch patent6 are U.S. Pat. Nos. 3,850,752; 4,255,329; 4,233,402; and 4,;208,479.
For performing the a~ay, kit6 can be provided where the various reagent6 are combinod in predetermined amount6 in combination with various ancillary materials for co~bination with the 6ample or for other u~es in the a66ay. In view of - 20 the wide spectrum of protocols and reagents, a wide variety of kits may be prepared. In general, where the method involve6 the addition of particles, the kits will include particle~
which have a ligand bound to the particle, either covalently or non-covalently. Also, there may be a label bound to the surface of the particle or disper~ed therein, particularly a dye, which may be colored in the visible range. The following references describe various labeling methods:
chemiluminescence (Maier, et al., U.S. Patent No. 4,104,029);
colored particles (Campbell, et al., U.S. Pat. No. 4,703,017);
colorable particles (deJaeger, et al., U.5. Pat. No.
4,837,168); or fluorescence (Langone and Van Vunakis, eds, Methods in EnzYmolooy 74; Part C (1981)). Radioisotopes may also be u~ed to label the particle6. (See, e.g., Langone and Van Vunakis, eds, Methods in EnzymoloqY 70; Part A (1980), which describes radioisotopic labeling methods.) In other instances, the particle may also be labelled with an enzyme.

WO 92/22797 PCI`/US92/036&0 21~ 2n~`

Where particles are not to be included, the reagents will normally involve labelled receptors or ligands, where the labels provide for a detectable signal and may provide for the inhibition of migration of the particles present in the as~ay medium. In addition to the labelled reagents, there may be ancillary reagents ~uch as buffers, stabilizers, detergent~, and as appropriate substrates for enzymes, bulking agents, and the like. Also likely to be included in such kits would be the wicking material, prepared in strip form or some other lo convenient shape. In preferred embodiment~ of the invention, the as~ay kit contain~ desiccant material; more preferably, the desiccant material is contained within the ~say device or housing.
The invention can be better understood by way of the following examples which are representative of the preferred embodiments thereof, but which are not to be construed as limiting the ~cope of the invention. `
EXANPLE I
~yme Antibody As~ay Extracted Lyme antigens are prepared from ~n~abu~d~on strain B-31 purchased from the American Type Culture Collection. The microorganism was grown in modified BSK II
Nedium as described below. It was incubated at 33-C and harvested by centrifugation. The harvested cells were washed three times with phosphate buffered saline (pH 7.4) containing S mM MgCl2, then extracted with 2% 6arcosyl in 10 mM tri6 buffered ~olution (pH 8.2) containing 1 mM EDTA. Thi6 extraction procedure was carried out until all the sarco~yl soluble material was extracted. The incoluble material was further blended with an Omni mixer. The blended material is hereinafter referred to as the "Lyme antigensn. It should further be noted that the same Lyme antigens or Lyme antigens from the same sourCe may be used to detect Lyme-specific antibodies in a variety of mammals, including cattle, dogs, cats and humans.

w~s2/22797PCT/US92/03680 ~11û920 EXANPLE II
PreDaration of Coniuaates While latex particles are used in the presently-described experiments, it should be appreciated that other particle6 may s be used successfully. In addition, while microparticles that are not conjugated with ligands are added to ligand-particle conjugates in various examples, it should be under~tood that there are various embodiments of the pre~ent invention in which such admixing is not necessary to the practice the present invention.
Unifor~ latex particles (nULPs~) are, in general, extremely uniform ~pheres of small diameter. Typical diameters range from less than about O.l~m to about 100 ~m.
Particles smaller than 5 ~m are usually prepared by emulsion polymerization.
a. Pre~r~ion of Liaand-Particle Con~uqa~e~
The basic process of ligand-particle conjugation, for example, via simple adsorption or covalent binding, is well known in the art, as i~ the use of colored latex particle~, which increase the resolution and readability of assays.
Various procedures are described, in general ter~, in Bangs, L.B., "Uniform Latex Particles,~ presented at a work~hop at the 41st National ~eeting, Amer. A~soc. Clin. Che~., 1989, and available in printed form from Seragen Diagnostic~ Inc., Indianapolis, IN; or Galloway, R.J., "Development of microparticle tests and immunoassays,~ Seradyn, Inc~, Indianapolis, IN. These articles, and references cited to therein, are hereby incorporated by reference.
One method of preparinq coated latex particles, for example, is the adsorption method. In general terms, one should: 1) utilize pure reagents; 2) clean the particles prior to coating; and 3) determine the quantitative surface covèrage of the particle and the ligand chemistry.
~or example, ligand-latex conjugates ("L-latexN) ~ay be prepared according to the following method: in the simplest ca e, the appropriate ligand is dissolved in a buffer W092/22797 PCT/US~2/0368~
2110~ 22-solution, added to a latex suspension, and stirred for times ranging from a few minutes to more than 24 hours. After equilibration, the latex is centrifuged and the supernatant containing any unadsorbed ligand is discarded. The latex is re-suspended in fresh buffer and centrifuged; the supernatant is again discarded. These steps are to be repeated until the latex is determined to be wached free of any residual un-adsorbed ligand. At this juncture, the latex coating process may be complete and the latex ready to use in latex agglutination assays.
Covalent coupling involves the permanent or covalent binding of a ligand or othér material to the latex particle surface. If covalent coupling is the method of choice, one must first couple the ligand to the latex particle6, then maintain the stability of the latex particle suspension, followed by preventing the protein from becoming denatur~d.
(For a general di~cu~ion of covalent coupling technique6, and citation to more detailed references, see Bangs, L.B., "Uniform L~tex Particles," which has been incorporated herein by reference.) While the foregoing di6cus~ion is in the context of latex particles, it will be appreciated that other particle~, including, without limitation, naturally-occurring (e.g.
plasmids) or synthetic particles (e.g. polymer6), and metal colloids or particles (e.g., gold sol particles), may be used.
These particles and their methods of extraction or preparation are well known in the art.
b. PreDaration of BSA-Latex Coniuaates Preparation of bovine serum albumin - latex conjugates ("BSA-latex") is similar to ligand-latex (L-latex) preparàtion, a6 described above, except that no ligand is used in the preparation, and BSA i6 u~ed instead. Alternatively, other proteins may be u6ed in place of the BSA, such a~ other albumins (including lactalbumin), ca6ein, globulin, non-~pecific immunoglobulin (which does not participate in theantigen-antibody reaction), and the like that can prevent nonspecific binding.
. .

21~3~-~

c. Mixture of Liqand-latex and BSA-latex L-latex and BSA-latex are mi~ed together in varying ratios, depending upon the test to be performed. For example, in preparing mixtures for use as set out in some of the Examples, L-latex and BSA-latex were mixed in approximately a 2:1 ratio to a 5:1 ratio, volume to volume, for u~e in the assays. Depending upon the nature of the as~ay, the ratio~
ean vary substantially, with areater amounts of protein-labeled latex resulting in greater reduetion of non~pecific binding. The amount of latex (or other partiele) that does not have ligand attached can be any amount that ia effective to appreeiably decrease nonspecifie binding, or false positives. Sueh amounts are readily determined by obvious empirieal methods.
d. Mixture of Liaand-latex and ~Nak~d" latex L-latex and naked latex are mixed together in varying ratios, depending upon the test to be performed, a~ noted above in regard to Ligand-latex/BSA-latex ~ixtures. As noted above, the ratios of L-latex to naked latex ean vary substantially, with greater a~ounts of protein-labeled latex resulting in greater reduetion of non~peeifie binding. The amount of latex (or other partiele) that does ~ot have ligand attaehed ean be any amount that i~ effeetive to appreeiably deerease nonspeeifie binding, or false positives. Sueh amounts are readily determined by obvious empirieal methods.
EXAMPLE III
Pre~aration of Bome~a Con~uaate 10 ml of blue earboxylated latex (preferably about 0.4 in diameter) having 50 mg/ml solid, obtained from Magsphere, is diluted to 0.5 mg/ml in 10% ~uerose and 20mM MES buffer, pH

5.5. Add Sulfo-NHS (Pieree) and EDAC (Sigma, St. Louis, M0) to a final eoneentration of 1.087 mg/ml and 2 mg/ml, respeetively. Mix by end-to-end rotation for about 30 minutes. The aetivated latex i~ washed five times with 10%
sucrose, 0.02% SDS in 20mM MES, pH 6.5, in an amieon W O 92~22797 PC~r/US92/03680 2 ~

concentrator with 0.2~m nylon membrane. Re-suspend to approximately 50 mg/ml with 10% 6ucrose, 2mM MES (pH 6.5), and sonicate four times for 15 seconds each time.
About 0.5 mg of extracted Bomel~ b~do~en antigenB are mixed in 0.01% SDS, 2mM ME~ buffer (pH 6.Sj, containing 10%
sucro~e and the 500 mg of activated latex ~uspen~ion. The latex and extracted Bon~a b~do~en are allowed to react for two hours at 22-C.
To neutralize Furface carboxyl groups not bound to the extracted Bome~a b~do~on antigen~, a solution of Tris buffer (pH 8.5), ie added to 20mM, followed by a solution of case~n (Sigma) and Zwittergent 3-12 (Calbiochem, San Diego, CA) of 0.5% and 0.1%, respectively. The Eon~a b~do~on-latex is diluted to a desired concentration containing 20% ~ucro~e, 20mM Tris (pH 8.5), 0.1% Zwittergent 3-12, 0.5% casein, and 0.3% BSA-latex, and i~ ready for printing onto the solid support or matrix.
EXANPLE IV
Reagent Preparation 1. Preparation of BSK-II Medium To 1 liter of fortified RPMI-1640 without glutamine add the following and mix well: 5 g Neopeptone (Difco) and 50 g BSA (Sigma A-4503; St. Louis, M0). Dissolve the neopeptone and BSA completely and then add the following: 2g TC
Yeastolate (Difco); 6g HEPES (Sigma); 0.7g sodium citrate; 5g glucose; 0.8g sodium pyruvate; 0.4g N-acetylgluco~amine (Sigma); and 2.2g sodium bicarbonate. Adjust the pH to 7.6-7.8.
Modified BSK-II medium was prepared according to the method set forth by Barbour, A.G., in Yale J. B. Med. 57: 521-525 (1984), which is incorporated herein by reference. The modified medium was prepared from fortified RPMI-1640 without glutamine ~nd 7% gelatin.

Wos2/227s~ 0 9 2 0 PCT/USs2/03680 2. Inoculation and Harvesting of Bo~e~a Cultures The equipment used included a 2.2 L Erlenmeyer fla6k; a vacuum equipped with a trap; 0.2 micron 1 liter filter unit6 (Nalgene 90mm diameter); and a laminar flow hood. The medium s was inoculated with Bome~a according to appropriate, safe laboratory procedures. The media flask~ are incub~ted at 33-35-C overnight as a precaution, to check for contamination.
Signs of contamination include the appearance of turbidity and/or an acidic pH shift.
EXAMPLE V
Assay PreDaration and Procedures 1. Preparation of solid pha~e one example of the use of the pre6ent invention in a solid phase assay format may proceed es~entially as follows.
The illustrated reaction device consists of a 601id --typically plastic -- housing containing a solid ~upport for the a6say. Generally, a ~e~brane strip such as nitrocellulo~e is used; preferably, an i~unochromatographic ~trip i6 u~ed.
For example, the right end of the membrane provides contact with the sample well. The ~ample well contain6 an absorbent pad which provides an even flow of the sample fluid from right to left along the membrane. ~obile particles are applied to a first zone of the membrane -- for example, latex beads --which particles are conjugated with the appropriate ligand or antiligand. For example, antigens extracted from Bon~Ma b~db~en were used in one experiment described herein.
Various methods of isolating and preparing viral or bacterial antigens are knGwn in the art. For example, and without limitation, Hepatitis A antigen may be prepared according to the method of Markus, et al., as described in U.S. Pat. No.
4,301,249, which is incorporated herein by reference.
In a second zone on the membrane, ligand or antiligand i8 immobilized. A third reagent may be used as well; for ex2mple, such an agent may be immobilized in a zone on the solid support. This agent may be capable of binding particle~
~ that migrate from the first zone after sample is added. This : .

W O 92/22797 PC~r/US92/0368~ .
9 ~ () third agent can act as a procedural control and may serve to indicate that the assay i~ complete, or that it has been properly performed, if, for example, a detectable response occurs in the zone in which the third agent is immobilized.
In a ~typical" chromatographic acsay test procedure, a liquid specimen is applied to the solid support proximal to the first zone. As the fluid moves via capillary action to the first zone of the me~brane, it mobilizes the mobile particles. The fluid continues to move the particles acro~s the membrane to the next zone or zones. If analyte iB pre6ent in the sample, a "sandwich" of solid phase-antigen/analyte/antigen-conjugated particles is for~ed and a detectable re6ult occurs. A6 the fluid continues to move the particles across the membrane, the fluid/particles come into contact with the third reagent, which is immobilized on the support. A detectable response should then occur in that zone, indicating that the test is valid, or that the te~t is complete.
2. Test Procedure Place the solid phase assay (or membrane alone) on a well-lighted, level surface. Place one drop (about 50~L) of liquid sample in/on the 6ample application area proximal to the first zone. The application area may also contain a buffer solution to further promote assay perfor~ance. One example of such a buffer comprises 2% ca~ein and 0.5%
Zwittergent in O.lM Tris (pH 8.0). Alternatively, the 6ample may be mixed with another substance -- e.g., saline ~olution -- prior to administration of the sample or sample mixture to the sample application area. Immediately thereafter, add several (preferably about 6) drops of developer solution to the sample application area. A useful developer solution contains the following, for example, in aqueous solution: 1%
Zwittergent, 1% Triton x-100, 0.02% SDS, 0.2% NaN3, and 50mM
Tris buffer (pH 8.5).
Allow the test to run for about 10 minutes and then read t~e results. Although a signal may appear in the third zone W~92/22797 PCT/US92/03680 _~LL~920 before the lo minutes elapse, the results are likely to be read with greater accuracy if one wait~ the full 10 minute6.
After the appropriate amount of time elapRe~, provided the appropriate signal is detected in the approprate zone or s zones, then the assay results may be read. For example, if a symbol appears in the third zone, then the test re~ult~ may be interpreted as valid and negative. If a 6ymbol appears in the second and third zones, then the re~ult~ may be interpreted as valid and positive.
Control solutions may further be provided for the purpose of comparison with as~ay re~ults or to te~t reagent viability.
For example, a positive control solution may comprise about 0.5mg/ml purified rabbit anti-Lyme antibody in 2mg/ml o*
rabbit gamma globulin and 50mM Tris (pH 8.0). A negative control may comprise 2mglml of rabbit gamma globulin in 50rM
Tris (pH 8.0). These solution~ may be provided in a kit for~
with any or all of the reagentC and as~ay component~ di~clo~ed herein.
Although the invention has been de~cribed in the context of particular embodiment~, it is intended that the scope of coverage of the patent not be limited to those particular embodiment~, but be determined by reference to the following claims.

Claims (28)

WE CLAIM:

1. An analytical device for the detection or determination of an analyte antibody in a bodily fluid comprising a layer of a plurality of substantially planar zones adjacent one another and in absorbent contact with one another, said layer including:
(a) a sample application zone;
(b) a conjugate zone containing antigen bound to mobile particles; and (c) a detection zone containing immobilized antigen, wherein the antigen is the same in both the conjugate and detection zones and is an antigen that binds with the analyte antibody, the liquid sample is capable of moving from the sample application zone through the conjugate zone and onto the detection zone, and if said analyte antibody is present in the sample, it is detected in the detection zone.

2. A device of Claim 1, wherein the mobile particles are colored plastic particles or a metal sol.

3. A device of Claim 2, wherein the antigen will bind to antibodies which are capable of binding to epitopes of Borrelia burgdorferi.

4. A device of Claim 3, wherein the mobile particles are colored polystyrene microparticles.

5. A device of Claim 4, wherein the layer is made from nitrocellulose.

6. A method employing the device of Claim 1 which comprises adding sample suspected of containing the analyte antibody to the sample application zone and waiting for sufficient time for the sample to traverse the layer through the detection zone, and reading the results in the detection zone.

7. A method of Claim 6, wherein the mobile particles are colored plastic particles or a metal sol.

8. A method of Claim 7, wherein the antigen used will bind to antibodies which are capable of binding to epitopes of Borrelia burgdorferi.

9. A method of Claim 8, wherein the mobile particles are colored polystyrene microparticles.

10. A method of Claim 9, wherein the layer is made from nitrocellulose.

11. A process for the determination of the presence or concentration of an analyte antibody in a fluid through the use of a chromatographic assay, comprising the steps of:
a) contacting a sample of said fluid with a labeled antigen for said analyte antibody in a conjugate zone of said chromatographic assay in order to form a mobile complex between said labeled antigen and said analyte antibody;
b) contacting said mobile complex with an immobilized antigen in a detection zone of said chromatographic assay, said detection zone being in absorbent contact with said conjugate zone, wherein said immobilized antigen is bound to a solid phase of said detection zone which is insoluble in said fluid, in order to form an immobile complex of said labeled antigen, said analyte antibody, and said immobilized antigen;
c) separating said labeled antigen not complexed with said analyte antibody and not bound to the solid phase of said detection zone from said solid phase;
d) measuring either the amount of labeled antigen bound to the solid phase of said detection zone or the amount of labeled antigen not bound thereto;
e) relating the amount of labeled antigen measured in step (d) to the amount of labeled antigen measured for a control sample prepared in accordance with steps a) through d), said control sample being free of said analyte antibody, to determine the presence of said analyte antibody in said fluid or relating the amount of labeled antigen measured in step (d) to the amount of labeled antigen measured for samples containing known amounts of said analyte antibody prepared in accordance with steps a) through d) in order to determine the concentration of the analyte antibody in said fluid;
wherein both said labeled and immobilized antigens are the same before they are labeled or attached to the solid phase of said detection zone, respectively.

12. A process of Claim 11, wherein said labeled antigen is present in said conjugate zone before said fluid is contacted with said chromatographic assay.

13. A process of claim 11, wherein said antigen reacts with antibodies which are capable of binding to epitopes of Borrelia burgdorferi.

14. A process of claim 11, wherein the label is an enzyme or a radioisotope.

15. A process of claim 14, wherein said label is alkaline phosphatase.

16. A chromatographic assay kit, comprising:
an immobilized antigen bound to a detection zone of a solid phase on which a chromatographic assay can be performed, said solid phase being insoluble in a fluid to be tested; and a mobile, labeled antigen present in a conjugate zone of said solid phase, wherein said immobilized and labeled antigens are present in sufficient amounts to perform at least one assay for an analyte antibody in said fluid, and wherein said immobilized and labeled antigens are the same before they are bound to said solid phase or are labeled, respectively.

17. A chromatographic assay kit of claim 16, wherein said immobilized and labeled antigens are antigens that bind to antibodies which are capable of binding to epitopes of Borrelia burgdorferi.

18. An assay kit of claim 16, wherein the label is an enzyme or a radioisotope.

19. An assay kit of claim 18, wherein the label is an enzyme.

20. An assay kit of Claim 19, wherein said label is alkaline phosphatase.

21. A process for the determination of the presence or concentration of an analyte antibody in a fluid through the use of a chromatographic assay, comprising the steps of:
a) simultaneously contacting a sample of said fluid with immobilized and labeled antigens, wherein the immobilized antigen is bound to a solid phase on which a chromatographic assay can be performed and which is insoluble in said fluid and wherein the labeled antigen is mobile and is provided in a measured amount in order to form an immobile complex between said immobilized and labeled antigens and said analyte antibody;
b) separating said fluid containing labeled antigen not complexed with said analyte antibody and not bound to said solid phase from said solid phase;
c) measuring the amount of labeled antigen bound to said solid phase or the amount of labeled antigen not bound thereto;
d) relating the amount of labeled antigen measured in step (c) with the amount of labeled antigen measured for a control sample prepared in accordance with steps (a) through (c), said control sample known to be free of said analyte antibody, to determine the presence of said analyte antibody in said fluid or relating the amount of labeled antigen measured in step (c) with the amount of labeled antigen measured for samples containing a known amount of said analyte antibody prepared in accordance with steps a) through c) to determine the concentration of said analyte antibody in said fluid;
wherein said immobilized and labeled antigens are the same before they are bound to said solid phase and labeled, respectively.

22. A process of claim 21, wherein said immobilized and labeled antigens bind to antibodies which are capable of binding to epitopes of Borrelia burgdorferi.

23. A process of claim 21, wherein the label is an enzyme or radioisotope.

24. A process of Claim 23, wherein said label is alkaline phosphatase.

25. A process for the determination of the presence or amount of an analyte antibody in a fluid through the use of a chromatographic assay, comprising the steps of:
a) contacting a sample of said fluid with an immobilized antigen, wherein said immobilized antigen is bound to a solid phase on which a chromatographic assay can be performed and which is insoluble in said fluid, in order to form an immobile complex between said immobilized antigen and said analyte antibody;
b) separating said fluid and said analyte antibody which is not bound to said solid phase from said immobile complex of said analyte antibody and said immobilized antigen;
c) contacting a measured amount of a mobile, labeled antigen with said immobile complex of said immobilized antigen and said analyte antibody to form an immobile complex composed of said labeled antigen, said analyte antibody, and said immobilized antigen;
d) separating said solid phase from said labeled antigen not bound thereto;
e) measuring either the amount of labeled antigen bound to said solid phase or the amount of labeled antigen not bound thereto;
f) relating the amount of labeled antigen measured in step (e) with the amount of labeled antigen measured for a control sample prepared in accordance with steps a) through e), said control sample being known to be free of said analyte antibody, to determine the presence of said analyte antibody, or relating the yyyamount of labeled antigen measured in said fluid in step (e) with the amount of labeled antigen measured for samples containing known amounts of said analyte antibody prepared in accordance with steps a) through e) to determine the concentration of analyte antibody in said fluid;
wherein said immobilized and labeled antigens are the same before they are bound to said solid phase or labeled, respectively.

26. A process of claim 25, wherein said immobilized and labeled antigens are both antigens that complex with antibodies which bind to epitopes of Borrelia burgdorferi.

27. A process of claim 25, wherein the label is an enzyme or a radioisotope.

28. A process of claim 27, wherein the label is alkaline phosphatase.