CA1104927A - Diagnostic immunochemical test material and procedure - Google Patents

Abstract

DIAGNOSTIC IMMUNOCHEMICAL TEST
MATERIALS AND PROCEDURE

ABSTRACT OF THE DISCLOSURE
Materials and procedure are disclosed for conducting a rapid diagnostic test for the detection of an immunochem-ically reactive substance. The test is suitable for detection of a specific antigen or antibody in serum or other bodily fluid. A substance capable of immunochemically reacting with the substance to be detected is immobilized on an insoluble carrier. The immobilized substance is used to harvest the substance to be detected from the bodily fluid.
The presence of the harvested substance is detected by the use of an antibody-enzyme conjugate capable of immunochemically binding the harvested substance. The immunochemically bound enzyme activity provides a direct measure of the amount of the harvested substance. Proper choice of carrier for the immobilized substance, method of immobilization, reagents and method of conducting the reaction sequence results in a rapid, sensitive, convenient and inexpensive technique, especially useful for detecting antibodies to Toxoplasma gondii and antigens of viruses, such as Hepatitis B.

Description

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This invention relates to the detection o antigen or antibody using ¦ immunochemical reactions. Sam?les of bodily ~luid may be tested for the 1~ presence of a particular antigen or antibody, from which a diagnostic 1l inference may be made.

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An immunochemical reaction is here defined as the specific binding l which takes place between an antigen and antibody. Antigens and antihodies ¦ are comps)nents of the immu~ity system whereby mammals including man protect themselves against infectious agents. An antigen is any substance Eoreign to the organism into which it has been introduced, which is capable ! o~ eliciting the protective immune re~ponse of that organism. Most antigens are proteinaceous materials in whole or in part, having high molecular weights. Antibodies are also proteinaceous macromolecules, elicited in lS response to the presence of a foreign antigen, in the organism. Antibodies have the property of being capable of binding with antigen molecules in highly specific combinations. The binding i5 characterized by its high degree of speciicity and low dissociation constant.
Normally, an animal has only those antibodies which are specifically directed against antigens to which it has been exposed in its environment.
However, an animal can be induced to form antibodies against other antigens by artlfically introducing them, for example by injection. Medical use is made of this phenomenon to immunize people against disease. It is also possible to cause a laboratory animal such as a rabbit or goat to make antibodies against specific substances. Such antibodies may be obtained from the blood o the animal and are exploited in highly speciîic assay techniques or the detection o the original antigenic substance. Virtually :`
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an~- prot~in can in principle be defectcd by means oE an immunochemical ! reaction.
Immunochemical reactions have been exploited in a variety of ways:
for the diagnosis oE disease: ~or the identification of a specific infecting l1 organism, as highly specific enzyme inhibitors, to determine the location ¦ of speci~ic proteins in tissues and within cells, and for the quantitative measurement of speciEic proteins Eor which no chemical -test is available, Since the reaction is a binding of one component ~antigen) to another (antibody~, there is no net cllange in the number of reactive groups as in an ordinary chemical reaction~ Analysis of an immunochemical reaction therefore requireS techniques for differentiating between bound and unbound component3.
A variety of methods has been employed in the prior art for the meaSurernent oE immunochemical reactions. Thege include hemagglutination, .~ ~ Lhdn ~t~ latex particle E~i~, agar gel diffusion, complement fixation, counterelectrophoresis, and the use of antibodies tagged with fluorescent dyes or radioisotopes. ¦
I ~emagglutination and Eluorescent antibody techniques have been ¦ applied in the detection oE antibodies against Toxoplasma ~ondii,p~ Toxoplasma !
is a protozoan parasite of man which lives primarily inside the cells of the host, SO that the organism is difficult to detect by microscopic means. The ~ast rnajority of Toxoplasma infections are asyr~ptomatic, However, an asymptomatically infected mother can pass the organism to the fetus, where Toxoplasma infection can cause a variety of birth defects: malformations, I hydrocephalus, mental retardatlon, eye diseases often leadin~ to blindness, and inEant mortality. A simple, inexpenSive screening test for pregnant Jes~rc~ble mothers would be highly ~ as a step toward the eradication oE
congen t 1l ~oxo~la~ . Although hernagglu~lnatlon and fluore9cent antibod~

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li techniques have been used in ~he diagnosis o~ Toxoplasma in~ection, the ¦I standard method has been the dye test. Serum antibody against Toxoplasma is detected in the dye test by taking advantage of the fact that live Toxoplasmal cells are partially lysed in the presence o~ antibody. Lysed celis are ! distinguishable from unlysed cells by the addi~ion of methylene blue whichonly stains intact organisms~ In practice the test is complicated by the additional requirement Çor an accessory factor~ thought to be complement, ¦ which must be obtained from the serum of Toxoplasma free clonors. Great ¦ attention-to detail is required in carrying out the tests successfully. More ¦ signi~icantly, the test is dangero`us to laboratory workers since it involves the use of live Toxoplasma. A number of laboratory infections have resulted in individuals who have perEormed the test (see Jacobs, L., "Serodiagnosis o~ Toxoplasmosis", in ~ Infections, S. Cohen and E. Sadun, eds., Blackwell Scientific Publications, 1976;. An improved ~;
immunochemical method for detect;ng Toxoplasma infection is therefore des~ra.6J~
highly e~.
Although immunochemical methods generally used in the prior art I can be highly sensitive, especially radioimmunoassay using radioisotope-'il tagged antibody, the widespread usefulness of immunochemical assays has ¦ been limited by three factors:
(~ ) the need ~or a convenient and inexpensive method of separating bound immunochemically reacted components from ; ` unbound components, ¦ (2) the need Eor a convenient and inexpensive way to Z5 1 measure the amount of immunochemical reactant bound, and 3~ the need for a procedure that can be carried out rapidly. I
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2~7 Tl~e first two o~ these ,li~ficulties have been overcome by recent il advances in the prior art, the tllird is overcome by the present invention.
The ~irst of these recent advances in the prior art is the development I of techniques for coupling an immunochemically reactive antigen or antibody I to an insoluble carrier material. An antibody imrnobilized on an insoluble carrier can, when exposed to a solution con~aining antigen, bind the antigen and render it, in turn, immobilized. The entire immunochemical reaction ¦ occurs on the carrier and the components which are bound on the carrier can be separated from the unreacted components by conventional techniques LO I for separating solid phase materials from a liquid phase. For example, if ¦ the carrier is in the form of beads e~- finely divided powder, separation can be accomplished by centrifugation or decantation. Qltern~tively, the immobilized phase may be the inner surface of the reaction vessel itself, or it may be in the form o~ a sponge or porous matri~ so that separation may be L5 carried out be simple decantation or by removal of the carrier, respectively.
The second advance has been the introduction of an enzyme-tagged antibody, which is a covalent conjugate of an antikody and an enzyme. Each retains its characteristic reactive properties: the antibody remains immunologically reactive and the enzyme retains its catalytic activity. When ~0 such a conjugate binds to an immobilized antigen, its presence can bedetected through the activity of the coupled enzyme, after separating unbound conjugate by appropriate means.
These two technlques have been used in combination to develop a very sensltive type of assay termed an enzyme-linked immunosorbent assay. The invention which is the subject of this report embodies these basic techniques.
For convenience, two types of enzyme-linked immunosorbent assay will be dlscussed and re~erred to as EL-1 and EL-2; see the diagram beiow:

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EL -1: C Ab Ag- Ab- Enz ¦ EL-2: C l~g ~b - AntiAb-Enz C stantls for an inert~ insoluble carrier;
¦j Ab symbolizes antibody;
¦¦ Ag symbolizes antigen;
C--Ab symbolizes carrier-bound antibody;
l ~--Ag symbolizes carrier-bound antigen;
jl Ab-Enz symbolizes the conjugate of an antibody with an enzyme ;
1¦ AntiAb-Enz symbolizes an antibody against immunoglobulin, 1l conjugated with an enzyme, ¦~ EL-1 is a technique for detecting the presence of an antigen. Antibodyagainst the antigen iS immobilized on an insoluble carrier. The immobilized antibody is then exposed to an antigen-containing fluid, The immobilized antibody harvests the anti~en from the solution by binding it in place. The carrier is then separated from the solution, washed free of contaminants, ~ l ¦ and exposed to a solution of the antibody conjugated to an enzyme. The principle Oe operation is that the conjugate ls able to bind only at sites occupied by the antigen, and that the number of sites so occupied determines the amount of conjugate which can bind. Each site where antigen is bound 1 i8 thuS tagged with bound enzyme whose presence is manifested by its ability to catalyze a reaction. The rate Or such reaction is proportional to the arnount ol enzyme present~and becomes a direct measure of the amount of antlgen bound, In EL-2, the component to be detected is an antibody. Antigen iS
immobilized on the carrier, Binding of the antibody is then measured by :j ~the subsequent binding of an anti-immunoglobulin-en~ym2 conjugate~ ~See 4 ¦ ~ Isdom, (:.D., ~n Clinical Chemistry, vol. ZZ, p. 1243 (ID76). ) ~' ' . '' .
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Il ~ntigen or antibocly molecllles may be immob~ ed on a solid carrier ¦¦ by a variety of methods kno~vn in the art, including covalent coupling, direct I adsorption, physical entràpment ancl attachment to a protein-coated surface.
!I For references describing this methodology, see Silman, I.H. and 1¦ Katchalski, E. in Annual Review of 3iochemistry, Vol. 35, p. 873 ~1966), I

¦ Melrose, G. J. H. J in Review of Pure and Applied hemistry, vol. 21, p. 83, (1971); and Cuatrecasas, PO and Anfinsen, C.B., in Methods in Enzymology, Vol. 22, (1971). `
The method oE attachment to a protein-coated surface is disclosed by pa~e"t/l~ G~
~ , Lai et al. ~German OS 2, 539, 65~, US. Y;~l;l})~ In this method, the internal and external surfaces of a microporous membrane are first coated with a water insoluble protein such as zein, collagen, fibrinogen, keratin, glutelin, polyisoleucine, polytryptophan, polyphenylalanine, polSrtyrosine, or copolymers of leucine with p~a~ino phenylalanine. Such ~15 1 a coating renders the membrane capable of immobilizing a wide variety of I biologically active proteins including enzymes, antigens, and antibodies. A
microporous structure is defined as one having more than 50% of its total , volume in-the form of pores ranging in size from 25 nanometers to 25 micrometers, preferably Erom 25 nanometers to 14 micrometers. A pore . .,.,, . .
6ize range from 25 nanometers to 5 micrometers is~ employed in most applications herein. Uncoated microporous membranes have as much as 70 tv 75% of their volume as pore space, The pores permit liquid flow through the membrane. After being coated by zein, for exarnple, the pore space Is reduced 5 to 10% with the result that the structure retains its essential properties of having ~ high proportion of its volume as pore space ¦ and permitting liquid flow through the pores, The structure has a large surtace area m contaot wlth any solution contained within the poreJ~

_7_ , . ' Such a coated membrane, having immobilized antigen or antibody, provides a compact, easy to manipulate carrier for the immobilized antigen or antibody. Its integral structure permits removal of bound from unbound components by simple mechanical means.
A difficulty attending the use of microporous membranes as carriers ~or immobili~ed antigen or antibody is that these structures may adsorb proteins nonspecifically.
Uncoated membranes of cellulose acetate and nitrate mixed esters can bind certain proteins and are also capable of exchanging bound for soluble protein. The physico-chemical basis for the binding is unclear~ Certain proteins appear to bind more readily than others. As a result, assays based on binding a specific antigen, antibody or conjugate in the presence of a mixture of proteins can result in high back-ground interference which may occur in an unpredictable manner.
A related dificulty is presented when coated membranes are used. Coated membranes, as disclosed by Lai, et al, are capable of binding proteins general]y, but the binding is neither as selective nor as variable as that displayed by uncoated membxanes. Consequently, the use of such mem~ranes to immobilize antibody or antigen in an EL-l or EI.~2 assay may also result in high ~ackground inter-erence due to the binding of unwanted protein species.
These difficulties have been effectively surmounted by the present invention.
Tn accordance with the present invention, there is provided a method for de-te~ting the presence OL one component of an immunochemical reaction between a first component that is selected from the group consisting of antibodies ,~
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and antigens, and a second component that binds immuno-chemically with the first component, comprising:
immobilizing the first component on the internal and external surfaces of a microporous membrane that has upper and lower external membrane sur~aoes ancl that is at least 50% microporous, the membrane having an interconnecting network of pores extending throughout il:s thickness between the upper and lower external membrane surfaces, providing internal membrane surfaces, the membrane having pore si~es in the range from 25 nanometers to 25 mi.crometers, and the network of pores permitting fluid flow through the membrane, thereby providing internal and external surface areas that are exposed to any fluid flow into and through the membxane rom the upper surface to the lower surface thereof, the aggregate internal surface area being many times largér than the external surface area, app].ying a solution of the second component to the upper surface of the membrane onto which the first component is immobilized and then passing it through the membrane, whereby the second component is brought into reactive contact with the immobilized first component on the external and internal surfaces of the membrane, to permit immunochemical binding of the second component to the immobllized.first component, then flowing a rinse solution through the membrane from the upper surface to discharge from the lower surface and to flush away residual solution of the second component, applying a solution of an enzyme labelled immunochemical reactant for the second component to the upper surface of the membrane and then passing it through the me~rane, whereby the labelled reactant is brought into reactive contact with the bound second component, ~lowing a rlnse solution through the membrane from its upper surfaGe ~a~
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to discharge from i.ts lower surface, to flush away any residual solution of the enzyme-labelled immunochemical reactant, and then detecting the presence on the surfaces of the membrane of the second component by observation of a colour change on the surfaces of the membrane produced by a reaction of the enzyme with a chromagen.
~ he present invention also includes a device for use in a diagnostic test for detecting the presence in a test solution of an immunochemically reactive substance selected from the group consisting of an antibody ancL an antigen tha~ are immunochemically reactive with each other, comprising: a microporous membrane having a pair of opposed external surfaces, one of which is an upper surace and the .
other of which is a lower surface, the membrane being formed with an intercommunicatiny network of pores extending throughout its thickness, with pore sizes in the range from 25 nanometers to ~5 micrometers, the membrane being at least ~ 50% porous and having a large internal surface area, the :~ pores providing passageways ~rom its upper surface to its 20 lower surface and permitting fluid flow through the membrane, the membrane having applied to its surfaces an immunochemically neutral protein dried in situ thereon, the membrane having immobilized thereto a biolo~ically active substance selected from the g.roup, a support structure that is formed with an opening, the membrane having its upper surface sealed to the suppor~ structure about the opening to aispose the mem~rane ~ across the opening, whereby a test solution applled in the opening to the upper surface of the membrane can flow through the membrane for contact with its internal surfaces 3Q ana the immunochemical reactant immobilized thereon, for discharge ~rom the lower surface of the membrane.
In addition, the present invention provides a test 9a ~

kit for use with a solutisn to be tested compris1.ng the device and reagent materials comprising, as a first reagent, a solution of a conjugate of an antibody and an enzyme, and as a second reagent material, a solution of a substrate for the enzyme moiety of the conjugate, and a rinse buffer solution.
In one of its specific embodiments, the present invention provides a method for detecting in a bodily fluid one componenk of a two component immunochemical reaction between a first component that is selected from the group consisting o~ antigens and anti~odies, second component from the group that binds immunochemically with the ~irst component, the method comprising: providing as a test device a plastic support structure having at least two generally ~up-shaped wells each of which is opened at its lower end and has a substantially circular transverse section, each of the wells having sealed thereto about its bottom opening a microporous membrane, each microporous membrane being disposed to have upper and lower external membrane surfaces and being at least 50~ microporous, each membrane having an intercommunicating netwo.rk of pores extending throughout its thickness ~etween its upper and lower membrane surfaces to provide internal surface areas, ha~ing pore sizes in the range from 25 nanometers to 25 micrometers, and permitting fluid flow therethrough, thereby providing internal and external surface areas that are exposed to any fluid flow into and through the porous membrane from the upper surface to the lower surface thereof, the aggregate internal surface area being many times larger than the external su.rface area, each membrane having immo~ilized thereto one component selected from the group, applying a sample of bod~ly fluid that may contain the complementary one of the components to .
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the upper surface of the membrane of a first one of the wells and then passing it through the membrane from the upper surface thereof to the lower surface thereof through the pores of the membrane for discharge from the lower surface of the membrane, the complementary component if present in the bodily fluid binding specifically to the immobilized component; applying to the upper surface of the membrane in a second cup a negative control fluid known to be free of the complementary component and then passing the negative con~rol fluid through the membrane from its upper surface to its lower surface or discharge from the lower surface of the membrane; passing washing fluid uni-directionally through each of the membranes to remove unbound materials; applying a solution of an enzyme conjugate for the complementary component to the upper surface of each of the membranes, the conjugate solution binding specifically to any of the complementary component that is hound on the membrane, and then flowing the enzyme conjugake solution through each of the membranes ~rom the upper surface of each respectively through the membrane for contact with its internal surfaces and for discharge from its respective lower surface; flowing washing fluid unidirectionall~
through each of the membranes to remove unbound conjugate solution; applying to the upper surface of each of the membrane means for detecting bound enzyme by observation of a colour change on the surfaces o~ the membrane produced by ; a reaction of the enz~me with a chromagen, and comparing ~ the two membranes to determine the substantial absence of enzyme from the mem~rane trea~ed with the negative control . solution and the presence or absence of enzyme on the ; membrane treated with the bodily fluld sample; the method - 9c-f~ .
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being capable of completion in less than one hour.
A test kit for effecting the latter process also is provided comprising a plastic support structure that is formed with at least two of the generally cup-shaped wells thereinl each having a bottom opening/ lo the pe:iphery of each of which one of the microporous membranes is sealed, each sealed membrane extending across ilts respective opening and having a majority of its volume as pore volume, each membrane having immobilized on its in~ernal and exte:rnal surfaces the immunochemical component a given one component from the group, one of the membranes serving as a test membrane for bodily fluid and the other of the membranes providing a negative control; each membrane beinq disposed to have an upper surface and a lower surface and an intercommunicating network of pores extending throughout its thickness, providing internal membrane surfaces, and ~ .
pro~iding passageways interconnecting the upper membrane -.
external surface and the lower membrane external surface, the pores having sizes in the range from 25 nanometers to 25 micrometers; a negative control reagent liquid that is free of the other immunochemical reactant component selected from the group; a solution of an enzyme con~ugate .~ that can immunochemically bind to the other component of the group, and means for permitting the detection of the : . presence of the enæyme in the conjugate on a membrane by observation o~ a colour change on the surfaces o~ the mem~rane produced by a reaction of the enzyme with a ~ ;
chromagen.
The present invention, therefore, is a diagnostic 3Q assay method and relates to the materials necessary to practice the method as well as to the method itself. The - 9d : .

method is applicable to the detection of an antigen or an antibody in a fluid sample. In one application of the invention, for example, it is used to detect the presence or absence of an antibody in a sample oi` serum. In another application, an antigen is detected in a sample of a bodily fluid.
The materials used for the practice of the invention include an antigen or antibody immobilized on the surface of an integral structure having a large surface available for contact with the sample and treated to minimize non-specific protein binding, means for mounting the structure so that sample, ~7ash solutions and reagent may be permitted to flow through the structure, a first reagent comprising a conjugate of an anti~ody with an enzyme and a second reagent comprising a substrate for the enzyme.
In the preferred embodiment, one component of the immunochemical reaction, either antigen or antibody, is immobilized on the zein-coated inkernal and external surfaces of a microporous mem~rane, as previously defined, whereby these surfaces are rendered immunochemically reactive.
The membrane is mounted so that the fluid to be tested, for example, serum, may be applied to one side of the membrane, allowed to flow through the pores of the men~rane and collected from the other side. The desired immunochemical ~; reaction between a component in the serum and the immobilized component takes place during the period of time in which the serum is in contact ~ith the reactive surfaces during passage through the membrane~
Once the desired immunochemical component is harvested from the test fluid, its presence on the mem~rane surface may be detected by the subsequent passage through the membrane of - 9e ;-~,, )~ . ' .

an antibody-enzyme conjugateO The antibody moiety of the conjugate is specific for the component to be detected, while the enzyme is chosen to be one whose activity is readily detected by methods well known in the art.
Peroxidase (enzyme no. 1.11.1.71 is preferred. The enzyme numbering scheme used herein is that set forth in Florkin et al, "Comprehensive ~iochemistry" Vol. 13, 3rd Ed., Elsevier Pub. Co. New York (1973~o Immunochemical binding of the conjugate to the harvested component occurs during passage of conjugate solution throug~ the membrane.
The conjugate will be immunochemically bound only at sites where the component to be detected is bound.

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A reagent solution conl:aining substrate for the bound en~yme is ne~t passed through the memhrane. Qualitatively, the prcsence o~ immuno-,¦ chemically bound peroxidase is detected by the use o~ a chrl~magenic substrat~
Il and the subsequent development of color. In the absence of nonspeciic ¦ binding any color which develops is due ~o the immunochemical binding of the conjugate, which in turn depends on the presence o the component to be ~ detected and demonstrates the existence of the component to be detected in 1I the test fluid.
¦ Quantitatively, the amount of color which is developed may be j measured, for example, by spectrophotometry. The amount of color developed is a measure o~ the amount of chromagenic substrate converted to product, which in turn is a measure of the amount of enzyrne bound. In the ! absence of nonspecific binding of the conjugate, the amount of conjugate hound is a measure of the amount of the component -to be tested harvested on the membrane, and hence a measure of the amount oE the component present in the test fluid.
In practice, a certain amount of nonspecific binding occurs, It is therefore necessary to employ a control sample, in which it is kr-own that the component to be detected is absent. ~n important feature of the invention is the adoption of techniques which minimize the amount of nonspecific binding and maximize the differences observed between positive samples and controls. In this regard, the use of a zein coal;ing on the microporous membranes, the use of a two stage immob~lization procedure, and the use ¦, of highly purified peroxidase in forrning the conjugate have been effectivelyl combirled in the preferred embodimen~ to reduce nonspecific binding. The procedures employed are critical to the practice of the invention and wlll be discussed in detail below~

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It has be~n discovered that immunochemical assays may be carried out according to the invention using extremely short incubation times. Complete tests can be run from start to finish in 20-30 minutes, as compared to several hours for prior methods. In addition, the invention provides advan-tages of simplicity and ease of operation, adaptability for routine use, and lack of requirement for a highly trained technician or expensive equipm~nt. Because of these advantages, the invention renders immunochemical assays more readily available for use in a wide range of clinical, industrial, and environmental tests. Examples of the invention's applicability include the routine detection of hepatitis B antigen in donated blood, the diagnosis of , and the testing o Eoodstuffs or microbial contamination or toxins.
Proper function of the invention is in part dependent upon the choice of a suitable surface upon which antigen or antibody is immobilized. The surface structure should permit 1uid flow into and through the structure and should present a large surface area relative to the volume of fluid con-tained within the structure. Such requirements are satisfied, to varying degrees, by such diverse structures as hollow fibre bundles, poxous refractory filters, microporous membrane filters, and packed columns. The choice of support in each case will depend upon the type of assay and the use to which i~ is put. For a wide variety of assays, where speed, convenience and economy must be considered, the use of a microporous mem~rane is preferred.
The method of immobilizing antigen or antibody to the surface of such a structure may in principle be any method suitable for the particular surface employed and material to .~
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be attached. The method of Lai, supra, is suitable for immobilization of biologically active materials to a wide variety of surfaces. In addition, the use of a coating has the unexpected advantage of providing a means for controlling nonspecific protein binding.
In the preferred emhodiment, ze;n~coated microporous membranes are employed. The term microporous membrane is here defined as having pores that fall within the size range from 25 nanomsters to about 25 micrometers and preFerably, from 25 nanometers to 14 micrometers. The coating may be applied without su~stantial loss of flow-through capability and with only a slight diminution of pore volume. Micro-porous membrane filters present the ~urther advantage that their external as well as their internal surfaces become coated and thus able to immobilize antibody. Particulate antigens, such as whole cells or cell fragments, can be bound - 12 ~

llC4~r~7 e~,-en tho-lgh they may be too large to enter the pores of the membrane, Preparations of antibody immobilized to zein-coated microporous membrane filters remain immunochemically reactiYe for long periods oE time uncler Il proper conditions. Samples may be stored under refrigeration or lyophilized ¦ asld stored in a controlled humidity environment.

i Antigens and antibodies used in the invention may be prepared by standard techniques well known in the art. Antibodies may be prepared I from the serum o~ animals such as rabbits, horses, or goats which have il beer immunized against the appropriate antigen. Antigens are purified ¦¦ from the source organism lay known techniques used in the Separation and purification of biological materials.
Since the structure having the irnmobilized component is -to be e~osed to a bodily fluid comprising a mixture of proteinaceouS rnaterials, any affinity between such materials and the structure's surface can result i in nonspeciic binding. Such binding could seriously interfere with the assay, For e~ample, the total amount of protein which could bind to the uncoated ¦¦ surfaces of a microporous membrane could exceed the amount bound by a specific immunochemical reaction. Similarly~ where a coated microporous membrane iS employed, the membrane may retain the ability to bind additional protein nonspecificallyO
It has been di8covered as a part of this invention that no~specific binding may be minimized by interposing a second stage immobilization step, in which an immunochemically neutral protein is immobilized to the ~ilter.
Immobilization therefore occurs in two stages according to the preferred 1 embodiment of the invention: a ~irst stage in which the desired immuno-chemical componen~ is immobilized, and a second stage, following the completi n oE the first, ~n which an immunochemically neutral protein such : l .
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l as Eetal calf serum or boville gamma globulin is next immobilized. The te~n immunochernically neutral is defined in terms of the specific components of the assay. Any protein which does not combine immuno-Il chemically with a component of the assay or with one of the reagents is 1¦ considered immunochemically neutral, even though such protein might be I¦ immunochemically reactive in another system. 'rhe combination o~ coating ¦¦ a microporous membrane by the method of Lai, immobilizing an ¦ immunochemical reactant in a first immobilization step, followed by I immobilizing an immunochemically neutral protein in a second immobilization LO step results is substantial reduction of nonspecific binding when themembrane bearing its immobilized components is exposed to a mixture of proteinaceous m ate rials .
The conjvgate o~ antibody with enzyme is made using techniques known in the prior art. (For references, see ~vrameas, S. and Uriel, J., L5 in Comptes Rendus ~Iebdomadaires des Seances de l'Acadernie des Sciences,vol. 262, p. 2543, (1966); Nakane, P.K. and Pierce, G.B., in ~ournal of Mistochemistry and Cytochemistry" vol. 14, p. 929, (1966); Nakane, P.K., il in Methods in Enzymology, vol. 37, p. 133, (1975). ) In an EL-1 type of ¦ assay, the antibody moiety of the conjugate should have the same ~0 ¦ immunological specificity as the immobilized antibody. In EL-2 where the¦ substance to be detected is an antibody, the immunochemically reactive ¦ moiety of the conjugate must be an antibody capable of binding immuno-chemically with the antibody to be tested. Such antibo~ies may be obtained by immunizing an animal with the antibody or immunoglobulin fraction of ~5 serum from the animal in which the antibody to be tested originated. For I e}~ample, where the antibody to be tested is a human antibody, a goat antibody¦ against human antibody is obtained from the serum of a goat immunized 1~ -14-against human immunoglo~ulin (antibody). The enzyme moiety may be any enzyme capa~le of catalyæing a reaction which can be detected by any method known to those skilled in the art, and which retains its activity after conjugation with antibody. Horseradish peroxidase (enzyme no. 1.11.1.7~
is preferred because of its convenience and suitability to a wide range of applications. It is well known that the enzyme catalyzes the oxidation of a variety of organic compounds in the presence of hydrogen peroxide. Many such organic substrates are chromagenic, i.e., undergo a colour change upon oxidation.
It has been found in the present invention that the purity of the enzyme preparation used in the ~ormation of conjugate has an effect on the degree of nonspecific binding.
The greater the purity of the enzyme preparation, the less the nonspecific binding. In part, the reduction is made possible because the total amount of conjugate protein required is reduced as the specific activity of the enzyme is increased. The opportunity ~or nonspecific ~inding is therefore reduced as well. In the preferred embodiment, the use of a highly purified peroxidase preparation has been found to significantly reduce the amount of colour reaction observed in control samples as compared with known positives.
The first reaction step in the assay procedure involves bringing together the immobilized component of the irnmunochemical reaction and the test fluid, which may contain the component to ~e detected, such that reactive contact occurs. It is at this step that the choice of a suitable carriex for the immobiliæed component, having a large surface area in relation to the volume contained within the structure becomes important~ As the test fluid flows through the `~.`1 . _ . .

structure, the pxobability that a solute molecule will contact one of the surfaces during liquid flow through the structure is very high. The probabil.ity of an immunochem-ically reactive contact is further increased as the probabil-ity of such collisions per unit of surface area is maximized.
It is believed that the probability of such collisions depends, in a general way, upon the parameters of pore size and flow rate, where solute - 15a -' ,"

concentra~ion and reaction temperature are held constant.
Thus, the more constricted the passageway through which the solute molecules pass, relative to their mean free path, the greater the likelihood of collision with the surface.
The lower the flow rate~ the longer the residence time of the solute within the structure itself, and the greater the probabillty of collision. In accordance with the fore~
going considerations, structures having large ratios of surface exposed to the volume of fluid flowing therethrough are employed in the present invention in an attempt to increase the probability of reactive contact with the surface and to reduce the time required to carry out the reactions involved.
The invention is descrihed further, by way of illustration, with reference to the accompanying drawings, wherein: ' Figure 1 is a perspective view of a device for use in the diagnostic assay of multiple samples, constructed in accordance with one embodiment of the invention;
Figure 2 is a fragmentary section, taken on the line 2-2 of Figure 1, looking in the direction of the arrows;
Figure 3 is a fragmentary top plan view thereof; and Figure 4 is a fragmentary top plan view of a device made in accordance with a different em~odiment of the inventionO
Referring now in detail to the drawings by numerals of referencel the de~ice 10 is formed from a molded plastic, preferably transparent and tou~h, base on a tray 12. The tray 12 has a bottom panel 13 and upstanding walls 14 that are formed with an internal lip 16. A holder 18 is de~achably seated on ~he lip 16. This holder is also formed from tough, , . ~ , .

i2~

transparent plastic and it in turn is formed with a plurality of generally cylindrical, cup-like recessed parts 20. The bottom opening of each cup 20 is covered with a membrane 22 that is heat sealed to the lower rim of the cup. In the embodiment of the invention shown in Figure 4, the holder 18' is formed with a much larger number of cups 20', thus permitting a single device to be used for a larger number of assays.
In use, a sample is poured into a cup. The sample flows through the membrane into the tray. As will be described in detail hereafter, the membrane is washed and treated with reagents r as needed, to detect the presence o~ absence of a particular material in the sample.
In the preferred embodiment, microporous membrane~
are employed as carrier for the immobilized component. The membrane is mounted in a convenient holder in order to permit fluid flow of sample, wash solutions :

~ I - 16a -~.~

and reagel~ts tl~rough the pore~ o~ the membr~le. Such mernbranes are a~-ailable in a variety of pore sizes. The optimum pore size will clepend on tlle requirements of the specific assay, includins the flolv characteristics of the sample to be tested and the amount of fluid to be processed. The incubation time is governed by the flo~,v rate through the filter and ma~- be controlled by a variety oE means known in the art. ~ simple and effective technique is to apply fluid dropwise to the upper sur~ace of the membrane so that the rate of flow oE fluid is governed only by the h~ dro3tatic pressure exerted by the drop as it rests on top of the membrane. IE the droplet is small enough, i. e., approaching the retention volume of the membrane, the - i' fluid will tend to ~low into the pores of the membrane arld be retained there by capillary attraction until displaced by additional fluid, Reaction time under these circumstances is controlled by t'ne residence time of the sarnple ! within the membrane pores, When the structure is mounted so that reactants and reagents can flour 1 -through the structure ~rom one side to the other, the entire seg~:lence of steps is easily and rapidly performed, Flrst, test fluid is per~itted to ~low , through the structure in order to harvest an~- of the component to be tested ~ -I present therein. Second, a solution of conjugate is permitted to flow through I at a controlled rate so that conjugate may immunochemically bind to any ¦ component to be tested harvested in the first step. Thirdg substrate solution ! is perrnitted to flow through the structure at a controLled rat~, in order to ' permit immunochemically bound enzyme to convert the substrate to a ~
measureable product,. Washing steps m~y be interposed between the threa reaction steps if desired. The incubation temperature ma~ be controlled within the range 15C to 45C to suit ;ndividual needs~ Room te~nperature is preferred Eor the sake of convenience, in a qualitati~-e assay. In the practice of the preferred embodiment, using To:{oplasma ant;gen imrrobilized _ . , .

Oll a microporolls n~embrar:e, tlle presence o~ antibody to Toxopla ,ma in ser~ull is readil~ detected using a series of 5 minute incul)ations for each step ~vitll a ~vaslling step interposed between each, so that t'ne entire sequence of reactions is completed in 20-30 minutes, as described in detail in Ex~mple 1.
There is a variety of chromagenic subsl:rates available for the Jes~a l /~
qV detection o peroxidase activity, For qualitatiYe tests, it is ~e~Pe~te to employ either a chromagen which precipitates on the mem~rane when acted upon by peroxidase. or one in which the peroxidase reaction product binds preferentially to the membrane,. An example of the former is 3-amino-ethylcarbazole. An exarnple of the latter is 4-amino antipyrine. Quantitative measurements may be carried out using a soluble chromagen product and . .
' measuring the development of color by suitable methods such`a~ spectro-photometry.
',~ The speed and operaSing convenience of the present invention ~nake l it eminently suited ~or routine analysis. All the compon~nts can be provided in a stable ~orm, dried or lyophillzed, so that a techrlician has only to dissolve the preweighed mixture of bu~er salts or reagents in apredetermined amount o~ water prior to use. A test kit embodying these advantages is l~ contemplated. For example, a test kit for the detection of Toxoplasma l~ antibodies in serum would comprise -the following: microporous membranes ~¦ having Toxoplasma antigen ~nmobilized thereon mounted in a suil:able holder for adding reaction components to the upper surface OI the membrane and for , collecting the materials from the under surface which have flowed t'nrough, 2S lyophilized antibody-enzyme conjugate, dried buffer salts preweighed, anddry substrate preweighed. Where the enzyme moiety of the conjugate is peroxidase, a substrate mixture containing 4-amino antipyrine, 4-hydro~cy-~enzy~e n~ 3-/4) benzoate, lyophilized glucose oxidase~, glucose and buffer saLts is advantageous. The action of glucose oxidase on glucose generates the '7 ~ drogen peroYiùe substr~te used in the pero:;ida~e reaction, t'n~reb~-elilrlinatincS the need to provide h~drogen pe~oxic~e, lvhich is di~ficult to stabilize îor long periods o~ storage. When oxidized 'oy pero~;ide in the presence of peroxidase~ 4-amino antipyrine forms a colored substance which preferentially adsorbs to the microporous rnembrane~ In this manner, all of the components o~ the test can be provided in stable, dry, water soluble form.
The described invention presents significant advances over ~he prior ~ art in terms of speed, operating simplicityr convenience and expense.
Specific e~amples of procedures embodying aspects of the invention will ne~
be presented, in order to further demonstrate the in~-ention.

, .
~ ! E:~ample 1 - ~Antibody I I Screening Assay ~' A. A microporous membrane filter having 3 micron nominal pore ii size ~Type SS> manufactured ~y Millipore Filter Corp., Bedford, ~Iass. ) was coated with zein according to the Eollowlng p ocedure. Zein ~79g) was dissolved in a solvent mixture containing 180 ml ethanol, 34~ ml n-butanol, ,, 80 ~nl ~ater and 30 ml cellosolve, by mixing in a ball mill until completely ~ -¦I dissolved, Filters were submerged in the zein solution, allowed to soak !I for 16 hQurs9 removed and air dried.
¦ B. Toxoplaama antigen was immobilized on coated membrane filters ¦ by immersing the coat. d filters overnigm at 4C in 1. 5 ml of an antigen I preparation containing 6mg - 12mg total protein, The filters were then air dried at room temperature, washed with MilliRO (Reglstered Trade ~ark 2~ ; of Millipore Corporation, Bedford, Mass. ) water and again aiI dried at room temperature~
The ~a antigen preparation was a lysate o~ fresh Toxoplasrna cells obtained from peritoneal exudates of infected mLce by techniques known in the art.

.~, ., 19- , '' ',, :
. I ' . . . .. . . . . . ..

C~. Tlle second-s~age immobilization ~as carried O-lt a~ter the immol~iliæation witll To-.;oplasm~ ;lntigen tvas c:ompleted ancl the ~ilters ~vere dry. The filters were placed in a beaker containin~ a solution oE bovine ~ r~
gamma globulin ~raction II at a concentration of 20 mg per ml in ~IilliPIO
water and allo~ved to soak îor appro~;imatelS 16 hours. The filters were T~iI
then air dried at room temperature, washed once with ~IilliP~O water and air dried again at room temperature.
D. Serum to be tested was obtained ~rorn patient's blood samples , which were allo~ved to clo$ to rernove red blood cells and clotting proteins.
The serum sample ~as diluted with an equal volume oE tris-saline buPfer, 5 (Tris-saline huFEer contained 0. l ~I 2-amino-2-hydro~cymethyl-1, 3-propanediol and 0.15 ~ sodium chloride, pH 8, 0), E. A coated membrane filter having immobilized Toxoplasma antigen was mounted in a suitable holder to permi$ added fluid to enter one side of li .
l the membrane, flow through the membrane pores and exit from the other ~l I~ side of the membrane. Serum (l00 ,ul, diluted l:l in tris-saline buEfer) was

3 passed into the membrane's pores and allowed to remain there ~or 5 minutes at room temperature. Serum was then washed out oE the filter by addition of Il 1 ml tris-saline buf~er.
¦~ F. The ~ilter was then treated by the addition of 100~ of antibody-¦, enzyme conjugate. The conjugate was composed of horseradish peroxidase 5 (Worthington, HPOFF) coupled to goat anti-human IgG ant~body (Meloy Laboratories~ Inc., ~pringEield, Va., lot No. G5l621, 34. 2 mg/ml). Coupling was carried out by the metaperiodate activation method, as descrihed by Nakane, P,K, and Ka~vaoi9 ~., in ~. Histochem. Cytochem., vol. 22, p. I084 ~1974). The conjugate solution added to the filter hacl 6.6 ,uglml total conJugate protein and was diluted 500-fold from a stock solution~ using l0% ~etal calf sçrum as diluent. The conjugate tvas incubated with the îilter for 5 minutes, lthen washed l,vith 1 ml tris-saline bu~er.
, G. Q~lalitative staining was done using 3-an ino-eth~Llcarbazole as the cllromagen. Tlle chromagen (10 mg) ~ as clissol;-ed in 6 ml dimeth~
sulfo~ide, rollowed by the ~urther addition of aO ml 0. 02 ~ sodillm ace~ate pH 5. O. Just before use, 0~ 5 ml o~ 3% (v/~-) h5~drogen peroxide was acided.
The solution was then allowed to react S minutes wi~n the components immobilized on the membrane filter while flowing through the filter, The presence of an~ibc dy to To~oplasma in the test serum was indicated b~- a red color developed on the test filter. A filter treated ~Yith a sample of normal serum remained white or faintly colored.

Example 2 . __ ~; ~n this experiment, the e~fect of varying the incubation times for the ,i ~
', serum incubation step (as described in Example I/E) and for the conju~ate i incubation step (Example I/F) was tested. The experimental procedure o~
~! Example I was followed, except that the second-stage immobilization ~step 1, I/C) was omitted. Test sarnples of serum were known to be negative ~lacking Toxoplasma antibody) or positive (containing Toxoplasma antibody) by .he !, fluorescentantibodytest, (Kelen, ~E., Ayllon-Leindl, L. andLabzoffsky, ¦I M.A., Canad. 3. Microbiol., vol 8, pp 545-554 tl962)).
¦ A. The time o~ serum incubation was varied, while conjugate 2~ I incubation time and aubstrate incubation time wère held constant. Results are shown in Table 1, where a minus sign (-) indicates lack of color development, and plus signs (+) indicate shades oE color on a scale îorm i ligb~ pink (+) to doric pink (++~

" .':
,~

. , .
"'' .. , . - . '' ~ ,' Table rr~lbe number j Serum I Ser~ Result ~ ~ontents ~ incubation I
l ! time I (nli~lUteS) !
1 negative 1 2 negative 15 3 positive ~ ++~

4 positive 5 +++
l O 5 positive 10 1 +~+
6 positive 10 +++
7 positive 1~ ++-~
8 positive 15 _ ++~

The 5 minute samples developed a5 much color as the 15 minute samples, indicating that a 5 minute incubation was adequate.
f ' e Xper;~
B. In -this _ 7 serum incubation tirne and substra~e 1, incubation time were held constant at S minutes, and conjugate incubation ,'~ time was varied ~rom 5 minutes to 15 minutes. The results are shown in ~! .
~, Table 2.
i' rrable 2 " Tube number Serum I Result , ,~ contentsincubation (minutes) . .
2 5 ' 1 negative 15 +
, . 2 negative 15 -t l l 3 positive 5 ++~
! 4 positive 5 positive 10 6 positive 10 ++-~
7 po sitive l S ~+ ~
l 8 positive_ _ 15 _ +~+
I
.
It was concluded that 5 minutes reaction times were adequate ïor both .
the se~um incubation and the conjugate incu~ation.

Exampl_ 3 This representative experiment was designed to test the effectiveness o~ the second-stage immobili~.~,ation, described in Example I/C, in reducing the hackground ~olor oï negative control samples. The serum sarnples employed were knowrl negative ~T ~coplasma antibody absent) or positive (having ~ma antibody ) samples as previously determined by the fluorescent antibody test, or the hemagglutination assay.
(Jacobs, L. and Lunde, M.N., J. Parasitol, vol. 43, pp 303-314 (1957)). The tests were carried out as described in Example 1, except that for one-step immobilization samples, step I/C was omitted, and for two-step immobilization samples, 100~ (W/v) fetal calf serum was substltuted for bovine gamma globulin in step I/C. The results are shown in Table 3. The symbols employed have the same meaning as in Example 2.

Table 3 ¦ Tube Sample Method of Result Inumber Immobilization . . . _ .
1 negative l-step +
2 negati~e l-step + .-3 negative 2-step 4 negative 2-skep negative 2-step _ 6 negative 2-step _ 7 positive 2-step ~
8 positive 2-step +++
~t was concluded that 2-step immobilization significantly decreased background colour in negative samples, and improved the colour differential between negative and positive samples.
The present invention relates to the detection of antigens and antibodies in bodily fluids for diagnostic pur-poses, exploiting immunochemical reactions. A combination of factoxs and principles has been brought to bear in reducing the time required to carry out the sequence o~ steps and in improving the operating simplicity and economy of the entire procedure.
In the preferred embodiment the use of zein or collagen coated microporous membxanes pxovides signific~nt advantages in the practice of , ~ ' ' .

. . .
: ~ - 23 ~

4~Z',' the invention. ~irst, immobilization of specific antigens or antibodies is readily effected. Second, the binding properties of coated micropo~ous membranes make them sufficiently manageable that nonspeci~ic binding can Il be suppressed to an acceptable level.
1¦ The method is applicable to any test fluid capable of flowing through ~¦ the type of structure described, or any fluid capable of being rendered ¦ Elowable therethrough, by dilution, prefiltration, and the like. The method Il is suita~le for use with either EL-l or EL-2 assays, i. e., eiLher an antigen!j or an antlbody may be detected in the test fluid. Adaptation of the inventionfor the assay of a particular antigen or antibody is primarily a matter of choosing the appropriate component to be immobilized on the structure, and choosin~ the proper immunochemical component to be conjugated to an enzyme.
While the invention has been described in connection with specific embodiments thereof, It will be understood that it is capable of further ¦ modifications and this application is intended to cover any variations, uses, ¦ or adaptations of the invention followIng in general, the principles of the ¦ invention and including such departures from the present disclosure as come ~ within known or customary practice within the art to which the invention ZO I pertains and as may be applIed to the essential features hereinbefore set ¦ forth, and as follows in the scope of the appended claims.
I ,~ ~ ~
! ~:

Claims (28)

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. A method for detecting the presence of one component of an immunochemical reaction between a first component that is selected from the group consisting of antibodies and antigens, and a second component that binds immuno-chemically with the first component, comprising:
immobilizing the first component on the internal and external surfaces of a microporous membrane that has upper and lower external membrane surfaces and that is at least 50% microporous, said membrane having an interconnecting network of pores extending throughout its thickness between said upper and lower external membrane surfaces, providing internal membrane surfaces, said membrane having pore sizes in the range from 25 nanometers to 25 micrometers, and said network of pores permitting fluid flow through the membrane, thereby providing internal and external surface areas that are exposed to any fluid flow into and through the membrane from the upper surface to the lower surface thereof, the aggregate internal surface area being many times larger than the external surface area, applying a solution of the second component to the upper surface of the membrane onto which the first component is immobilized, and then passing it through the membrane, whereby the second component is brought into reactive contact with the immobilized first component on said external and internal surfaces of the membrane, to permit immunochemical binding of the second component to the immobilized first component, then flowing a rinse solution through the membrane from said upper surface to discharge from said lower surface and to flush away residual solution of the second component, applying a solution of an enzyme-labelled immunochem-ical reactant for said second component to said upper surface of the membrane and then passing it through the membrane, whereby said labelled reactant is brought into reactive contact with the bound second component, flowing a rinse solution through the membrane from its upper surface to discharge from its lower surface, to flush away any residual solution of the enzyme-labelled immunochemical reactant, and then detecting the presence on the surfaces of the membrane of the second component by observation of a colour change on the surfaces of the membrane produced by a reaction of the enzyme with a chromagen.

2. The method of claim 1, wherein the detecting step comprises applying to and then passing through the membrane on which the second component is immunochemically bound, a solution of a conjugate of an enzyme with an antibody to the second component, to immunochemically bind the conjugate, and then detecting the presence of the enzyme on the membrane.

3. The method of claim 2, wherein the step of detecting the presence of the enzyme on the membrane includes applying a solution of a substrate for the enzyme to the membrane and then passing it through the membrane, and measuring the extent of enzyme catalyzed reaction with the substrate, in order to determine the amount of antibody-enzyme conjugate bound to the second component, in order to determine the amount of the second component bound, thereby providing a measure of the amount of said second component in the solution of the second component.

4. The method of claim 1 wherein the microporous membrane is coated with an immunochemically neutral protein to suppress nonspecific binding to the membrane.

5. The method of claim 4, wherein the step of detecting the presence of the enzyme on the membrane includes adding a substrate for the enzyme and measuring quantitatively the extent of any enzyme catalyzed reaction, in order to determine the amount of antibody-enzyme con-jugate immunochemically bound to the second component, in order to determine the amount of the second component bound, thereby providing a measure of the amount of the second component in the solution of second component.

6. The method of claim 2, wherein the microporous mem-brane, prior to the immobilization therein of the first component, is precoated with an immunologically neutral protein, said coated membrane having the first component immobilized thereon, then the second component is brought into reactive contact with the first component by applying the solution of the second component to the upper surface of the membrane, then flowing it through the membrane and over the coated external and internal surfaces of the membrane, to be discharged from the lower external surface of the membrane, whereby the second component is brought into reactive contact with the first component upon appli-cation to and flow through the membrane.

7. The method of claim 6, wherein the second component is an antibody to Toxoplasma gondii, the first component is an antigen of Toxoplasma gondii, and the solution of the second component is serum.

8. The method for detecting the presence of one component of an immunochemical reaction between a first component that is selected from the group consisting of an antibody and an antigen, and the second, other component from said group, which second component binds immunochemically with the first component, comprising:
applying to a microporous membrane having upper and lower external membrane surfaces, and an intercommunicating network of pores extending throughout its thickness, providing internal membrane surfaces, and permitting fluid flow through the membrane, said membrane having a coating on its internal and external surfaces of an immunochemically neutral protein to suppress nonspecific binding, and pore sizes in its uncoated state in the range from 25 nanometers to 25 micrometers, the coated membrane being at least 50%
microporous, a solution of a biologically active first component selected from said group, and flowing said solution through the porous membrane from the upper surface to the lower surface thereof for discharge from said lower surface, thereby immobilizing said first component on the membrance surfaces, then applying a solution of the second, other component from said group to the upper surface of the membrane and then flowing it through the membrane on which the first component is immobilized, for contact with the external and internal coated surfaces on which the first component is immobilized, whereby the solution of the second component is brought into reactive contact with the immobilized first component, to permit immunochemical binding of the second component to the immobilized first component, then flowing a rinse solution through the membrane from its upper surface to discharge from its lower surface, to flush away residual solution of the second component, then applying a solution of an enzyme-labelled immuno-chemical reactant for said second component to the upper surface of the membrane and passing it through the membrane, then flowing a rinse solution through the membrane from its upper surface to discharge from its lower surface, to flush away residual solution of the enzyme-labelled immuno-chemical reactant, and then detecting the presence on the surfaces of the membrane of the second component by observing a colour change on the surfaces of the membrane produced by a reaction of the enzyme with a chromagen.

9. The method of claim 8, wherein the first component is an antigen of Toxoplasma gondii, the second component is an antibody to Toxoplasma gondii, and the solution of the second component is serum.

10. A method of nonimmunochemically immobilizing two proteins on the internal and external surfaces of a micro-porous membrane having pore sizes in the range from 25 nanometers to 25 micrometers in its uncoated state providing passageways and permitting fluid flow through the membrane, comprising:
coating the internal and external surfaces of the membrane with a protein selected from the group consisting of zein and collagen, to provide a coated membrane that is at least 50% microporous, immersing the coated membrane in a solution of a biologically active first protein selected from the group consisting of the antigen of and the antibody to Toxoplasma gondii, in order to immobilize the first protein, and immersing the coated membrane having the biological-ly active first protein immobilized thereon in a solution of an immunochemically neutral second protein selected from the group consisting of fetal calf serum and bovine gamma globulin fraction II, in order to immobilize the second protein while the first protein remains immobilized and re-tains its biological activity.

11. The method of claim 10, wherein the first immobilized protein is an antigen.

12. The method of claim 10, wherein the first immobilized protein is an antibody.

13. In an immunochemical assay for detecting the presence in a solution of an immunochemical selected from the group consisting of the antigen of and the antibody to Toxoplasma gondii, comprising a specific binding reaction between a first immunochemical component immobilized on the coated surfaces of a microporous membrane having pore sizes in the range from 25 nanometers to 25 micrometers in its uncoated state, and that is at least 50% microporous providing passageways and permitting fluid flow through the membrane, in a test fluid, wherein nonspecific binding occurs as a side reaction and wherein the coating is a protein selected from the group consisting of zein and collagen, a method for suppressing the nonspecific binding comprising:
treating the coated membrane, having the first component immobilized thereon, by immobilizing to the coated membrane, a protein immunochemically neutral in the assay and selected from the group consisting of fetal calf serum and bovine gamma globulin fraction II, whereby, when the second component is brought into reactive proximity to the immobilized first component, a specific immunochemical reaction takes place and nonspecific binding is suppressed.

14. The method of claim 13 wherein the first component is an antibody and the second component is an antigen.

15. The method of claim 13 wherein the first component is an antigen and the second component is an antibody.

16. A device for use in a diagnostic test for detecting the presence in a test solution of an immunochemically reactive substance selected from the group consisting of an antibody and an antigen that are immunochemically reactive with each other, comprising:
a microporous membrane having a pair of opposed external surfaces, one of which is an upper surface and the other of which is a lower surface, said membrane being formed with an intercommunicating network of pores extending throughout its thickness, with pore sizes in the range from 25 nanometers to 25 micrometers, said membrane being at least 50% porous and having a large internal surface area, said pores providing passageways from its upper surface to its lower surface and permitting fluid flow through the membrane, said membrane having applied to its surfaces an immunochemically neutral protein dried in situ thereon, said membrane having immobilized thereto a biologically active substance selected from said group, a support structure that is formed with an opening, said membrane having its upper surface sealed to said support structure about said opening to dispose said membrane across said opening, whereby a test solution applied in said opening to the upper surface of the membrane can flow through the membrane for contact with its internal surfaces and the immunochemical reactant immobilized thereon, for discharge from the lower surface of the membrane.

17. The device of claim 16 wherein said support structure is formed from a synthetic plastic material and is formed with a well, said opening being disposed at the bottom of the well, and said membrane being mounted across the opening and sealed to the wall of the well about said opening and thereby to said plastic support structure.

18. The device of claim 17, wherein said support structure comprises a pair of generally cup-shaped wells each of which is open at its lower-end and has a substan-tially circular transverse section, each of which has a membrane sealed to its lower end across the open end of the well as in claim 16, and wherein each of said membranes has immunochemical reactant immobilized thereto.

19. The device of claim 16, 17 or 18, wherein the immobilized biologically active substance is an antigen of Toxoplasma gondii.

20. A test kit comprising a device in accordance with claim 16 or 18, for use with a solution to be tested, said kit including reagent materials comprising, as a first reagent, a solution of a conjugate of an antibody and an enzyme, and as a second reagent material, a solution of a substrate for the enzyme moiety of the conjugate, and a rinse buffer solution.

21. A method for detecting in a bodily fluid one component of a two component immunochemical reaction between a first component that is selected from the group consisting of antigens and antibodies, second component from said group that binds immunochemicallyy with said first component, said method comprising:
providing as a test device a plastic support structure having at least two generally cup-shaped wells each of which is opened at its lower end and has a substantially circular transverse section, each of said wells having sealed thereto about its bottom opening a microporous membrane, each microporous membrane being disposed to have upper and lower external membrane surfaces and being at least 50%
microporous, each membrane having an intercommunicating network of pores extending throughout its thickness between its upper and lower membrane surfaces to provide internal surface areas, having pore sizes in the range from 25 nano-meters to 25 micrometers, and permitting fluid flow there-through, thereby providing internal and external surface areas that are exposed to any fluid flow into and through the porous membrane from the upper surface to the lower surface thereof, the aggregate internal surface area being many times larger than the external surface area, each said membrane having immobilized thereto one component selected from said group, applying a sample of bodily fluid that may contain the complementary one of said components to the upper surface of the membrane of a first one of said wells and then passing it through the membrane from the upper surface thereof to the lower surface thereof through the pores of the membrane for discharge from the lower surface of the membrane, said complementary component if present in said bodily fluid binding specifically to said immobilized component;
applying to the upper surface of the membrane in a second cup a negative control fluid known to be free of said complementary component and then passing said negative control fluid through the membrane from its upper surface to its lower surface for discharge from the lower surface of the membrane;
passing washing fluid unidirectionally through each of said membranes to remove unbound materials;
applying a solution of an enzyme conjugate for said complementary component to the upper surface of each of said membranes, said conjugate solution binding speci-fically to any of said complementary component: that is bound on said membrane, and then flowing said enzyme conjugate solution through each of said membranes from the upper surface of each respectively through the membrane for contact with its internal surfaces and for discharge from its respective lower surface;
flowing washing fluid unidirectionally through each of said membranes to remove unbound conjugate solution;
applying to the upper surface of each of said membtane means for detecting bound enzyme by observation of a colour change on the surfaces of the membrane pro-duced by a reaction of the enzyme with a chromagen, and comparing the two said membranes to determine the substantial absence of enzyme from the membrane treated with the negative control solution and the presence or absence of enzyme on the membrane treated with the bodily fluid sample;
said method being capable of completion in less than one hour.

22. The method of claim 21, wherein said support structure is formed with a plurality of generally cup-shaped wells, and each of said wells has one of said membranes sealed across its bottom opening respectively, wherein only one of said membranes is a negative control membrane.

23. The method of claim 21, wherein said membranes are unsupported on their lower external surfaces.

24. A test kit for use in practicing the method of claim 21 comprising:
a plastic support structure that is formed with at least two of said generally cup-shaped wells therein, each having a bottom opening, to the periphery of each of which one of said microporous membranes is sealed, each said sealed membrane extending across its respective opening and having a majority of its volume as pore volume, each said membrane having immobilized on its internal and external surfaces the immunochemical component a given one component from said group, one of said membranes serving as a test membrane for bodily fluid and the other of said membranes providing a negative control;
each said membrane being disposed to have an upper surface and a lower surface and an intercommunicating network of pores extending throughout its thickness, providing internal membrane surfaces, and providing passage-ways interconnecting the upper membrane external surface and the lower membrane external surface, said pores having sizes in the range from 25 nanometers to 25 micrometers;
a negative control reagent liquid that is free of the other immunochemical reactant component selected from said group;
a solution of an enzyme conjugate that can immuno-chemically bind to said other component of said group, and means for permitting the detection of the presence of the enzyme in said conjugate on a membrane by observation of a colour change on the surfaces of the membrane produced by a reaction of the enzyme with a chromagen.

25. The test kit of claim 24, wherein each of said membranes also has on its surface a coating dried in situ from a solution of an immunochemically neutral protein, to suppress nonspecific binding.

26. The test kit of claim 24, wherein each of said membranes is unsupported on its lower surface.

27. The test kit of claim 24, wherein said support member is formed with at least three generally cup-shaped wells, and wherein said kit includes a positive control reagent solution containing said complementary immuno-chemically reactive component.

28. The test kit of claim 24, wherein said means for detecting the presence of enzyme includes means for producing an enzyme-chromagen reaction that produces an insoluble, coloured reaction product in situ on the surfaces of the membrane.