CA1119340A - Method of coupling a protein to an epoxylated latex and the products formed therefrom - Google Patents

Abstract

ABSTRACT

The invention is to a latex-protein conjugate and method of its preparation of a latex having free surface epoxy groups with a protein having a free amino group to form a new amine bond. Methods of using said latex-protein conjugate in diagnostic tests and enzymes detection are also described.

Description

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METHOD OF COUPLING A PROTEIN TO AW EPOXYLATED LATEX
AND THE PRODUCTS FORMED THEREFROM

The invention relates to a method of coupling a latex having surface epoxide groups to a protein or protein component by the formation of an amine bond. Such carrier ;
bound proteins are particularly useful in carrying out immunological tests which wre based upon the antigen-~O antibody reaction.The antigen-antibody reaction is the basis for all immuno-logical test methods. Special proteins called antibodies are produced by an animal in response to the presence of an antigen, that is a foreisn substance usually a protein, in ~, the body fluids of the animal. This normal body response to a foreign protein has led to the development of a number of techniques which are used to diagnose various human and animal diseases or disorders.
In vitro tests for the presence of a suspected antigen or antibody in a body fluid are carried out by adding the immunological counterpart to a vial of the body fluid, i.e., add antigen if the test is for the presence of antibody or add antibody if the test is for the presence of antigen.
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If the suspected protein is present, the resulting antigen-antibody reaction is generally manifested by precipitation or agglutination ofthe antigen-antibody complex.
In some instances the antigen-antibody complex is slow to form and the particles that are formed are too small o to be observed with certainty. In such cases, detectabi-lity of the antigen-antibody reaction can be improved by utilizing a carrier. When the antigen or antibody is coated onto the surface of a carrier the reaction that occurs with the immunological counterpart products a visible mass or agglutant. The proteinic antigen or antibody may be adsorbed onto the surface of carriers such as erythrocites, bacterial cells, bentonite, polystyrene latex particles, anionic phenolic resins, or finely divided diazotized amino cellulose.
It has been found, however, that chemical binding of the antigen or antibody molecule to the carrier is superior to physical adsorption. U.S. Patent 3,857,931 teaches that proteinic antigen~sor antibodies can be chemically bound to a polymer latex carrier having surface carbonyl groups by an amide bond formed in the presence of a water-soluble carbodiimide coupling agent. U.S. Patent 3,806,417 de-scribes a method of binding a protein to a compound conta-ining an epoxide group and connecting said intermediate with a carrier material. A disadvantage of this method is that the protein must first be reacted with a compound having both an epoxy and olefinic group.

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The resulting conjugate is then polymerized with another olefinic monomer and bis-olefinic crosslinking agent to form the carrier bound enzyme.
The following references describe methods of bonding a protein to a polymer carriers: United States Patents 3,639,558; 3,853,708; 3,841,970; 3,844,892;
3,821,084, and 4,086,199.
In particular, where a latex system is employed as the carrier the prior art teaches coupling methods requesting the use bifunctional coupling agents or of coupling agents which forms a linkage between the reactive groups on the protein and those on the carrier particles. The problem with these methods is that the coupling agents are unable to distinguish between the reactive groups on the protein and those on the carrier; therefore protein to protein coupling and even intramolecular protein coupling may occur. Such undesirable linkage can result in conformational changes in the protein molecule leading to changes in activity.
Accordingly, the invention provides a method for coupling a protein having a reactive group containing a labile hydrogen to a latex wherein the latex particles range in size from about 0.15 to 1.5 micrometers in diameter and have an inner core and an outer shell, said inner core being formed by the polymerization or copolymerization of one or more hard monomers and said outer shell being formed by the copolymerization of one or more hard monomers and a copolymerizable ethylenically unsaturated compound having free epoxy groups said core and shell optionally containing copolymerized soft monomer which comprises reacting said latex and said protein at a pH 7.0-9.0 for a time sufficient to form a covalent bond between the reactive group of the protein and the free epoxy group of the latex and removing the unreacted protein from the latex-protein conjugate.
Thus, the present invention is directed to a process for covalently ' '; ~
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-, ' -' bonding a protein having a reactive group containing a labile hydrogen atom to a latex having surface epoxide groups. Preferably the reactive group is a free amino group, but other reactive groups such as carboxyl, phenol, hydroxyl, and thiol would also be operable. The coupling occurs when the amino groups of the protein react with the epoxide groups of the latex at a pH of about 7.0-9.0, to form a new carbon to nitrogen bond, hereafter called an amine bond, binding the protein to the surface of the latex. This process offers a number of advantages over methods of preparing protein-latex conjugates known to the prior art. Specifically, the present method is relatively easy and convenient to carry out. It does not subject the protein to any harsh reaction conditions which may result in distortion or denaturation of the protein molecule. This is especially important since such conformational changes can result in loss of activity. The present method also makes it possible to discharge the residual epoxy groups following binding to the proteins. The method of this invention has the further advantage of providing carrier bound proteins with uniform particle size distribution.
As used herein the term latex refers to an aqueous colloidal dispersion of a water insoluble polymer. Latexes used in the practice of the present invention usually consist of substantially spherical polymer particles ; 20 having a diameter of between 0.15 and 1.5 micrometers, with from 0.45 to 0.90 micrometers ~lM) being preferred. Particle morphology consists of an inner core and a surrounding shell. The core is polymerized or copolymerized from hard monomers as, for example, styrene. The shell is formed by the copolymerization of one or more hard monomers with a copolymerizable ethylenically unsaturated ` compound having a three-membered epoxy ring. Optionally soft monomer may be copolymerized into the core and/or shell along with the hard monomer to prevent fracturing of the particles. As used herein the term "hard monomer" refers to ` - 4 ~

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those monomers which form polymers having a glass transition temperature above room temperature. The term "soft monomer" refers to those monomers which form polymers having a glass transition temperature below room tempera-ture. Preferred emulsion polymerizable monomers for the o core are hard monomers which can be polymerized and/or copol~merized with each other in any proportions and/or with other monomers to yield non-film forming polymers.
As used herein,"non-film forming"refers to those polymer particles of the latex which are not film forming or do 15 not coalesce under conditions of use. These monomers include monovinylidene carbocyclic monomers, e.g., sty-rene, a-methylstyrene, ar-(t-butyl)styrene, ar-methylsty-rene, ar, ar-dimethylstyrene, ar~chlorostyrene, ar-(t-amyl) styrene, ar-b~omostyrene, ar-fluorostyrene, ar-cyanostyrene, 20 ar-methoxystyrene, ar-ethylstyrene, ar-hydroxymethyl-styrene, ar-ethoxystyrene, ar-chloro-ar-methylstyrene, ar, ar-dichlorostyrene, ar, ar-difluorostyrene, vinyl naphthalene, and other such emulsion polymerizable monomers having not more than 26 carbon atoms; esters of a, ~ -ehtylenically 25 unsaturated carboxylic acids which polymerize to form non-film forming polymers, e.g., methyl methacrylate, chloroethyl methacrylate, n-butyl methacrylate, ethyl methacrylate, isobutyl methacrylate, isopropyl methacrylate, phenyl metha-crylate, butyl chloroacrylate, cyclohexyl chloroacrylate, 30 ethyl chloroacrylate , methyl chloroacrylate, isopropyl chloro-"~,~

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, ' ~119340 acrylate and other such esters capable of being poly-merized to form hard polymers a, ~ -ethylenically un-saturated esters of non-polymerizable carboxylic acids, e.g., vinyl benzoate, vinyl toluate, vinyl ar-ethyl-benzoate, allyl ar-ethylbenzoate, Yinyl pivalate, vinyl trichloroacetate and other such monomers wherein the unsaturated moiety has from 2 to 14 carbon atoms and the acid moiety has from 2 to 12 carbon atoms; a,~ -ethyl-enically unsaturated nitriles, e.g., such nitriles having not more than 12 carbon atoms; other polymerizable vinyl monomers such as vinyl chloride, vinyl bromide and the like. Of the foregoing monomers, the monovinylidene carbo-cyclic aromatic monomers, particularly styrene and mixtures of styrene, methyl methacrylate and acrylonitrile, are especlally preferred.
Soft monomers which may be optionally added include the alkyl acrylates such as ethylacrylate, butylacrylate, or

2-ethylhexyl acrylate; the higher alkyl methacrylates such as hexyl methacrylate or 2-ethylhexyl methacrylate; and the coniugated dienes such as isoprene or butadiene.
Preferred monomer for the copolymer of the shell is a mixture of one or more hard monomers as described above and a copo-lymerizable ethylenically unsaturated compound having a three-membered epoxy ring.
Representative epoxy monomers include unsaturated alkyl glycidyl esters, unsaturated alkyl glycidyl ethers, unsa-. ~ . .

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turated cycloalkyl glycidyl ethers, unsaturated alkyl-substituted phenyl glycidyl ethers, and the monoepoxide compounds of the diene type monomers.
Suitable glycidyl esters include glycidyl methacrylate, glycidyl acrylate, glycidyl ester of crotonic acids and 1~ long chain unsaturated fatty acids and the like; un-saturated alkyl glycidyl ethers include vinyl glycidyl ether, isopropenyl glycidyl ether, oleyl glycidyl ether, allyl and methallyl glycidyl ethers and the like; unsa-turated cycloalkyl and phenyl glycldyl ethers include 15 4-vinyl cyclohexyl glycidyl etherj p-vinylbenzyl glycidyl ether,` o-allyl phenylglycidyl ether, and the like; the monoepoxide compounds of the diene type monomers include butadiene monoepoxide, chloroprene monoepoxide, 3,4-epoxy-l-pentene, 4,5-epoxy-1-pentene, 4,5-epoxy-2-pentene, 4,5-20 epoxy-l-hexene, 3,4-epoxy-1-vinylcyclohexene and divinyl-benzene monoxide, and the like.
The exact amount of the epoxy monomer needed to prepare latexes of this invention is dictated by the population of surface epoxy groups needed for optimal coupling with 25 the desired protein. For example, for coupling human chorionic gonadotropin to a styrene-glycidyl methacrylate latex a coupling site density of about one epoxy unit per 5 to 40 square angstrom units (A02) gives satisfactory results with about one epoxy unit per 8 to 20 A02 being preferred. This same coupling site density would also be ~. . .

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operable for other proteins such as, for example, insulin.
In preparing latex bound proteins for use in immunolo-gical testing it has been found that monodispersed latexes, that is latexes having substantially uniform particle sizes, are preferred because uniformity of size assures o an equal statistical distribution of antigen or antibody molecules on the surface of the latex particles. For a given weight of polymer, the total surface area of latex will lncrease with a decrease in the size of the particles and vice versa. Thus, in a latex containing a distribution lS of various particle sizes the smaller particles will have a greater surface area and, consequently, more total reaction sites than the larger particles. The unequal distri-bution of antigen or antibody on the latex particles will lead to unequal agglutination of particles and poorly defined diagnostic results.
Another advantage of using uniform latex particles as diagnostic agents is that they are better suited for instrumental analysis. Particles of the same size will flocculate, agglutinatef or settle at the same rate whereas different sized particles will agglutinate at variable rates. Thus an instrumental method based on the absorption or transmission of light through an agglutinating latex suspension will be more accurate, more reproducible, and easier to s.andardize and read with uniform latex particles than with latexes having varied particles sizes. Conjugates . .

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of human chorionic gonadotropin bound to styrene-glycidyl methacrylate latex using the present invention have showed substantially no change in immunological activity after storage ~or as long as twelve months at about 4-6C.
o The following examples describe the preparation of three monodispersed styrene-glycidyl methacrylate latexes.
Commercially available monodispersed polystyrene latexes were capped with a mixture of styrene and glycidyl metha-crylate. Unless otherwise indicated amounts are expressed as parts by weight.

Example 1 ~he following ingredients were added into a one liter three-necked flask equipped with a stirrer, a nitrogen inlet tube and a condenser.
Dry Wet Weight Weight Parts Parts 25 Polystyrene latex (particle size 0.76 micrometer) 60.5 188 Dihexyl sodium sulfosuccinate 0.3 6 Potassium persulfate 0.3 6 Sodium bicarbonate 0.3 6 30 Styrene 45 45 -~ Glycidyl methacrylate 15 15 Water - 134 . ' .~

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The reaction mixture was stirred and the flask was purged with nitrogen for about 10 to 20 minutes. The temperature of the reaction was brought to 65C and held at that temperature for four hours while main-taining a positive nitrogen pressure. The resulting latex lo was cooled and filtered. The average particle size of the latex was determined by electron microscopy and found to be about .91 ,uM in diameter.

Example 2 Using the general procedure outlined in Example 1 above the following ingredients were used to prepare a mono-dispersed latex having an average particle size of about .59 ~M in diameter.
Dry Wet Weight Weight Parts Parts Polystyrene latex (particle size 0.503 micrometer) 30.1 95 25 Dihexyl sodium sulfosuccinate0.15 3 Potassium persulfate 0.15 3 Sodium bicarbonate 0.15 3 Styrene 15 15 Glycidyl methacrylate 15 15 Water - 266 _ _ .. . . ..

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Example 3 The following ingredients were used to prepare a mono-dispersed latex having an average particle size of .20 ~M
in diameter. The same general procedure was used as descri-bed above.
o Dry Wet ~eight Weight Parts Parts Polystyrene latex (particle size 0.176 micrometer) 30 300 15 Dihexyl sodium sulfosuccinate0.15 3 Potassium persulfate 0.15 3 Sodium bicarbonate 0.15 3 Styrene 15 15 Glycidyl methacrylate 15 15 20 ~later ~ 61 The epoxy content of the latex expressed as milliequi-valents oxirane groups per gram (-CH-CH2/gram) and as square angstrom units per one oxirane group was determined for each latex prepared above. The results are shown in Table I below:

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Latex Particle0 /0~ ~O~mequiv Ex.No. Size(yM)A /-CH-CH2 -CH:-CH2/gram 1 .907 15.0 0.07 o 2 .592 9.4 0.17 3 .201 17.0 0.278 Milliequivalent of oxirane groups per gram of polymer were determined by treating the latex containing a known amount of polymer with known equivalents of hydrogen iodide. The residual hydrogen iodide remaining in the latex was titrated with standardized silver nitrate solution to determine the amount of hydrogen iodide that reacted with the epoxy groups. Milliequivalents of epoxy groups were then determined from this informatiGn.
The latexes prepared above were found to be suitable for use in the practice of the present invention. Samples from each of the latex preparations described above were coupled with human chorionic gonadotropin, hereafter called HCG.
This hormone is a glycoprotein and governs the production and secretion of progesterone by the corpus luteum. It is normally secreted by the chorionic tissue of the placenta during pregnancy. Latex-bound HCG is useful in the detection of pregnancy.

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Example 4 A solution containing 5000IU of HCG in 7 ml of pH 8.0 phosphate buffer ~I=0.05; 0.1 M NaCl; 1:10,000 thime-rosal) was added with stirring to a siliconized 25 ml round bottomed flask already containing 1.61 grams of o styrene-glycidyl methacrylate prepared as described in Example 1 above. The resulting latex suspension was stirred 4 days in a cold room (5C). The reaction mix-ture was washed by membrane filt~ation for about 3 hours in a Diafl ~ filter cell with 145 ml of phosphate buffer. The washed latex and rinse was dialyzed in cellophane against pH 8.2, 0.1 M glycine buffer. After two days of dialysis 16.4 grams of the styrene-glycidyl methacrylate latex-HCG product was obtained having a detection sensi-tivity of 0.9 IU/HCG/ml..

Example 5 This product was prepared in an identical manner asExample 4 except for the following excepticns.The reaction mixture was treated after 3 days with 1 ml of pH 8.2 glycine buffer to discharge the residual epoxy groups.
After stirring for 1 more day in the cold, 9.9 grams of the reaction mixture was washed by membrane filtration using 188 ml of pH 7.0 phosphate buffer (I=0.05;0.1 M NaCl).
The resulting product and rinse were treated with 13 mg of ' ,' ' : : ~ `'"' .. ~ .

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sodium azide as a preservative. The resulting product had a detection sensit-vity of 1.8 IU/HCG~ml..

Example 6 o Latex prepared according the procedure of Example 2 above (3.08 grams of latex, 0.46 gram polymer) was placed in a 25 ml round bottomed flask equipped with a Teflo ~ coated magnetic stirring bar. A solution contai-ning 5000I~ of HCG in 8 ml of cold, pH 8 phosphate buffer(I=0.05; 0.1 M NaCl; 0.01 percent thimerosal~ was added slowly to the flask. The reaction was stirred gently for 90-96 hours at about 5C. The latex-HCG conjugate was washed with 110-115 ml of phosphate buffer by ultrafiltra-tion for about 70-110 minutes. The combined latex-HCG and rinses were dialyzed in cellophane at 5C for 24 hours.
Product prepared by this procedure showed a detection sensi-tivity of 0.9-1.8 IU HCG/ml..
Using the procedures already described HCG was coupled to latex prepared in Example 3 above. Using the general pro-cedures and methods already described additional samples of HCG-latex conjugate were prepared. The factors which were found to significantly affect the quality of the final product are summarized below.

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~ -15-The examples a~ove illustrate the operation of the present invention. Other factors which have been observed to effect the coupling of H~G to the styrene-glycidyl methacrylate monodispersed latexe~ described above were the age of the latex and the means of stor~ge of the latex o prior to coupling. The latex should be stored under refri-geration and used in coupling reactions as soon as conve-nient. The latex prepared in Example 2 above was found to behave satisfactorily in the coupling reaction with HCG
after storage for as long as one month under refrigeration.
In preparing immunologically active latex-HCG conjugates it was found that the source of HCG was an important variab]e in determiningsensitivity. HCG preparations from various commercial sources should be checked to see which is most satisfactory for the particular applications.
Although varying amounts of protein may be coupled to the latex, attaching maximum amounts of HCG to the latex did not produce optimal results. Although other amounts were operable, the preferred amount of HCG used in the coupling reaction was about 1,000-25,000 I.U. per gram of polymer with about 11,000 I.U./gram polymer particularly preferred.
The preferred reaction concentration of polymer solids was about 4-5~.
In general, the coupling reaction is carried out at a pH
of from about 7.5-8.5 with a pH of about 8.0 preferred.
A phosphate buffer is generally used to couple HCG to the latex.

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S Borate buffers are generally unsatisfactory with proteins such as HCG which have a high carbohydrate content. It was also discovered that the coupling reaction time could be reduced to about one day or less by conducting the reaction at room temperature instead of 5 C as 9hown ln lo the examples above. However, where no special refrige-rated facility is used, appropriate control of potential microbial contaminants is required.
Following coupling of the latex to the HCG, it is essential that unreacted HCG and other impurities be removed from the reaction mixture. Ultrafiltration followed by cello-phane dialysis or high-speed centrifugation followed by cellophane dialysis gave a satisfactory product. Hollow-fiber dialysis although still operable was less effective in removing the excess HCG from the reaction mixture.
If the final latex-HCG conjugate is buffered with the pre-ferred 8.2 glycine buffer, it is unnecessary to discharge the residual epoxy groups. If, however, a neutral buffer system is desired, an extra step to discharge the residual epoxy groups is desirable (as illustrated in Example 5 above).

In general, it was found that aging the final latex-~CG
conjugate significantly improved the immunological activity of the product. The aging period required to obtain optimal immunological activity depends on the temperature at which 3 the aging occurs, i.e. the lower the temperature the longer ,,~ .~.

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the aging period. It was found that product aged for 3to 5 days at about 25C gave satisfactory performance.
Product stored at about 5C required storage for about two weeks to achieve optimal activity.
The latex-HCG conjugate may be combined into a kit o containing all the necessary reagents for immunologically testing for pregnancy. Such a kit would contain a first reagent composition comprising the latex-HCG conjugate described above and a second reagent composition compri-sing buffered anti-HCG serum, generally obtained from New Zealand white rabbits. See J.Clin.Endocr. 33, 988 (1971) The basic reagents may also be combined with ancillary components such as an opaque glass slide with recessed mixing areas and an applicator stick.
It may also be desirable when the conjugate is intended for immunological purposes to dye the latex particles prior to coupling with the protein to aid in the detection of agglutination. This is readily accomplished by mixing the latex with a dye solution containing a solvent which is ; able to penetrate the latex particles. For example, Calco ' 2 Oil Blue N dye (American Cyanamid) dissolved in benzene was found to give satisfactory results. The benzene was removed in an evaporator prior to coupling with the protein.
The following examples illustrates the coupling of styrene-glycidyl methacrylate latex to insulin.

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, ., Example 8 Latex prepared as described in Example 1 above (~.5 grams of latex, 0.75 grams of polymer) was placed in a flask to which 2.5 ml of insulin solution (15 mg of insulin) lo and 5 droPs of O.lN sodium hydroxide were added. The final pH was 8. The reaction mass was stirred at 5C for 90 hours whereupon 4.4 grams of the latex reaction mixture (4.8grams) was washed by membrane filtration in a Diafl ~ filter cell using 0.1 ,u Millipore filter and pH 8 borate buffer. A
hydrodynamic pressure up to about 50 p.s.i.g. was used and the effluent was scanned by U.V. for the presence of uncoupled insulin. The filtration was continued until no insulin was detected in the effluent. Thin layer electro-phoresis confirmed the insulin was chemically bound to the la~ex.

Example 9 Latex prepared as described in Example 1 above (6.3 grams of latex, 0.92 grams of polymer) was added to a solution containing 7.33 mg of horseradish peroxidase in 10 ml of pH 8 phosphate saline buffer. The mixture was covered and stirred for about 5 days at 5C. The reaction mixture was diluted (2-3x) with buffer and centrifuged at 18,000 rpm for about 35 minutes. The supernatant was discarded and , .

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the latex-peroxidase residue was resuspended in pH 7 phosphate saline buffer with 1% surfactant added. The centrifugation and resuspension was repeated two more times using pH 7 phosphate saline buffer without surf-actant. The latex-peroxidase conjugate was filtered o through a thin mat of glass wool to obtain the final product. The resulting conjugate was tested for enzymatic peroxidase activity using a modification of the method of Steinman et al. fJ.Cell Biol. 55, 186 ~1972)7 Three samples of product plus one sample of product super-natant were allowed to react for different time intervalswith substrate containing dianisidine dihydrochloride and hydrogen peroxide in a pH 7 phosphate saline buffer.
Reaction mixtures were filtered and the filtrates were scanned at 400 nanometers. The results were as follows:

Reaction Time Sample (min:sec? OPtical Density~
1 4:55 0.53 2 5:17 0.62 3 9:17 0.65

4 (supernatant) 10:13 0.005 ~scanned at 400 nanometer The data show very little enzymatic activity (oxidation 3 of dianisidine) in the supexnatant reaction mixture (sample ~4). Thus, enzymatic activity of the latex-peroxidase ,~ . . - . .

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product was due essentially to enzyme immobilized on the latex particles and not to soluble residual peroxidase in the supernatant.

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Claims (12)

C L A I M S

1. A method for coupling a protein having a reactive group containing a labile hydrogen to a latex wherein the latex particles range in size from about 0.15 to 1.5 micrometers in diameter and have an inner core and an outer shell, said inner core being formed by the polymerization or copolymerization of one or more hard monomers and said outer shell being formed by the copoly-merization of one or more hard monomers and a copoly-merizable ethylenically unsaturated compound having free epoxy groups said core and shell optionally containing copolymerized soft monomer which comprises reacting said latex and said protein at a pH 7.0-9.0 for a time suffi-cient to form a covalent bond between the reactive group of the protein and the free epoxy group of the latex and removing the unreacted protein from the latex-protein conjugate.

2. The method of Claim 1 wherein the latex is a mono-dispersed latex and the reactive group on the protein is amino.

3. The method of Claim 1 wherein the latex is styrene-glycidyl methacrylate.

4. The method of Claim 3 wherein the protein is human chorionic gonadotropin.

5. The method of Claim 3 wherein the protein is insulin.

6. The method of Claim 3 wherein the protein is an enzyme.

7. The method of Claim 4 wherein the coupling site density of the styrene-glycidyl methacrylate latex is about one epoxy unit per 5 to 40 square angstrom units of particle surface area and the amount of human chorionic gonadotro-pin used to prepare the latex-protein conjugate is 1,000 to 25,000 international units per gram of polymer.

8. A protein-latex conjugate formed by the method of Claim 2.

9. The protein-latex conjugate of Claim 8 wherein the protein moiety is human chorionic gonadotropin and the latex moiety is styrene-glycidyl methacrylate.

10. The protein latex conjugate of Claim 8 wherein the protein moiety is insulin and the latex moiety is styrene-glycidyl methacrylate.

11. The protein-latex conjugate of Claim 8 wherein the latex is dyed.

12. A kit for immunologically testing for pregnancy which comprises a first reagent containing a monodispersed styrene-glycidyl methacrylate latex with human chorionic gonadotropin bound thereto and a second reagent containing anti-human chorionic gonadotropin serum.