GB2041940A - Method of Coupling a Protein to an Epoxylated Latex and the Products Formed Therefrom - Google Patents

Abstract

A latex-protein conjugate is formed by reacting two-layer particles of a latex having free epoxy groups with a protein having a reactive group. The conjugates may be used in diagnostic tests and enzyme detection.

Description

SPECIFICATION Method of Coupling a Protein to an 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 are based upon the antigen-antibody reaction.

The antigen-antibody reaction is the basis for all immunological test methods. Special proteins called antibodies are produced by an animal in response to the presence of an antigen, that is a foreign 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.

If the suspected protein is present, the resulting antigen-antibody reaction is generally manifested by precipitation or agglutination of the antigen-antibody complex. In some instances, the antigenantibody complex is slow to form and the particles that are formed are too small to be observed with certainty. In such cases, detectability 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 erythrocytes, 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 Specification No. 3,857,931 teaches that proteinic antigens or 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 Specification No. 3,806,41 7 describes a method of binding a protein to a compound containing 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.

The resulting conjugate is then polymerised with another olefinic monomer and bis-olefinic crosslinking agent to form the carrier-bound enzyme. The bonding of a protein to a polymer carrier is described in U.S. Patent Specifications Nos. 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 reactants which have been used are bifunctional coupling agents or coupling agents which form 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 and, as a result, 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.

The method of the present invention covalently couples a protein having a reactive group containing a labile hydrogen atom to a latex; the method comprises reacting the protein with latex particles having an inner core, an outer shell and a diameter of from 0.15 to 1.5 Mm, wherein the inner core has been formed by the polymerisation or copolymerisation of a hard monomer and the outer shell has been formed by the copolymerisation of a hard monomer and a copolymerisable ethylenicallyunsaturated compound having free epoxy groups, and removing unreacted protein from the latexprotein conjugate which is formed.

Preferably, the reactive group on the protein is a free amino group, but other reactive groups, such as carboxyl, hydroxyl (phenolic or aliphatic) and thiol may be used. The coupling occurs when the reactive groups of the protein react with the epoxide groups of the latex, usually at a pH of 7.0 to 9.0 when the reactive groups are amino groups, to form a new carbon to nitrogen bond, hereinafter referred to as 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 consist of substantially spherical polymer particles having a diameter of from 0.15 and 1.5 micrometers, with from 0.45 to 0.90 micrometers (ym) being preferred. Particle morphology consists of an inner core and a surrounding shell. The core is polymerized or copolymerized from hard monomers, 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 those momomers which form polymers having a glass transition temperature above 1 OOC., usually above 200 C., and preferably above 300 C. The term "soft monomer" refers to those monomers which form polymers having a glass transition temperature below 30 C., usually below 2O0C., and preferably below 1 00 C. Preferred emulsion-polymerisable monomers for the core are hard monomers which can be polymerised and/or copolymerised with each other in any proportions and/or with other monomers to yield polymers which are non-film-forming, i.e.

polymers which do not form films or do not coalesce under conditions of use. Monomers which may be used to form the core include monovinylidene carbocyclic monomers such as styrene, ring-substituted styrenes in which examples of suitable substituents are methyl (once or twice), ethyl, tert-butyl, tertamyl, fluorine (once or twice), chlorine (once or twice), bromine, cyano, hydroxy, methoxy, ethoxy or a combination of methyl and chlorine, vinylnaphthalene and other emulsion-polymerisable monomers having not more than 26 carbon atoms; esters of ,-ethylenically unsaturated carboxylic acids which polymerise to form non-film-forming polymers, e.g., methyl methacrylate, chloroethyl methacrylate, nbutyl methacrylate, ethyl methacrylate, isobutyl methacrylate, isopropyl methacrylate, phenyl methacrylate, butyl chloroacrylate, cyclohexyl chloroacrylate, ethyl chloroacrylate, methyl chloroacrylate isopropyl chloroacrylate and other such esters capable of being polymerised to form hard polymers;; -ethylenically unsaturated esters of non-polymerisable carboxylic acids, e.g., vinyl benzoates optionally carrying a ring substituent such as ethyl, vinyl toluate, allyl benzoates optionally carrying a ring substituent such as ethyl, vinyl pivalate, vinyl trichloroacetate and other such monomers wherein the unsaturated group has from 2 to 14 carbon atoms and the acid has from 2 to 1 2 carbon atoms; a,-ethylenically unsaturated nitriles having, for example, not more than 12 carbon atoms;-and other polymerisable vinyl monomers such as vinyl chloride or vinyl bromide. Of the foregoing monomers, the monovinyiidene carboxylic aromatic monomers, and particularly styrene and mixtures of styrene, methyl methacrylate and acrylonitrile, are especially preferred.

Soft monomers which may be optionally added include the alkyl acrylates such as ethyl acrylate, butyl acrylate or 2-ethylhexyl acrylate; the higher alkyl methacrylates such as hexyl methacrylate or 2ethylhexyl methacrylate; and the conjugated dienes such as isoprene or butadiene.

The preferred monomer for the copolymer of the shell is a mixture of one or more hard monomers as described above and a copolymerisable ethylenically unsaturated compound having a threemembered epoxy ring.

Representative epoxy monomers include unsaturated alkyl glycidyl esters, unsaturated alkyl glycidyl ethers, unsaturated 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 long chain unsaturated fatty acids; unsaturated alkyl glycidyl ethers include vinyl glycidyl ether, isopropenyl glycidyl ether, oleyl glycidyl ether, allyl and methallyl glycidyl ethers; unsaturated cycloalkyl and phenyl glycidyl ethers include 4-vinyl cyclohexyl glycidyl ether, p-vinylbenzyl glycidyl ether o-allyl phenylglycidyl ether; and the monoepoxide compounds of the diene type monomers include butadiene monoepoxide, chloroprene monoepoxide, 3,4-epoxy-1 -pentene, 4,5-epoxy-1 - pentene, 4,5-epoxy-2-pentene, 4,5-epoxy-1 -hexene, 3,4-epoxy- 1 -vinylcyclohexene and divinylbenzene monoxide.

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 the desired protein. For example, for coupling human chorionic gonadotropin to a styrene-glycidyl methacrylate latex a coupling site density of one epoxy unit per 5 to 40 square angstrom units ( 2) gives satisfactory results with one epoxy unit per 8 to 20 2 being preferred. This same coupling site density would also be operable for other proteins such as, for example, insulin.In preparing latex-bound proteins for use in immunological testing it has been found that monodispersed latexes, that is latexes having substantially uniform particle sizes, are preferred because uniformity of size ensures an equal statistical distribution of antigen or antibody molecules on the surface of the latex particles. For a given weightof polymer, the total surface area of latex will increase with a descrease in the size of the particles, and vice versa. Thus, in a latex containing a distribution 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 distribution 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, agglutinate 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 standardize and read with uniform latex particles than with latexes having varied particles sizes. Conjugates of human chorionic gonadotropin bound to styrene-glycidyl methacrylate latex using the present invention have showed substantially no change in immunological activity after storage for as long as twelve months at about 4-60C.

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 methacrylate. Unless otherwise indicated amounts are expressed as parts by weight.

Example 1 The following ingredients were added to a one liter three-necked flask equipped with a stirrer, a nitrogen inlet tube and a condenser.

Dry Weight Wet Weight Parts Parts 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 Styrene 45 45 Glycidyl methacrylate 15 15 Water 134 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 650C and held at that temperature for four hours while maintaining a positive nitrogen pressure. The resulting latex was cooled and filtered. The average particle size of the latex was determined by electron microscopy and found to be about 0.91 m.

Example 2 Using the general procedure outlined in Example 1, the following ingredients were used to prepare a mono-dispersed latex having an average particle size of about 0.59 Hm.

Dry Weight Wet Weight Parts Parts Polystyrene latex (particle size 0.503 micrometer) 30.1 95 Dihexyl sodium sulfosuccinate 0.15 3 Potassium persulfate 0.15 3 Sodium bicarbonate 0.15 3 Styrene 15 15 Glycidyl methacrylate 15 15 Water 266 Example 3 The following ingredients were used to prepare a mono-dispersed latex having an average particle size of 0.20,us. The same general procedure was used as described above.

Dry Weight Wet Weight Parts Parts Polystyrene latex (particle size 0.176 micrometer) 30 300 Dihexyl sodium sulfosuccinate 0.15 3 Potassium persulfate 0.15 3 Sodium bicarbonate 0.15 3 Styrene 15 15 Glycidyl methacrylate 15 15 Water 61 The epoxy content of the latex expressed as milliequivalents oxirane groups per gram and as square angstrom units per oxirane group was determined for each latex prepared above. The results are shown in Table I below: Table I Latex Particle A2/oxliane mequiv oxfrane Ex. No. Size (/1M) group grnups/gram 1 .907 15.0 0.07 2 .592 9.4 0.17 3 .201 17.0 0.278 Milliequivalents 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 information. 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.

The following Examples illustrate the invention.

Example 4 A solution containing 50001U of HCG in 7 ml of pH 8.0 phosphate buffer (1=0.05: 0.1 M NaCI; 1:10,000 thimerosal) was added with stirring to a siliconized 25 ml round-bottomed flask already containing 1.61 grams of styrene-glycidyl methacrylate prepared as described in Example 1. The resulting latex suspension was stirred 4 days in a cold room (50C). The reaction mixture was washed by membrane filtration for about 3 hours in a Diaflo 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, 1 6.4 grams of the styrene-glycidyl methacrylate latex-HCG product were obtained having a detection sensitivity of 0.9 lU/HOG/mI.

Example 5 This product was prepared in an identical manner as Example 4 except for the following steps: 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 were washed by membrane filtration using 188 ml of pH 7.0 phosphate buffer (1=0.05; 0.1 M NaCI). The resulting product and rinse were treated with 13 mg of sodium azide as a preservative. The resulting product had a detection sensitivity of 1.8 lU/HCG/ml.

Example 6 Latex prepared according to Example 2 (3.08 grams of laiex, 0.46 gram polymer) was placed in a 25 ml round bottomed flask equipped with a Teflon coated magnetic stirring bar. A solution containing 50001U of HCG in 8 ml of cold, pH 8 phosphate buffer (1=0.05; 0.1 M NaCI; 0.01 percent thimerosal) was added slowly to the flask. The reaction was stirred gently for 90-96 hours at about 500The latex-HCG conjugate was washed with 1 10--1 15 ml of phosphate buffer by ultrafiltration for about 70-110 minutes. The combined latex-HCG and rinses were dialyzed in cellophane at 50C for 24 hours. The product prepared by this procedure showed a detection sensitivity 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 procedures 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.

For example, factors which have been observed to effect the coupling of HCG to the styrene glycidyl methacrylate monodispersed latexes described above were the age of the latex and the means of storage of the latex prior to coupling. The latex should be stored under refrigeration and used in coupling reactions as soon as convenient. 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 variable in determining sensitivity. 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 l.U. per gram of polymer with about 11,000 l.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.

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 iess by conducting the reaction at room temperature instead of 50C as shown in the Examples above. However, where no special refrigerated 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 cellophane dialysis or high-speed centrifugation followed by cellophane dialysis gave a satisfactory product. Hoilow-fibre 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 Example 4 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-HCG conjugate significantly improved the immunological activity of the product. The aging period required to obtain optimal immunological activity depends on the temperature at which the aging occurs, i.e. the lower the temperature, the longer the aging period. It was found that product aged for 3 to 5 days at about 250C gave satisfactory performance. Product stored at about 50C required storage for about two weeks to achieve optimal activity.

The latex-HCG conjugate may be combined into a kit 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 comprising buffered anti-HCG serum, generally obtained from New Zeaiand 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 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 illustrate the coupling of styrene-glycidyl methacrylate latex to insulin.

Example 8 Latex prepared as described in Example 1 (2.5 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) and 5 drops of 0.1 N sodium hydroxide were added. The final pH was 8. The reaction mass was stirred at 50C for 90 hours, whereupon 4.4 grams of the latex reaction mixture (4.8 grams) was washed by membrane filtration in a Diaflo filter cell using 0.1 y Millipo:e 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 electrophoresis confirmed the insulin was chemically bound to the latex.

Example 9 Latex prepared as described in Example 1(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 5 C. The reaction mixture was diluted (2-3x) with buffer and centrifuged at 18,000 rpm for about 35 minutes. the supernatant was discarded and 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 surfactant. The latex-peroxidase conjugate was filtered 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., J. Cell. Biol. 55, 1 86 (1 972). Three samples of product plus one sample of product supernatant were allowed to react for different time intervals with 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: Table II 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 of dianisidine) in the supernatant reaction mixture (sample 4). Thus, enzymatic activity of the latex-peroxidase product was due essentially to enzyme immobilized on the latex particles and not to soluble residual peroxidase in the supernatant.

Claims (14)

Claims

1. A method for covalently coupling a protein having a reactive group containing a labile hydrogen atom to a latex, which comprises reacting the protein with latex particles having an inner core, an outer shell and a diameter of from 0.15 to 1.5,us, wherein the inner core has been formed by the polymerisation or copolymerisation of a hard monomer (as hereinbefore defined) and the outer shell has been formed by the copolymerisation of a hard monomer (as herein before defined) and a copolymerisable ethyienically-unsaturated compound having free epoxy groups, and removing unreacted protein from the latex-protein conjugate which is formed.

2. A method according to claim 1 wherein the core and/or the shell have been formed by copolymerisation with a soft monomer (as hereinbefore defined).

3. A method according to claim 1 or claim 2 wherein the latex is a monodispersed latex.

4. A method according to any preceding claim wherein the reactive group on the protein is an amino group.

5. A method according to claim 4 wherein the reaction is carried out at a pH of from 7.0 to 9.0.

6. A method according to any preceding claim wherein the latex is styreneg!ycidyl methacrylate.

7. A method according to any preceding claim wherein the protein is human chorionic gonadotropin.

8. A method according to any of claims 1 to 6 wherein the protein is insulin.

9. A method according to any of claims 1 to 6 wherein the protein is an enzyme.

10. A method according to any of claims 1 to 5 wherein the latex is styrene-glycidyl methacrylate having a coupling site density of one epoxy unit per 5 to 40 square angstrom units of particle surface area and the protein is human chorionic gonadotropin which is used in an amount of 1 ,000 to 25,000 international units per gram of polymer.

11. A method according to any preceding claim wherein the latex is dyed.

12. A method according to claim 1 substantially as described in any of Examples 4 to 9.

13. A protein-latex conjugate formed by a method according to any preceding claim.

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