CH639777A5 - Process for binding a protein to an epoxydised latex and related products - Google Patents

Abstract

The process in question enables a protein with a reactive group containing a mobile hydrogen to be bound to a latex in which the particles have a diameter of approximately 0.15 to approximately 1.5 micrometres and have an internal nucleus and an external layer. The internal nucleus is formed by the polymerisation or copolymerisation of one or more "hard" monomers and the external layer is formed by the copolymerisation of one or more "hard" monomers and a copolymerisable compound containing an ethylene unsaturation having epoxide groups. The internal nucleus and the external layer can contain, if desired, a copolymerised "soft" monomer. The process comprises reacting the said latex and the said protein at pH 7.0-9.0 for a sufficient length of time to enable a covalent bond to form between the reactive group of the protein and the free epoxide group of the latex, with the non-reacted protein being removed from the latex-protein conjugate.

Description

The invention relates to a process for binding a protein or a protein component to a latex having epoxy groups on the surface by forming an amine bond.

These support-bound proteins are particularly useful in conducting immunological assays based on the antigen-antibody reaction.

The antigen-antibody reaction is the basis of all immunological test procedures. Particular proteins called antibodies are produced by animals in response to the presence of an antigen, that is, a foreign substance, usually a protein, in the biological fluids of the animal.

This normal response of the body to a foreign protein has led to the development of numerous techniques that are used to diagnose various diseases or ailments in humans or animals.

In vitro assays to determine the presence of a suspected antigen or antibody in a biological fluid are carried out by adding the immunological counterpart io to a sample of biological fluid; that is, an antigen is added if the experiment serves to determine the presence of an antibody, or an antibody is added, if the experiment is carried out to determine the presence of an antigen. If the suspected protein is present, the resulting antigen-antibody reaction 15 is generally manifested by precipitation or agglutination of the antigen-antibody complex.

In some cases the antigen-antibody complex forms slowly and the particles formed are too small to be observed with certainty. In such cases, the determinability of the antigen-antibody reaction can be improved by using a support. When the antigen or antibody is fixed on a surface of a support the reaction which takes place with the immunological counterpart 25 produces a visible mass or agglutinate. Protein antigen or antibody can be absorbed on a surface of a support such as erythrocytes, bacterial cells, bentonite, polystyrene latex particles, phenolic anionic resins, or finely divided diazotized amino cellulose. However, it has been found that chemically binding the antigen or antibody molecule to a support provides results superior to physical adsorption. American patent 3 857 931 illustrates that protein type antigens or antibodies can be chemically bonded 35 to a support latex having surface carboxylic groups by the formation of amine bonds in the presence of a water-soluble binding agent of the carbo-diimidic type. U.S. Pat. No. 3 806 417 discloses a process for binding a protein to a compound containing an epoxide group and connecting said intermediate with a support material. A disadvantage of this process is that the protein must first be reacted with a compound having both an epoxide group and an olefinic group. The resulting conjugate product is then polymerized with another olefinic monomer and with a bis-olefinic agent forming cross-bonds to give rise to the enzyme bound to the support.

The following American patents describe procedures for binding a protein to a support of a polymeric nature.

50 rica: 3 639 558; 3 853 708; 3,841,970; 3,844,892; 3 821084 and 4 086 199.

In particular, when a latex is used as a support, the state of the art illustrates bonding processes which require the use of bifunctional binding agents or forming a bond between the reactive groups on the protein and those on the support particles.

The problem associated with these processes consists in the fact that the binding agents are not able to distinguish between the reactive groups on the protein and those on the substrate.

60 port; therefore a bond between protein and protein and even an intramolecular protein bond can occur. Such unwanted bonds can give rise to con-formal modifications in the protein molecule which entail changes in activity.

65 This invention relates to a process for covalently bonding a protein having reactive groups containing hydrogen atoms movable to a latex having epoxy groups on the surface. Preferably the group

3

639777

po reactive is an amino free group, but other reactive groups such as the carboxyl, the phenolic group, the hydroxyl group and the thiol group would also be usable. The bond is formed when the amino groups of the protein react with the latex epoxy groups, usually at a pH of about 7.0-9.0, to form a new carbon nitrogen bond, henceforth called an amine bond, which binds the protein to the surface of the latex. This process offers numerous advantages over the previously known protein-latex conjugate product preparation processes.

Specifically, this method turns out to be feasible in a simple and convenient way. It does not subject proteins to severe reaction conditions which can lead to distortions or denaturation of the protein molecule. This is particularly important because such conformational changes can result in loss of activity. This process also makes it possible to discharge the residual epoxy groups after the formation of the protein bond. The process of this invention has the further advantage of providing proteins bound to a support with a uniform particle size distribution.

As used herein, the term latex "refers to a colloidal aqueous dispersion of a water insoluble polymer. The latexes that are used in practice in the case of this invention consist of substantially spherical polymeric particles having a diameter between 0.15 and 1.5 micrometers, and preferably between 0.45 and 0.90 micrometers. The morphology of the particles consists of an inner core and an outer layer. The inner core is made up of polymers or copolymers of "hard" monomers such as for example styrene. The external surface is formed by copolymerization of one or more "hard" monomers with a copolymerizable compound containing unsaturations of the ethylene type and having a three-term epoxy ring. Optionally, a "soft" monomer can be copolymerized in the core inside and / or on the outer surface together with the "hard" monomer to prevent fracture in the particles. As used here, the term "hard monomers" wounds to those monomers which form a polymer having a glass transition temperature above room temperature. The term "soft monomers" refers to those monomers which form polymers having a glass transition temperature below room temperature.

Preferred monomers for the emulsion polymerization of the inner core of the latex particles are the "hard" monomers which can be polymerized or copolymerized with each other in any proportion and / or with other monomers to give rise to polymers which they do not form films. As it is used here, the term "non-film forming" refers to those latex polymer particles that do not form films or do not represent themselves under conditions of use. These monomers include carbocyclic monovinyliden monomers, e.g. styrene, a-methyl-styrene, ar- (t-butyl) styrene, ar-methylstyrene, ar, ar-dimethylsti-kidney, ar-chlorostyrene, ar- (t-amyl) styrene, ar-bromostyrene, ar-fluorostyrene, ar-cyanostyrene, ar-methoxisterene, ar-ethyl-styrene, ar-hydroxymethylstyrene, ar-ethoxystyrene, ar-chlorine-ar - methylstyrene, ar, ar-dichlorostyrene, ar-ar -difluorostyrene, vinyl naphthalene, and other emulsion polymerizable monomers having no more than 26 carbon atoms; carboxylic acid esters with a, ß-ethylene unsaturations which polymerize to give rise to polymers which do not form films, for example, methyl methacrylate, chloroethyl methacrylate, n-butyl methacrylate, ethyl methacrylate, isobutyl methacrylate, isopropyl methacrylate, phenyl methacrylate , butyl chloroacrylate, cyclohexyl chlorine-

acrylate, ethyl chloroacrylate, methyl chloroacrylate, isopropyl chloroacrylate and other esters capable of polymerizing giving rise to "hard" polymers; esters with a, ß-ethylenic unsaturations of non-polymerizable carboxylic acids, for example, vinyl 5 benzoate, vinyl toluate, vinyl ar-ethyl benzoate, allyl ar-ethyl-benzoate, vinyl pivalate, vinyl trichloroacetate and other monomers where the unsaturated portion has from 2 to 14 carbon atoms and the acid portion has 2 to 12 carbon atoms; nitriles with a, ß-ethylene unsaturations, for example nitriles io which have no more than 12 carbon atoms; other polymerizable monomers such as for example vinyl chloride, vinyl bromide and the like. Among the monomers mentioned above, aromatic monovinylidencarbocyclic monomers, in particular, styrene 15 and mixtures of styrene, methyl methacrylate and acrylonitrile are particularly preferred.

The "soft" monomers that can be added include alkyl acrylates such as ethyl acrylate, butyl acrylate,

0 2-ethylhexyl acrylate; alkyl methacrylates with higher 20 number of carbon atoms such as hexyl methacrylate or

2-ethylhexyl methacrylate; and conjugated dienes such as isoprene or butadiene.

The preferred monomer for the copolymer of the outer layer of particles is a mixture of one or more monomers.

25 "hard" as described above and of a copolymerizable compound having an unsaturation of ethylene type with a three-membered epoxy ring. Examples of epoxy monomers include unsaturated alkyl glycidic ethers, unsaturated alkyl glycidic ethers, unsaturated cycloalkyl glycidic ethers, 30 phenyl glycid ethers where the phenyl portion is replaced with unsaturated alkyls, and monopoxides of diene-type monomers.

Suitable glycidic esters include glycidyl methacrylate, glycidyl acrylate, glycidic esters of crotonic acid and long chain unsaturated fatty acids and the like; unsaturated alkyl glycidic ethers include vinyl glycid ether, isopropenyl glycid ether, oleyl glycid ether, Thalli glidicyl ether, methyl allyl glycidyl ether and the like; unsaturated glycid ethers that contain mild and phenyl cycloalkyl-40 groups include 4-vinyl cyclohexyl glycidyl ether, p-vinylbenzyl glycidyl ether, o-allyl phenylglycidyl ether, and the like;

The mono-epoxy compounds of the diene-type monomers include butadiene monoepoxide, chloroprene mono-epoxide, 3,4-epoxy-1-pentene, 4,5-epoxy-1-pentene,

45 4,5-epoxy-2-pentene, 4,5-epoxy-1-hexene, 3,4-epoxy-1-vinylcyclohexene and divinylbenzene monoxide and the like.

The exact amount of epoxy monomer required to prepare the latexes of this invention is determined by the density of surface epoxy groups necessary to form a bond under optimal conditions with the desired protein. For example, to bind human chorionic gonadotropin to a styrene-glycidyl methacrylate latex a density of ciria binding sites an epoxy unit for from 50 to 400 (nm.) 2, gives satisfactory results since a density of about one epoxy unit for 80 to 200 (nm.) 2. This same binding density would also be effective for other proteins such as insulin.

In the preparation of proteins linked to a 60 latex to be used in immunological assays it has been found that monodisperse latexes, i.e. latexes that have particles of substantially uniform size, are preferable because uniformity of size ensures an equal statistical distribution of antigen molecules or antibody on the surface of the particles themselves. For a given weight of polymer, the total surface area of the latex increases with decreasing particle size and vice versa. Thus, in a latex that contains a distribution of particles

639777

4

of various sizes, the smaller particles will have a higher surface area and consequently a higher number of reaction sites than larger particles. The unequal distribution of the antigen or antibody on the latex particles will result in an unequal agglutination of the particles and therefore not well defined diagnostic results. Another advantage of using latex with a uniform distribution of particles as a diagnostic agent is that it is much more suitable for instrumental analysis. Particles with the same dimensions flocculate, agglutinate or settle with the same speed, while particles with different dimensions agglutinate with variable speeds. Therefore, an instrumental process based on the absorption or transmission of light through a suspension latex that agglutinates, will be more accurate, more reproducible and more easily standardized and measurable with a latex having uniform sized particles than with a latex having particles with variable dimensions. Conjugates of human chorionic gonadotropin bound to a styrene-glycidyl methacrylate latex using the process of this invention have shown that no substantial change in immunological activity occurs after storage for a period of 12 months at about 4-6 ° C.

The following examples describe the preparation of three monodisperse styrene-glycidyl methacrylate latexes. Commercially available monodisperse polystyrene latexes were coated with a mixture of styrene and glycidyl methacrylate. Unless otherwise indicated, quantities are expressed as parts by weight.

Example 1

The following ingredients were added to a three-necked vessel equipped with a stirrer, a nitrogen feed tube and a condenser.

Weight

Weight

dry wet

Set off

Set off

Polystyrene latex

(particle diameter 0.76

micrometers)

60.5

188

Diesyl sodium sulfosuccinate

0.3

6

Potassium persulphate

0.3

6

Sodium bicarbonate

0.3

6

Styrene

45

45

GlidiciI methacrylate

15

15

water

-

134

The mixture was stirred while a stream of nitrogen was passed through the container for about 10-20 minutes. The reaction temperature was maintained at 65 ° C for four hours under nitrogen pressure. The resulting latex was cooled and filtered. The average size of the latex particles was determined by electron microscopy. The particle diameter was found to be approximately 0.91 nM.

Example 2

Using the general procedure described in Example 1, the following ingredients were used to prepare a monodisperse latex having particles with an average diameter of about 0.59 (iM.

Weight Wet dry weight Parts Parts

Polystyrene latex

(particle diameter 0.508 mi-

micrometers)

30.1

95

Diesyl sodium sulfosuccinate

0.15

3

Potassium persulphate

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 monodisperse latex having particles with an average diameter of 0.20 [J, M. The same general procedure as described above was used.

Weight Wet dry weight Parts Parts

Polystyrene latex (particle diameter 0.176

micrometers)

30

300

Diesyl sodium sulfosuccinate

0.15

3

Potassium persulphate

0.15

3

Sodium bicarbonate

0.15

3

Styrene

15

15

Glycidyl methacrylate

15

15

water

-

61

For each latex prepared as described above, the content of epoxy groups of the latex was determined by expressing it as milliequivalents of oxiranic groups per gram (-CH-CH2 / gram) and as angstromqua-dred units for each oxiranic group. The results are presented in Table I below.

TABLE I

Latex E.g.

Particle size

(P.m)

TO

ao2 / -ch-ch2

mequiv -CH-CHk / gram

1

0.907

15.0

0.07

2

0.592

9.4

0.17

3

0,201

17.0

0.278

The milliequivalents of oxiranic groups per gram of polymer were determined by treatment of the latex

5

I

15

20

25

30

35

40

45

50

55

60

65

5

639777

containing a known amount of polymer with a known number of hydrogen iodide equivalents. The residual hydrogen iodide that remained in the latex was titrated with a standard silver nitrate solution to determine the quantity of hydrogen iodide that had reacted with the epoxy groups. The milliequivalents of epoxy groups were then determined from this information.

The latexes prepared according to what described above proved useful for the use in the practice of this invention. Samples of each of the latex preparations described above were paired with human chorionic gonadotropin, hereinafter referred to as HCG. This hormone is a glycoprotein that 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 for determining pregnancy.

Example 4

A solution containing 5000 IU of HCG in 7 ml of a phosphate buffer pH 8.0 (I = 0.05; NaCl 0.1 M; time-rosal 1:10 000) was added under stirring to a 25 ml silicone flask containing 1.61 grams of stere-glycidyl methacrylate prepared as described in Example 1. The resulting latex suspension was stirred for 4 days in a cold room (5 ° C). 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 the rinse waters were dialysed in cellophane against a 0.1 M buffer of glycine at pH 8.2. After two days of dialysis, 16.4 grams of conjugated product of styrene-glycidyl methacrylate and HCG having a determination sensitivity of 0.9 IU / HCG / ml were obtained.

Example 5

This product was prepared identically to Example 4 with the following exceptions. The reaction mixture was treated after 3 days with 1 ml of glycine buffer at pH 8.2 to discharge the residual epoxy groups. After stirring for a further cold day, 9.9 grams of the reaction mixture were washed by filtration through a membrane using 188 ml of phosphate buffer at pH 7.0 (I = 0.05; NaCl 0.1 M). The resulting product and the rinse waters were treated with 13 mg. sodium azide as a preservative. The resulting product had a determination sensitivity of 1.8 IU / HCG / ml.

Example 6

A latex prepared according to the procedure of Example 2 (3.08 grams of latex, 0.46 grams of polymer) was placed in a 25 ml flask equipped with a magnetic stirrer covered with Teflon®. A solution containing 5000 IU of HCG in 8 ml of cold phosphate buffer at pH 8 (I = 0.05; NaCl 0.1 M; timerosal 0.01 percent) was slowly added to the reaction vessel. The reaction mixture was slowly stirred for 90-96 hours at about 5 ° C. The latex-HCG conjugate was washed with 110-115 ml of phosphate buffer by ultrafiltration for about 70-110 minutes. The latex-HCG conjugate and the washing waters were combined and dialysed in cellophane at 5 ° C for 24 hours. The product prepared according to this procedure showed a sensitivity of determination of 0.9-1.8 IU HCG / ml.

Using the procedure already described, the HCG was bound to the latex prepared in Example 3. Using the general procedures and the procedures already described other samples of latex-HCG conjugate were prepared. The factors that significantly affect the quality of the final product are summarized below.

The examples above illustrate how this invention operates. Other factors affecting ac-5 coupling of HCG with the monodisperse styrene-glycidyl methacrylate latexes described above are the age of the latex and the ways of storing the latex before mating. The latex should be stored under refrigeration and used in the coupling reactions as soon as possible. The latex prepared in Example 2 performed satisfactorily in the coupling reaction with the HCG after a one month storage period under refrigeration. .

In the preparation of logically active immuno-15 latex-HCG conjugates it was found that an important variable in determining sensitivity is the source of HCG. Preparations of various commercial origin must be checked to see which one is most satisfactory for the particular application. Although 20 variable amounts of protein can be bound to the latex, it has been found that coupling the maximum amount of HCG to the latex does not produce the best results. Although other quantities also gave acceptable results, the amount of HCG which was preferred in the coupling reaction was about 25 1000-25 000 IU per gram of polymer, 11 000 IU / gram of polymer being the particularly preferred amount. The preferred concentration of solid polymers in the reaction was about 4-5%.

In general, the coupling reaction is carried out at a pH of about 7.5-8.5, a pH of about 8.0 being preferred. A phosphate buffer is generally used to couple HCG to latex. Borate buffers are generally unsatisfactory with proteins such as HCG which have high carbohydrate content. It has also been found 35 that the reaction period can be reduced to about a day or less by carrying out the reaction at room temperature instead of 5 ° C as shown in the examples above. However, when no particular refrigeration conditions are used, appropriate control of the 40 potential contaminants of microbial origin is necessary. After coupling the latex with the HCG, it is essential that unreacted HCG and other impurities are removed from the reaction mixture. Ultrafiltration followed by dialysis through cellophane or high speed centrifugation followed by dialysis through cellophane gave a satisfactory product. Dialysis through hollow fibers although still usable was less effective in removing excess HCG from the reaction mixture.

If the final latex-HCG conjugate is buffered with the preferred glycine buffer at pH 8.2, it is no longer necessary to discharge the residual epoxy groups. If, however, it is desired to use a neutral buffer system, it is preferable to carry out a further step to discharge the residual epoxy groups (as illustrated in example 5 above). 55 In general, aging of the final latex-HCG conjugate has been found to significantly improve the immunological activity of the product. The aging period required to obtain optimal immunological activity depends on the temperature at which aging is performed, i.e., the lower the temperature the longer the aging period. It has been found that a product aged from 3 to 5 days at about 25 ° C gives satisfactory results. A product aged at 5 ° C required a conservation of about 2 weeks to achieve 65 optimal activity.

The HCG latex conjugate can be incorporated into an analytical kit containing all the reagents necessary for the immunological determination of pregnancy. this

639777

6

the kit will contain the latex-HCG conjugate described above as the first reagent, and an anti-HCG buffered serum, generally obtained from white New Zealand rabbits, as the second reagent. See J. Clin. Endoer. 33, 988 (1971). The base reagents can also be accompanied by subsidiary equipment such as an opaque slide with mixing areas at intervals and a stick for application.

It may also be appropriate, when the conjugate is used for immunology purposes, to dye the latex particles before coupling with the protein in order to facilitate the determination of allutination. This is easily accomplished by mixing the latex with a solution of a dye containing a solvent capable of penetrating the latex particles. For example, it has been found that the Calco Oil Blue N (American Cyanamid) dye dissolved in benzene gives satisfactory results. The benzene was removed in an evaporator before coupling with the protein.

The following examples illustrate the coupling of styrene-glycidyl methacrylate latex with insulin.

Example 8

The 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 were added of sodium hydroxide 0.1N. The final pH was 8. The reaction mass was stirred at 5 ° C for 90 hours after which 4.4 grams of the latex resulting from the reaction mixture (4.8 grams) were washed by filtration through a membrane in a Diaflo filter cell ® using a 0.1 ji Millipore filter and a borate buffer at pH 8. A hydrodynamic pressure of about 3.4 atmospheres was applied above atmospheric pressure (50 psig) and the effluent was examined by UV to determine the presence of unpaired insulin. Filtration is continued until no more insulin was found in the effluent. Thin layer electrophoresis confirmed that insulin was chemically bound to latex.

Example 9

A 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 a phosphate buffered saline at pH 8. The mixture was covered and stirred for about 5 days at 5 ° C. The reaction mixture was diluted (2-3 times) with the tam-5 and centrifuged at 18,000 rpm for about 35 minutes. The supernatant was discharged and the latex-peroxidase residue was again suspended in a phosphate saline buffer at pH 7 with the addition of 1% of a surfactant. Centrifugation and suspension were repeated two more times using a phosphate saline buffer at pH 7 without surfactant. The latex-peroxidase conjugate was filtered through a thin glass wool to obtain the final product. The resulting conjugate was tested for peroxidase enzymatic activity using a modification of the Steinman et al. Process [J. Cell. Biol. 55, 186 (1972) 7. Three samples of the product plus a sample of the supernatant were reacted for different time intervals with a substrate containing dianisidine dihydrochloride and hydrogen peroxide in a tam-20 place saline phosphate at pH 7.

The reaction mixture was filtered and the filtrates were examined at 400 nanometers. The results were as follows:

25

Sample Reaction time Optical density *

(min: sec)

4:55 0.53

5: 17 0.62

9: 17 0.65

10: 13 0.005

35

* tested at 400 nanometers

The data show very little enzymatic activity (oxidation-40 dianisidine) in the supernatant reaction mixture (sample 4). Therefore the enzymatic activity of the latex peroxidase product was essentially due to the enzyme immobilized on the latex particles and not to residual peroxidase soluble in the supernatant.

30 2

3

4 (supernatant)

v

Claims (13)

639777 1. Method for binding a protein having a reactive group containing a hydrogen mobile with. a latex where the particles have a diameter from about 0.15 to about 1.5 micrometers and have an inner core and an outer layer, this inner core being formed by the polymerization or copolymerization of one or more "hard" monomers and this outer layer being formed by the copolymerization of one or more "hard" monomers and a copolymerizable compound containing an ethylene unsaturation having epoxy groups, which includes reacting said latex and said protein at pH 7.0-9.0 for a time sufficient to give rise to the formation of a covalent bond between the reactive group of the protein and the free epoxy group of the latex and to remove the unreacted protein from the latex-protein conjugate. 2. Process according to claim 1, characterized in that the inner core and the outer layer contain a copolymerized "soft" monomer. 2 Method according to claims 1 and 2, wherein the latex is a monodisperse latex and the reactive group on the protein is an amine group. 4. Process according to claims 1 and 2, wherein the latex is styrene-glycidyl methacrylate. 5. The process of claim 4 wherein the protein is human chorionic gonadotropin. 6. The method of claim 4 wherein the protein is insulin. 7. Process according to claim 4, wherein the protein is an enzyme. 8. Process according to claim 5, where the density of the styrene-glycidyl methacrylate latex binding sites is about one epoxy unit for 50 to 400 (nm.) 2 of surface area of the particles and the amount of human chorionic gonadotropin used to prepare the latex-protein conjugate it is from 1000 to 25 000 international units per gram of polymer. 9. Protein-latex conjugate formed according to the process of claim 3. 10. A protein-latex conjugate according to claim 9, wherein the protein portion is human chorionic gonadotropin and the latex portion is styrene-glycidyl methacrylate. 11. A protein-latex conjugate according to claim 9, wherein the protein portion is insulin and the latex portion is styrene-glycidyl methacrylate. 12. A protein-latex conjugate according to claim 9, wherein the latex is colored. 13. Use of the compounds produced by the process according to claim 1, for the immunological determination of pregnancy, characterized by the use of a conjugate, comprising a first reagent containing a monodisperse styrene-glycidyl methacrylate latex with alloyed human chorionic gonadotropin and a second reagent containing a human chorionic gonadotropin antiserum.