NL7901200A - Coupling protein to latex particles having epoxy gps. - for use in immunological analysis esp. pregnancy testing - Google Patents

Abstract

Coupling of protein (I) having a reactive gp. which includes a labile H atom., to latex particles of dia. 0.15-1.5 mu comprises reacting at pH 7-9 to form covalent bonds between (I) and free epoxy gps. in the lates. Unfixed (I) is then removed. The latex particles comprise a core, made by (co)polymerising >=1 'hard' monomer, and an outer sheel made by copolymerising >=1 'hard' monomer and an ethylenically unsatd. monomer having free epoxy gps. Both core and shell may include a 'soft' comonomer. Specifically (I) includes NH2 gps., esp. it is human chorionic gonadotropin (HCG), insulin or an enzyme, and the latex is esp. styrene-glycidyl methacrylate. The conjugates are useful in antigen-antibody testing, esp. pregnancy testing. Severe conditions which might denature (I) are avoided; the particles are of uniform size distribution, so are partic. suitable for automated analysis.

Description

The Dow Chemical Company of Midland, Michigan, United States, R. America

Method for coupling a protein to an epoxylated latex and the products obtained.

The invention relates to a method for coupling a latex with surface eporydic groups to a protein or protein component by forming an amine bond. Such carrier-bound proteins are particularly useful in the conduct of immunological tests based on the anti-no-antibody action.

The antigen-antibody reaction is the basis for all immunological test methods. Special proteins, referred to as antibodies, are produced by an animal in response to the presence of an antigen, ie 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 used for the diagnosis of various human and animal diseases or disorders.

In vitro tests for the putative presence of an antigen or antibody in a body fluid are performed by adding the immunological counterpart to a vial containing the body fluid, which means that antigen is added if the assay serves to demonstrate the presence of anti -20 body or antibody is added if the test serves to detect the presence of antigen. If the putative proteins are present, the resulting antigen-antibody response will generally be manifested by precipitation or agglutination of the antigen-antibody complex. In some cases, the antigen-antibody complex is slowly formed and the particles formed are too small to be observed with certainty. In such cases, the detectability of the antigen-antibody response can be enhanced using a carrier. When the antigen or antibody is coated on the surface of a 7901200 2 support, the reaction occurring with the immunological counterpart results in a visible mass or agglutinate. The protein antigen or antibody can be adsorbed on the surface of carriers such as erythrocites, bacterial cells, bentonite, polystyrene latex-5 particles, anionic phenolic resins or finely divided diazotized amino cellulose. However, it has been found that the chemical binding of the antigen or antibody molecule to the support is superior to physical adsorption. U.S. Pat. No. 3,857,931 discloses that protein antigens or antibodies can be chemically bound to a poly-10 latex support with surface earbonyl groups by an amide bond formed in the presence of a water-soluble carbodiimide coupling agent. US patent 3,806.U17 describes a method of binding a protein to a compound containing an epoxy group and joining this intermediate to a support material. A drawback of this method is that the protein must first be reacted with a compound having such an epoxy group as an olefinic group.

The resulting conjugate is then polymerized with another olefinic monomer and a bis-olefinic crosslinking agent to form the enzyme-bound enzyme. Methods for binding a protein to polymer supports are described in U.S. Pat. Nos. 3,639,558, 3,853,708, 3.8H1,970, 3,88,892, 3,821,081, and U.086,199.

In particular, when a latex system is used as a carrier, the prior art describes coupling methods which require the use of bifunctional coupling agents, or of coupling agents, which form a bond between the reactive groups on the protein and those on the carrier particles. The difficulty with these methods is that the coupling agents cannot distinguish between the reactive groups on the protein and those on the cell carrier; therefore, a protein-to-protein coupling and even an intramolecular protein coupling can occur. Such undesired binding can result in conformational changes in the protein molecule, leading to changes in activity. The invention relates to a method for covalently binding a protein with a reactive group, containing a labile hydrogen atom, to a 7901200 3 latex with surface epoxide groups. Preferably, the reactive group is a free amino group, but other reactive groups, such as carboxyl, phenol, hydroxyl and thiol, can also be used. The coupling occurs when the amino groups of the protein react with the epoxide groups of the latex, usually at a pH of about 7.0-9.0, to form a new carbon-nitrogen bond, referred to below as an amine binding, whereby the protein binds to the surface of the latex. This process offers a number of advantages over the hitherto known methods for the preparation of protein-latex conjugates. More particularly, the method according to the invention is relatively easy to carry out. In the method of the invention, the proteins are not exposed to drastic reading conditions, which could result in a distortion or denaturation of the protein molecule. This is particularly important because such conformational changes can result in loss of activity. The method according to the invention also makes it possible to remove the remaining epoxy groups after binding to the proteins. The method of the invention has the further advantage of providing support-bound proteins 20 with a uniform particle size distribution.

The term latex used herein refers to an aqueous colloldal dispersion of a water-insoluble polymer. The latices used in the method according to the invention consist of substantially spheroidal polymer particles with a diameter of between 0.15 and 1.5 micrometers, with a diameter of 0.1 * 5-0.90 micrometers ( ^ uM) is preferred. The particle morphology consists of an inner core and a surrounding shell. The core is polymerized or copolymerized from hard monomers, such as styrene. The shell is formed by copolymerizing one or more hard monomers with a copolymerizable ethylenically unsaturated compound with a three-membered epoxy ring. Optionally, soft monomer can be copolymerized in the core and / or shell together with the hard monomer to prevent particle breakage. The term "hard monomer" as used herein refers to monomers which form polymers having glass transition temperature above room temperature. The term "soft monomer" refers to monomers which form polymers with a 7901200 lb -¾k glass transition temperature below room temperature. Preferred emulsion polymerizable monomers for the core are hard monomers which can be polymerized and / or copolymerized with each other in arbitrary proportions and / or with other monomers to form non-film-forming polymers. The term "non-film-forming" as used herein refers to polymer particles of the latex, which are non-film-forming or do not coalesce under the conditions of use. These monomers include carbocyclic monovinylidene monomers, for example styrene, α-methylstyrene, ar- (t-butyl) styrene, ar-methyl-10 styrene, ar-ar-dimethylstyrene, ar-chlorostyrene, ar- (t-anyl) styrene, ar- bromostyrene, ar-fluorostyrene, ar-eyanostyrene, ar-methoxystyrene, ar-ethylstyrene, ar-hydroxymethylstyrene, ar-ethoxystyrene, ar-chloro-ar-methylstyrene, ar.ar-dichloro-styrene, ar.ar-difluorostyrene, vinyl-naphthalene, and other such emulsion-polymerizable monomers having 15 carbon atoms or more, esters of α, β-ethylenically unsaturated carboxylic acids, which polymerize to form non-film-forming polymers, for example, methyl methacrylate, chloroethyl methacrylate, n-butyl methyl acrylate, ethyl methacrylate, isobutyl methacrylate , isopropyl, methacrylate, phenyl methacrylate, butyl chloroacrylate, cyclohexyl chloro-acrylate, ethyl chloroacrylate, methyl chloroacrylate, isopropyl chloroacrylate and other such esters, which can be polymerized to form g of hard polymers, α.β-ethylenic. unsaturated esters of non-polymerizable carboxylic acids, for example, vinyl benzoate, vinyl toluate, vinyl ar-ethyl benzoate, allyl ar-ethyl benzoate, vinyl pivalate, vinyl trichloroacetate and other such monomers, in which the unsaturated portion is 2-lb carbon atoms and the acid portion is 2-12 carbon atoms. Atoms contain α, β-ethylenically unsaturated nitriles, for example nitriles with no more than 12 carbon atoms, other polymerizable vinyl monomers such as vinyl chloride, vinyl bromide and the like. Of the above-mentioned monomers, particularly preferred are the carbocyclic aromatic monovinylidene monomers, in particular styrene and mixtures of styrene, methyl methacrylate and acrylonitrile.

Soft monomers that may be optionally used include the alkyl acrylates, such as ethyl acrylate, butyl acrylate, or 2-ethylhexyl acrylate, the higher alkyl methacrylates, such as hexyl methacrylate or 2-ethylhexyl methacrylate, and the conjugated dienes, such as 7901200 isoprene or butadiene.

A preferred monomer for the shell copolymer is a mixture of one or more hard monomers as described above and a copolymerizable ethylenically unsaturated compound 5 with a three-membered 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 monomer compounds of diene type monomers.

Suitable glycidyl esters include glycidyl methacrylate, glycyl acrylate, glycyl cylinders of crotonic acids and unsaturated chain fatty acids and the like, unsaturated alkyl glycidyl ethers, vinyl glycidyl ether, alkyl propyl ether, phenyl ethyl cycloethyl ether, allyl glyceryl ether, allyl glyceryl ether, allyl glyceryl ether, allyl glyceryl ether, allyl glyceryl ether, allyl ethyl ether, vinyl cyclohexyl glycidyl ether, p-vinylbenzene glycidyl ether, o-allyl phenyl glycidyl ether and the like, the monoepoxide compounds of the diene-type monomers include butadiene monoepoxide, chloroprene monoepoxide, 3-epoxy-1-pentene, 1-epoxy-1-epoxy 4,5-epoxy-2-pentene, .5-epoxy-1-hexene, 3-epoxy-1-vinyl-cyclohexene and divinylbenzene monoxide and the like.

The exact amount of the epoxy monomer required for the preparation of the latices of the invention is determined by the population (amount) of surface epoxy groups required for optimal coupling with the desired proteins. For example, in the coupling of human choronic gonadotropin to a styrene-glycidyl methacrylate latex, a coupling site density of about one epoxy unit per 5-0 square square unit units (A) gives satisfactory results, with about one epoxy unit per 8-20 A is preferred. This same coupling site density would also be useful for other proteins, such as insulin. In the preparation of latex-bound proteins for use in the immunological test, it has been found that mono-dispersed latices, that is, latices of substantially uniform particle sizes, are preferred because uniformity of particle sizes provides a uniform statistical distribution of antigen. or ensures antibody molecules on the 790 1 2 0 0 <* -¾ 6 surface of the latex particles. For a given weight of polymer, the total surface area of the latex will increase with a decrease in the size of the particles and vice versa. Thus, in a latex containing a distribution of varying particle sizes, the smaller particles will have a larger surface area and, consequently, more reaction sites in total than the larger particles. The uneven distribution of antigen or antibody on the latex particles will lead to uneven particle agglutination and poorly defined diagnostic results.

Another advantage of the use of uniform latex particles as diagnostic agents is that they are more suitable for the instrumental analysis. Particles of the same size will flocculate, agglutinate or sediment at the same rate, while particles of different sizes will agglutinate at variable rates. Therefore, 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 latices with particles of varying sizes. Conjugates of human chorone gonadotropin bound to styrene-glycidyl methacrylate latex using the method of the invention showed virtually no change in immunological activity after storage of up to 12 months at about H-6 ° C.

The following examples describe the preparation of three mono-dispersed styrene-glycidyl methacrylate latices. Commercially available mono-dispersed polystyrene latices were capped with a mixture of styrene and glycidyl methacrylate. Unless otherwise indicated, the parts are parts of the penis.

Example I

The following ingredients were added to a 1 liter three neck flask equipped with stirrer, nitrogen inlet tube and cooler.

7901200 ö * · »7

Dry weight Wet weight Share Share

Polystyrene latex (particle size 0.76 μm) 60.5 188 5 Dihexyl sodium sulfosuccinate 0.3 6

Potassium sulphate 0.3 6

Sodium hydrogen carbonate 0.3 6

Styrene k5 ^ 5

Glycidyl Methacylate 15 15 10 Water - 13¾

The mixture was stirred and the flask purged with nitrogen for about 10-20 minutes. The reaction temperature was attempted at 65 ° C and this temperature was maintained for k 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, where it was found that the diameter was about 0.91 µM.

Example II

The general procedure 20 described in Example I was repeated using the ingredients indicated below to prepare a monodispersed latex with an average particle size (diameter) of about 0.59 µm.

Dry weight Wet weight Share Share 25 Polystyrene latex (particle size 0.503 micron) 30.1 95

Dihexyl sodium sulfosuccinate 0.15 3

Potassium persulfate 0.15 3

Sodium Toy Dryer Carbonate 0.15 3 30 Styrene 15 15

Glycidyl methacrylate 15 15

Water - 266

Example III

The following ingredients were used to prepare a mono-dispersed latex with an average particle size (disaster) of 0.20 µM. The same general procedure as above 790 1 2 0 0 δ r '

Written applied.

Dry weight Wet weight Pélen 'Share

Polystyrene latex 5 (particle size 0.176 μm) 30 300

Dihexyl sodium sulfosuccinate 0.15 3

Potassium persulfate 0.15 3

Nat ri umhy dr o noc arb on aat 0,15 3

Styrene 15 15 10 Gly cidyl methacrylate 15 15

Water - 61

The epoxy content of the latex, expressed as ⁄ milliequivalents of oxiran groups per gram (-CH-CHg / gram) and as square angstrom units per oxiran group, was determined for each latex prepared above. The results are shown in Table A below.

Table A

Latex of Particle Size Μ yQx ^ .0S mequiv.

example (, uM) A / / -CH-CHg -GH-CHg / gram.

20 I 0.907 15.0 0.07 II 0.592 9, 0.17 III 0.20201 17.0 0.278

The number of milliequivalent oxiran groups per gram of polymer determined by treatment of the latex containing a known amount of polymer, with known equivalents of water or iodide. The residual hydrogen iodide remaining in the latex was titrated with standardized silver nitrate solution to determine the amount of hydrogen iodide that had reacted with the epoxy groups. The milliequivalents of epoxy groups were then determined from this information. The latices prepared above were found to be suitable for use in the store of the invention. Samples of each of the latex formulations described above were coupled with human choronic gonadotropin, referred to below as HCG. This hormone is a glycoprotein and controls the formation and separation of progesterone by the corpus luteum. It is normally secreted by the chorione tissue from the placenta during pregnancy. HCG bound to latex 7901200 Ί 9 is useful for detecting pregnancy.

Example IV

A solution containing 5000 IU HCG in 7 ml phosphate buffer with a pH of 8.0 (I = 0.05; 0.1 M NaCl; 1: 10,000 thimerosal), 5 ml stirred added to a 25 ml round bottom flask} which already contained 1.61 g of styrene-glycidyl methacrylate prepared as described in Example I. The resulting latex suspension was stirred in a cold room (5 ° C) for U days. The reaction mixture is washed by membrane filtration for about 3 hours in a 10 Diaflo (registered trademark) filtered with 1 ^ 5 ml of phosphate buffer.

The washed latex and the rinsing liquid were dialyzed in cellophane against 0.1 M glycine buffer with a pH of 8.2. After 2 days of dialysis, 16.4 g of the styrene-glycidyl methacrylate-latex HCG product was obtained with a detection sensitivity of 0.9 IU / HCG / ml.

Example V

This product was prepared in the same manner as described in Example IV with the following exceptions. The reaction mixture was treated after 3 days with 1 ml of glycine buffer with a pH of 8.2 to remove the remaining epoxy groups. After stirring for 1 more day in the cold, 9.9 g of the reaction mixture were washed by membrane filtration using 188 ml of phosphate buffer with a pH of 7.0 (I = 0.05; 0.1 M NaCl). The resulting product and the rinsing liquid were treated with 13 mg of sodium azide as a preservative.

The resulting product had a detection sensitivity of 1.8 IU / HCG / 25 ml.

Example VT

Latex prepared according to the procedure of Example II (3.08 g latex, 0.16 g polymer) was placed in a 25 ml round bottom flask equipped with a Teflon (registered trademark) coated magnetic stir bar. A solution containing 5000 IU HCG in 8 ml cold phosphate buffer with a pH of 8 (I * 0.05; 0.1 M NaCl; 0.01% thimerosal) was slowly added to the flask. The reaction mixture was stirred calmly for about 90-96 hours at about 5 ° C. The latex HCG conjugate was washed with 110-115 ml phosphate buffer by ultrafiltration for about 70-110 minutes. The combined latex HCG and rinsing liquid were dialyzed in cellophane at 5 ° C for 2¼ hours.

790 1 2 00 «✓ -¾ 10

The product prepared according to this procedure showed a detection sensitivity of 0.9-1.8 IU / HCG / ml.

Using the procedures already described, HCG was coupled to the latex prepared in Example III. Further samples of HCG-latex conjugate were prepared using the general procedures and methods already described. The factors which were found to significantly influence the quality of the final product are listed below.

The above examples illustrate the implementation of the sprout according to the invention. Other factors that have been found to affect the coupling of HCG to the styrene-glycidyl methacrylate monodispersed latices described above were the age of the latex and the manner of storing the latex before the coupling was performed. The latex should be stored under cooling and used in the coupling reactions as soon as possible.

The latex prepared in Example II was found to behave satisfactorily in the coupling reaction with HCG after 1 month storage under refrigeration.

In the preparation of immunologically active latex-20 HCG conjugates, the source of HCG was found to be an important variable in the determination of sensitivity. HCG formulations from varying technical sources should be investigated to determine which is most satisfactory for the special applications. Although varying amounts of proteins can be coupled to the latex, the adherence of maximum amounts of HCG to the latex did not give optimal results. While other amounts are useful, the preferred amount of HCG used in the coupling reaction is about 1000-25,000 I.U. per gram of polymer, with an amount of 11,000 I.U./gram of polymer being particularly preferred. The preferred reaction concentration of polymer solids was about U-5%

Generally, the coupling reaction is carried out at a pH of about 7.5-8.5, with a pH of about 8.0 being preferred. A phosphate buffer is generally used for the coupling of HCG to the latex.

Borate huffers are generally unsatisfactory with proteins such as HCG, which have a high carbohydrate content. It was also found that the coupling reaction time could be reduced to about 1 day or less by carrying out the reaction "at room temperature instead of at 5 ° C as in the above examples.

However, if no special refrigerated facilities are used, appropriate control of potential microbial infections is necessary.

After coupling the latex to HCG, it is essential to remove unreacted HCG and other impurities from the reaction mixture. Ultrafiltration followed by cellophane dialysis or high speed centrifugation followed by cellophane dialysis gave a satisfactory product. Hollow fiber dialysis, while still useful, is less effective in removing excess HCG from the reaction mixture.

If the final latex-HCG conjugate is buffered with the preferred glycine buffer (pH 8.2), it is not necessary to remove the remaining epoxy groups. However, if a neutral buffer system is human, an additional step is required to remove the remaining epoxy groups (as illustrated in Example V).

In general, it was found that aging of the latex HCG final conjugate significantly improved the immunological activity of the product. The aging period necessary to obtain optimal immunological activity depends on the temperature at which aging takes place, that is, the lower the temperature, the longer the aging period. It has been found that a product aged for 3-5 days at about 25 ° C showed satisfactory behavior. A product stored at about 5 ° C required storage for about 2 weeks to obtain optimal activity.

The latex HCG conjugate can be combined in a pack containing all necessary reagents for the immunological pregnancy challenge. Such a pack may contain a first reagent formulation comprising the above-described latex HCG eonjuume and a second reagent formulation comprising buffered anti-HCG serum generally obtained from New Zealand white rabbits (see J. Clin. Endocr. 33, 988 (1971)} The base reagents may also be combined with auxiliary components, such as an opaque glass slide with receding mixing areas and an applicator stick.

790 12 00 12

When the conjugate is intended for immunological purposes, it may also be desirable to color the latex particles before coupling them with proteins to facilitate detection of the agglutination. Pit can be easily accomplished by mixing the latex with a dye solution containing a solvent which can penetrate into the latex particles. For example, Calco Oil Blue N dye (American Cyanamid), dissolved in benzene, has been shown to give satisfactory results. The benzene was removed in an evaporator before coupling with the proteins.

The following examples illustrate the coupling of styrene glycidyl methacylate latex to insulin.

Example VIII

Latex as prepared as described in Example 15 I (2.5 g of latex, 0.75 g of polymer) was charged into a flask containing 2.5 ml of insulin solution (15 mg of insulin) and 5 drops of 0.1 K sodium hydroxy -the were added. The final pR was 8. The reaction mass was stirred at 5 ° C for 90 hours, after which g of the latex reaction mixture (U, 8 g) was washed by membrane filtration in a Diaflo (registered trademark) filter cell using a 0.1 µm Millipore filter and a borate buffer with a pE of 8. A bydrodynamic pressure of up to about 3.5 kg / cm was applied and the effluent was examined by ultraviolet radiation for the presence of unlinked insulin. Filtration was continued until no more insulin could be detected in the effluent. The thin layer electrophoresis revealed that the insulin was chemically bound to the latex.

Example IX

Latex prepared as described in Example I (6.3 g latex; 0.92 g polymer) was added to a solution containing 7.33 mg horseradish peroxidase in 10 ml phosphate salt buffer with a pE of 8 The mixture was covered and stirred at 5 ° C for about 5 days. The reaction mixture was diluted (2-3 times) with buffer and centrifuged at 18,000 rpm for about 35 minutes. The supernatant was discarded and the latex peroxidase residue was resuspended in phosphate salt buffer with a pH of 7 to which (1% surfactant was added. The centrifugation and resuspension 79 0 1 2 0 0 '

Claims (11)

1. Method for coupling a protein with a reactive group, containing a labile hydrogen atom, to a latex, wherein the latex particles vary in size (diameter) from about 0.15 to 1.5 micrometers and an inner core and a outer shell, wherein the inner core is formed by polymaisation or copolymerization of one or more bar of the monomers and the outer shell is formed by copolymerization of one or more hard monomers and a copolymerizable ethylenically unsaturated compound with free epoxy groups, the core and shell may optionally contain copolymerized soft monomer, characterized in that the latex and the protein are reacted at 790 1 2 0 0 1H with a pH of 7.0-9 * 0 for a sufficiently long time to produce a covalently bond between the reactive group of the protein and the free epoxy group of the latex and remove the unreacted proteins from the latex-protein conjugate. 2. Method as claimed in claim 1. 'characterized. that the latex is a mono-dispersed latex and the reactive group on the protein is an amino group, Process according to claim 1, characterized in that the latex is styrene-glycidyl methacrylate. 10 b. Method according to claim 3, characterized in that the protein is human choronic gonadotropin. The method according to claim 3, characterized in that the protein is insulin. 6. A method according to claim 3, characterized in that the protein is an enzyme. A method according to claim U, characterized in that the coupling site density of the styrene-glycidyl methacrylate latex is approximately one epoxy unit per 5-10 square angstrom units particle area and the amount of human choronic gonadotropin used to prepare the latex-protein conjugate is 1000-25000 international units per gram of polymer. A protein-latex conjugate obtained according to the method of claim 2. The protein-latex conjugate according to claim 8, characterized in that the protein portion is human choronic gonadotropin and the latex portion is styrene-glycidyl methacrylate. Protein-latex conjugate according to claim 8, characterized in that the protein portion is insulin and the latex portion is styrene-glycidyl methacrylate. Protein-latex conjugate according to claim 8, characterized in that the latex is colored. 12. A suit for the immunological determination of pregnancy, comprising a first reagent containing a monodispersed styrene-glycidyl methacrylate latex with attached human choronic gonadotropin and a second reagent containing anti-human choronic gonadotropin serum. 7901200 g *. ~ »0« r 13 · Methods, products and pack as described in the description and / or examples. 790 1 2 0 0