Immunological diagnostic reagent

The invention relates to diagnostically useful reagents, a process for the manufacture thereof and diagnostic methods utilizing said reagents.

The diagnosis of pathological states or other conditions in human beings as well as animals is often accomplished by the application of immunological principles. These principles are utilized to determine the presence of antibodies or antigens in the body fluids of the subject. An antigen is a foreign c substance, wijih, when introduced into the subject, causes the production of certain soluble substances designated as antibodies. Any foreign substance, e.g., protein, which is not normally present in a given subject can cause the formation of antibodies when introduced into the subject under proper conditions.

The antibodies, after their formation, react with the antigen and thus, in the case of a microbial or viral invader, protect against infections.

Immunological testing procedures are based upon the antigen-antibody reaction which usually is manifested by an 20 insolubilization or agglutination.

Generally, the presence of an antigen or antibody is confirmed or diagnosed by contacting the corresponding antibody or antigen with a body fluid of the subject, usually .

30/27.12.71 urine, blood serum or a specially treated blood extract, although other body fluids can also be used, The presence of the antibody or antigen in the subject is noted if an insoluble antigen-antibody complex forms.

Because some complexes for׳m very slowly and have very small particle sizes, it is necessary to utilize carriers in order to make it possible to visually dlcern them. Among the carriers which have been employed are sheep and human erythrocytes, bacterial cells, bentonite, latex particles, e.g. polystyrene, anionic phenolic resins and finely divided diazotized amino cellulose.

The French Patent Specification No. 2002.101 discloses the use of carbodilmides to couple proteinaceous materials to carrier particles such as red blood cells, gelatin and so on. However, one of the disadvantages of the reagents disclosed in said French Patent Specification is that the coating on the carrier is a double coating.

Furthermore, the carriers are degradable and in some cases might even be serologically active.

The British Patent Specification No. 1.192.784 relates to reagents for use in the determination of proteins and polypeptides. The reagents comprise antibodies chemically bound to polymer carriers obtained[ by cross-linking substances such as dextran, starch, dextrin and other polysaccharides and polyvinylalcohol with a bi-functional substance such as epichlorohydrin. However, the reagents disclosed in said British Patent Specification are Specifically Intended for use in a radlo-immuno-assay and are not suitable for an agglutination assay.

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The British Patent Specification

No. 1143.938 discloses the use for immunological determinations of polystyrene latex physically coated With antigens or antibodies. These reagents have the disadvantage of a less strong and uniform coating on the particles than the chemical bonding, with the consequence of less reliable results in diagnostic tests. Furthermore, physical bonding has a less wide applicability than chemical bonding since some antigens and antibodies are not amenable to physical bonding.

The known carriers which physically bind serologically determinant materials are limited in their applicability and usefulness in immunological diagnostic procedures because they suffer from a number of disadvantages. Among the important disadvantages are in many instances a lack of sensitivity and in many cases poor stability.

These defects are primarily due to the fact that when latex particles are physically coated with the serologically reactive material an equilibrium between the free and associated material exists. This results in competitive inhibition between the free and the latex bound antibody or antigen for the corresponding antigen or antibody. Furthermore, many negatively charged proteins can not be physically coated to inert latex particles without hydrolysis or enzymatic degradation. This treatment may result in conformational changes of structure which can be detrimental to the specificity of the reaction. Small peptides do not lend themselves even under these conditions to physical coating to inert polymers which limits the applicability of this procedure for many immunologic diagnostic tests.

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There is thus a need for a carrier which is serologically inert, will form, with a wide spectrum of serologically determinant materials, a diagnostically useful reagent which is stable, specific, sensitive and provides an easily ascertainable visual evaluation in the minimum of time.

According to tie present invention serologically determinant materials are bound through an amide linkage to aarboxylated butadiene styrene, acrylic acid, methacrylic acid, etc., type of polymers. The reagents of the Instant invention have not the disadvantages of the physically coated reagents described in the British Patent Specification 1143.938. Furthermore, they have the advantage that, in opposition to the disclosure in the French Patent Specification No. 2002.181, the coating on the carrier is realised in a single operation and that the polymeria carriers are of smaller size than those of the British Patent Specification No. 1192.784 and thus suitable for agglutination assays.

This invention thus provides a water-insoluble immunological diagnostic reagent having a specific gravity of about that of water comprising discrete particles of a serologically inert latex polymer having a particle size of about 0.01 to about 0.9 microns and being selected from the group consisting of aarboxylated styrene butadienes, aarboxylated polystyrenes, carboxylated polystyrenes with amino groups, acrylic acid polymers, acrylonitrile polymers, methacrylic acid polymers, acrylonitrile butadiene styrenes, polyvinyl acetate acrylates, polyvinyl pyridines, vinyl chlorideacrylates; said latex polymer having condensed thereto through an amide linkage a known serologically determinant material. This invention further relates to a process for the manufacture of such a reagent which process is characterized in that a serologically determinant material is reacted witih a serologically inert latex polymer containing reactive groups a - \ ׳ ג

which are formable into an amide linkage, in the presence of a water-soluble carbodlimide at a temperature of from about 5°C to about 40°C.

It has been discovered that many serologically determinant materials, e.g., polysaccharides, intact protein molecules and peptides, which previously could not be satisfactorily physically attached to polymeric latex carriers, can be covalently linked by the formation of an amide bond to a certain group of serologically inert, latex polymeric

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b carriers by chemical means.

The phrase serologically determinant materials as used in the context of this invention refers to those materials which can be determined in human and animal body fluids by utilization of immunological principles. This invention comprehends within its scope all those serologically determinant materials which can be chemically linked by the formation of an amide bond to a serologically inert latex carrier particle, using a carbodiimide condensing agent.

Typical suitable serologically determinant materials are isolated human and animal antibodies, serum components, toxins, bacterial and viral components, hormones, enzymes, alkaloids, cellular and tissue extracts, small molecular weight substances, e.g., insulin, angiotension and urokinase, and the like. Specific representative materials which are eminently suitable for use in diagnostic tests are human chorionic gonadotropin, human gamma globulin and human albumin.

The amount of serologically determinant material linked to the serologically inert latex polymeric carriers is usually from about 0.01% to 15.0% by weight. However, each particular serologically determinant material is utilized in an amount in which it is most successfully employed in a diagnostic test, therefore, each material is combined with the carrier in a ratio suitable for its specific requirements. This invention therefore comprehends within its scope the use of an amount of serologically determinant material in combination with a serologically inert latex polymeric carrier sufficient to provide a diagnostically effective reagent.

As used within the context of this invention, serologically inert latex polymers or serologically inert latex polymeric carrier particles includes latex polymers which are waterinsoluble, have a particle size in the range of from about 0.01 microns to about 0.9 microns, a specific gravity near that of water so that after coupling with the serologically determinant material, the specific gravity of the particles is about 1.0, enabling them to remain permanently in aqueous suspension; the particles must be inert with respect to immunological diagnostic tests, they also must have sufficient surface charge density so that when coupled to the serologically determinant material, their repulsive forces are enough to prevent aggregation and the particles must have active groups which are capable of forming an amide linkage with a serologically determinant material by the condensation of a primary or secondary amine group and a carboxyl group. Thus, the polymeric carriers can have either carboxyl groups, amine groups or groups convertible into them or any combination of these groups. Typical suitable groups on the polymeric carriers are those containing an active hydrogen e.g., -COOH, -CONHg, a nitrile group, a secondary amine group, a primary amine group or any combination thereof.

The chemical reaction forming the amide linkage is carried out according to this invention in the presence of a watersoluble carbodiimide condensing agent. The degree of coupling with the serologically determinant material by means of carbodiimides is dependent upon the density of the reactive groups in the polymer. The density of the reactive groups is not critical to the operability of this invention as long as a sufficient amount of reactive groups should be present to provide coupling of a sufficient amount of serologically determinant material to be useful in diagnostic teats.

Typical suitable carrier particles are those supplied commercially as an aqueous latex suspension, usually in concentrations of about 40% to about 60% solids. Many types of latex polymers are suitable for use in this invention provided they meet the criteria set forth above. This invention comprehends the use of all the suitable latexes.

Typical suitable latex polymers are carboxylated styrene butadienes, carboxylated polystyrenes, carboxylated polystyrenes with amino groups, acrylic acid polymers, methacrylic acid polymers, acrylonitrile butadiene styrenes, polyvinyl acetate acrylates, polyvinyl pyridines/ vinyl chloride-acrylates,-a&amp;d־ 4he like! Some commercially available latexes which are suitable for use in this invention are Amsco Res 4150, Amsco Res 5011 (American Mineral Spirits Co.)ן Dow Latex 815, Dow Latex 816, Dow Latex 620, Dow Latex 859 (The Dow Chemical Co.);

Hycar 1512, Hycar 1877X8, Hycar 2600x120 (Goodrich Chemical Co.); Gelva 900, lytron 612, Lytron 624 (Monsanto); Rhoplex LC40 5216, Amberlite Ultrafine (Rohm and Haas).

According to this invention, ths serologically determinant materials are covalently coupled to discrete particles of the carrier by utilizing as a' condensing agent water-soluble monocarbodlimides represented by the formula r-N=C=N-R wherein R is: cycloalkyl having from 5 to 6 carbon atoms in the ring; alkyl of from 2 to 12 carbon atoms, e.g,, ethyl, n-propyl, isopropyl, n-butyl, sec.-butyl, iso-butyl, tert.-butyl, amyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl and dodecyl; monoarylsubstituted lower alkyl radicals, e.g., benzyl- a- and β-phenylethyl; monoaryl radicals, e.g., phenyl; morpholino; piperidyl; morpholinyl substituted lower alkyl radicals, e.g., ethyl morpholinyl; piperidyl substituted lower alkyl radicals, e.g., ethyl piperidyl; di-lower alkylamino; lower alkyl radicals; pyridyl substituted lower alkyl radicals, e.g., a, β and γ methyl or ethyl pyridyl; acid addition salts; and quaternary amines thereof.

The carbodiimides may be prepared in accordance with the general method of E. Schmidt, F. Hitzler and E. Lahde, Ber, 71. 1935 (1938) from the corresponding thioureas by oxidation with mercuric oxide in acetone. The thioureas may be prepared from the corresponding amine by reaction with carbon disulfide in the case of symmetrical thioureas. The unsymmetrical thioureas may be prepared by an amine with an isothiocyanate. The carbodiimides are also preparable from the corresponding ureas.

As stated above, water-soluble carbodiimides are most suitable for use in the present invention. If the carbodiimde bears a terminal amino group, it may be made water'soluble by forming an acid addition salt with a hydrohalic acid, e.g., HOI, HBr or HI; sulfuric acid, sulfonic acids, nitric acid, phosphoric acid and phosphonic acids; also the tertiary amino groups can be made water soluble by quaternization with a suitable quaternization agent, e.g., methyl tosylate, methyl bromide, methyl iodide, benzyl bromide, ethyl iodide, ethyl bromide, benzyl iodide, ethyl tosylate, methyl sulfate, ethyl sulfate and the like.

All of the foregoing are examples of compounds which are suitable as condensing agents useful in the practice of this invention.

The serologically determinant material and the carrier are reacted, according to this invention, in aqueous medium preferably at room temperature (about 20°C to about 25°C). Temperatures from about 5°C to about 40°C, however, are suitable for the reaction. In order to insure a chemical coupling of the serologically determinant material to the carrier, a sufficient amount of condensing agent is used to insure that all the available amide bonds are formed. Generally, about 0.05 percent to about 2.0 per cent by weight of a watersoluble carbodiimide, based on the weight of the particles, is suitable, usually, however, about 1 percent by weight is used.

The pH of the reactions is important since it should not be such as will denature any protein reactants. Usually a pH of from 5 to 7.5 is suitable. This pH is maintained by the use of appropriate conventional inorganic buffer systems such as phosphate buffers or the like.

The reaction is completed in from about 5 minutes to about 24 hours. Generally, about 4 or 5 hours is sufficient.

The resulting product is a water-insoluble material suspended in an aqueous medium buffered to a pH of about 5.0 to 8.5 depending on the particular system used and the stability requirements of the serologically determinant material. The specific gravity of the product is about the same as water with the result that the suspension of the product is stable. The products can be isolated, e.g., by centrifugation, and are white, somewhat thixotropic viscous clay-like materials. Chemically, the product is a monolayer of serologically determinant material condensed to discrete particles of the serologically inert carrier by means of an amide linkage. The serologically determinant material in the product is either a single active entity or if contaminants are present they do not interfere with the specificity of the reaction.

All such products which are formed from the reactants which meet the criteria set forth above and which in themselves meet the criteria set forth above are comprehended by this invention.

As examples of specific products whithin the scope of this invention are human chorionic gonadotropin linked through an amide linkage to a carboxylated butadiene styrene copolymer containing a monomer.ratio of 45% ^-butadiene, 55% styrene and having a density of carboxyl groups which is about 1% to about 5%, usually about 5% by weight; human albumin linked through an amide linkage to a carboxylated butadiene styrene copolymer containing a monomer ratio of 45% butadiene, 55% styrene and having a density of carboxyl groups which is about 1% to about 5%, usually about 3% by weight.

Once ths product is formed, it can be utilized in specific diagnostic tests utilizing immunological principles. It can be used in any convenient concentration depending on the specific test, however, concentrations of from about 1.0% to about 2,5% by weight are suitable with 1,3% by weight preferred. Thus, for example, the product formed when human chorionic gonadotropin is coupled to the carrier particle can be used as a diagnostic reagent to determine if a woman is pregnant. This can be accomplished, for example, by placing a drop of test urine on a clean glass slide, mixing a drop of ant-i-human chorionic gonadotropin serum, then adding a drop of the HCGcarrier product in aqueous suspension. Within two minutes the results of the test are observed and are about 90-98% accurate.

The advantages of such a test are its simplicity, speed, specificity, accuracy and lack of false positives. This is because there is no interference from other proteins caused by non-specific layering.

As another example, the product formed when gamma globulin is coupled to the carrier particle can be used as a diagnostic reagent to determine if a patient has rheumatoid c

arthritis. This can be accomplished, for example, by placing the buffered test sera on a glass slide and mixing in a drop of the gamma globulin-carrier reagent in an aqueous suspension.

Within one minute, the results of the test are observed and are about 80¢ accurate.

The reagent materials useful in the immunological testing procedures can conveniently be packaged for commercial purposes, e.g. in a diagnostic reagent kit containing two separate containers, one with the appropriate antiserum and the 10 other containing in aqueous suspension, the serologically determinant material linked to the serologically inert carrier through an amide bond. The concentration of the aqueous subpension of the serologically determinant material linked to the serologically inert carrier through an amide bond can be any convenient concentration. However, from about 1.0% to about 2.5% by weight is preferred.

The following examples illustrate the invention.

Example 1 (a) 577.000 I.U. of human chorionic gonadotropin (HOG) powder are dissolved in 65 ml of sterile saline and heated for 1 hour at 80°C, then cooled to room temperature. 60 ml of the cooled HOG solution are poured rapidly into a reaction flask containing 500 ml of a carboxylated styrene butadiene (Dow No. 816) latex having a pH of 9.5, a specific gravity of 1.050 and a viscosity of 100 ops. using a Brookfield No. 1 at 20 rpm. The concentration of the j/fi^ex is adjusted to 78-82

Ϊ0 mgm/ml. Ab soon as the HOG is dispersed evenly in the latex, 1.8 gms l-cyclohexyl-5-[2-morpholinyl-(4)-ethyl]-carbodiimide metho-p-toluenesulfonate dissolved in 180 ml of water are added. The reactants are mixed for 2 hours at room temperature, then centrifuged at 25,000 x g at 10°C,

The supernatants are poured off and the resulting latex pellets are milled in about 400 ml of water for about 10 minutes and centrifuged for about 1.5 hours at 25,000 x g at 10°C. The supernatant is then discarded and the pellets are resuspended in 400 ml of buffer at pH 8.2 containing 0.1 M

Tris—HC1, 0,85 percent NaCl and 0,1 M ε-amino-n—caproic acid.

The buffered mixture is milled and centrifuged for 1.5 hours at 25,000 x g at 10°C. The supernatant is poured off and the latex pellets are recovered. The pellets have particles of an average size of about 0.20-0.25 μ, contain 0.75 percent of

HOG by weight, and have a specific gravity of 1.01*

The product is suitable for packaging into a diagnostic

C reagent test kit having two containers, one of which contains the buffered HOG - carrier reagent and the other contains the anti-HCG serum reagent. Suitable concentrations and amounts of reagents in the containers in a kit are 2.2 ml of the HOGcarrier reagent at a concentration of 13.5 mgm/ml in a buffer at pH 8,2 composed of Tria-HCl, NaCl and ε-amino—n-caproic acid and 0.01 percent Thimersal. The anti-HCG serum is diluted approximately 1:60 in a buffer solution at pH 7.9-8.2 containing 0.1 M tris, 0.85 percent NaCl, 0.1.M ε-amino-n-caproic acid, 1 percent Bovine albumin and 0.1 percent NaNg.

The anti-HCG serum is derived from rabbits by means well known to the art. Thus, rabbits are immunized with HCG, then the anti-serum is collected, tested for titer, then stored for use.

Example 2 ml of a 6 percent human albumin suspension is diluted in 50 ml of distilled water and stirred for 10 minutes. 8 ml of aarboxylated butadiene styrene copolymer, 50 percent solids (Dow 816) are added followed by the addition of 37 ml of 0.1 M phosphate buffer at pH 5.5 and the mixture is stirred for 10 minutes. 160 ml of 1 percent aqueous l-cyclohexyl-3-[2-morpholinyl-(4)-ethy]J-carbodiimide metho-p-toluenesulfonate is added. The reaction mixture is stirred overnight at room temperature. The product is recovered by centrifugation. It is a white claylike mass composed of particles having a particle size of about 0.2 μ, a specific gravity of 1.01 and contains 10% human albumin by weight.

The product is suspended in saline solution buffered with 0.1 M Tris-HCl to a pH 7.2-7.4 and containing 0.1% carboxymethyl cellulose. This forms a milky white suspension suitable for packaging into diagnostic kits.

The product is packaged into a diagnostic reagent test kit having two containers, one of which contains an aqueous buffered albumin-carrier suspension of 1.8 ggm per ml and the other which contains properly diluted goat ;antihuman albumin serum. Each container contains about 1 to 2 ml of reagent.

The test for human albumin in meconium utilizing the reagents prepared according to Example 2 is carried out as follows:

ml of antihuman albumin serum are delivered into a glass test tube, then 1 ml of meconium solution (1:50 by volume) in saline is added with mixing and incubated at J7°C for 10 min. Two drops of the albumin-carrier suspension are then added with mixing. After one hour at 37°C, the results of the test are read; agglutination indicates less than 16pg/ml albumin present in the meconium and non-agglut!nation indicates albumin at levels of 16pg/ml or higher. This level of !™man albumin in the meconiums is abnormal1y high and further tests can then be run to determine the existence of an abnormal or diseased condition, e.g. cystic fibrosis.

Example 3

Denatured gamma globulin is prepared by suspending Cohn Fraction II in distilled water to a 1% solution. The suspension is left to stand at room temperature overnight then centrifuged 5 at 10,000 rpm. for 30 minutes. The supernatant is poured off and filtered through Whatman No. 1 paper.

ml of a 1:10 water suspension of acrylonitrile latex (Hycar-Latex 1571) is mixed with 20 ml of denatured 1% gamma globulin prepared as above. 10 ml of 1% aqueous l-cyclohexyl-310 [2-morpholinyl-(4)-ethyl]-carbodiimlde metho-p-toluenesulfonate are then added. The reaction mixture is heated in a 56°C water bath for 10 minutes. The resulting suspension is centrifuged at 15,000 rpm. for 1 hour. The supernatant ia poured off and the residue ia resuspended in 50 ml of 0.1 M Glycine-Saline buffer at pH 8.2. The suspension is again centrifuged and the residue, particles of a white clay-like material containing 0.01 to 1¢ gamma globulin and with an average particle size of about 0.09 μ and a specific gravity of 1.01, is recovered.

The gamma globulin-carrier particles are suspended in a 20 0.1 M Glycine-Saline buffer at pH 8.2 and utilizediin a diagnostic immunological test for rheumatoid arthritis as follows:

Lambda of test sera are delivered onto a clean glass slide. One drop of 0.1 M Glycine-Saline buffer at pH 8.2 is added and mixed with a wooden applicator stick. Two drops of 25 the prepared reagent are added and then the reagents are mi again. The slide is tilted back and forth gently for one minute. The results of the test are then observed; an agglutination on the slide is indicative of a serum containing rheumatoid factor, non-agglut!nation denotes the serum is free from rheumatoid factor.