GB2027031A

GB2027031A - Diagnostic Reagent Comprising a Proteinaceous Material Bonded to a Latex

Info

Publication number: GB2027031A
Application number: GB7831929A
Authority: GB
Original assignee: F Hoffmann La Roche AG
Current assignee: F Hoffmann La Roche AG
Priority date: 1978-08-02
Filing date: 1978-08-02
Publication date: 1980-02-13
Also published as: JPS5522192A; EP0008682A1; DK325879A; AU4925679A

Description

SPECIFICATION Diagnostic Reagent The present invention relates to a new activated latex polymer and a process for its manufacture. Furthermore, 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 more and more accomplished by the application of immunological principles, utilized to determine the presence of antibodies or antigens in a body fluid of the subject. Within the various known testing procedures, like the agglutinationtest, the radioimmunoassy, the enzymeimmunoassay, the immunofluorescence or the immunodiffusion, the agglutinationtest is considered as being the cheapest and the most simple and quick one. In an agglutinationtest, carriers are utilized in order to make it possible to visually or photometrically discern the formed antigenantibody complexes which have very small sizes. Among the carriers which have been employed are sheep and human erythrocytes, bacterial cells, bentonite, latex particles, e.g. polystyrene, or carboxylated copolymers of styrene and butadiene, anionic phenolic resins and finely divided diazotized amino cellulose. The known carriers are however limited in their applicability and usefulness in immunologica! diagnostic procedures since the corresponding immunological reagents show in many instances a lack of sensitivity and/or a poor stability. This is of importance particularly in case the immunological reagent comprises sensitive antigens or antibodies bound to the carrier. In particular, it has not been possible to develop an agglutinationtest for the determination of myoglobin in the urine of patients with suspected myocardial infarction, because the known immunological carriers are not satisfying for attaching myoglobin under retention of its immunological properties or do not show a sufficient stability. The known methods for determining myoglobin in urine like the radial immunodiffusion are expensive and cumbersome. In particular, they do not allow a quick estimation of myoglobin in urine, which the physician could carry out at the bedside of the patient with suspected myocardial infarction. There is thus a need for a carrier which will form, with a wide spectrum of immunologically active materials, in particular with myoglobin, a diagnostically useful reagent which is stable, specific, sensitive and provides an easily ascertainable visual or photometric evaluation in the minimum of time. The present invention relates to an activated latex manufactured by treating discrete particles of a latex carrier to which a water-soluble proteinaceous material is covalently bound with an activating agent. Furthermore, the present invention relates to a water-insoluble immunological reagent having a specific gravity of about that of water, comprising discrete particles of a latex carrier to which a proteinaceous material is covalently bound having condensed thereto, through a covalent link, an immunologically active material. Furthermore, the present invention relates to a process for the manufacture of such a waterinsoluble immunological reagent, comprising reacting an immunologically active material with an activated latex manufactured by treating discrete particles of a latex carrier, to which a proteinaceous material is covalently bound, with an activating agent. Antigens or antibodies attached to the protein coated latex spheres of the invention, being in a very natural environment display high levels of biological activity and have favourable properties concerning the observed inhibition of latex agglutination by normal human serum. As used within the context of this invention, "latex carrier" or "core polymer" includes latex polymers which are water-insoluble, 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 coating with a proteinaceous material and coupling with the immunologically active material the specific gravity of the particles is about 1.0 (0.95-1.05), enabling them to remain permanently in aqueous suspension. The core polymer must be inert with respect to immunological diagnostic tests and must have active groups which are capable of forming a covalent linkage with a proteinaceous compound. For example, the latex carriers can have carboxyl groups, amine groups or groups convertible into them.Typical suitable groups on the latex carriers are those containing an active hydrogen, e.g. -COOH, -CONH2, a nitrile group, a primary amine group or a.secondary amine group. Typical suitable latex 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. Preferred latex polymers are carboxylated latex polymers like carboxylated styrene butadienes, carboxylated polystyrenes, carboxylated polystyrenes with amino groups, acrylic acid polymers, methacrylic acid polymers, acrylonitrile polymers, acrylonitrile butadiene styrenes, polyvinyl acetate acrylates, polyvinyl pyridines, vinyl chloride-acrylates and the like. Some commercially available latexes which are suitable for use in this invention are Amsco Res 4150, Amsco Res 3011 (American Mineral Spirits Co.); Dow Latex 815, Dow Latex 816, Dow Latex 620, Dow Latex 859, Dow Latex CL 241 (The Dow Chemical Co,); Hycar 1512, Hycar 1877X8, Hycar 2600X120 (Gbodrich Chemical Co.); Gelva 900, Lytron 612, Lytron 624 (Monsanto); Rhoplex LC40 3216, Amberlite ultrafine (Rohm and Haas) Particularly preferred latex polymers are carboxylated styrene butadienes.The term "proteinaceous material" encompasses all aminoacid polymers suitable for covalent attachment of immunologically active materials. Preferred proteinaceous materials are water-soluble proteins, polypeptides or peptides. Examples of such proteinaceous materials are albumine and globulins. Particularly preferred are bovine serum albumine and bovine gamma globulin. The term "immunologically active material" refers to components of physiological fluids, cell and tissue extracts for which an immunological counterreactant is available or can be produced. Typical immunological materials are primary amines, aminoacids, peptides, proteins, lipoproteins, glycoproteins, sterines, steroides, lipoides, nucleic acids, enzymes, hormones, vitamines, polysaccharides and alcaloides. Examples of such immunologically active substances are given in the following table:

Table I

CollagenAmyloid

CatalasePeroxidase

Secretory IgA

Secretory IgM

Sectretory IgASecretory IgM I. Microorganisms bacteria 1. Gram-positive cocci Streptococci (pyogenes, fecalis and viridans) Staphylococci (aureus and albus) Pneumocci (D. pneumoniae) 2. Gram-negative cocci Neisseria (gonorrhoeae and meningitidis) 3. Gram-positive aerobic bacilli Bacillus anthracis Corynebacterium diphtheriae Erysipelothrix Listeria monocytogens 4. Gram-positive anaerobic bacilli Clostridia (botulinum, perfringens, welchii and tetani) 5. Gram-negative anaerobic bacilli Bacteroides 6. Gram-negative intestinal bacilli Escherichia Klebsiella Enterobacter Proteus Pseudomonas Salmonella Shigella 7. Gram-negative nonintestinal bacilli Pasteurella (pestis and tularensis) Hemophilus influenzae Brucella (melitensis, abortus and suis) Bordetella pertussis Malleomyces 8. Spirochetes Treponema pallidum Leptospira Borrelia 9. Mycoplasma 10. Mycobacteria 11. Vibrio 12. Actinomyces Protozoa 1.Intestinal Protozoa Amoebae 2. Flagellates Trichomonas Leishmania Trypanosomes Toxoplasma 3. Spotozoa Plasmodia (vivax, falciparum, malariae and ovale) 4. Intestinal nematodes Pinworms Hookworms Whip worms 5. Tissue nematodes Trichinella Filaria (Wuchereria bancroftii) Dracunculus 6. Trematodes Schistosomes Intestinal flukes Tissue flukes 7. Cestodes Tapeworms 8. Toxoplasma (T. gondii) Fungi 1. Sporotrichum 2. Cryptococcus 3. Blastomyces 4. Histoplasma 5. Coccidioides 6. Candida Viruses and Rickettsia 1. Rickettsia 2. Viruses Canine hepatitis Shope papilloma Influenza A & B Fowl plague Herpes simplex Adenoviruses Polyoma Rous sarcoma Vaccinia Poliovirus Measles Canine distemper Leukemia Mumps Newcastle disease Sendai ECHO Foot and mouth disease Psittacosis Rabies Extromelia Arbor viruses II.Tissue Antigens Including Organ Specific Antigens Polysaccharides Hyaluronidase Tetanus toxin Egg ovalbumin Ovine serum albumin Kidney Liver Skin Heart (Myoglobin) Gastrointestinal tract Prostate Embryonic antigens (alpha I fetoprotein) Tumor antigens (carcinoembryonic antigen) Muscle III Hormones Pituitary hormones Insulin Glucagon Thyroid hormone Chorionic gonatropin Chorionic growth hormone-prolactin Human placental lactogen IV Enzymes Pancreatic chymotrypsinogen Procarboxypeptidase Deoxyribonuclease Ribonuclease Glyceraldehyde -3-phosphate dehydrogenase V. Blood Cell Antigens, Blood Group Substances and Other Isoantigens Platelets Megakaryocytes Leucocytes Erythrocytes Blood group substances Forssman antigen Histocompability antigens VI.Plasma Proteins Fibrin and fibrinogen Plasminogen and plasmin Albumin Immunoglobulins

a-1-antitrypsin Complement factors Ceruloplasmin Gc-globulin Haptoglobin a-2-macroglobulin beta -2-microglobulin Orosomucoid Prealbumin Transferrin VII. Milk Proteins Lactoferrin Lysozyme Secretory component VIII. Saliva Proteins Secretory component IX Urine Proteins X. Pathologic Proteins Myeloma protein Macroglobulinaemic proteins Dysoglobulinaemic proteins Bence Jones I, II proteins C-reactive protein Cryoglobulins XI.Antibodies Including Autoantibodies Antinuclear factor Thyroid autoantibodies Anti-Tamm-Horsfall protein Cold agglutinins Rheumatoid factor Adrenal autoantibodies Autoantibody to gastric parietal cells in pernicious anemia Anti-colon Anti-liver Anti-kidney Autoantibodies to spermatozoa Anti-heart Muscle autoantibodies in myasthenia gravis Autoantibodies to nervous tissue Autoantibodies against fibrous tissue and vascular components Autoantibodies against platelets and megakaryocytes Antibodies against trophoblasts Antibodies to microorganisms Antibodies to animal antigens Antibodies to drugs A particularly preferred immunologically active material is myoglobin, human placental lactogen, antibodies against IgG, or streptolysine 0. The novel activated latex of the present invention can be manufactured by a) reacting an aqueous latex suspension with a proteinaceous material and b) reacting the resulting product with an activating agent. Step a) can be performed in a conventional manner depending on the active functional groups on the latex and the proteinaceous material used. In a preferred embodiment, in which the used latex is a carboxylated polymer, the reaction of step a) which involves the formation of an amide bond, is performed in the presence of a coupling agent like a water-soluble carbodiimide, the Woodward reagent K (N-ethyl-5-phenylisoxazolium-3'-sulfonate) or a water-soluble chloroformiate.

R-N=C=N-R' I Particularly preferred coupling agents are water-soluble carbodiimides represented by the formula wherein R or R' are: 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, nbutyl, sec.-butyl, isobutyl, tert.butyl, amyl, hexyl, heptyl, octyl, nonyl, undecyl and dodecyl; monoaryl-substituted lower alkyl radicals, e.g., benzyl-alpha - and/5-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., alpha . beta , and y methyl or ethyl pyridyl; acid addition salts; and quaternary amines thereof. According to a preferred embodiment of this invention, in step a) the proteinaceous material and an aqueous suspension of the carboxylated core latex are reacted preferably at room temperature (about 20[deg]C to about 25[deg]C). Temperatures from about 0[deg]C to about 40[deg]C, however, are suitable for the reaction. In order to insure a chemical coupling of the proteinaceous material to the core latex, a sufficient amount of the coupling agent is used to insure that sufficient amount of amide bonds is formed. Generally, about 0.005 percent to about 6.0 percent by weight of a water-soluble carbodiimide, based on the weight of the particles, is suitable, usually, however, about 0.05 to 2.0 percent by weight is used. The pH of the reaction can very from 2 to 7 and is preferably from 4 to 5. The order of adding the proteinaceous material, the carboxylated core latex and the coupling agent is not critical. However it is preferred to react in a first step the core latex polymer with the water-soluble coupling agent and to add in a second step the proteinaceous material. The ratio of core latex to proteinaceous material in the resulting product can vary within broad limits and is preferably 2 to 150., a ratio of 10 to 50 being particularly preferred. In step b) of the process of the present invention the latex of step a) comprising discrete particles of a latex carrier to which a proteinaceous material is covalently bound is activated by means of an activating agent. Activating agents suitable for the purpose of the invention are, in general, polyfunctional compounds, having two or more of the reactive groups: azo, sulfonic acid, fluoro groups activated by nitro groups, azide, imine, and reactive chloro groups such as chloro groups attached to a ring having appropriate resonance structures. These reactive groups are capable of reacting with the primary amino, sulfhydryl (mercapto), carboxylic and hydroxyl groups in the proteinaceous materials constituting the surfaces of the carrier particles and the immunologically active materials. A representative list of such activating agents is: bis-diazobenzidine, bis-diazobenzidine disulfonic acid, tetraazo-p-phenylenediamine, difluorodinitrobenzene, di-fluorodinitrodiphenyl sulfone, a carbodiimide of formula I, toluylene diisocyanate, cyanuric chloride, dichloro-striazine, N-t-butyl-5-methylisoxazolium perchlorate, a dialdehyde, an alpha, betasaturated aldehyde, and mixtures thereof. particularly preferred activating agents are bis diazo-benzidine disulfonic acid and toluylene diisocyanate The conditions of activation depend on the specific activating agent employed and can vary within broad limits. However the pH is preferably from 3 to 9 and the temperature lies between 0.C and 50[deg]C. The activating agent is preferably added in big excess, this excess being removed after reaction, for example by washing in the cold. After excess activating agent has been removed by washing, the such activated latex is reacted with the immunologically active material. Although the activated latex can be stored it is preferred to react it with the immunologically active material immediately after activation. The immunologically active material is preferably added in excess under conditions which do not affect significantly the biological activity of the immunologically active material. The pH is preferably from 4 to 9 and the temperature between 0[deg]C and 50[deg]C. The final product is preferably washed several times in buffer and the excess immunologically active material recovered. The amount of immunologically active material linked to the novel activated latex is usually from about 0.01% to 15.0% by weight. However, each particular immunologically active material is utilized in an amount in which it is most successfully employed in a diagnostic test. Therefore each material is combined with the activated carrier in a ratio suitable for its specific requirements. This invention therefore comprehends within its scope the use of an amount of immunologically active material in combination with an activated latex sufficient to provide a diagnostically effective reagent. Once the 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 0.5% to about 2.5% by weight are suitable with 1.2% by weight preferred. Thus, for example, the product formed when human myoglobin is coupled to the novel activated latex, can be used as a diagnostic reagent to determine myoglobinuria in a patient with suspected myocardial infarction. This can be accomplished, for example, by placing a drop of test urine on a clean glass slide, mixing it with a drop of appropriately diluted anti-human myoglobin serum, then adding a drop of the myoglobin-latex in aqueous suspension. within a few 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. As another example, the product formed when sheep anti human IgG is coupled to the novel activated latex can be used as a diagnostic reagent to determine gamma globulin in serum or other body fluids. As a further example, the product formed when human placental lactogen is coupled to the novel activated latex can be used as a diagnostic reagent to determine placental lactogen in human serum or other body fluids. The immunological reagent of the present invention can be used for the determination of immunologically active materials in a direct or in an indirect (inhibition) agglutinationtest or in the kenetic photometric method disclosed in the German Patent Application no 2749956. In the direct test the sample and the latex particles coated with the counterreagent for the immunologically active material to be determined are mixed and that agglutination observed visually or photometrically. The test is positive in case an agglutination takes place. In the indirect test the sample is mixed with the counterreagent (e.g. antiserum) for the material to be determined and then with latex particles coated with the same material. The agglutination is observed visually or photometrically. The test is positive in case no agglutination occurs. The immunological reagents of the present invention can conveniently be packaged for commercial purpose e.g. in a diagnostic reagent kit. In case of a direct test the reagent kit for the determination of a known immunologically active material contains in a container a water-insoluble immunological reagent having a specific gravity of about that of water comprising discrete particles of a latex carrier to which a proteinaceous material is covalently bound having condensed thereto through a covalent link an immunological counterreagent for the immunologically active material to be determined. In case of an indirect test the reagent kit for the determination of a known immunologically active material contains in a first container a waterinsoluble immunological reagent having a specific gravity of about that of water comprising discrete particles of a latex carrier to which a proteinaceous material is covalently bound having condensed thereto through a covalent link the immunologically active material to be determined; and in a second container an immunological counterreagent for the immunologically active material to be determined. The following examples illustrate the invention.

Example 1 Preparation of Bovine Serum Albumine (BSA) Coated Latex Carboxylated styrene butadiene latex (Dow CL 241 ) was washed and feed from ammonia through treatment with the ionexchange resin Dowex AG 50-X8; Bio Rad (styrene divinyl benzene polymer latice carrying nuclear sulfonic acid groups) in the sodium form. 18 ml of this latex CL 241 in the Na±form (solid content 78 mg/ml) was adjuted to pH 4.75. With rapid stirring a solution of 30 mg CMC (1cyclohexyl-3-[2-morpholinyl-(4)-ethyl] carbodiimide-metho-p-toluenesulfonate) in 6 ml of water was added to the suspension. After 3 minutes 6 ml of a 1 % BSA solution was added. After the addition of the protein the pH of the suspension was 5.7. The mixture was kept stirring at room temperature for 24 hours. It was then centrifuged for 40 minutes at 44'000 xg. The supernatant was discarded and the pellet suspended in about 40 ml of water, homogenized with a mixer and recentrifuged. Washing was repeated four times with 0.1 M borate buffer pH 8.5 and then twice with distilled water. The latex was finally taken up in 60 ml of water and stabilized by the addition of 120 Microl of 20% sodium azide solution.

Example 2 Preparation of Human Immunoglobulin G (IgG) Coated Latex 6 ml ionexchanged (treatment see example 1) latex CL 241 (79.5 mg/ml) was adjusted to pH 4.75 and activated by the addition of 2 ml of a CMC-solution containing 8 mg CMC. After three minutes the latex suspension was poured into 8 ml of a rapidly stirring solution of human IgG in borate buffer. 120 mg commercial IgG had been suspended in 12 ml 0.015 M borate buffer pH 8.5, stirred for an hour and the insoluble material removed by centrifugation. The optical density of the solution at 280 nm was 6.44 corresponding to a protein concentration of approximately 5.1 mg/ni. The reaction mixture was stirred at room temperature during 24 hours, it was then centrifuged 30 minutes at 45'000 xg. From the optical density reading of the supernatant at 280 nm it could be seen that virtually all the IgG was bound to the latex.In order to remove unsedimented latex spheres the supernatant was filtered through a Nucleopore membrane of 0.1 Micro effective pore size before the optical density was measured. The latex was washed four times with about 40 ml of 0.1 M borate buffer pH 8.5, then twice with distilled water. It was finally taken up in 20 ml of water and stabilized by the addition of 40,u1 of 20% sodium azide solution.

Example 3 Preparation of Bis-diazobenzidine-2,2'disulfonic Acid (BDBDS) 0.11 g (1.0n Mol) anhydrous sodium carbonate was dissolved in 3 ml distilled water. To this solution 0.34 g (1.0 nMol) benzidine-2,2'disulfonic acid (BDS) was added. After a clear solution was obtained 0.142 g (2.05 nMol) sodium nitrite was also added. The reaction vessel was immersed in an icebath and 0.6 ml 30% hydrochloric acid (4.9 nMol) was added to the rapidly stirred solution. After 5 minutes 14.6 mg (0.15 nMol) sulfamic acid was added and the suspension kept stirring in the icebath. Minutes later crystalline BDBDS precipitated from the yellow solution. Upon storage of the suspension at 0[deg]C the BDBDS precipitate transformed into beautiful fine needles. For the activation of the protein coated latexes the BDBDS suspension obtained as described above was first centrifuged and either this supernatant or a suspension of the solid BDBDS in 0.01 N hydrochloric acid was used.

Example 4 Activation of Bovine Serum Albumin (BSA) Coated Latex With Bis-diazobenzidine-2,2'disulfonic Acid (BDBDS) a) To 5 ml BSA coated latex 2 ml BDBDS supernatant of example 3 was added at 0[deg]C. After combining, the pH of the suspension was raised from 2.0 to 8.5 through the addition of 1 ml 1 M sodium borate buffer of pH 8.1 and drops of 1 N sodium hydroxide. The mixture was stirred for about 10 minutes and then centrifuged 30 minutes at 45'000 xg and 2[deg]C. The supernatant was decanted and saved for the colour determination of BDBDS. The yellowish pellet was suspended in about 12 ml icecold 0.1 M borate buffer pH 8.5 and the latex suspension recentrifuged for 30 minutes.The pellet was suspended in 0.5 ml of icecold borate buffer to be combined with the ligand protein solution. b) To a rapidly stirred suspension of 5 ml BSA coated latex 0.5 ml BDBDS suspension in 0.01 N hydrochlorid acid of example 3 was added at 0[deg]C. (After centrifugation of the BDBDS precipitation mixture the crystalline BDBDS had been suspended in about 15 ml of 0.01 N hydrochloric acid). The pH of the reaction mixture was raised to 8.5 through the addition of 1 ml 1 M borate buffer pH 8.1. After 15 minutes the reaction mixture was diluted to about 12 ml with icecold distilled water. It was centrifuged for 30 minutes at 45'000 xg and 2[deg]C. The supernatant was decanted and saved for the colour determination of BDBDS with a-naphthol.The activated latex was washed with about 12 ml icecold 0.1 M borate buffer pH 8.5, recentrifuged and suspended in 0.5 ml cold borate buffer, to be combined with the ligand protein solution.

Example 5 Activation of Immunoglobulin G (IgG) Coated Latex With Bis-diazobenzidine-2,2'-disulfonic Acid (BDBDS) To a stirred suspension of 5 ml human IgG coated latex 1.5 ml BDBDS supernatant was added at 0[deg]C. After combining, the pH of the suspension was raised to 8.5 by addition of 1 ml 1 M sodium borate buffer of pH 8.1 and drops of 1 N sodium hydroxide. The mixture, which developed some gasbubbles (obviously from liberated nitrogen) was stirred for about 10 to 15 minutes and then centrifuged for 30 minutes at 45'000 xg and 2[deg]C. The supernatant was decanted and the pellet washed with about 12 ml icecold 0.1 M borate buffer pH 8.5, suspending it with a glassrod. The latex was centrifuged at 2[deg]C for 25 minutes, the washing buffer decanted and the pellet suspended in 0.2 ml icecold borate buffer.

Example 6 Activation of BSA Coated Latex With Toluylene-2,4-diisocyanate (TDIC) To a stirred suspension of 5 ml BSA coated latex 0.5 ml 1 M sodium borate buffer pH 8.1 and 1 ml dioxane was added, followed by 0.2 ml of a solution of 20,u1 TDIC in 4 ml dioxane. The pH of the reaction mixture was 9.25. After about 45 minutes the latex was diluted with water to about 12 ml and centrifuged for 30 minutes at 45'000 xg. The pellet was resuspended in 0.1 M borate buffer pH 8.5 and the latex washed in about 12 ml of this buffer. After centrifugation the pellet was directly suspended in the ligand protein solution using a glassrod.

Example 7 Activation of IgG Coated Latex With Toluylene2,4-diisocyanate (TDIC) The procedure of example 6 was repeated with IgG in place of BSA.

Example 8 Coupling of Human Placental Lactogen (HPL) to Bovine Serum Albumin (BSA) Coated Latex Activated With Bis-diazobenzidine-2,2'disulfonic Acid (BDBDS) The activated, washed residue from 5 ml BSA coated latex as described above in example 4 a) was suspended with a glassrod in 1 ml of an icecold 1 % HPL solution in 0.1 M sodium borate buffer pH 8.5. The reaction was allowed to proceed in the dark at 4[deg]C for 48 hours, while the sample was rapidly stirred on a magnetic stirrer. At the end of this time the latex was centrifuged (30 minutes, 45'000 xg, 2[deg]C), the supernatant taken off with a pipet and saved for reutilization of excess HPL and the pellet suspended in about 12 ml 0.1 M glycine buffer pH 8.2. It was recentrifuged and washed three more times with the same amount of that buffer. Finally it was taken up in 5 ml of glycine buffer stabilized with 0.04% sodium azide. 20 It[ of this latex, mixed with 60 Microl PBS and 20 Microl rabbit anti HPL antiserum (1:30 in PBS) showed visible agglutination after 25 seconds. If 0.5,ug HPL was premixed with the antiserum no agglutination could be detected for 4 1/2 minutes.

Example 9 Coupling of Human Placental Lactogen (HPL) to Human Immunoglobin (IgG) Coated Latex Activated With Bis-diazobenzidine-2,2'disulfonic Acid (BDBDS) The activated, washed residue from 5 ml human IgG coated latex as described above in example 5 was suspended with a glassrod in 0.2 ml icecold 0.1 M borate buffer pH 8.5. 1 ml of a 1 % HPL solution in water was added and the coupling reaction allowed to proceed in the dark at 4[deg]C for 72 hours, the sample being stirred with a magnetic stirrer. The latex was then centrifuged, the supernatant pipetted off and saved for reutilization of excess HPL and the pellet suspended in about 12 ml glycine buffer. It was recentrifuged and suspended three more times in glycine buffer, to be finally taken up in 5 ml of this buffer. 20,ul of this latex, mixed with 60 Microl PBS and 20 Microl rabbit anti HPL antiserum (1:80 in PBS) showed visible agglutination after 15 seconds. If 0.5 Microg HPL was premixed with the antiserum no agglutination could be detected for 5 minutes.

Example 10 Coupling of Human Myoglobin to Bovine Serum Albumine (BSA) Coated Latex Activated With Bis-diazobenzidine-2,2'-disulfonic Acid (BDBDS) 5 ml BSA coated latex was activated with 1 ml BDBDS suspension in 0.01 N hydrochlorid acid and the latex treated as described in example 4 b). The pellet of the washed, activated latex was suspended with a glassrod in 1 ml 0.1 M borate buffer pH 8.5 containing 20 mg human myoglobin. The reaction was allowed to proceed in the dark at 4[deg]C for 60 hours, while the suspension was stirred on a magnetic stirrer. The latex was then centrifuged, the supernatant pipetted off for reutilization of excess myoglobin and the' pellet suspended and washed in about 12 ml of glycine buffer. Washing of the latex was repeated three more times in the same buffer.It was finally taken up in 5 ml of the same glycine buffer stabilized with 0.04% sodium azide. 20 Microl of this latex, mixed with 40 Microl veronal acetate buffer pH 8.2 (48 mM in each, sodium acetate and sodium barbital, pH adjusted with glacial acetic acid), 20 Microl normal human urine and 20 Microl rabbit anti human myoglobin antiserum (diluted 1:20 in veronal acetate) showed visible agglutination in 40 seconds. With urine containing 5 Microg/ml myoglobin no agglutination could be detected for 9 minutes.

Example 11 Coupling of Sheep Anti Human Immunoglobulin G (IgG) Antibodies to Bovine Serum Albumine (BSA) Coated Latex Activated With Bis-diazobenzidine-2,2'-disulfonic Acid (BDBDS) 5 ml BSA coated latex was activated with 0.5 ml BDBDS suspension in 0.01 N hydrochloric acid and the latex treated as described above in example 4 b). The pellet of the washed, activated latex was suspended with a glassrod in 0.5 ml icecold 0.1 M borate buffer pH 8.5 and 1.0 ml antibody solution in 0.1 M sodiumbicarbonate pH 8.5 was added. The antibody solution had been prepared by affinity chromatography of serum on human IgG sepharose. The reaction was allowed to proceed in the dark at 4[deg]C for 72 hours, the sample being stirred magnetically. The latex was then centrifuged and the supernatant with excess antibody saved for reutilization.From the optical density in the supernatant it was calculated that about 2.1 mg of antibody was bound. The latex was washed four times with glycine buffer and finally taken up in 5 ml of that buffer stabilized with 0.04% sodium azide. 20 Microl of this latex mixed with 80 Microl veronal buffered saline (145 mM sodium chloride, 3.13 mM barbital, 1.82 mM sodium barbital) containing 0.2 Microg human IgG gave specific agglutination visible in 25 seconds.

Example 12

(BDBDS) Coupling of Streptolysine 0 to Bovine Serum Albumine (BSA) Coated Latex Activated With

5 ml BSA coated latex was activated with 2 ml BDBDS supernatant as described before in example 4 a). The activated washed residue was suspended with a glassrod in 1 ml of an icecold streptolysine 0 preparation in phosphate buffered saline (PBS) pH 8.5 showing a titer of 1160 in the hemolytic assay (at 50% lysis of cells). After a reaction time of 48 hours the latex was centrifuged and the supernatant tested in the hemolytic assay. It showed a titer of 6 now, indicating that most of the streptolysine had reacted and was bound to the latex. The latter was resuspended and washed four times with 0.1 M glycine buffer pH 8.2, to be finally taken up in 5 ml of this buffer stabilized with 0.04% sodium azide. 25 Microl of this latex, mixed with 50 Microl PBS and 25 Microl rabbit anti streptolysine antiserum (already diluted from Merieux) showed visible agglutination after 35 seconds.

Example 13 Coupling of Human Placental Lactogen (HPL) to Bovine Serum Albumine (BSA) Coated Latex Activated With Toluylene-2,4-diisocyanate (TDIC) The activated, washed residue from 5 ml BSA coated latex as described in example 6 was suspended with a glassrod in 1 ml of a 1 % HPL solution in water. The reaction of the activated latex with HPL was allowed to proceed at 4[deg]C for 96 hours, while the sample was stirred.with a magnetic stirrer. The latex was then centrifuged and excess HPL recovered from the supernatant. It was resuspended and washed four times in glycine buffer (about 12 ml each) to be finally taken up in 5 ml of this buffer stabilized with 0.04% sodium azide. 20 Microl of this latex, mixed with 60 Microl PBS and 20 Microl rabbit anti HPL antiserum (1:40 in PBS) showed visible agglutination in 10 seconds. If 1 Microg HPL was premixed with the antiserum agglutination occured only after 1 1/2 minutes leading to a very fine precipitate after 5 minutes.

Example 14 Coupling of Human Placental Lactogen (HPL) to Human Immunoglobuline G (IgG) Coated Latex Activated With Toluylene-2,4diisocyanate (TDIC) The activated, washed residue from 2.5 ml human IgG coated latex as described in example 7 was suspended with a glassrod in 0.5 ml of a 1 % HPL solution in water. It was treated the same way as described above for the TDIC activated BSA latex, being finally taken up in 2.5 ml of glycine buffer stabilized with 0.04% sodium azide. 20Microl of this latex, mixed with 60,u1 PBS and 20 Microl rabbit HPL antiserum (1:40 in PBS) showed visible agglutination in 15 seconds. If 0.5 Microg HPL was premixed with the antiserum no visible signs of agglutination could be detected for 6 minutes.

Claims

1. Activated latex manufactured by treating discrete particles of a latex carrier to which a proteinaceous material is covalently bound with an activating agent.

2. Latex according to claim 1, characterized in that the particles of latex carrier have a size in the range from about 0.01 microns to about 0.9 microns.

3. Latex according to claim 1 or claim 2, characterized in that the specific gravity of the particles of latex carrier is about 1.0.

4. Latex according to any one of claims 1 to 3, characterized in that the latex carrier is a carboxylated latex.

5. Latex according to claim 4, characterized in that the carboxylated latex is a carboxylated styrene butadiene copolymer.

6. Latex according to any one of claims 1 to 5, characterized in that the proteinaceous material is bovine serum albumin.

7. Latex according to any one of claims 1 to 5, characterized in that the proteinaceous material is immunoglobulin G.

8. Latex according to any one of claims 1 to 7, characterized in that the ratio of carrier latex to the proteinaceous material is between 2 and 50.

9. Latex according to claim 8, characterized in that the ratio of carrier latex to the proteinaceous material is between 10 and 30.

10. A water-insoluble immunological reagent having a specific gravity of about that of water comprising discrete particles of a latex carrier to which a proteinaceous material is. covalently bound having condensed thereto through a covalent link an immunologically active material.

11. Reagent according to claim 10, characterized in that the particles of the latex carrier have a size in the range from about 0.01 microns to about 0.9 microns. <

12. Reagent according to claim 10 or claim 11, characterized in that the latex carrier is a carboxylated latex.

13. Reagent according to claim 12, characterized in that the carboxylated latex is a carboxylated styrene butadiene copolymer.

14. Reagent according to any one of claims 10 to 13, characterized in that the proteinaceous material is bovine serum albumine.

15. Reagent according to any one of claims 10 to 13, characterized in that the proteinaceous material is IgG.

16. Reagent according to any one of claims 10 to 15, characterized in that the ratio of carrier latex to the proteinaceous material is between 2 and 50.

17. Reagent according to claim 16, characterized in that the ratio of carrier latex to the proteinaceous material is between 10 and 30.

18. Reagent according to any one of claims 12 to 17, characterized in that the immunologically active material is myoglobin.

19. Reagent according to any one of claims 12 to 17, characterized in that the immunologically active material is human placental lactogen.

20. Reagent according to any one of claims 12 to 17, characterized in that the immunologically active material is an antibody against IgG.

21. Reagent according to any one of claims 12 to 17, characterized in that the immunologically active material is streptolysine 0.

22. Process for the manufacture of an activated latex according to any one of claims 1 to 9 comprising treating discrete particles of a latex carrier to which a proteinaceous material is covalently bound with an activating agent.

23. Process according to claim 22, characterized in that the activating agent is bisdiazotized bensidine-2,2'-disulfonic acid.

24. Process according to claim 23, characterized in that the activating agent is toluylene-2,4-diisocyanate.

25. Process for the manufacture of a reagent according to any one of claims 10 to 21 comprising reacting an immunologically active material with an activated latex manufactured by treating discrete particles of a latex carrier to which a proteinaceous material is covalently bound with an activating agent.

26. Method for the determination of an immunologically active material in a sample, wherein the sample is contacted with a waterinsoluble immunological reagent having a specific gravity of about that of water comprising discrete particles of a latex carrier to which a proteinaceous material is covalently bound having condensed thereto through a covalent link an immunological counter-reagent for the immunologically active material to be determined, and observing whether an agglutination takes place.

27. Method according to claim 26, wherein the immunologically active material to be determined is gammaglobulin and the immunological counterreagent is sheep anti-humang IgG.

28. Method for the determination of an immunologically active material in a sample, wherein the sample is contacted with an immunological counterreagent for the immunologically active material to be determined and with a water-insoluble immunological reagent having a specific gravity of about that of water comprising discrete particles of a latex carrier to which a proteinaceous material is covalently bound having condensed thereto through a covalent link the immunologically active material to be determined; and observing whether an agglutination takes place.

29. Method according to claim 20, wherein the immunologically active material to be determined is myoglobin and the immunological counterreagent is myoglobin antiserum.

30. Reagent kit for the determination of a known immunologically active material containing in a container a water-insoluble immunological reagent having a specific gravity of about that of water comprising discrete particles of a latex carrier to which a proteinaceous material is covalently bound having condensed thereto through a covalent link an immunological counterreagent for the immunologically active material to be determined.

31. Reagent kit according to claim 30, wherein the immunologically active material to be determined is gammaglobulin and the immunological counterreagent is sheep antihuman IgG.

32. Reagent kit for the determination of a known immunologically active material containing in a first container a water-insoluble immunological reagent having a specific gravity of about that of water comprising discrete particles of a latex carrier to which a proteinaceous material is covalently bound having condensed thereto through a covalent link the immunologically active material to be determined; and in a second container an immunological counterreagent for the immumologically active material to be determined.

33. Reagent kit according to claim 32, wherein the immunologically active material to be determined is myoglobin and the immunological counterreagent is myoglobin antiserum.

34. Use of an activated latex according to any one of claims 1 to 9 as a carrier in immunological determinations.

35. Use of a reagent according to any one of claims 10 to 20 in immunological determinations.