CS261508B1 - Mouse Lymphocyte Lybridoma Producing Monoclonal Antibody for Specific Tumor Antigen of MSB1 - Google Patents

Abstract

The purpose of the solution is to prepare an isotype- and idiotype-homogeneous antibody of high purity. The stated purpose is achieved by using the mouse lymphocyte hybridoma MSB—NTP—1, producing a monoclonal antibody recognizing a specific tumor antigen of the lymphoblastoid cell line, denoted by the abbreviation MSB1. The mouse lymphocyte hybrid has applications in veterinary medicine.

Description

4 281508

BACKGROUND OF THE INVENTION The present invention relates to a mouse lymphocytic hybridoma producing an antibody that recognizes a specific tumor antigen, lymphoblastoid cell, designated MSB1.

Up to now, antibodies to the lymphoblastoid cell line MSB1, which carry the surface of the diagnostic antigen "MAT-SA", have been prepared by applying live MSB cells to the experimental animals (rabbit, sheep). For example, to induce a sufficiently high antibody response, it is necessary to inject multiple times at different dosages and intervals of time.The serum of such immunized animals serves as a source of antibodies, especially in the diagnostic examination of poultry breeding. the mixture of antibodies whose distribution in the producer is varied is very diverse, qualitatively and quantitatively varying from one sampling to the next and is unrepeatable, and in addition, the serum contains mainly desirable antibodies against the surface antigens that occur on normal cells sera can be treated by complex sputum clearance procedures so that the serum contains antibodies only against the specific tumor antigen. This effect leads to large losses in serum and a significant decrease in the specific antibody titer. An understandable process of satiety can be standardized, so that even the high heterogeneity of conventional sera is overwhelmed. Furthermore, the usefulness of the diagnostic reagents thus prepared is also reduced by the fact that their specific polyclonal antibodies are highly heterogeneous in terms of the interaction with surface cell antigens. In addition, preparation of a sufficient amount of lymphoblastoid cells MSB1 for immunization purposes is demanding in terms of workplace equipment, skilled personnel and financial means.

MSB-NTP-1 hybrids were prepared in a manner known per se by cloning a hybridoma group in sputum agar, resulting in mouse 2-amino-hydroxy-8-azapurine (8-azaguanine) resistant NSO-spleen resistant spleen cells. BALB / c mice in the ancillary cell line, immunized with mephoblastoid cell lineage MSB1 (G. Khuhler, C. Milstein, GW Butecher, JCHoward: Nature 286, 550 (1977)].

The above drawbacks are substantially obviated by the invention, which is based on the mouse lymphocyte hybridoma MSB-NTP-1, producing a monoclonal anti-substance subclass IgG1, kappa, recognizing the specific tumor antigen of the lymphoblastoid cell line designated MSB1. These hybrids are stored in the Virological Institute of the Slovak Academy of Sciences, Mlýnská dolina1, 817 03 Bratislava. An advantage of the hybridoma is that it produces an iso-homogeneous antibody, the so-called clonal antibody, which recognizes a specific one; The antigen of the lymphoblastoid cell line designated by the abbreviation MSB1 can be used to identify a specific tumor antigen in the differential diagnosis of tumor diseases. The single cell lymphocyte hybridoma MSB-NTP-1 grows in vivo in the peritoneal cavity of BALB / c mice with concomitant production of aspites. It also grows in vitro in culture cells suitable for animal cells. Also, after freezing and storage in liquid nitrogen and after re-thawing, it retains its ability to produce a monoclonal antibody recognizing a specific tumor antigen of the lymphoblastoid cell line designated MSB1. Unlike conventional anisotropic, non-specific, ballast antibodies, it does not. Example 1

2.107 cells of mouse 2-amino-hydroxy-8-azapurine (8-azaguaninj resistant cell line, which are combined with 1 * 10 < 8 > -soluble lymphoid cells immunized with 1 ml of 50% by weight polyethylene glycol) are used to prepare the hybrid cell line. 3.108 MSB1 lymphoblastoid cells, within 14 days, under the selective pressure of 4-amino pteroyl-glutamic acid (aminopterin), hybrid cell lines that produce monoclonal antibodies recognizing the specific tumor antigen of lymphoblastoid cell line designated MSB1. then cloned by mixing 0.5% w / w agar with 5.10e to 10 x 10 6 mouse spleen cells in RPMI 640, containing 100 mg / ml of calcium nitrate tetrahydrate, 2000 mg / ml. 1 D-glucose, 100 mg / ml of magnesium sulfate heptahydrate, 400 mg / ml of potassium chloride, 1512 mg / ml of dibasic sodium phosphate, 6000 mg / ml of dihydrogen monohydrate sodium phosphate, 100 mg / ml of 2-amino-5-guanidino-valeric acid (arginine), 50 mg. ml-1 2-aminosuccinic acid (asparagine), 20 mg ml 1-2-amino succinic acid (aspartic acid), with 2 S 1 and 3 50 mg.ml "1 3 ', 3'-dithiobis (2- 2-aminoglutaric acid (glutamine), 300 mg / ml of 2-aminoglutaramic acid (glutamine), 1 mg / ml of 2-aminogutaraminyl 2 - mercaptopropionyl-aro-acetic acid (glutathione ie glutamyl-cysteinyl-glycine), 10 mg / ml aminoacetic acid (glycine), 15 mg / ml acid-amino-1-thiazole: 1-4 propionic (histidine), 50 mg / ml of 2-amino-3-methylvaleric acid (isoleucine), 50 mg / ml of 2-amino-4-methylvinyl (leucine), 40 mg / ml. 1-lysine acid, 15 mg / l of 2-amino-4- (methylthio) -butyric acid (methionine), 15 mg / ml 2-amino-3-phenylpropionic acid ( phenylalanine), 20 mg / ml 2-pyrrolidinecarboxylic acid (proline), 30 mg / ml 2-amino-3-hydroxyproic acid 2-amino-3-hydroxy-butyric acid (threonine), 5 mg / ml 2-amino-3-hydroxypropionic acid (serine), 20 mg / ml. Of 2-amino-3-hydroxy-butyric acid (threonine), 5 mg / ml of 2-amino-3- (3-indolyl) propionic acid (tryptophan), 20 mg / ml of 2-amino- 3- (4-hydroxy-phenyl) -propionic (tyrosine), 20 mg. ml of 2-amino-3-methylbutylic acid (valine), 0.2 mg / ml of 5- (2-oxoimidazolidino [4,5-c] thio-2-yl) acid pentanoic (biotin), 0.005 mg.ml “1 cobalt [- (5,6-dimethylbenzimidazol)] -cobalt-cyanocobamide vitaminBl2> 0,250 mg.ml“ 1 DN- (2,4-dihydroxy-3,3-diine) Calcium 8-butylbutyryl-2-amine propionate (calcium pantothenate), 3 mg. ml of "1'-hydroxyethyltrimethylammonium n-chloride (cboline chloride), 1 mg.ml" of N- (2-aniono-4-hydroxy-pteridine-6H-methyl) -piperidine-benzoic acid (folic acid), 35 mg mV1 (1.2.3.5, 4.6 / cyclobexondex.d) (myonosite), 1 mg / ml pyridine-3-carboxylate (.beta.-t-thiamine), 1 mg / ml. of 4-aminobenzoic acid (p-aminobenzoic acid), 1 mg .ml of 1-3-hydroxy-4,5-bis (hydroxymethyl) -2-methylpyridine chloride (pyridoxine chloride), 0.2 mg / ml. 7,8-dimethyl-10- (1'-D-ribityl / iso-aloxazine) (ribo avalanche), 1 mg. rol “1 3- (4-amino-2-methylpyrimidyl-5-methyl) -5- (2-hydroxyethyl) -4-methylthiazole (thiamine chloride), 2000 mg.mll of sodium bicarbonate with 2 2-amino-glutaramic acid (glutamine), 5.10-5 moles of 2-mercaptoethanol, 100 ggml "1 OL-2-deoxy-2- (methylanine) - glucopyranosyl- (1-2) -O-L-deoxy -3-C-furoyllyxof uranosyl- (1-4-) -1,3-deoxy-1,3-diguanidoscyloinozyme sulphate (streptomycin sulphate), 100 units per millimeter of x- (N-phenylacetaamido-penicillic acid ( penicillin G), 1 mmole of N-2-hydroxyethylpiperazine-N'-2-ethane sulfonic acid (Hepesu) is dissolved in three times redistilled water, then taken up three times with redistilled water to a volume of 900 ml of solution, which is filled with 100 ml of normal The resulting pH of the culture medium thus prepared is 7.2, and after the agar has set, 100% by weight of the cells in 0.25% by weight of agar are applied to a layer of 0.5% by weight of agar. grow up colonies that are expand in RPMI 640 medium supplemented with 2 mmol of 2-aminoglutaramic acid (glutamine), 5 µl of 5 moles of 2-mercaptoethanol, 100 µg. ml of "O-L-2-deoxy-2- (methylamino) -glu-copyranosyl- (1-2) -O-L-deoxy-3-C-formyl-lyxofuranosyl- (1-4-1, 3-deoxy-1,4-diguanido-scylnolosine sulfate (streptomycin sulfate), 100

Claims (1)

Dissolve 7 units per milliliter of 6- (n-phenylacetamido-penicillinic acid (penicillin G), 1mmol of N-2-hydroxyethylpiperazine-N '- 2-ethanesulfonic acid (Hepesu) in water redistilled water and make up to 900 times with three times distilled water. ml of a solution which is supplemented with 100 ml of a normal inactivated equine serum The resulting pH of the prepared culture medium is 7.2 In 1 ml of culture medium, 3 to 700,000 hybridomas of MSB-NTP-1 harboring 10 to 100 µg of monoclonal IgG1, kappa, antibody are stored recognizing the specific tumor antigen of the lymphoblastoid cell line designated by the abbreviation MSB1 EXAMPLE 2 The procedure of Example 1 is followed except that untreated conventional fetal fetal serum is used in the culture medium. unable to grow and produce monoclonal antibodies after fusion MSB-NT mouse lymphocyte hybridoma P-1sa is cultured at 37 ° C. The mean generation time is 22 hours, the modal number of chromosomes is 24 months after the fusion of 70chromosomes. The antibody produced is a finer immunoglobulin recognizing a specific tumor antigen of the lymphoblastoid cell line, abbreviated as MSB1, class IgCl_, kappa. The monoclonal antibody produced by the murine lymphocyte hybridoma MSB-NTP-1 can be used in veterinary practice for the treatment and differential diagnosis of tumorous disease in poultry. OBJECT OF A Mice Lymphocytic Hybrids MSB-NTP-1, producing a moncklonal antibody against the SPECIFIED specific antigen of the cell line MSB1, subclass IgG1, kappa. Severografia, n. P. Plant 7, Most Price 2,40 Kcs