CS259233B1 - Mouse Lymphocytes Hybridoma Producing Monoclonal Antibody Against MSBI Eymphoblastoid Cell Line - Google Patents

Abstract

The aim 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 surface antigens of the lymphoblastoid cell line denoted by the abbreviation MSBI. The mouse lymphocyte hybridoma has application in veterinary medicine.

Description

259233

The present invention relates to a mouse lymphocyte hybridoma producing a monoclonal antibody-recognizing surface antigens of the lymphoblastoid cell line designated MSB1.

So far, antibodies to the lymphoblastoid cell line MSB1, which carries on its surface the diagnostic antigen "MATSA", have been prepared by applying live MSB1 cells to experimental animals / rabbit, sheep /. Since a single dose of cells is not sufficient to induce a sufficiently high antibody response, they need to be injected multiple times at different doses and time intervals. The serum of such immunized animals serves as a source of antibodies in particular in the diagnostic examination of poultry farms. The mentioned immunization process has several disadvantages. A heterogeneous mixture of antibodies is obtained, the distribution of which in the producer's organism changes, is very diverse, qualitatively and quantitatively varies from one to the other and is unrepeatable. In addition, serum contains unwanted antibodies by the counter-antigen that occurs! on normal cells. As a result, sera need to be treated by complex normal spleen cell saturation procedures so that the serum contains antibodies only against the surface antigens of the lymphoblastoid cell line designated by the abbreviation MSB1.

This modification results in large serum losses and a significant decrease in the specific antibody titer. An understandable saturation process cannot be standardized, as a result of which the high heterogeneity of conventional sera becomes even greater. Furthermore, the usefulness of the thus prepared diagnostic serum is also reduced by the fact that its specific polyclonal antibodies are very diverse in terms of their interaction with surface cell antigens. In addition, the preparation of a sufficient number of lymphoblastoid MSB1 cells for immunization purposes is demanding in terms of workplace equipment, skilled personnel, and funding.

MSB-NTP-2 hybrids were prepared in a known manner by cloning a hybridoma group in soft agar resulting from the fusion of murine 2-amino-hydroxy-8-azapurine / 8-azaguanine / resistant NSO-labeled spleen cells and mouseBALB / c spleen cells immunized lymphoblastoid cell line MSB1 / G. Kohler, C. Milstein, G. W. Butcher, J. C. Howard: Nature 286, 550 (1977).

The above-mentioned disadvantages are substantially eliminated by the invention, which is based on the mySom lymphocyte hybridoma MSB-NTP-2 producing the monoclonal antibody subclass IgG1, kappa, recognizing the surface antigens of the lymphoblastoid cell line designated MSB1. This hybridoma is stored in the Virological Institute of the Slovak Academy of Sciences, Mlýnská dolina 1, 817 03 Bratislava. The advantage of the hybridoma is that it produces an isotype-homogeneous antibody, the so-called monoclonal antibody, which recognizes the surface antigens of the lymphoblastoid cell line designated MSB1 and thus can be used in diagnostic assays seeking antibodies against Marek's disease associated tumor antigens. Single cell lymphocyte hybrid MSB-NTP-2 grows in vivo in the peritoneal cavity of BALB / c mice while forming ascites.

It also grows in vitro in culture media suitable for animal cells. Also, after freezing and storage in liquid nitrogen and thawing, it retains its ability to produce a monoclonal antibody recognizing the surface antigens of the lymphoblastoid cell line indicated by the abbreviation MSB1. Unlike conventional antisera, it does not contain unsuitable, non-specific, ballast antibodies. Example 1

Is 2.10 used to prepare the hybrid cell line? cells of mouse 2-amino-6-hydroxy-8-azapurine / 8-azaguanine / resistant myeloma cell line, which was made up with 1 ml of 50% wt. polyethylene glycol combines with 1.10 splenic lymphoid cells immunized

O 3.10 MSBl lymphoblastoid cells. Within 14 days, 4-amino-pteroylglutamic acid / aminopterin / hybrid cell lines that produce monoclonal antibodies recognizing the surface antigens of the lymphoblastoid cell line designated by the MSB1 abbreviation are grown under selective pressure. 3 259233

These cells are then cloned by mixing 0.5 wt. agar with 5 * 10 < 6 > 10 < 10 > mouse spleen cells in RPMI140 medium containing 100 mg / ml calcium nitrate tetrahydrate, 2000 mg / ml D-glucose, 100 mg / ml magnesium sulfate heptahydrate, 400 mg / ml potassium chloride, 1212 mg / ml of sodium phosphate dibasic, 6,000 mg / ml of sodium dihydrogen phosphate monohydrate, 100 mg / ml of 2-amino-5-guanidinovalene / arginine / 50 mg / ml 2-amino-succinic acid (asparagine), 20 mg / ml of 2-amino succinic acid / aspartic acid /, 50 mg / ml of 3 *, 3'-dithiobis / 2-aminopropanoic acid / cystine / 20 mg 2-aminoglutaric acid / glutamic acid 1, 300 mg.m-2-aminoglutaramic acid / glutamine / 1 mg.ml-1 2-aminoglutaraminyl-2-mercaptopropionyl aminoacetic acid / glutathione. -cysteinyl-glycine / 10 mg / ml of amino-acetic acid / glycine / 15 mg / ml of α-amino-1-imidazole-propionic acid / histidine / 50 mg / ml of acid 2-amino-3-methylvaleric (isoleucine), 50 mg.ml-1 of 2-amino-4-methylvaleric acid (leucine), 40 mg / ml of 2,6-diaminohexanoic acid / lysine / 15 mg.ml-1 2-amino-4- (methylthio) butyric acid / methionine acid, 15 mg of 2-amino-3-phenylpropionic acid / phenylalanine / 20 mg / ml of 2-pyrrolidinecarboxylic acid / proline / 30 mg 2-amino-3-hydroxypropionic acid / serine / 20 mg / ml 2-amino-3-hydroxybutyric acid / threonine / 5 mg / ml 2-amino-3- / 3-indolyl / propionic acid tryptophan / 2-amino-3- (4-hydroxyphenyl) -propionic acid (20 mg / ml) / 2-amino-3-methylbutyric acid 20 mg / ml / l (va-lino), 0.2 mg / ml of 5- [2-oxoimidazolidino] 4,5-c-thiol-2-yl / pentanoic / biotin /, 0.005 mg / ml cobalt / W- / 5 , 6-dimethylbenzimidazolyl-cobalt (5-cyanocobamide of vitamin B, - /, -1 0.250 mg.ml of calcium calcium / 2,4-dihydroxy-3,3-dimethylbutyryl) - calcium / calcium pantothenate / 3 mg.ml 2-hydroxyethyltrimethylammonium chloride N, N-2-amino-4-hydroxypteridin-6-ylmethyl) -p-aminobenzoic acid (folic acid), 1 mg / ml of folic acid (35 mg / ml). 3.5 (4.6) cyclohexanedexole (myoinositol), 1 mg (1-pyridine-3-carboxyamide / nicotinamide), 1 mg of 4-aminobenzoic acid (p-aminobenzoic acid), 1 mg. ml-3-hydroxy-4,5-bis (hydroxymethyl) -2-methylpyridine chloride (pyridoxine chloride), 0.2 mg / ml of 7,8-dimethyl-10 ~ / 1 '-D-trimethyl / isoaloxazine (riboflavin), 1 mg / ml of 3- (4-amino-2-methylpyrimidyl-5-methyl) -5- (2-hydroxyethyl) -4-methylthiazole / thiamine chloride (2000 mg / ml) sodium bicarbonate-5 supplemented with 2 mmol of 2-aminoglutaramic acid / glutamine / 5.10 mol of 2-mercaptoethanol, 100 µg / ml of O-KL-2-deoxy-2- (methylamino) glucopyranosyl (1-2) -O-KL-deoxy-3-C-formyllyxo-furanose-1- (1-4) -1,3-deoxy-1,3-diguanidoscyloinositic sulfate / streptomycin sulphate /, 100 units per million of 6- (N-phenylacetoamido) -penicilic acid / penicillin G (1 mmol) of N-2-hydroxy acid Dissolve ethylpiperazine-N "-2-ethane sulfonic acid / Hepesu in three times redistilled water and make up to 900 ml of solution three times with redistilled water, supplemented with 100 ml of normal inactivated horse serum. The resulting pH of the culture medium thus prepared is 7.2. Upon solidification of the agar, a layer of 0.5 wt. of 100 hybrid cells in 0.25 wt. agare. After 10 days, colonies are grown and grown in RPMI 640 medium supplemented with 2 mmol of 2-amine-glutaric acid / glutamine (5 * 10 < -1 > mol of 2-mercaptoethanol, 100 [mu] m < -1 >-KL-2-deoxy-2). - (1- (1-I) -O- (XL-deoxy-3-forms of llyxofuranosyl- [1-4] -1,3-deoxy-1,4-diguanidoscinosine sulphate / streptomycin sulphate) methylamino-glucopyranose; Dissolve 100 units per milliliter of 6- (N-phenylacetylammonium) -penicilic acid / penicillin G (1 mmol) of N-2-hydroxyethylpiperazine-N "-2-ethanesulfonic acid / Hepesa in three redistilled water and make up to 3 times with redistilled water. per volume of 900 ml of solution, which is supplemented with 100 ml of normal inactivated equine, The resulting pH of the culture medium thus prepared is 7.2 In 1 ml of culture medium there are 350-700 thousand hybridomas of MSB-NTP-2 producing 10-100 µg of monoclonal IgG1, kappa , an antibody recognizing the surface antigens of the lymphoblastoid cell line designated by the abbreviation MSB 1. Example 2

The procedure is as in Example 1 except that untreated conventional calf fetal serum is used in the culture medium. The fused cells transferred to the culture medium thus prepared are unable to grow and produce monoclonal antibodies.

Claims (1)

OBJECT OF THE INVENTION Mouse lymphocyte hybridoma MSB-NTP-2, producing a monoclonal antibody against MSB1, a lymphoblastoid cell line, IgG1 subclass, kappa.