CS259232B1 - Myoma lymphocyte hybridoma producing a monoclonal antibody against poultry lymphocyte surface antigens - 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-3 producing a monoclonal antibody recognizing the surface antigens of the lymphoblastoid cell line designated by the abbreviation MSB1 and normal spleen cells of poultry. The mouse lymphocyte hybridoma has applications in veterinary medicine.

Description

The invention relates to a murine lymphocyte hybridoma producing a monoclonal antibody recognizing the surface antigens of the lymphoblastoid cell line designated MSB1 and normal spleen cells of poultry.

To date, antibodies to the lymphoblastoid cell line MSB1, which carries the diagnostic MATSA antigen on its surface, have been prepared by administering live MSB1 cells to test animals (rabbit, sheep). Since a single dose of cells is not sufficient to elicit a sufficiently high antibody response, they need to be injected multiple times at different doses and time intervals. The serum of the animals immunized in this way serves as a source of antibodies, in particular to the diagnostic examination of poultry. This immunization procedure has several drawbacks. A heterogeneous mixture of antibodies is obtained, the distribution of which varies in the producer organism, is very heterogeneous, qualitatively and quantitatively varies from one collection to the next and is unique. In addition, serum contains undesirable antibodies against surface antigens, which also occur on normal cells. As a result, sera need to be sophisticated by sophisticated saturation procedures so that the serum only contains antibodies against the surface antigens of the lymphoblastoid cell line designated MSB1 and normal spleen cells of poultry.

This treatment results in large serum losses and a significant decrease in the specific antibody titer. Understandably, the saturation process cannot be standardized, as a result of which the high heterogeneity of conventional sera is even deepened. In addition, the usefulness of the diagnostic serum thus prepared is 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 sufficient quantities of MSB1 lymphoblastoid cells for immunization is demanding in terms of equipment, skilled personnel and financial resources.

The MSB-NTP-3 hybridoma was prepared according to a known method by cloning a group of hybridomas in soft agar resulting from the fusion of mouse 2-amino-6-hydroxy-8-azapurine / 8-azaguanine / NSO-resistant myeloma line cells and BALB mouse inbred line spleen cells. / c, immunized with the lymphoblastoid cell line MSB1 / G. Kohler, C. Milstein,

Butcher, G.W., Howard, J.C., Nature 286, 550 (1977) //.

These disadvantages are substantially eliminated by the invention, which is based on the murine lymphocyte hybridoma MSB-NTP-3, producing a monoclonal antibody recognizing the surface antigens of the lymphoblastoid cell line designated by the abbreviation MSB1 and normal spleen cells, subclass IgG1, kappa. This hybrid is deposited in the Virologift Institute of the SAS, Mlýnská dolina 1, 817 03 Bratislava.

The advantage of the hybridoma is that it produces an isotype-homogeneous antibody, the so-called " a monoclonal antibody that recognizes the surface antigens of the lymphoblastoid cell line designated M3B1 and normal spleen cells of poultry. The single cell lymphocyte hybridoma MSB-NTP-3 grows in vivo in the peritoneal cavity of BALB / c mice while producing ascites.

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

Example 1

For the preparation of the hybrid cell line, 2.10? mouse 2-amino-6-hydroxy-8-azapurine / 8-azaguanine / resistant myeloma cell line cells which are treated with 1 ml of p

% wt. polyethylene glycol was coupled to 1.10 spleen lymphoid cells of mice immunized

Q

3.10 MSB1 lymphoblastoid cells. Within 14 days they grow under selective acid pressure

4-amino-pteroylglutamic / aminopterin / hybrid cell lines that produce monoclonal antibodies recognize the surface antigens of the lymphoblastoid cell line designated by the abbreviation MSB1 and normal-ovine poultry cells.

259232 6 ph

These cells are then cloned by mixing 0.5 wt. agar with 5.10 to 10.10 mouse spleen cells in RPMI 1640 medium containing 100 mg.ml ^-1 calcium nitrate tetrahydrate, 2,000 mg.ml ¹ D-glucose, 100 mg.ml ¹ magnesium sulfate heptahydrate,

400 mg.ml ¹ potassium chloride, 1 512 mg.ml ¹ sodium hydrogen phosphate, 6 000 mg.ml ^-1 sodium dihydrogen phosphate monohydrate, 100 mg.ml ^-1 2-amino-5-guanidinovaleric acid (arginine), 50 mg.ml ¹ 2-Aminosuccinamic acid (asparagine), 20 mg.ml ^-1 of 2-aminosuccinic acid (aspartic acid), 50 mg.ml ^-1 of 3 ', 3'-dithiobis (2-aminopropanoic acid), (cystine), 20 mg ml ^-1 of 2-aminoglutaric acid (glutamic acid), 300 mg.ml ^-1 of 2-aminoglutaramic acid (glutamine), 1 mg.ml ^-1 of 2-aminoglutaraminyl-2-mercaptopropionyl aminoacetic acid (gjutation) i.e. glutaminyl-cysteinyl-glycine (10 mg.ml ^-1 aminoacetic acid / glycine), 15 mg.ml ^-1 α-amino-1-imidazole-propionic acid (histidine), 50 mg.ml ^-1 of 2-amino- 3-methylvaleric (isoleucine), 50 mg.ml ^-1 of 2-amino-4-methylvaleric acid (leucine), 40 mg.ml ^-1 of 2,6-diaminohexanoic acid (lysine), 15 mg.ml ^-1 of 2- amino-4- (methylthio) butyric (methionine), 15 mg.ml ^-1 of 2-amino-3-phenylpropionic acid (phenylalanine), 20 mg.ml ^-1 of 2-pyrrolidinecarboxylic acid (proline), 30 mg.ml ^-1 2-amino-3-hydroxypropionic / serine /, 20 mg.ml ¹ of 2-amino-3-hydroxybutyric acid / threonine / j 5 mg.ml of 2-amino-3- / 3-indolyl / propionic acid / tryptophan /. 20 mg.ml of 2-amino-3- / 4-hydroxyphenyl / propionate / tyrosine / 20 mg.ml ¹ of 2-amino-3-methylbutyric / valfhu / 0.2 mg.ml ^"1 of 5 / 2-oxoimidazolidino [4,5-c] thiol-2-yl (pentanoic) biotin],

0,005 mg.ml ” ¹ cobalt <X / K- (5,6-dimethylbenzimidazolyl) - - cobalt β-cyanocobamide of vitamin Bj ^ /,

0.250 mg.ml ^-1 DN- (2,4-dihydroxy-3,3-dimethylbutyryl) -p-2-aminopropionate (calcium pantothenate), 3 mg.ml ^-1 1-hydroxyethyltrimethylammonium chloride (choline chloride), 1 mg.ml " ¹ N- (2-amino-4-hydroxypteridin-6-ylmethyl) -p-aminobenzoic acid (folic acid), 35 mg.ml" ¹ / 1,2,3,5 (4,6) cyclohexanedexole) / myoinositol (1 mg.ml ^-1 pyridine-3-carboxyamide (nicotinamide)), 1 mg.ml ^-1 4-aminobenzoic acid (p-aminobenzoic acid), 1 mg.ml ¹¹ 3-hydroxy-4,5-bis (hydroxymethyl) / 2-metylpyridínchloridu / pyridoxínchloridu / 0.2 mg.ml ^"1 7,8-dimethyl-10 / l-D-ribityl / izoaloxazinu / riboflavin /, 1 mg.ml" ¹ 3/4-amino -2-methyl-pyrimidyl-5-methyl (-5- (2-hydroxyethyl) -4-methylthiazole / thiamine chloride), 2000 mg.ml ¹ sodium bicarbonate supplemented with 2 mmol of 2-aminoglutaramic acid (glutamine), 5.10 mol 2-mercaptoethanol,

100 ^-1 0 pg.ml-BL-2-deoxy-2 / methylamino / ~ glucopyranosyl / L-2 / -0 - «- L-deoxy-3-C-formyllyxofuranozyl- / L-4 / -l, 3-deoxy-1,3-diguanidoscyloinositsulfate (streptomycin sulfate), 100 units per milliliter of 6- (N-phenylacetoamido) -penicillanic acid (penicillin G), 1 mmol of N-2-hydroxyethylpiperazine-N'-2-ethane sulfonic acid / Hepesu Dissolve in three times redistilled water and make up to three times with redistilled water to a volume of 900 ml of solution, which is supplemented with 100 ml of normal inactivated horse serum.

The resulting pH of the culture medium thus prepared is 7.2. After the agar had solidified, 0.5 wt. agar plated 100 hybrid cells in 0.25 wt. agar. After 10 days colonies come up, which are propagated in RPMI 1640 supplemented with 2 mmol of 2-amimoglutarámovej / glutamine /, 5.10 ^- ^ mol of 2-mercapto $ butanol, 100 jug.ml ¹ 0-KL-2-deoxy ~ 2 - (methylamino) -glucopyranosyl- (1-2) -O-α-deoxy-3-C-formyllyxofuranosyl- (1-4) -1,3-deoxy-1,4-diguanidoscyloinosulfate (streptomycin sulfate), 100 units per milliliter 6- (N-phenylacetic-copper) -penicillanic acid () penicillin G), 1 mmol of N-2-hydroxyethylpiperazine-N'-2-ethane sulfonic acid (Hepesi) is dissolved in three times distilled water and made up to three times with distilled water to volume of 900 ml of solution to be supplemented with 100 ml of normal inactivated horse serum.

The resulting pH of the culture medium thus prepared is 7.2. 1 ml of culture medium contains 350 to 700 thousand MSB-NTP-3 hybridomas producing 10 to 100 µg of monoclonal IgG1, kappa, an antibody recognizing the surface antigens of the lymphoblastoid cell line designated MSB1 and normal spleen cells.

Example 2

The procedure is as in Example 1 except that non-tested conventional calf fetal serum is used in the culture medium. The cells transferred to the culture medium thus prepared after the fusion are not able to grow and produce monoclonal antibodies.

MSB-NTP-3 mouse lymphocyte hybridoma is cultured at 37 ° C. The mean generation time is 19.4 hours, the modal number of chromosomes is 24 months after the fusion of 78 chromosomes. The monoclonal antibody produced is a murine immunoglobulin recognizing the surface antigens of the lymphoblastoid jacket line designated by the abbreviation MSB1 and normal spleen cells of the poultry class IgG1, kappa.

The monoclonal antibody produced by the murine lymphocyte hybridoma MSB-NTP-3-MO can be used in veterinary practice in the examination and differential diagnosis of poultry cancer.

Claims (1)

Mouse lymphocyte hybridoma MSB-NTP-3, producing a monoclonal antibody against surface antigens of lymphocyte lymphocytes, IgG1 subclass, kappa.