CS259230B1 - Murine hybridoma producing monoclonal antibodies against Newcastle disease virus - 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 NDV-NTP-1, producing a monoclonal antibody binding Newcastle disease virus. The mouse lymphocyte hybridoma has applications in veterinary medicine.

Description

Ž59230

The invention relates to a mouse lymphocyte hybridoma producing a monoclonal antibody against Newcastle disease virus.

Until now, Newcastle disease virus antibodies have been prepared by injecting virussa in experimental animals, especially rabbits and sheep. Since the virus dose is insufficient to elicit a sufficiently high antibody response, it is necessary to re-inject it multiple times at different doses and time intervals. The serum of such immunized animals serves as a source of antibodies in particular for the diagnostic examination of poultry breeds. The mentioned immunization procedure has several disadvantages. A heterogeneous mixture of antibodies is obtained, the distribution of which varies in the producer's organism, is very diverse, qualitatively aquatic, varies from one sampling to another and is unrepeatable.

In addition, serum contains many antibodies to undesirable ingredients that are found in viral material! used for immunization. As a result, sera need to be treated with complex procedures to obtain heterogeneous and non-standard preparations. The usefulness of such a prepared diagnostic serum is also reduced by the fact that its specific polyclonal antibodies are highly heterogeneous in their interaction with the virus. In addition, pre-immunization virus preparation is costly.

NDV-NTP-1 hybrids were prepared in a known manner by cloning the mSkkomagare hybridoma family, resulting from the fusion of murine 2-amino-6-hydroxy-8-azapurine / 8-azagua'in / cell-resistance-labeled NSO and spleen BALB spleen cells. / c, immunized with purified NDV virus strain L Kansas / 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 mouse cell hybridoma NDV-NTP-1, producing the monoclonal antibody of the Newcastle disease-binding IgG1, kappa subclass. These hybrids are stored in the Virological Institute of the Slovak Academy of Sciences, Mlýnská dolina 1, 817 03 Bratislava. The advantage of a hybrid is that it produces an isotype and idiotype-homogeneous antibody, the so-called monoclonal antibody, that specifically binds to Newcastle disease virus, and thus can be used to identify it in samples from different poultry farms.

Single cell lymphocyte NDV-NTP-1 hybrids grow in vivo in the peritoneal cavity of BALB / c mice while forming ascites. Suitable animal cells are also grown in vitro in culture media. Also, after freezing and storage in liquid nitrogen and after thawing, it retains its ability to produce a Newcastle disease virus-binding monoclonal antibody. It does not contain unsuitable, ballast antibodies, unlike conventional antisera. Example 1

For the preparation of the hybrid cell line, 2 x 10 7 cells of mouse 2-amino-6-hydroxy-8-azapurine / 8-azaguanine / resistant myeloma cell line were used and 1 ml of 50% hirot. polyethylene glycol combines with 1.10 spleen lymphoid cells of mice immunized with 10 µg of virus. Within 14 days, under the selective pressure of 4-amino-pteroyl-glutamic / aminopterin / hybrid cell line, which produce monoclonal antibodies, they grow against the Newcastle disease virus that bind to it but do not affect its biological activity.

These cells are then cloned to mix 0.5 wt. agar with 5.10 ® to 10.10 ® mouse spleen cells in RPMI 640 medium containing 100 mg / ml calcium nitrate tetrahydrate - 1 - 1 - 1 calcium, 2000 mg / ml D-glucose, 100 mg / ml magnesium sulfate heptahydrate 400 mg / ml of potassium chloride, 1112 mg / ml of dibasic sodium phosphate, 6000 mg of sodium dihydrogen phosphate monohydrate, 100 mg / ml of 2-amino-5-guanidinovaleric acid / arginine / 50 mg. ml-2-amino-succinic acid / asparagine /, 20 mg / ml 2-amino succinic acid 3 259230 / aspartic acid / 50 mg / ml 3 *, 3'-dithiobis / 2-aminopropanoic acid / cystine / 20 mg / ml 2-aminoglutaric acid / glutamic acid /, 300 mg / ml 2-amino-glutaramic acid / glutamine / 1 mg / ml 2-aminoglutaraminyl-2-mercaptopropionyl-amino-acetic acid / glutathione ie. glutaminyl-cysteinyl-glycine (10 mg / ml of aminoacetic acid / glycine), 15 mg / l of α-amino-1-imidazole-4-propionic acid / histidine / 50 mg / ml of 2-acid. of amino-3-methylvaleric (isoleucine), 50 mg / ml 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 / l of 2-amino-3-phenylpropionic acid / phenyl-alanine /, 20 mg / l of 2-pyrrolidinecarboxylic acid / proline /, 30 mg / ml of 2-amino-3-hydroxypropionic acid / serine / 20 mg / ml of 2-amino-3-hydroxybutyric acid / threonine / -1-15 mg / ml of acid 2 -amino-3, 3-indolyl / propionic / tryptophan /, 20 mg / l of 2-amino-3- (4-hydroxyphenyl) propionic acid / tyrosine / 20 mg / l of 2-amino-3-methylbutyric acid (valine), 0.2 mg / ml of 5- (2-oxoimidazolidino [4,5-c] thiol-2-yl) pentanoic / biotin (0.005 mg / ml cobalt alpha / afla) [5,6-dimethylbenzimidazolyl] -cobalt-beta-cyano bamidVitamin B 4, 0.250 mg / ml of DN- / 2,4-dihydroxy-3,3-dimethylbutyryl / -beta-2-aminopropionate calcium / calcium pantothenate /, 3 mg / ml of 2-hydroxyethyltrimethylammonium chloride / oholin chloride / 1 mg / ml of N- (2-amino-4-hydroxypteridin-6-ylmethyl) -p-aminobenzoic acid (-1-1) / 35 mg / ml / 1,2,3,5 / 4,6 (cyclohexanedexol) / myoinositol /, 1 mg / ml of pyridine-3-carboxyamide / nicotinamide / 1 mg / l of 4-aminobenzoic acid / p-aminobenzoic acid / 1 mg / ml of 3-hydroxy-4,5 -bis / hydroxymethyl / 2-methylpyridine chloride / pyridoxine chloride /, 0.2 mg / ml of 7,8-dimethyl-10- (1'-D-ribityl / isoaloxazine / riboflavin), 1 mg / ml of 3- / 4-amino-2-methylpyrimidyl-5-methyl / -5- (2-hydroxyethyl) -4-methylthiazole / thiamine chloride, 2000 mg / l of sodium bicarbonate supplemented with 2 mmol of 2-aminoglutaramic acid / glutamine / 5.10-5 mol of 2-mercaptoethanol, 100 µg / ml of 10-alpha-L-2-deoxy-2- (methylamino) -glucopyranosyl - 1 - 2 / -O-alpha-L-deoxy-3-C -formyllyxofuranosyl- [1-4] -1,3-deoxy-1,3-diguanidose oyloinozitsulphate / streptomycin sulphate /, 100 units per milliliter of 6- (N-phenylacetoamido) -penicillic acid / penicillin G /, 1 mmol of N-2-hydroxyethylpiperazine-N'-2-ethane sulphonic acid / Hepesu / are dissolved in three times redistilled water and make up to 900 ml of solution three times with redistilled water and make up to 100 ml with 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-amino-glutaramic acid / glutamine /, 5.10-mol of 2-mercaptoethanol, 100 µg / ml of 10-alpha-L-2-deoxy. 2- / Methyl-amino-glucopyranosyl 1- (1-2) -O-afla-L-deoxy-3-C-formyllyxofuranose-1- (1-4) -1,3-deoxy-1,3-diguanidoscyloinositic sulfate / streptomycin sulfate Dissolve 100 units per milliliter of 6- (N-phenylacetamido / -penicillic acid / penicillin G), 1 mmol of N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid / Hepes / in three redistilled water and make up to three times with distilled 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. In 1 ml of culture medium, there are 350 to 700 thousand NDV-NTP-1 hybridomas producing 10-100 µg of monoclonal IgG1, kappa, antibody. Example 2

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

The mouse lymphocyte hybridoma NDV-NTP-1 is cultured at 37 ° C. The mean generation time is 22.4 h, the modal number of chromosomes is 24 months after the 73 chromosome fusion. The produced monoclonal antibody is murine immunoglobulin IgG1 class, kappa.

Claims (1)

OBJECT OF THE INVENTION Mouse lymphocyte. NDV-NTP-1 hybridoma producing monoclonal antibodies against Newcastle disease virus, IgG1 subclass, kappa.