CS260264B1 - Mouse lymphocyte hybridoma producing monoclonal antibodies neutralizing 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-2, producing a monoclonal antibody neutralizing Newcastle disease virus. The mouse lymphocyte hybridoma has application in veterinary medicine.

Description

The invention relates to a murine lymphocyte hybridoma producing roonoclonal antibodies neutralizing Newcastle disease virus.

To date, antibodies to Newcastle disease virus have been prepared by injecting the virus into test animals, particularly rabbits and sheep. Since a single dose of virus is not sufficient to elicit a sufficiently high antibody response, it is necessary to inject it several times at different doses and at different time intervals. The serum of the animals thus immunized serves as a source of antibodies, in particular for the diagnostic examination of poultry holdings. This immunization procedure has several drawbacks. (A heterogeneous mixture of antibodies is obtained, the distribution of which varies in the producer organism, is very diverse, qualitatively and quantitatively varies from one collection to the next and is unrepeatable. In addition, the serum contains a large number of antibodies against undesirable admixtures found in As a result, the sera need to be sophisticated by complex procedures to obtain heterogeneous and non-standard preparations, and the usefulness of the diagnostic sera thus prepared is reduced by the fact that its specific polyclonal antibodies are very diverse in terms of virus interaction. virus for immunization purposes is costly.

The NDV-N'I'P-2 hybridoma was prepared in a known manner by cloning a group of macromages in macar agar resulting from the fusion of cells of the mouse 2-amino-6-hydroxy-8-azapurine (8-azaguanine) resistant NSO-labeled myeloma line and spleen cells of an inbredin line of BALB / c mice immunized with purified NDV, strain L Kansas [G. Kohler, C. Milstein, G.W. Butcher, J.C. Howard: Nature 286, 550 (1977)].

These disadvantages are substantially eliminated by the invention, which is based on the murine lymphocyte hybridoma NDV-NTP-2, producing a monoclonal antibody of the IgM class, kappa, neutralizing Newcastle disease virus. This hybrids are stored in the Institute of Virology of SAS, (Mlýnská dolina 1, 817 03 Bratislava).

The advantage of the hybridoma is that it produces an isotype and idiotype homogeneous specific antibody, the so-called " a monoclonal antibody that specifically neutralizes the Newcastle disease virus and thus can be used not only for diagnostic purposes but also promising for preventive and therapeutic purposes. single-cell lymphocyte hybrids of NDV-NTP-2 hybrids grow 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 after thawing, it retains its ability to produce a monoclanal antibody neutralizing Newcastle disease virus. It does not contain unsuitable, ballast antibodies, unlike conventional antisera.

Example 1

For the preparation of the hybrid cell line, a 2 IQ ⁷ cell of a 2-amino-8-hydroxy-8-azapurine (8-azaguanine) resistant myeloma cell line was used, which was treated with 1 ml of 50 wt. Polyethylene glycol is coupled to 1. 10® spleen lymphoid cells of mice immunized with 10 µg of virus. Over the course of 14 days, under the selective pressure of 4-amino-pteroylglutamic acid (aminopterin), hybrid cell lines producing monoclonal antibodies against Newcastle disease virus grow and interact with its biological activity. by mixing 0.5% by weight of agar with 5-10 * 10 < 10 > mouse spleen cells in a RPMI 1640 medium containing

100. mg. ml ^-1 calcium nitrate tetrahydrate,

000 mg. ml of ¹ D-glucose,

100 mg. ml ^-1 magnesium sulfate heptahydrate,

400 mg. ml ¹ potassium chloride, il 512 mg. ml ¹ disodium phosphate,

Θ 000 mg. ml ¹ sodium dihydrogen phosphate monohydrate,

100 mg. ml ¹ of 2-amino-5-guanidinovaleric acid (arginine), mg. ¹ ml of 2-amínosukcínovej (asparagine), mg..ml ^-1 of 2-a.mínojant ^ rovej (aspartic acid), mg. ml ¹ of 3 ', 3'-dithiobis (2-aminopropanoic acid) (cystine), mg. ml ¹ of 2-aminoglutaric acid (glutaric acid),

300 mg. ¹ ml of 2-aminoglutaric (glutamine), '1 mg. ml ¹ of 2-aminoglutaraminyl-2-mercaptopropionyl-aminoacetic acid (glutathione, ie glutaminyl-cystelnyl-glycine), mg. ml ¹ aminoacetic acid (glycine),

260284 mg. ml ^-1 of a-amino-imidazol-a-propionic acid (hlstidínu), m; ^_1 .ml of 2-amino-3-methylpentanoic acid (isoleucine), mg. ml ¹ of 2-amino-4-methylvaleric acid (Leucine), mg. ml- ¹ of 2,6-diamino-hexanoic acid (lysine), 15 mg. mr ¹ of 2-amino-4- (methylthio) butyric acid (methionine), mg. ml ^-1 of 2-amino-3-phenylpropionic acid (phenylalanine), mg. ml ^-1 of 2-pyrrolidinecarboxylic acid (proline), mg. ml ¹ of 2-amino-3-hydroxyproplonic acid (serine), mg. ml ^-1 of 2-amino-3-hydroxybutyric acid (threonine), mg. ml ^"1 of 2-amino-3- (3-hydroxybutyric acid (threonine), mg. ¹ ml of 2-amino-3- (3-indolyl) propionic acid) (tryptophan), '20 mg. ml ¹ of 2-amino-3- (4-hydroxyphenyl) -propionic acid (tyrosine), mg ml ^-1 of 2-amino-3-methylbutyric acid (valine),

0.2 mg. ml ¹ of 5- (2-oxoimidazolino (4,5-c) thiol-2-yl) pentanoic acid (biotin),

0.005 mg. ml of ¹ cobalt and [α- (5,6-dimethylbenzimidazole)) - cobalt / 1-cyanocoibamide vitamin B12),

0.250 mg. ml ¹ DN- (2,4-dihydroxy-3,3-dimethylbutyryl) - β-2-aminopropionate (calcium pantothenate), ⁱ 3 mg. ml ^-1 2-hydroxyethyltrimethylammonium chloride (choline chloride), mg. ml ^-1 of N- (2-amino-4-hydroxypteridin-6-ylmethyl) -β-aminobenzoic acid (folic acid), mg. ml ¹ (1,2,3,5 / 4,6 / cyclohexanedexole) (myoinozltol), mg. ml of ¹ pyridine-3-carboxyamide (nicotinamide), mg. ml ^-1 of 4-aminobenzoic acid p-aminobenzoic acid), 1 mg. ml ¹

3-hydroxy-4,5-bis (hydroxymethyl) -2-methylpyridine chloride (pyridoxine chloride),

0.2 mg. ml ^{1 of} 7,8-dimethyl-10- (1'-D-ribytyl) isoaloxazine (riboflavin), mg. ¹ ml of 3- (4-amino-2-methyl-pyrimi'dyl-5-methyl) -5- (2-hydroxyethyl) -4-methylthiazole (thiamine),

000 mg. ml ^"1 of sodium bicarbonate supplemented with 2 mmol of 2-amínoglutarámovej (glutamine), ⁵ .10 M 2-mercaptoethanol,

100 jUg. 0 ml of ^{1-al-2-deoxy-I2} (methylamino) -glutapyranozyl (1-2) -OaL-deoxy-3-Cf ormyllyxof uranozyl- (1-4) -1,3-deoxy-l, 3-diguanidoscyloinozitsulfátu (streptomyeínsulfátuf.

100 units per milliliter of 6- (N-phenylacetoamidine-penicillanic acid (penicillin mmol of N-2-hydroxyethylplperazine-N'-2-ethanesulfonic acid (Hepesu)) is dissolved in three times with distilled water and made up to three times with distilled water to a volume of 900 ml 100 ml of normal inactivated horse serum is added and the pH of the culture medium thus prepared is 7.2.After the agar has set, 100 hybrid cells in 0.25% agar are applied to the 0.5% agar layer. colonies are grown which are expanded in RPMI 1640 medium supplemented with 2 mmol 2-aminoglutaramic acids (glutamine), 10 ⁵ moles of 2-mercaptoethanol,

100 µg. ml ¹ OaL-2-deoxy-2- (imethylamino) -glucopyranosyl- (1-2) -OaL-deoxy-3-formyllyxofuranosyl- (1-4) -1,3-deoxy-1,3-diguanidoscyloinosulfate (streptomycin sulfate) .

100 units per milliliter of 6- (n-phenylacetamido) -penicillanic acid (penicillin G), mmol of N-2-hydroxyethylpiperazine-N'2-ethanesulfonic acid (Hepesu) are dissolved in three times redistilled water and made up to three times with distilled water to a volume of 900 ml of the 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 NDV-NTP-2 hybridomas producing 10 to 100 µg of monoclonal IgM, kappa antibody.

Example 2

The procedure is as in the example 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 unable to grow and produce monoclonal antibodies.

NDV-NTP-2 mouse lymphocyte hybridoma is cultured at 37 ° C. The mean generation time is 22.4 h, the modal chromosome count is 24 months after the fusion of 75 chromosomes. The monoclonal antibody produced is a mouse immunoglobulin of the IgM class, kappa.

The monoclonal antibody produced by the murine lymphocyte hybridoma NDV-NTP-2 can be used in veterinary practice to deceive the virus in poultry farming and promising for preventive and therapeutic purposes.

Claims (1)

The mouse lymphocyte hybridoma NDV-NTP-2 producing a monoclonal antibody does not invent the tralizing Newcastle disease virus, class IgM, kappa.