CS259231B1 - Mice lymphocytes hybridoma producing monoclonal antibody inhibiting Newcastle disease virus hemagglutination activity - 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-3, producing a monoclonal antibody inhibiting the hemagglutination activity of Newcastle disease virus. The mouse lymphocyte hybridoma has application in veterinary medicine.

Description

The invention relates to a murine lymphocyte hybridoma producing monoclonal antibodies against Newcastle disease virus with hemagglutinin inhibitory activity.

To date, antibodies to Newcastle disease virus have been prepared by injecting the virus into test animals, particularly rabbits and sheep. Because not enough! a single dose of virus to elicit a sufficiently high antibody response, need to be injected multiple times at different doses and time intervals. The serum of such immunized animals serves as a source of antibodies mainly to 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 many antibodies to unwanted admixtures found in the 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 the diagnostic serum thus prepared is also reduced by the fact that its specific polyclonal antibodies are very diverse in terms of virus interaction. Moreover, the preparation of the virus for immunization purposes is expensive.

The NDV-NTP-3 hybridoma was prepared in a known manner 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 spleen cells of inbred ΒΑΧ mouse line. , Β / c, immunized with purified NDV, strain L Kansas / G. Kohler, C. Milstein, G.W. Butcher, J.C. Howard: Nature 286, 550 (1977) //.

These drawbacks substantially overcome the invention, which is based on the murine lymphocyte hybridoma NDV-NTP-3, producing a monoclonal antibody of subclass IgG1, kappa, which inhibits the hemagglutinating activity of Newcastle disease virus.

The advantage of the hybridoma is that it produces an isotype and idiotype homogeneous specific antibody, the so-called " a monoclonal antibody that specifically inhibits the hemagglutinating activity of Newcastle disease virus and thus can be used to identify it in examined samples from different poultry holdings. The single cell lymphocyte hybridoma NDV-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, thawed and stored in liquid nitrogen and after thawing again retains its ability to produce a monoclonal antibody binding Newcastle disease virus. It does not contain unsuitable, ballast antibodies, unlike conventional antisera.

Example 1

To prepare a hybrid cell line, 2.10 mouse 2-amino-6-hydroxy-8-azapurine (8-azaguanine / resistant myeloma cell line) cells are used, which are treated with 1 ml p

% wt. polyethylene glycol was coupled to 1.10 spleen lymphoid cells of mice immunized with 10 µg of virus. Within 14 days, under the selective pressure of 4-amino-pteroylglutamic acid / aminopterin / hybrid, cell lines that produce monoclonal antibodies against Newcastle disease virus that bind to it and affect its biological activity will grow. These cells are then cloned by mixing 0.5 wt. agar 5.10® 10.10® and mouse spleen cells in RPMI 1640 containing 100 mg.ml ^-1 -1 -1 nitrate tetrahydrate, calcium -1, 2000 mg.ml D-glucose, 100 mg.ml magnesium sulfate heptahydrate , 400 mg.ml 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), mg.ml ^{- 1} 2-aminosuccinic acid (asparagine), 20 mg.ml ^-1 of 2-aminosuccinic acid (aspartic acid), 50 mg.ml ^-1 of 3 ', 3'-dithiobis (2-aminopropanoic acid) (cystine), mg.ml ^-1 2-aminoglutaric acid (glutamic acid), 300 mg.ml ^-1 2-aminoglutaramic acid (glutamine), 1 mg.ml ^-1 2-aminoglutaraminyl-2-mercaptopropionyl-aminoacetic acid (glutathione). glutaminyl-cysteinyl-glycine (10 mg.ml ^-1 aminoacetic acid / glycine), 15 mg.ml alpha-amino-1-imidazole-4-propionic acid (histidine), 50 mg.ml ^-1 2-amino- 3-methylvaleric (isouecin), 50 mg.ml ^-1 of 2-amino-4-methylvaleric acid (leucine), 40 mg.ml ^-1 of 2,6-diaroinohexanoic acid (lysine), 15 mg.ml ^-1 of 2- amino-4- (methylthio) -raasylic acid (methionine), 15 mg.ml 2-amino-3-phenylpropionic acid (phenylalanine), 20 mg.ml ^-1 2-pyrrolidinecarboxylic acid (proline), 30 mg.ml ^-1 acid 2-amino-3-hydroxypropionic acid (serine), 20 mg.ml ^-1 of 2-amino-3-hydroxybutyric acid (threonine), mg.ml ^-1 of 2-amino-3- (3-indolyl) propionic acid / tryptophan) 20 mg.ml ^-1 of 2-amino-3- (4-hydroxyphenyl) -propionic acid (tyrosine), 20 mg.ml ^- 2-amino-3-methylbutyric acid (valine), 0,2 mg.ml ¹ 5- (2-oxoimidazolidino (4,5-c) thiol-2-yl) pentanoic acid (biotin),

0,005 mg.ml ^-1 cobalt alpha / alpha- (5,6-dimethylbenzimidazolyl) - - cobalt-beta-cyanocobamide vitamin B ₁₂ /, 0,250 mg.ml ¹ DN- (2,4-dihydroxy-3,3-dimethylbutyryl) calcium beta-2-aminopropionate (calcium pantothenate), 3 mg.ml ^- 2-hydroxyethyltrimethylammonium chloride (choline chloride), mg.ml ^-1 N- (2-amino-4-hydroxypteridin-6-ylmethyl) -p-aminobenzoic acid (folic acid), 35 mg.ml ^- (1,2,3,5) 4,6 (cyclohexanedexole) (myoinositol), 1 mg.ml ^-1 pyridine-3-carboxyamide (nicotinamide), 1 mg.ml * 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 ^-1 3- (4-amino-2-methyl-pyrimidyl-5-methyl) -5- (2-hydroxyethyl) -4 methylthiazole / thiamine /, 2000 mg.ml ¹ of sodium bicarbonate supplemented with 2 mmol of 2-aminoglutarámovej / glutamine /, 5.10 ^-5 M 2-mercaptoethanol, 100 jug.ml 0-.alpha.-L-2-deoxy-2- / methylamino / -g Lukopyranosyl- (1-2) -O-alpha-L-deoxy-3-C-formyllyxofuranosyl- (1-4) -1,3-deoxy-1,3-diguanidoscyloinosulfate (streptomycin sulfate), 100 units per milliliter of 6- (N-phenylacetoamido) -penicillanic acid (warbler G), 1 mmol of N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (Hepes) is dissolved in three times distilled water and made 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. After the agar had solidified, 0.5 wt. agar plated 100 hybrid cells in 0.25 wt. agar. After 10 days, colonies are grown which are expanded in RPMI 1640 medium supplemented with 2 mmol of 2-aminoglutaramic acid -5-1 (glutamine), 5.10 mol of 2-mercaptoethanol, 100 µg of 0-alpha-L-2-deoxy- 2- (methylamino) -glucopyranosyl- (1-2) -O-alpha-L-deoxy-3-C-formyllyxofuranosyl- (1-4) -1,3-deoxy-1,3-diguanidoscyloinosulfate (streptomycin sulfate), 100 N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid naimililiter 6- (N-phenylacetamido) -p-penicillanic acid / penicillin GZ, 1 mmol of N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (Hepes) is dissolved in three times distilled water and made up to three times with distilled water which is 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-3 hybridomas producing 10 to 100 µg of monoclonal IgG1, kappa, antibody.

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 unable to grow and produce monoclonal antibodies.

NDV-NTP-3 mouse lymphocyte hybridoma is cultured at 37 ° C. The mean generation time is 22.4 h, the modal number of chromosomes is 24 months after the fusion of 70 chromosomes. The monoclonal antibody produced is a mouse immunoglobulin of the IgG1 class, kappa.

The monoclonal antibody produced by the murine lymphocyte hybridoma NDV-NTP-3 can be used in veterinary practice to detect Newcastle disease virus in poultry farms.

Claims (1)

Mouse lymphocyte hybrid NDV-NTP-3, producing a monoclonal antibody that inhibits the haemagglutination activity of Newcastle disease virus, an IgG1 kappa subclass.