CS265305B1 - Hybridomas producing monoclonal antibodies differentiating feline encephalitis complex virus - Google Patents

Abstract

The aim of the solution is to prepare idiotypic homogeneous antibodies distinguishing tick-borne encephalitis complex viruses. The stated purpose is achieved by using mouse lymphocyte hybridomas KEN-10-2-1 and/or NEK-10-2-2, producing antiviral monoclonal antibodies. The mouse lymphocyte hybridoma has applications in medicine.

Description

265305 2

The invention relates to hybridomas producing monoclonal antibodies distinguishing virus-complexes of tick-borne encephalitis.

So far, specific Polyclonal Antibodies have not been able to distinguish between tick-borne encephalitis viruses in serological techniques. Affinity Chromatography Methods Specific polyclonal antibodies were insufficient for differentiation of tick encephalitis viruses. Also, monoclonal antibodies from the murine lymphocyte hybridoma, designated by the working name KEMA, produced into the microculture medium and into ascites fluids, were unable to differentiate tick-borne encephalitis viruses.

Hybridoma KEN-10-2-1 and NEK-10-2-2 were prepared in a known manner by cloning groups of hybridomas in soft agar resulting from cell fusion of 2-amino-6-hydroxy-8-azopurine (8-aza-guanine) -resistant resistant cells. myeloma line labeled NSO and spleen cells in inbred lineage BALB / c, immunized with tick-borne encephalitis virus (strain 4,072), obtained from a sick man in 1966 in the Sverdlovsk region, respectively. tick-borne encephalitis virus (Skalica strain) which was obtained in Czechoslovakia in 1974 from Clethrionomys glareolus g. Kóhler, C. Milstein: Nature 256, 495 (1975); G. Galfre, SC Howe, C. Milstein , GW Butcher, JC Howard: Nature 286, 550 (1977); M. Grešíková, M. Mrciak, V. Brtek, M. Sekey: 2. Internat.Arbeitskolloquium Ober Naturherde von Infektionskrankheiten in Zentraleuropa, Graz, 105 (1976) J.

The above drawbacks are substantially obviated by the invention, which is based on the mouse lymphocyte hybridoma KEN-10-2-1, producing monoclonal IgG antibodies and / or in the murine cell hybridoma NEK-10-2-2 producing monoclonal antibodies of the IgG class, which are capable of distinguishing tick-like viruses encephalitis. These hybridomas are stored in the SAC Virological Institute, Mlýnská dolina 1, 817 83 Bratislava. The advantage of hybridoma KEN-10-2-1 is that it produces idiotype-homogeneous antibodies, which, unlike specific polyclonal and monoclonal antibodies produced by murine lymphocyte hybridoma KEMA, distinguish tick-borne encephalitis virus from other viruses of this complex. The advantage of NEK-10-2-2 is that it produces isotype-homogeneous antibodies, called monoclonal antibodies, which, unlike specific polyclonal antibodies and monoclonal antibodies produced by mouse lymphocyte hybridoma KEMA, distinguish Powassan virus from other members of the tick-borne encephalitis virus complex. Single cell lymphocyte hybridomasKEN-10-2-1 and NEK-10-2-2 growth in vivo in the peritoneal cavity of BALB / c mice with concomitant production of aspites. Growth also in vitro in culture media suitable for animal cells. Also, freezing and storage in liquid nitrogen and after thawing retain their ability to produce antiviral monoclonal antibodies. Unlike conventional antisera, they do not contain unsuitable, ballast antibodies. The monoclonal antibodies retain their activity for at least 24 months with appropriate storage, depending on their origin. Example 1 7

For the preparation of the hybrid cell line, 2.10 cells of mouse 2-amino-6-hydroxy-8-azapurine (8-azaguanine) resistant myeloma cell line were used, which was made up with 1 ml of 50% w / w. polyethylene glycol combine with 1.10 spleen mouse lymphoid cells immunized with the virus of the Russian spring-summer encephalitis (strain 4,072) and the mouse immunized with the tick-borne virus (the Skalica strain. They grow under the selective pressure of 4-amino-pteroylglutamic acid (aminopterin) hybrid cell line within 14 days that produce monoclonal antibodies against Russian spring-summer encephalitis and tick-borne encephalitis viruses, and the clones are then cloned by mixing 0.5% by weight of agar with 5.10 ® to 10.10 ® mouse spleen cells in RPMI 640 medium containing 100 mg.ml-1 calcium nitrate tetrahydrate, -1-1-1,000 mg.ml D-glucose, 100 mg.ml magnesium sulfate heptahydrate, 400 mg.ml potassium chloride, 1212 mg.ml-1 dibasic sodium phosphate, 6,000 mg / ml of sodium phosphate monohydrate monohydrate, 100 mg / ml of 2-amino-5-guanidine-valeric acid (arginine), 50 mg / ml of 2-amino succinic acid. (asparagine), 20 mg.ml-1 2-amino succinic acid (aspartic acid), 50 mg.ml -1 '3', 3'-dithiobis / 2-aminopropanoic acid (cystine), 20 mg.ml 1 265305 2-aminoglutaric acid (glutamic acid), 300 mg / ml 2-aminoglutaramic acid (glutamine), 1 mg / ml 2-aminoglutaraminyl-2-mercaptopropionyl-aminoacetic acid (glutathione ie glutaminyl-cysteinyl-glycine), 10 mg.ml of aminoacetic acid (glycine), 15 mg / ml of α-aminolimidazole-4-propionic fhistidine), 50 mg / ml of 2-amino-3-methylvaleric acid (isoleucine), 50 mg / ml of acid 2-amino-4-methylvaleric (leucine) -1 -1 40 mg.ml of 2,6-diamohohexanoic acid (lysine), 15 mg.ml of 2-amino-4- (methylthio) -butyric acid (methionine) 15 mg of 2-amino-3-phenylpropionic acid (phenylalanine), 20 mg / l of 2-pyrrolidinecarboxylic acid (proline), 30 mg / ml of 2-amino-3-hydroxypropionic acid (sero), 20 mg / ml of 2-amino-3-hydroxybutyric acid (threonine), 5 mg / ml 1-2-amino-3- (3-indolyl) propionic acid (tryptophan), 20 mg / ml 2-amino-3- [4- hydroxyphenyl] propionic acid (tyrosine), 20 mg / ml 2-amino-3 7 - 0.2 mg.ml of 5- [2-oxoimidazoline, 5-thiol-2-yl] pentanoic acid (biotin), 0.005 mg.ml cobalt alpha £ [alpha] - [5,6-dimethylbenzimidazolyl, [beta] -cobalt [beta] -cyanocobamide (vitamin B.), [0125] 0.250 mg.ml calcium calcium / 2,4-dihydroxy-3,3-dimethylbutyryl [beta] -2-aminopropionate (calcium pantothenate), 3 mg / ml 2-hydroxyethyltrimethylammonium chloride (choline chloride), 1 mg / ml of N- (2-amino-4-hydroxypteridin-6-ylmethyl) -p-aminobenzoic acid (folic acid), 35 mg / ml. 1,2,3,5 / 4,6 / cyclohexanedexol / (myoinositol), 1 mg.ml pyrxdine-3-carboxyamide (nicotinamide), -1-11 mg.ml 4-aminobenzoic acid (p-amoxobenzoic 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-ribityl / isoaloxazine) ( riboflavin), 1 mg.m 11 11 3- (4-Amino-2-methylpyrimidyl-5-methyl) -5- (2-hydroxyethyl) -4-methylthiazole (thiamine chloride), 2,000 mg / l of sodium bicarbonate supplemented with 2 amino acids 2-α-minoglutaramic acid (glutamine), 5.10 mol 2-mercaptoethanol, 100 µg / ml 0-alpha-L-2-deoxy-2- (methylamoxo) -glucopyranosyl- / 1 - 2 / -O-alpha-L- deoxy-3-C-formyl-lyxofuranosyl- (1-N) -1,3-deoxy-1,3-diaguanidoscyloinositic sulfate (streptomycin sulfate), 100 units per milliliter of 6- (N-phenylacetoamido) -penicillic acid (penicillin G), Dissolve 1 mmole of N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (Hepesu) in tricrystallized 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. After the agar has set, 100 hybrid cells in 0.25% by weight are applied to the 0.5% agar layer. agare. After 10 days, colonies are grown and grown in RPMI-5 640 medium supplemented with 2 mmol of 2-aminoglutaramic acid (glutamine), 5.10 mol of 2-mercapto-ethanol, 100 µg / ml of 0-alpha-L-2. -deoxy-2- (methylamino) -glucopyranosyl- [1-92] -O-alpha-L-deoxy-3-C-formyllyxofuranosyl- [1- 54] -1,3-deoxy-1,3-diguanidoscyloinositite sulphate ( Dissolve 100 units per millimiter of 6- (N-phenylacetamido) -penicilic acid (penicillin G), 1 mmol N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (Hepesu) in three redistilled water and make up to redistilled. water to a volume of 900 ml solution, 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 KEN hybridomas and NEKs producing 10-100 µg of monoclonal IgG antibody. Example 2

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

Mouse lymphocyte hybridoma KEN-10-2-1 and NEK-10-2-2 are cultured at 37 ° C. The median generation time is 22 h, the modal number of chromosomes is 24 months after the fusion of 83 (KEN-10-2-1) or 92 (NEK-10-2-2) chromosomes. Monoclonal antibodies produced are murine immunoglobulins of the IgG class.

Monoclonal antibodies produced by murine lymphocyte hybridomas KEN-10-2-1 aNEK-10-2-2 can be used in medicine for the safe virological diagnosis of complex cell encephalitis viruses. Table 1 lists the activity of monoclonal antibodies KEN and NEK in the indirect 265305 4 immunofluorescence reaction with encephalitis virus and other flaviviruses. Table 2 shows the reaction of the KEN monoclonal antibody in the indirect immunofluorescence assay with encephalitis virus group. Table 3 shows the response of the monoclonal antibody NEK-10-2-2 in the reaction of complement binding and immunofluorescence with encephalitis virus viruses.

Tabulkal

Activity of monoclonal antibodies KEN and EQS in the response of indirect immunofluorescence by encephalitis virus and other flaviviruses

MP Label Antibody titers with viruses KEY YF DENGUE 1 DENGUE 2 VEE KEN-KM-1 32 Z 2 4 2 4 2 42 Z 2 KEN-KM-2 16 4 2 4 2 4 2 4 2 4 2 KEN-KM-3 64 <2 <2 4 2 4 2 4. 2 KEN-AT-1 10 240 410 410 410 410 Z10 KEN-AT-2 2 560 4110 410 4 10 410 4 10 KEN-AT-3 640 £ 10 410 4 10 410 4 10 NEK-KM-1 32 z 2 4 2 Z 2 4 2 4 2 NEK-KM-2 32 Z 2 2 Z 2 Z 2 4 2 NEK-KM-3 64 W 2 W 2 4 2 W 2 4 2 EQS -AT-1 10 240 410 410 - <10 410 4 10 NEK-AT-2 5 120 + 10 4 10 410 4 10 4 10 NEK-AT-3 640 <10 4 10 4 10 4 10 * —10 IAT Checks Against of the KE 320 virus. 10 4 10 410 4 10 410 IAT against YF virus Z 10 4 10 '160 4 10 4 10 Virus IAT against virus E 10 320 4 20 4 20 4 20 4 10 IAT against DENGUE 4 10 40 40 320 320 of 10 IAT hybridoma MAK-VEE Z 10 <10 4 10 4 10 Z 10 2 560

Explanation: KM - culture medium AT - ascitic fluid IAT - immune ascitic fluid MP - monoclonal antibody KE - tick-borne encephalitis virus JE - Japanese encephalitis virus YF - yellow fever virus VEE - Venezuelan encephalitis virus com PK - polyclonal antibodies

Table 2

PM KEN reaction in indirect immunofluorescence assay with tick-borne encephalic virus (KE) viruses.

Claims (1)

Mouse lymphocyte hybridomas KEN-10-2-1 and / or NEK 10-2-2, producing monoclonal antibodies of the IgG class, distinguishing tick-borne encephalitis complex viruses.