CS268403B1 - Mouse b-lymphocytar hybridome production series hbs-01 a0 35/86 - Google Patents

Abstract

The solution concerns a new production mouse the HBS-01 hybridoma line producing a specific monoclonal antibody subclass IgG1 against viral surface entigen hepetitis B (HBsAg)

Description

The present invention relates to a novel hybridoma production line producing a specific monoclonal antibody against hepatitis B virus surface antigen (HBsAg). The hybridoma was constructed by fusing a mouse myeloma lineage cell and a mouse lymphocyte sensitized with HBs antigen. '

Diagnostic sera against HBsAg are an essential component of a set of immunoreactivities necessary for the eerological diagnosis of human disease with viral hepatitis 8 and for detecting the presence of hepatitis B virus in the blood and blood derivatives in the transfusion service. These aerials are part of the HBsAg detection kits / SEVATEST ELISA HBsAg - MACRO I or ELISA HBsAg MICRO 1 /, which are used in healthcare in routine blood testing.

The diagnostic sera used to determine HBsAg by the above assays are hyperimmune rabbit and goat antisera, prepared by re-immunization of animals with purified HBsAg prepared from human reptiles of HBsAg carriers. The preparation of HBsAg is very demanding on the material, methodological and organizational support of the production process. The disadvantage of this method of preparing hyperimmune antisera is that some animals do not respond well to HBsAg immunization, resulting in production losses. In addition to the desired antibodies against HBsAg, unwanted antibodies against human serum proteins may be present in hyperimmune antisera and must be removed from the serum by saturation. Standardization of individual batches of diagnostic sera is difficult and always incomplete with the current method of preparation, because it follows from the very nature of the current method of preparation of immune sera that two production series of the same properties cannot be obtained. ·

Another disadvantage of HBsAg testing performed with polyclonal era to date is that erroneous results due to lower sensitivity or unexpected properties of diagnostic sera cannot be ruled out. _F

Many of the above-mentioned problems of the previously used method of preparation of diagnostic sera as well as a number of shortcomings resulting from inappropriate properties of the polyclonal diagnostic sera used so far can be eliminated by introducing the preparation of murine monoclonal antibodies against HBsAg produced by B-lymphocyte hybridomas. The main benefit of the introduction of monoclonal antibodies is the simplification and acceleration of the production process, saving purified HBs antigen, standardizing the preparation of individual batches of diagnostic sera, increasing the sensitivity and specificity of testing and reducing the time required to perform a diagnostic test.

The source of the monoclonal antibody against HBsAg is the hybridoma line HBS-01, deposited in the hybridoma collection of the Institute of Sera and Vaccines, Prague 10, W. Piecka 108 under the designation HBS-01.

Said hybridoma was constructed in a manner known from the literature /S.Fazekas de St. Groth, 0. Scheidigger: Production of monoclonal antibodies: Strategy and tactics, 0. Immunol. Meth. 35: 1, i960; 0. Kalil. 0. Crevet, M. Fellous, 3. Drouet, A.M. Courouce, C. Ropers: Production of anti-HBs monoclonaus anticorps. Ann.Immunol. (Inst. Pasteur/ 132c, 319, 1961) by cloning a set of hybrid cells resulting from the fusion of mouse cells of the myeloma line P3-X63-Ag8.653 and sensitized lymphocytes obtained from the spleens of 8ALB / c mice immunized with HBsAg.

The B-cell hybridoma line HBs-01 produces completely homogeneous antibodies, so-called monoclonal antibodies, which react specifically with HBsAg and not with human plasma proteins. The HBS-O1 hybridoma can be grown in vitro or in vivo cell culture conditions in the peritoneal cavity of BALB / c mice or stored for a long time at liquid nitrogen temperature. The monoclonal antibody produced by the HBs-01 hybridoma is specific for HBsAg and free of other unwanted antibodies.

HBS - 01 hybridoma cells show an ultrastructural image of typical myeloma cells with free and membrane - bound polyribosomes. Under cell culture conditions, they grow as

CS 268403 B1 weaker cell adherence (semi-suspension culture) in MEM or RPMI 1640 grade supplemented with 2-mercaptoethanol (5 x 10 8 M), 10% calf serum for tissue cultures and antibiotics / 100 .mu.l penicillin, 10 .mu.g treptomycline and 40 .mu.g gentamicin in 1 ml / pre 37 ° C. The average generation time of the production line HBS -01 boat 20 hours. The monoclonal antibody produced is mouse Immunoglobulin class IgG 1, with an isoelectric point of 6.6 to 6.8.

Example 1

In order to propagate the HBe-01 hybridoma production line and entrain the monoclonal antibody, 1 χ 10 ⁵ cells / 1 ml of culture medium was seeded in a culture vessel. After 3 days of cell growth, a culture medium enriched with monoclonal antibodies generated by cells of the hybridoma production line was obtained from the culture vessel. Monoclonal antibodies reacted with β-purified HBsAg in a radioimmunoassay up to a dilution of 10 ⁶

Example 2 g An anti-HBsAg monoclonal antibody was tested in vivo after inoculating 5 x 10 cells of the hybridoma production line HBS-01 into the perltoneal cavity of a BALB / c mouse, which had been injected in the same manner with 0.5 ml of paraffin oil 14 days in advance. Monoclonal antibodies against HBeAg were included in the ascitic fluid obtained from the perltoneal cavity of mice 12 days after cell inoculation. Monoclonal antibodies reacted and purified 9

HBsAg in a radioimmunoassay up to 1 and 10 dilutions. 5 ml of ascitic fluid with an immunoglobulin content of 12 mg / ml were obtained.

Claims (1)

PR R E 0 M E T of the invention • Mouse B-lymphocyte hybridoma production line HBS - 01 AO 35/86 producing an IgG1 subclass monoclonal antibody against hepatitis B virus surface antigen.