CS252792B1 - Mouse Lymphocytic Hybridoma HBs 720 ^ A? - Google Patents

Abstract

The solution concerns a mouse lymphocyte hybridoma producing an antibody against the surface antigen of hepatitis ES, which is stored in the Hybridoma Collection of the Institute of Molecular Genetics of the Czechoslovak Academy of Sciences under the designation HESs 7 2 C A.?. The monoclonal antibody of this hybridoma itself is suitable for detecting the surface antigen in an enzyme immunoassay.

Description

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Mouse Lymphocyte Hybridoma HBs 720 ^ A?

The invention relates to a murine lymphocyte hybridoma producing an antibody against the ES hepatitis surface antigen, which is deposited in the Hybridoma Collection of the Institute of Molecular Genetics of the Czechoslovak Academy of Sciences under the designation HESs 7 2 C A. The monoclonal antibody of this hybridoma itself is suitable for the detection of surface antigen in an enzyme-linked immunoassay.

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The present invention relates to a novel hybridoma, i.e. a hybrid cell line which has been constructed by fusing cells of the myeloma line Sp2 / O and washing spleen lymphoid cells producing antibodies against the hepatitis 3 virus surface antigen. in the diagnosis of the disease.

To date, antibodies to the surface antigen have been produced by injecting purified surface antigen repeatedly into production animals, most commonly rabbits (goats).

The serum of the immunized animals collected after a period of antigen treatment serves as a source of antibodies. This procedure, which is called conventional immunization, has 2 major unavoidable disadvantages. For each immunization, a surface antigen is required, the purification of which is a very costly and health-risk procedure. Antibodies against the surface antigen, but also antibodies against human serum proteins, especially against human serum albumin, these ballast antibodies need to be removed.

The disadvantages of conventional sera against surface antigen are eliminated by the use of hybridoma technology. The hybridoma-monoclonal antibody product is directed against a single antigenic determinant of the surface antigen, and monoclonal antibody production does not require antigen immunization of animals. The use of a monoclonal antibody further enables the sensitivity of the SL13A detection method, which is used to diagnose the disease, to be increased.

It is known from the published results that the hepatitis 3 virus surface antigen is not the only antigenic component, but is a complex of several antigens. The group-specific determinant a is found in all isolates. The other 2 pairs of specific determinants d / y and r / w do not usually occur together and are considered alleles (Le Bovier,

G.L .: The heterogeneity of Australia antigen, J. Infect.

Dis. 123: 671 (1971)

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Monoclonal antibodies against the group-specific determinant and are preferred for diagnostic use

Substantial progress in the diagnosis of heoatitis B, which results in both sensitivity and increased assay reliability, is achieved when cloned hybrid cell lines producing a monoclonal antibody against a group specific and surface antigen determinant are available. Such a hybridoma is deposited in the Collection of Hybridomas of the Institute of Molecular Genetics of the Czechoslovak Academy of Sciences in Prague, Plemingovo nám. 2, by HBs 720 ^ ^

The hybridoma was obtained by a method known in the literature (Kunitomo, K., Iahara, S., Saji, H., Hosoi, T. Monoclonal antibodies to hepatitis B surface antigen (HBsAg) and a tool for the au tomated HBsAg screening. Vox Sanguinis 4b: 104) 1983) »by cloning a population of hybrid cells obtained by hybridizing cells from the spleen of BAL3 / c mice that have been immunized with the surface antigen of Heoatitis 3 virus and cells of the mouse myeloma line Sp2 / 0.

The advantage of hybridomas is that they produce homogeneous monoclonal antibodies that react with a surface antigen. The HBs 720-γγ hybridoma can be cultured in vitro in animal cell media and is adapted for growth in vivo in the oeritoneal cavity of BALB / c mouse mice. From cans that are stored in liquid nitrogen, antibody production can be started without additional antigen. The antibody produced by clone HBs 720 ^ A ^ reacts specifically with a determinant of hepatitis B virus surface antigen.

Example

In order to propagate the hybridoma cells in vivo, 2x10 ^b hybridoma cells were injected into the peritoneal cavity of a 3ALB / c mouse. This mouse was injected with 0.7 ml paraffin oil 14 days prior to the administration of the hybridoma cells into the peritoneal cavity. After 12 days growth of hybridomas in peritoneal

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- 4 cavities were sacrificed and the ascitic fluid produced was collected. • A total of 5 ml of ascitic fluid containing 10 mg / ml of immunoglobulin was obtained. The monoclonal antibody reacted specifically with the surface antigen of the Heoatitis B virus in the enzyme and radioimmunoassay.

Immunoglobulin was petrified from the ascites fluid by the sulfate method and microtiter plates were coated with this immunoglobulin at a concentration of> 0 µg / ml. Samples examined for the presence of the Heoatitis B virus surface antigen and horseradish peroxidase surface antigen polyclonal antibody conjugate were then added and the enzyme immunoassay was completed in the manner used to detect the surface antigen using the 1L1SA HtísAg Micro 1 diagnostic kit (Ú50L). The use of monoclonal antibody allowed the detection of 1.5 ng of surface antigen / ml, which represents a two-fold increase in assay sensitivity compared to a system using conventional polyclonal antibodies.

The basic culture medium for HBs 72C .A "hybridoma cells is Hagl's minimal essential medium with Hanks salt mixture supplemented with non-essential amino acids, the photo medium (referred to as H-MZMd - Institute of Molecular Genetics GtíAV) is supplemented with penicillin for action of hybridoma. - inactivated veal dusk (3ioveta, Ivanovice na Hané). Hybridoma is cultured at 37 ° C · HBs 720 ^ Αγ hybridoma produces IgG1 subclass antibody, isoelectric point is pH 5j2-5> 4 ·

The monoclonal antibody produced by clone HBs 720 ^ Αγ reacts specifically with the surface antigen of the Heoatitis B virus. This hybridoma can be industrially used as a source of anti-surface antigen antibody in diagnostic kits.

Claims (1)

PH OF THE SOUL OF THE INVENTION HBs 720 ^ mouse lymphocyte hybridoma producing a monoclonal antibody of IgG1 subclass against hepatitis B virus surface antigen Printed by Moravian Printing Works,