CS258886B1 - Mouse lymphocyte hybridoma producing antibodies against the viral hepatitis B antigen - Google Patents

Abstract

The present invention relates to a mouse lymphocyte hybridoma producing antibodies against the core antigen of the hepatitis B virus, which is stored in the Hybridoma Collection of the Institute of Molecular Genetics of the Czechoslovak Academy of Sciences under the designation HBc 18 H5G1. The antibody is suitable for detection of the core antigen, for affinity purification of the core antigen and for detection of antibodies against the core antigen in human cells.

Description

The invention relates to a murine lymphocyte hybridoma producing antibodies against the hepatitis O virus cortical antigen.

The hybrid cellular union was constructed by fusing myeloma union mouse SP2 / 0 cells and mouse spleen lymphold cells producing antibodies to the hepatitis B virus root / marrow / antigen. The root antigen is a structural component of hepatitis B virus. accepted as CORE HBcAg.

Determination of levels of anti-cortical antigen in human sera is essential for the diagnosis and monitoring of hepatitis B. This investigation is not yet possible under clinical conditions in Czechoslovakia, as a detection kit containing purified and purified anti-cortical antigen is not available.

This deficiency removes the murine lymphocyte hybridoma producing antibodies against the hepatitis virus core antigen according to the invention. The antibodies can be used in the enzymatic immunoassay for the detection of boron antigen, as well as for affinity purification of the cortical antigen, allowing the purification of high purity cortical antigen from hepatitis B patients.

The monoclonal antibody can also be used in a modified enzymatic immunoassay to detect antibodies against cortical antigen in human sera. Significant progress in the diagnosis and monitoring of hepatitis B is achieved when a cloned hybrid line is available, producing antibodies against the hepatitis B virus cortical antigen.

Said hybridoma was obtained by a method known in the literature (Kohler, G., Milstein, C., Nature 256: 495-497,

(1975) cloning a population of hybrid cells obtained by hybridizing cells from the spleen of BALB / c mice that were immunized with the hepatitis B virus cortical antigen and mouse myeloma line SP2 / 0 cells. The advantage of the HBc18H1-G1 hybridoma is that it produces antibodies specifically reacting with the hepatitis B virus cortical antigen. This monoclonal antibody can be used to detect the cortical antigen, to affinity purify the cortical antigen, and to diagnose anti-cortical antigen antibodies in human sera. The use of this antibody allows the development of a diagnostic kit for the detection of antibodies against cork antigen achieving optimal parameters.

The HBc18H1G1 hybridoma can be cultured in vitro in animal cell media and is adapted for growth in vivo in the peritoneal cavity of BALB / c mouse mice. From cans that are stored in liquid nitrogen, antibody production can be initiated.

Example:

In order to propagate the hybridoma cells in vivo, 1.5 and 10 ⁶ hybridoma cells were injected into the peritoneal cavity of the BALB / 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 of hybridoma growth in the peritoneal cavity, the mouse was sacrificed and unproduced ascites fluid was collected. A total of 5 ml of ascites fluid containing approximately 3 mg / ml of immunoglobulin was obtained. The monoclonal antibody reacted specifically with the cork antigen in an enzyme-immunoassay.

Immunoglobulin was purified from the ascites fluid by the sulphate method. Baroque microtiter plates were coated with a monoclonal antibody produced by HBc 18 H ^ Gi clone at a concentration of 30 µg / ml, then samples containing various concentrations of hepatitis B virus cortical antigen were added and finally a horseradish peroxidase polyclonal antibody conjugate and enzyme immunoassay were added. ** 3 ·· was completed in a standard manner. This assay allowed the detection of 10 ng of cork antigen / ml.

The basic culture medium for HBc 18 H ^ G ^ hybridoma cells is Eagle's minimal essential medium with Hanks salt mixture supplemented with non-essential amino acids. This medium (referred to as the H-MEMd-State of Molecular Genetics of the Czechoslovak Academy of Sciences) is supplemented with inactivated calf serum (Bioveta, Ivanovice na Hané), penicillin and streptomycin for hybridoma culture. The hybridoma is cultured at 37 ° C. The HBc18H1G1 hybridoma produces an IgG2A subclass, the isoelectric point being pH 7.3-7.6.

The monoclonal antibody produced by the HBc 18 HgG.j clone reacts specifically with the hepatitis B virus cortical antigen. This antibody can be used to detect cortical antigen, to affinity purify the cortical antigen, and to detect human anti-cortical antigen antibodies in an enzyme immunoassay. This hybridoma can be used industrially as a source of anti-cortical antigen antibody in diagnostic kits *

Claims (1)

SUBJECT OF THE INVENTION Mouse lymphocyte hybridoma producing antibodies against the hepatitis B virus cortical antigen, deposited in the Collection of Hybridomas of the Institute of Molecular Genetics of the Czechoslovak Academy of Sciences, under the designation HBc 18 HgG |