CS255642B1 - Mouse lymphocytar hybridome forming antibodies against cortical antigen of hepatitis-b virus - Google Patents

Abstract

The solution relates to mouse lymphocyte antibody-producing hybridoma against hepatitis B virus cortical antigen, which is deposited in the Institute's Hybridoma Collection molecular genetics of the Czechoslovak Academy of Sciences under the designation IMG-CZAS HBc 18 E ^ B. The antibody is Suitable for detection of cortical antigen for affinity purification of cortical antigen and for the detection of anti-cortical antibodies antigen in human sera.

Description

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

The hybrid cell line was constructed by fusing cells of the mouse myeloma line SP2 / 0 and mouse spleen lymphoid cells producing antibodies against the hepatitis B virus root / marrow / antigen. The root antigen is a structural component of the hepatitis B virus virion. commonly 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 examination is not yet possible under clinical conditions in Czechoslovakia, as a detection kit containing purified cortical antigen and antibodies against it is not available.

This deficiency is eliminated by the murine lymphocyte hybridoma producing antibodies against the hepatitis B virus cortical antigen of the invention deposited in the Collection of Hybridomas of the Institute of Molecular Genetics of the Czechoslovak Academy of Sciences, Prague 6, Flemingovo nam. 2, under the designation IMG-HBc Czasy ^E 18 ^B u · I2 Said antibodies can be used in the enzyme immunoassay for the detection of cortical antigen as for affinity purification cortical antigen, which allows a highly purified pure crustal antigen from the liver of patients for hepatitis type B.

The monoclonal antibody can also be used in a modified enzyme immunoassay for the detection of anti-cortical antigen antibodies in human sera. Substantial progress in the diagnosis and monitoring of hepatitis B is achieved when a cloned hybrid line producing antibodies against the hepatitis B virus cortical antigen is available.

The hybridoma was obtained by a method known in the literature (Ktthler, G., Milstein, C., Nature 256: 495-497, 1975) by cloning a population of hybrid cells obtained by hybridizing cells from the spleen of BALB / c mice immunized with the hepatitis virus root antigen. B and cells of the mouse myeloma line SP2 / 0. The advantage of the HBc 18 Ε ^Β ^ ^3e 'hybridoma produces antibodies specifically reacting with the hepatitis B virus cortical antigen. This monoclonal antibody can be used to detect 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.

Hybridoma HBc ^E ^^ 18 ^ 2 ^B ^ ^{3e mo well} cultured in vitro in a suitable media for animal cells, and adapted to grow in vivo in the peritoneal cavity of BALB / c. From cans that are stored in liquid nitrogen, antibody production can be initiated.

Example

To expand the hybridoma cells in vivo, 1.5 x 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 the produced ascites fluid was collected. A total of 5 ml of ascites fluid was obtained containing approximately 5 mg / ml of immunoglobulin. The monoclonal antibody reacted specifically with the cortical antigen in an enzyme immunoassay.

Immunoglobulin was purified from the ascites fluid by the sulphate method. The wells of the microtiter plates were coated with the monoclonal antibody produced by clone HBc 18 ^e 11 ^e 12 ' ^{at a concentration of 30} µg / ml. Samples containing various concentrations of the hepatitis B virus cortical antigen were then added, and finally the horseradish peroxidase polyclonal antibody conjugate antibody against cortical antigen was added and the enzyme immunoassay was completed in a standard manner. This assay allowed the detection of 10 ng of cork antigen / ml.

Essential culture medium for HBc 18 ^E 21 ^B 12 ^Ea hybridoma cells 94's minimal essential medium with Hanks salt mixture supplemented with non-essential amino acids. This medium (referred to as H-MEMd - The Institute 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 HBc 18 hybridoma produces a subclass antibody

IgG3, isoelectric point is pH 9. _Λ

The monoclonal antibody produced by the HBc 18 E ^^ B ^ ₂ 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 antibodies against cortical antigen in an enzyme immunoassay. This hybridoma can be industrially used as a source of anti-cortical antigen antibody in diagnostic kits. ,.

SUBJECT OF THE INVENTION

Claims (1)

SUBJECT OF THE INVENTION Mouse lymphocyte hybridoma IMG-CZAS HBc 18 Ε ^ Β · ^, producing antibodies against the hepatitis B virus cortical antigen