CS265494B1 - Mouse lymphocytar hybridome producing antibodies against antigen of b-hepatitis virus (hbeag) - Google Patents

Abstract

The present invention relates to mouse lymphocytic hybridoma producing antibodies against HBeAg, which is deposited in the Collection of Hybridomas Institute of Molecular Genetics of the Czechoslovak Academy of Sciences under the designation IMG CZAS HBe A2C12F9B1. The antibody is suitable for detection and affinity purification HBeAg.

Description

The invention relates to a murine lymphocyte hybridoma producing antibodies against the hepatitis B virus e 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 hepatitis B virus e antigen (HBeAg). The E antigen is produced by proteolytic cleavage of the core antigen in an infected liver cell, followed by leaching of the e antigen into the blood.

Determination of HBeAg in the blood is essential for diagnosis and especially for monitoring the course of hepatitis B. These tests have so far been performed using human polyclonal antibodies. However, the assay using human polyclonal anti-HBeAg antibodies achieves low sensitivity and specificity of the reactions.

This deficiency is eliminated by the murine lymphocyte hybridoma producing antibodies against HBeAg according to the invention, deposited in the collection of hybridomas of the Institute of Molecular Genetics of the Czech Academy of Sciences, Flemingovo 2, under the designation HBe ₂ C ₂₂ F _g B ₂ ·

Said antibodies can be used in an enzymatic immunoassay to detect HBeAg, as well as affinity purification of HBeAg, allowing the purification of high purity e antigen.

Substantial progress in the diagnosis and monitoring of hepatitis B is achieved when a cloned hybrid line is available, producing! antibodies against hepatitis B virus e antigen

Said hybridoma was obtained by a method known in the literature (Kčhler, G., Mistein,

(Nature 256: 495-497, 1975) by cloning a population of hybrid cells obtained by hybridizing cells from the spleen of BALB / c mice that were immunized with HBeAg and mouse myeloma line SP2 / 0 cells. HBeAg was prepared by digesting HBeAg, which was purified from bacteria containing a recombinant expression plasmid.

An advantage of the HBe ^A 2 ^C 12 ^F 9 ^B 1 hybridoma is that it produces antibodies specifically reacting with HBeAg. This antibody can be used to detect HBeAg and to affinity purify HBeAg.

The use of this antibody allows the development of a diagnostic kit for the detection of HBeAg achieving optimal parameters.

The HBe ^A 2 ^C 12 ^F 9 ^B 1 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. canned products that are stored in liquid nitrogen can be started by producing an antibody.

Example

To amplify hybridoma cells in vivo, 1x10 ⁶ hybridoma cells were injected into the peritoneal cavity of BALB / c mouse mice. 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 15-30 days of hybridoma growth in the peritoneal cavity, the mice were sacrificed and the ascites fluid collected.

On average, 4 ml of ascites fluid was obtained containing approximately 4 mg / ml of immunoglobulin. The monoclonal antibody reacted specifically with HBeAg in an enzyme-linked immunoassay.

Immunoglobulin was purified from the ascites fluid by the sulfate method. The wells of the microtiter plates were coated with the monoclonal antibody produced by the clone HBe ^A 2 ^C 12 ^F 9 ^{B 1 at a} concentration of 50 µg / ml. Samples containing different concentrations of HBeAg were then added, and finally, a polyclonal anti-HBeAg conjugate with horseradish peroxidase was added and the enzyme immunoassay was completed in a standard manner.

This assay allowed the detection of 10 ng HBeAg / ml.

The basic culture medium for HBe ^A 2 ^C 12 ^F 9 ^B 1 hybridoma cells is Eagle's minimum 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 HBe ^A 2 ^C 12 ^F 9 ^B 1 hybridoma produces an IgG2B isotype antibody, the isoelectric point is pH 6.7 to 7.3.

The monoclonal antibody produced by clone HBe ^A 2 ^C 22 ^F 9 ^B 1 reacts specifically with HBeAg.

This antibody can be used for the detection and affinity purification of HBeAg. This hybridoma can be industrially used as a source of HBeAg antibody in diagnostic kits.

Claims (1)

Mouse lymphocyte hybridoma IMG CZAS HBe ^A 2 ^C 12 ^F 9 ^B 1 'producing antibodies against hepatitis B virus e antigen (HBeAg).