Preparation Of The Monoclonal Antibody Against BCG HSP65

Mycobacterial 65kD Heat shock protein (HSP65) is one of the most important antigens of BCG, it was one of protein chaperones (contributing to protein folding, stability and transport). More recent studies have shown that mycobacterial Hsp65 exhibited strong immunogenicity and contained strong T cell epitopes that could be used as a convenient and safe carrier molecule for presenting foreign polypeptide epitopes to the immune system in vivo in the absence of adjuvants.In order to detect and pure the HSP65 or fusions of it, we must establish hybridoma cell lines producing anti-HSP65 antibody to get anti-HSP65 antibodies. Female BALB/c mice immunized with the purified antigen of HSP65, and the spleen cells of them were fused with SP2/0 cells to raise hybridomas. After inoculation for several times, the positive of clones were screened by indirect ELISA and the characteristics of the antibodies were detected.Five hybridoma cell lines were established and four of them, 4G2, 6C7 , 7F4 and 10B12 secreted high quality antibodies steadily. And the antibodies secreted were stable to environmental factor such as temperature, acid and alkali, etc. Among them, 4G2, 6C7, 7F4, 10B12 were IgG2a subtype, k light chain, others were IgG1 subtype and k light chain.The study has practical value in analyzing the bio-characteristics of HSP65, and we can use them establish an efficient platform to detect and pure the HSP65 or the fusions of it.