Studies on stabilization of a human-mouse heterohybridoma producing sperm immobilizing antibody by recombinant DNA technology.

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For stabilizing the gene coding for sperm immobilizing monoclonal antibody (IgM, lambda from a human-mouse hetero-hybridoma (H6-3C4), the human rearranged immunoglobulin mu-chain genes were cloned from the hybridoma. The cloned V region of the heavy chain (VH) gene was ligated to human immunoglibulin gamma1-heavy chain constant region (C gamma 1) genes. This resulted in the heavy-chain class-switched from mu-chain to gamma 1-chain of H6-3C4 asntibody.The class-switched heavy-chain gene as well as the cloned lambda-chain gene were introduced into mouse myeloma cell line X63Ag8.653 by protoplast fusion and electroporation respectively.Five stable transformants were established. The culture supernatants were electrophoresed on a SDS-PAGE and the proteins were transblotted to the nitrocellulose membranes. Followed by staining with peroxidase conjugated anti-human gamma-chain or lambda-chain antibodies. Under non-reducing condition, all transformants secreted the complete form of human IgG molecules with a molecular weight of 160Kd. Under reduciht condition, the 54Kd band of human gamma-chain and 25Kd band of human lambda-chain were observed. The titers of sperm immobilizing antibody of the culture supernatants of the transformants were in the range of 10-20 units in SI50 titer.The amounts of monoclonal antibodies secreted from the transformants were about 0.2-0.5umg/ml. The SI50 titers and IgM concentrations in the supernatant of H6-3C4 hybridoma were about 5,000 units and 2.0mug/ml, respectively. If we considered the lower complement-dependent sperm immobilizing activity of IgG1 as compared to IgM, the affinity of the recombinant antibody appeared to be the same as that of the original H6-3C4 monoclonal antibody. Thus we succeeded in establishing the stable transformants producing human IgG1 which fully retained the specificity of the original sperm immobilizng human monoclonal IgM.