Comparison of three heparin bovine serum albumin binding methods for production of antiheparin antibodies.

IgG antiheparin antibody secreting hybrid cells have not been successfully raised so far. Therefore, we compared three different conjugating procedures for preparation of LMMH-protein conjugates. Albumin was chosen as carrier protein. Only the conjugate prepared by reductive amination induced antiheparin antibody production in mice. Conjugates prepared by the carbodiimide method and Mannich reaction did not induce heparin immunogenicity and did not induce antibody production in mice. Antiheparin antibody production in mice and by hybrid cells was detected by assaying serum and culture supernatant. LMMH-tyramine, labeled with 125I, was bound by the antibodies and immunoprecipitated by goat anti-mouse IgG antibodies. The tracer was competitively inhibited by nonlabeled heparin for binding to the heparin antibodies. Hybridoma cells producing the specific antiheparin antibody were cloned and subcloned up to monoclonality. Six hybrids were selected and designated H1.18, H1.21, H3.1, H3.17, H3.18, H4.24. In conclusion, six monoclonal antiheparin antibodies were raised using a LMMH-albumin conjugate prepared by reductive amination.