PRODUCTION OF TYPE-II COLLAGEN SPECIFIC MONOCLONAL-ANTIBODIES

Immunoassays are used for the specific measurement of type II collagen , a major.cartilage protein, which is lost in osteoarthritic joints. P oor immunogenicity and species dependent immune response to type II co llagen make it difficult to obtain specific antibodies required for im munoassay development. In addition, type II collagen antibodies exhibi t reactivity to structurally dissimilar antigens such as actin, myoglo bin, thyroglobulin and ssDNA, complicating the isolation of specific a ntibodies. It is therefore necessary to characterize the antibody reac tivity against both noncollagenous antigens and different collagen typ es. In this study, immune response to type II collagen was improved by conjugation to carrier proteins, KLH and BSA. Hybridomas were generat ed by fusions of lymphocytes derived from lymph nodes or spleens with X63-653-Ag8 myeloma cells. Compared to spleens, the utilization of lym ph nodes as a source of lymphocytes resulted in a 23% higher number of hybridomas secreting type II collagen antibodies. Hybridomas secretin g polyreactive antibodies were identified based on their reactivity to thyroglobulin and eliminated. Extensive testing of the remaining mono clonal antibodies with other structurally dissimilar antigens and vari ous types of collagen for reactivity, allowed us to isolate specific m onoclonal antibodies to type II collagen. We emphasize the importance of characterization of the reactivity of type II collagen monoclonal a ntibodies before employing them for immunoassays.