The objective of this project is to produce and characterize some specific murine monoclonal antibodies against dynorphin A (1-17) and dynorphin B (1-13) (or rimorphin). The hybridomas thus raised will be used as consistent and cost-effective sources of the specific monoclonal antibodies which will supersede the antisera currently produced by some research groups. Using radioimmunoassay (RIA) and solid-phase enzyme-linked immunosorbent assay (ELISA), these monoclonal antibodies can be widely applied for the quantitation of the dynorphins in both normal and diseased neural tissues. These antibodies can also be utilized for the localization of dynorphin-immunoreactive pathways in the nervous system using immunocytochemistry. The long-term goal of this study is to provide a better immunological means to elucidate the mechanisms of processing of proenkephalin B and the metabolism of dynorphins in different animal species, with regard to their possible role in neurotransmission and neuromodulation.Furthermore, the functional significance and the differential actions of dynorphin A and dynorphin B in the physiology of analgesia, sedation, visceral reflex, and neuroendocrine regulation can be examined.In this proposed study, BALB/c mice will be immunized with dynorphins conjugated to thyroglobulin for a month, and the immune spleen cells then isolated for in vitro immunization for 4 days. The Iymphocytes are then fused with myeloma cells (P3 x 63-Ag8.653); the hybridomas will be isolated by HAT medium and cloned. The monoclonal antibodies are screened for titer and typed for subclasses, using ELISA, and for the degree of cross-reactivity with other related opioid peptides, using RIA. The specific monoclonal antibodies will be produced from ascites fluid by injecting the hybridomas into BALB/c mice, and purified by hydroxylapatite column chromatography. The specific hybridoma cells will be stored frozen as permanent sources of the antibodies.National Institute of Mental Health (NIMH)
