Comparison of three immunoassays in the screening and characterization of monoclonal antibodies against arginine-vasopressin.
A method for screening monoclonal antibodies (McAbs) to neuropeptides was evaluated using 8-arginine-vasopressin (AVP) as a model. Mice were immunized with AVP-thyroglobulin conjugate and their spleen cells were fused with X 63-Ag8.653 mouse myeloma cells. The resulting hybridoma supernatants were screened for specific antibody production using 3 different assays: solid phase enzyme radioimmunoassay in Terasaki plates (Ter-ELISA), liquid phase radioimmunoassay (LPRIA) and an immunohistochemical technique. From 2 independent fusions, 7 McAbs specific for AVP were obtained. They belonged to the IgG1 subclass and reacted more strongly to the ring part of the nonapeptide. The screening strategy proposed relies upon a crude selection of conjugate-reacting hybridomas, followed by neuropeptide-specific hybridoma identification using both LPRIA (with radioiodinated synthetic peptide) and an immunohistochemical technique (to detect natural neuropeptide). During subcloning steps Ter-ELISA is then chosen, to select for specific clones and to eliminate those reacting with the carrier thyroglobulin.
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D001127:Arginine Vasopressin / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D006825:Hybridomas / Q000276:immunology; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D011863:Radioimmunoassay
D000911:Antibodies, Monoclonal; D001127:Arginine Vasopressin