Subtype specific recognition of human alpha(2)C2 adrenergic receptor using monoclonal antibodies against the third intracellular loop

Monoclonal antibodies (Mabs) against human alpha(2)C2-adrenergic receptor (alpha(2)C2-AR) were raised in mice and characterized, Bacterially expressed fusion protein consisting a sequence from the putative third intracellular loop (amino acids 213-343) of human alpha(2)C2 and glutathione-S-transferase (GST) was used as antigen. Results from mass spectrometry of purified thrombin cleaved alpha(2)C2 polypeptide suggested that the epitope region would lie near the aminoterminal end of the 3rd intracellular loop of human alpha(2)C2-AR. Elevation of Mabs was detected with Western blotting from mouse blood samples. Three alpha(2)C2 specific cell clones were expanded to in vitro production in hollow fiber systems. The specificity of the Mabs was further determined by immunoprecipitation and immunocytochemistry. Scatchard analysis of thrombin digested, purified, Europium-labelled antigen (amino acids 213-343 of alpha(2)C2) revealed binding affinity constants of 0.4 x 10(9), 0.7 x 10(9) and 1.6 x 10(9) M-1 and K(d)s of 2.6, 1.4 and 0.6 nM for the three Mabs 2B1, 3G3 and 7G1, respectively. (C) 1997 Academic Press.