Molecular characterization of antipeptide antibodies against the 5-HT(1A) receptor: Evidence for state-dependent antibody binding

Differential immunohistochemical labeling is often observed using different antibodies against the same protein. Two polyclonal antipeptide antibodies against the 5-HT(1A) receptor have been generated by our group. The S1A-170 (aa 170-186) and 258 (aa 258-274) are specific for sites in the second extracellular loop and third intracellular loop, respectively. Comparison of the labeling patterns of these two antibodies and other antipeptide antibodies against the 5-HT(1A) receptor revealed that although similar populations of cells were labeled, individual antibodies favor certain staining patterns. Immunocytochemistry and western blotting results of transfected cell lines and brain tissue revealed the following: (1) both the S1A-170 and S1A-258 are specific for the 5-HT(1A) receptor when used for immunocytochemistry in transfected HEK-293 and COS-1 cells; (2) when expressed in cultured cell lines, the 5-HT(1A) receptor is differentially glycosylated dependent on cell type, and the S1A-258 is specific for only certain species on immunoblots; and (3) the S1A-258 and L₅B₇ label common bands at 40 and 70 kDa on immunoblots of hippocampal proteins, but show opposite staining intensities. These results provide evidence for the immunocytochemical specificity of both the S1A-170 and S1A-258 and suggest that the discrepancies noted in immunohistochemistry may be due in part to different molecular conformations.