Production and characterization of monoclonal antibodies to pituitary adenylate cyclase activating polypeptide type I receptor

Pituitary adenylate cyclase activating polypeptide type I receptor (PACAPr) belongs to the novel subfamily of the G-protein coupled receptors with a l ong extracellular N-terminus, which functions as a major binding site for t he PACAP. Three different N-terminal fragments of rat PACAPr were overexpre ssed in Escherichia coli and purified using His-tags or maltose-binding pro tein as anchors for affinity chromatography. The purified and refolded prot eins were used for the production and screening of monoclonal antibodies (M Abs) to PACAPr. Fifteen hybridoma cell lines producing MAbs specific to PAC APr were generated and characterized. Epitope analysis by competitive enzym e-linked immunoadsorbent assay (ELISA) indicated the presence of two groups of overlapping epitopes in the N-terminal fragment of PACAPr. Reactivity o f MAbs with SDS-denaturated and native rat PACAPr was demonstrated by immun oblotting and flow cytometric analysis using transiently transfected COS ce lls and stably transfected CHO cells expressing rat PACAPr. Each antibody w as examined by immunoblotting for the ability to cross react with the human PACAPr in human neuroblastoma NB-OK cells and most of them were shown to r ecognize human PACAPr as effectively as rat PACAPr. MAbs against the N-term inal extracellular domain of PACAPr can be used for the immunochemical stud y of the receptor-ligand interaction and for the investigation of PACAPr di stribution in normal and tumor tissues.