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 long extracellular N-terminus, which functions as a major binding site for the PACAP. Three different N-terminal fragments of rat PACAPr were overexpressed in Escherichia coli and purified using His-tags or maltose-binding protein as anchors for affinity chromatography. The purified and refolded proteins were used for the production and screening of monoclonal antibodies (MAbs) to PACAPr. Fifteen hybridoma cell lines producing MAbs specific to PACAPr were generated and characterized. Epitope analysis by competitive enzyme-linked immunoadsorbent assay (ELISA) indicated the presence of two groups of overlapping epitopes in the N-terminal fragment of PACAPr. Reactivity of MAbs with SDS-denaturated and native rat PACAPr was demonstrated by immunoblotting and flow cytometric analysis using transiently transfected COS cells and stably transfected CHO cells expressing rat PACAPr. Each antibody was 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 recognize human PACAPr as effectively as rat PACAPr. MAbs against the N-terminal extracellular domain of PACAPr can be used for the immunochemical study of the receptor-ligand interaction and for the investigation of PACAPr distribution in normal and tumor tissues.
Zvirblienë
A
A
Institute of Biotechnology, Vilnius, Lithuania.
Zvirblis
G
G
Cao
Y J
YJ
Fahrenholz
F
F
D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000276:immunology*; D000915:Antibody Affinity; D001667:Binding, Competitive / Q000276:immunology; D016466:CHO Cells / Q000276:immunology; D019556:COS Cells / Q000276:immunology; D006224:Cricetinae; D003429:Cross Reactions / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay; D018604:Epitope Mapping; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D008934:Mitogens / Q000276:immunology; D010446:Peptide Fragments / Q000096:biosynthesis / Q000235:genetics; D051381:Rats; D051236:Receptors, Pituitary Adenylate Cyclase-Activating Polypeptide; D051237:Receptors, Pituitary Adenylate Cyclase-Activating Polypeptide, Type I; D011979:Receptors, Pituitary Hormone / Q000276:immunology* / Q000378:metabolism; D011994:Recombinant Proteins; D014162:Transfection / Q000276:immunology; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; D008934:Mitogens; D010446:Peptide Fragments; D051236:Receptors, Pituitary Adenylate Cyclase-Activating Polypeptide; D051237:Receptors, Pituitary Adenylate Cyclase-Activating Polypeptide, Type I; D011979:Receptors, Pituitary Hormone; D011994:Recombinant Proteins