Abstract
It is quite difficult to generate monoclonal antibodies that recognize the three-dimensional structures of the antigens of interest. To address this limitation, we developed a new hybridoma technology termed "optimized stereospecific targeting (SST)". Here we aimed at generating stereospecific monoclonal antibodies against a G protein-coupled receptor (GPCR). The optimized SST technique enabled the efficient production of conformation-specific monoclonal antibodies against human corticotropin-releasing hormone receptor 1 (huCRHR1). Hybridoma cells secreting stereospecific monoclonal antibodies were selectively cloned by a limiting dilution method and the target monoclonal antibodies were purified by protein A column chromatography. They specifically cross-reacted with native huCRHR1 expressed on the surface of CHO cells, whereas they showed no affinity for MDA-MB-231 cancer cells, which abundantly express EphA2 on the cell surface. Furthermore, immunofluorescence analysis revealed that treatment of huCRHR1-expressing CHO cells with 4% paraformaldehyde led to a decrease in the affinity of purified monoclonal antibodies for intact huCRHR1 on the cell surface. In addition, purified monoclonal antibodies showed no cross-reactivity with huCRHR1 expressed on Sf9 insect cells. These results strongly suggest that monoclonal antibodies generated by the optimized SST technique feature specific binding to the intact form of the target GPCR on mammalian cells.