Target-specific monoclonal antibodies detect the GPCRs PAR1 or DRD2 expressed on live cell surfaces by Flow Cytometry 2260881

Abstract

Abstract Introduction G protein-coupled receptors (GPCRs) are the largest family of multi-transmembrane proteins, and major targets for drug discovery. Antibody staining/detection of GPCR targets on live cells is important for studying receptor expression, localization, and dynamics in their native, functional state. Due to their complex structure and dynamic conformations, it is very challenging to develop antibodies against GPCRs. Protease-activated receptor-1 (PAR1) and Dopamine receptor D2 (DRD2) are Class A GPCRs. PAR1 is a promising target for cancer therapeutics, while DRD2 is a pivotal target for therapeutic intervention in the treatment of neuropsychiatric disorders. Methods Based on multiparameter analyses of PAR1 and DRD2 sequences and structures, we designed unique immunogens and immunized mice. After detection of PAR1 or DRD2 antibodies in immunized mouse sera, immune mouse B cells were isolated to generate PAR1- or DRD2-specific monoclonal antibodies (mAbs) using hybridoma technology. The PAR1- or DRD2-mAbs were characterized for binding specificity, affinity and live cell staining using multiple assays. Results Using unique fit-for-target immunization platforms, PAR1 or DRD2 specific antibodies were elicited in mice that could bind to the target antigen by ELISA and target protein expressed on live cell surface by flow cytometry. Subsequently, PAR1 or DRD2 specific mAbs were developed from immunized mouse B cells. PAR1- or DRD2-specific mAbs had high binding specificity and affinity to the relevant target antigen as measured by ELISA and surface plasmon resonance (SPR). Flow cytometry analyses demonstrated that the mAbs could potently bind to PAR1- or DRD2 expressed on live cell surfaces. Conclusion As antibody staining of GPCRs on cell surface is a foundational and indispensable technique, these PAR1 and DRD2 specific, potent and live cell binding mAbs can be very useful tools for PAR1 and DRD2 research and drug discovery. Funding Source n/a Topic Categories Technological Innovations in Immunology (TECH)