Development of monoclonal antibody targeting membrane protein utilizing modified Ecobody technology

This study presents a preliminary report on the development of rabbit monoclonal antibodies targeting the extracellular domain of rat G protein-coupled receptor 54 (GPR54). The extracellular domain of GPR54 (GPR54 ECD) was fused with the albumin-binding domain of Streptococcus sp. protein G, and subsequently expressed in Escherichia coli, purified, and used to immunize a rabbit. Antigen-specific B cells were isolated using fluorescently labeled peptides corresponding to the GPR54 extracellular domain 2 region. Antibody genes were amplified from the sorted B cells, cloned into vectors, and transformed into competent cells. Combinatorial pairing of light and heavy chain genes, followed by cell-free protein synthesis, led to the identification of four antibody pairs with reactivity toward the GPR54 ECD based on enzyme-linked immunosorbent assay. Western blotting confirmed the ability to detect target proteins with minimal cross-reactivity. This study highlights the possible use of the combinatorial pairing of antibody genes to isolate rare antigen-specific monoclonal antibodies.