Conformation-specific monoclonal antibodies recognizing the native structure of G protein-coupled receptor (GPCR)

Class-Switching of B Lymphocytes by DNA and Cell Immunization for Stereospecific Monoclonal Antibodies against Native GPCR

“…The optimized SST technique was used to generate hybridoma cells secreting the target conformation-specific monoclonal antibodies, as previously reported [8][9][10]. In brief, the spleen was aseptically removed from a mouse after DNA immunization and cell immunization.…”

“…Human CRHR1-expressing CHO-K1 cells (2.0-3.5 × 10 4 cells/200-350 µL) were incubated in a 96-well culture plate in complete E-RDF medium at 37 • C in a humidified atmosphere containing 5% CO 2 for one to two days. Each well was washed with PBS, and hybridoma supernatant or 1/50-1/1600-fold diluted mouse serum after DNA and cell immunization was added as a primary antibody with incubation for 2 h at 4 • C. After washing with PBS, 1/10,000-diluted goat anti-mouse IgG (H + L) antibody conjugated with HRP was added as a secondary antibody, and the mixture was incubated for 1 h at 4 • C. Each well was washed at least five times with PBS, and the absorbance was measured at 490 nm using o-phenylenediamine as a substrate [9,10]. ∆OD values at 490 nm (OD490 nm-blank OD490 nm) denote specific binding of stereospecific monoclonal antibodies to the intact antigen.…”

“…B cell and myeloma cell complexes were then selectively fused by electrical pulses to produce hybridoma cells that can secrete conformation-specific monoclonal antibodies (Figure 1). The critical points of the technique are DNA immunization, B-cell selection by antigen-expressing myeloma cells, and selective fusion of B cell and myeloma cell complexes [8][9][10]. DNA immunization must be a privileged method to selectively sensitize the target B lymphocytes that produce antibodies recognizing the native structures of the target antigen.…”

“…We previously reported a novel technology termed optimized stereospecific targeting (SST) [8][9][10], which consists of three important steps: (1) DNA immunization;…”

“…(2) B-cell selection by antigen-expressing myeloma cells; and (3) selective fusion of B cell and myeloma cell complexes, to generate hybridoma cells that secrete stereospecific monoclonal antibodies. The optimized SST technique features the selective production of conformation-specific monoclonal antibodies against membranous proteins such as receptors with high efficiency [9,10].…”