Human RP105 monoclonal antibody enables the production of antigen-specific antibody

Antibody detection is a critical approach in clinical contexts. Detecting antibodies, particularly those directed against donor HLA in organ transplantation and self-antigens in autoimmune diseases, is crucial in diagnosis and therapy. Radioprotective 105 (RP105, CD180 ), a type I transmembrane protein with structural similarities to Toll-like receptor 4, is expressed in immune-competent cells, such as B cells. Studies involving mice have reported that the antimouse RP105 antibody strongly activates B cells and triggers an adjuvant effect against viral infections. However, the antihuman RP105 antibody (ɑhRP105) weakly activates human B cells. In this study, we established new culture conditions under which human B cells are strongly activated by ɑhRP105. When combined with CpGDNA, this activation induced a heightened production of interleukin 6. Under these culture conditions, specific antibody production against blood group carbohydrates, ɑGal, and SARS-CoV-2 within supernatants from human B-cell cultures was successfully detected. Furthermore, comprehensive analysis employing LC/MS/MS analysis and single-cell RNA sequencing revealed that ɑhRP105 triggered a different activation stimulus from CpGDNA. These findings could be applied to developing a straightforward approach for identifying antibody-producing B cells in cases involving transplant rejection and autoimmune diseases. PBMCs from 3 healthy volunteers were stimulated with ɑhRP105 for 36 hours. Activated and non-activated CD19+ B-cells were stained with six different hashtag antibodies (contorol: #1, 3, and 5 vs stimulate:#2, 4, and 6), anti-CD19, and anti-CD20. Hashtagged cells from three patients were sorted, pooled, and analyzed by single-cell RNA-seq.