Anti human igm

The rRVFV-N-based IgM capture ELISA followed the same steps as with the inactivated virus-based ELISA system with some modifications. For each serum sample to be tested, 4 wells were coated with 1:500 dilution of anti-human IgM (Cappel, MP Biochemicals). Then 1:100 diluted human serum was added to the 4 wells. Two wells were added with 50 ng rRVFV-N protein (positive antigen) and the other two wells were added with 50 ng of rSFTSV-N protein (negative antigen). A 1:10,000 dilution of anti-RVFV rabbit hyperimmune serum was added to all four wells, after which a 1:10,000 diluted peroxidase conjugated anti-rabbit IgG (American Qualex, Califonia, USA) was then added. Color development was done by adding ABTS substrate.

Cell lines and primary cells were treated with acalabrutinib and stimulated with plate-bound anti-human IgM (MP Biomedicals; Santa Ana, CA). Plates were prepared by incubating a 10 μg/mL IgM solution in PBS for 6 to 12 hours at 4°C, and then rinsing with PBS. Whole cell lysates were prepared as previously described [24 (link)], followed by polyacrylamide gel electrophoresis and transfer of proteins to nitrocellulose membranes. The following polyclonal antibodies were used to detect protein on immunoblots: anti-phospho-PLCG2 (Tyr 1217, Cat. #3871), anti-PLCG2 (Cat. #3872), anti-phospho-IKBA (Ser32, Cat. #2859), anti-IKBA (Cat. #4812), anti-phospho-ERK1/2 (Thr202/Tyr204, Cat. #9101), anti-ERK1/2 (Cat. #9102), anti-phospho-AKT (Thr308, Cat. #9257), and anti-AKT (Cat. #9272), anti-phospho-NFKB P65 (Ser536, Cat. #3031), anti-NFKB P65 (Cat. #3034)(Cell Signaling Technologies; Danvers, MA), anti-phospho-BTK (Tyr223, Cat. #ab68217, Abcam, Cambridge, MA), and anti-BTK (cat. #B3187, Sigma-Aldrich).