Anti igm

Wells of a high-binding microtiter plate (Greiner Bio-One cat # 655061) were coated with 50 μL anti-MBP (New England Biolabs) at 3 μg/mL in TBS pH 7.4, and then blocked with 100 μL of blocking solution (3% non-fat milk in TBST). The plate was washed three times using wash buffer (TBS containing 0.2% Tween 20). 50 μL of 2 μg/mL MBP fused full-length nucleocapsid protein and MBP proteins in blocking solution were added to respective wells. The plates were incubated for 1 hour at 37°C. The plates were washed 3 times, then 50 μL of heat-inactivated serum at 1:40 dilution was added to wells containing full-length N protein and MBP proteins were added respectively and further incubated for 1 hour at 37°C. The plates were washed with wash buffer, then 50 μL of alkaline phosphatase-conjugated secondary goat anti-Human anti-IgG (Sigma Cat), anti-IgA (Abcam), and anti-IgM (Sigma) at 1:2500 dilution was added and incubated for 1 hour at 37°C. The plate was washed, and 50 μL p-Nitrophenyl phosphate substrate (SIGMA FAST) was added to the plate and absorbance measured at 405 nm using a plate reader (Molecular Devices SpectraMax ABS Plus Absorbance ELISA Microplate Reader). Appropriate control sera were included in the study.

Mouse spleens were harvested, disaggregated into single-cell suspension, and follicular B cells were purified by negative selection using biotinylated CD9 (KMC8, BD Biosciences), CD43 (S7, BD Biosciences) and CD93 (AA4.1, eBioscience) antibodies and streptavidin-coated MACS beads (Miltenyi Biotec) according to the manufacturer’s instructions. Purified follicular B cells samples were collected unstimulated or after stimulation for 48 h with 10 µg/ml anti-IgM (115-006-020, Jackson ImmunoResearch), 10 µg/ml LPS (Sigma) or both anti-IgM and LPS (10 µg/ml each).