Biotinylated anti mouse igg antibody

ELISPOT assays were performed on the day of CLN isolation to examine antigen-specific IgG producing cells. Immobilon-IP PVDF-based 96-well plates (Millipore) were coated with 100 µg/well of HSA overnight at 4 °C. The plates were blocked with 1% bovine serum albumin for 1 h at room temperature (RT) and then washed 4 times with PBS containing 0.05% Tween 20 (PBST). Lymphocytes from CLNs were incubated with 4 × 10⁵ cells/well for 3 days at 37 °C in a 5% CO₂ incubator. ELISPOT plates were washed 3 times with PBST and 100 µl of biotinylated anti-mouse IgG antibody (Southern biotech) diluted 1:500 in PBST was added to each well. After 2 h incubation at RT, plates were washed 4 times with PBST and 100 µl of streptavidin-alkaline phosphatase (Southern biotech) diluted 1:1,000 in PBST was added for 2 h. Following washing, ELISPOT plates were developed with Vector blue substrate kit (Vector laboratories Inc.). To stop the reaction, the plates were washed and dried. Spot patterns were analyzed using an ImmunoSpot image analyzer (Cellular Technology). Data are presented as the mean numbers of antigen-specific HSA-specific IgG producing cells per 10⁶ lymphocytes from quadruplicate wells.