Quantifying Antigen-Specific Antibody-Secreting Cells

Antibody-secreting cells (ASCs) in blood and bone marrow were analyzed by performing the enzyme-linked immunospot assay (ELISPOT) as described in detail elsewhere [21 (link)]. Briefly, 96-well MultiScreen_HTS HA filter plates (Millipore) were coated with 10 µg/mL of anti-monkey IgG (H&L) antibody (Rockland) to determine the total ASCs or with 10 µg/mL of polio virus–specific antigens (Sanofi Pasteur) to determine the antigen-specific ASCs. After the overnight coat at 4°C, the plates were washed with PBS/0.05% Tween 20 (PBS-T) followed by PBS and blocked for 2 hours with complete RPMI at 37°C. The lymphocytes were then plated with 3-fold serial dilutions and kept in a 5% CO₂ incubator at 37°C for 5 hours. The plates were then washed with PBS followed by PBS-T and incubated with biotin-conjugated anti-monkey IgG (Rockland) for 2 hours at room temperature. The plates were washed with PBS-T and incubated for 3 hours at room temperature with Horseradish Peroxidase Avidin D (Avidin D-HRP; Vector labs). The plates were then washed with PBS-T followed by PBS and developed with 3-amino-9-ethylcarbazole (AEC) substrate (BD Biosciences) according to the manufacturer’s protocol. Plates were then dried, and spots were imaged and counted using Immunospot Cellular Technology Limited (CTL) counter and Image Acquisition 4.5 software (Cellular Technology).