Enumeration of Antigen-Specific Antibody-Secreting Cells

B cell ELISPOT assays were performed as previously described (18 (link)), with minor modifications. Briefly, MultiScreen 96-well filter plates (Merck Millipore, Darmstadt, Germany) were coated with 10 μg/ml inactivated influenza virus or anti-human IgG Fc antibody (Sigma) overnight at 4°C for enumeration of influenza virus-specific antibody-secreting cells or total IgG-secreting cells, respectively. The wells were washed with PBS and blocked with R10 medium for 2 h at 37°C. Whole PBMCs or PBMC-derived fluorescence-activated cell sorting (FACS)-sorted cells (described in the section below) were plated and incubated overnight in a 5% CO₂ incubator at 37°C. The plates were washed with PBS-Tween 20 (PBST), followed by incubation with biotinylated anti-human IgG and horseradish peroxidase (HRP)-conjugated streptavidin (BD Pharmigen). Spots were developed using 3-amino-9-ethylcarbazole (AEC) substrate (BD Pharmigen). To stop the reaction, the plates were washed with water. Spots of antibody-secreting cells were counted using an ELISPOT reader (Bioreader 4000, BIOSYS, Germany). The number of spots is reported as the number of antigen-specific cells or IgG antibody-secreting cells per million cells.