Multiscreen ip filter plate

ELISpot assays were performed on isolated PBMCs before and after vaccination. MultiScreen-IP Filter plates (Millipore) were coated with 10 μg/mL of human anti-IFN-γ coating antibody (clone 1-D1K, Mabtech) in a carbonate buffer (Sigma-Aldrich) and stored at 4 °C overnight. The coated plates were washed three times with PBS and blocked with R10 media for a minimum of 1 h at 37 °C. After the blocking. 1.25 × 10⁵ PBMCs were added into each well as per the assigned layout. PBMCs were stimulated with a SARS-CoV-2 S1 pool (Wuhan strain) of 15-mer, with 11 amino acid overlap containing amino acid sequence 1–692 (PepTivator^®) at a final concentration of 1 µg/mL. Each assay was performed in duplicate and incubated for 16–18 h at 37 °C with 5% CO₂. Plates were developed by washing them six times with PBS/T, followed by the addition of 1 μg/mL of anti-IFN-γ detector antibody (7-B6-1-Biotin, Mabtech) to each well. After a 2-h incubation, plates were washed again, and 1:1,000 SA-ALP was added for 1 h at RT. After a final wash step, plates were developed using BCIP NBT-plus chromogenic substrate (Mabtech). ELISpot plates were counted using an Immunospot Microanalyzer (Cellular Technology Limited). Responses were averaged across duplicate wells, and the mean response of the unstimulated (negative control) wells were subtracted. Results are shown as SFCs/10⁶PBMCs.

Detection of total IgM and NP-specific IgM producing cells was performed using enzyme-linked immunosorbent spot (ELISpot) assay. MultiScreen-IP filter plates (Millipore) were pretreated with 70% ethanol and washed in sterile PBS. Plates were coated with 5 μg/ml anti-mouse IgM (Southern Biotech) or 5 μg/ml of NP (25), conjugated with BSA (Biosearch Technologies), diluted in PBS, and incubated overnight at 4°C. The following day, plates were washed in sterile PBS, blocked in complete RPMI medium with 50-μM 2-mercaptoethanol and 10-mM HEPES for 1 h at 37°C, and the indicated cell numbers added in triplicate. Plates were incubated for 17 h at 37°C in 5% CO₂. Cells were then removed by washing in PBS and 0.1 μg/well of biotinylated anti-mouse IgM (Mabtech) diluted in PBS was added to the wells. After 2 h of incubation at RT, plates were washed and developed with 100 μl of 5-bromo-4-chloro-3-indolyl phosphate/NBT-plus substrate (Mabtech). The reaction was stopped when distinct spots could be observed, by rinsing the plates extensively in tap water. Spots were counted by ELISpot reader (CTL) and analyzed using the Biospot suite (CTL).

Sterile 96-well Multiscreen-IP filter plates with a PVDF membrane (Millipore Corp, Bedford, MA, USA) were coated with either anti-human IgG (15 μg/mL, Mabtech, Nacka Strand, Sweden) or recombinant HBsAg (10 μg/mL) overnight at 4℃. Numbers of total IgG + secreting cells were determined by wells coated with capture anti-human IgG, while numbers of HBsAb secreting B cells were examined by wells coated with HBsAg. Wells with no coating were used as negative controls. Plates were washed with sterile PBS and blocked with RPMI-1640 medium/10%FBS for 2 h at room temperature. Serially diluted pre-activated cells were added at 5000 or 2500 cells/well for IgG coated wells, 100,000 cells/well for no coat wells, and 100,000, 200,000, or, 400,000 cells/well for HBsAg-coated wells. The culture plates were incubated for 18 h at 37 °C, 5% CO₂. Each condition was performed in duplicates. Frequency of antibody-secreting cells was measured after washing with PBS and incubation with biotin-labeled anti-IgG mAb (Mabtech, cat#3850-2 H, Sweden) and horseradish peroxidase (HRP)-conjugated streptavidin (Mabtech, Nacka Strand, Sweden). An automated ELISpot image analyzer (Cellular Technology Limited, Hongkong, China) was used to analyze and count the spots. Frequency of HBsAb-secreting B cells per well was calculated by subtracting background values from negative control wells.