Mt78 145

ELISpot was performed according to manufactures instructions (Human IgG ELISpotBASIC HRP, Mabtech). Briefly, PVDF ELISpot plates (MAIPS4510, Millipore) were Ethanol treated and coated with capture antibody (MT91/145, Mabtech) at a concentration of 15 µg/ml in PBS and incubated overnight at 4 °C. Total PBMCs or sorted CD11c^hi B cells (CD19⁺CD11c^hi) or plasma cells (CD19⁺CD27⁺⁺CD38⁺⁺) from SLE donors (n = 4) were added to wells at indicated concentrations in complete media and incubated for 16 h at 37 °C. The plates were then washed and incubated with detection mAb (MT78/145, Mabtech) at a concentration of 1 µg/ml for 2 h at RT, followed by streptavidin-horseradish peroxidase conjugate for 1 h at RT and developed with precipitating TMB substrate (Mabtech) for 15 min. The images were captured using an Immunospot reader (Cellular Technology Limited).

The presence of DBL-TH-specific MBCs was determined with an ELISPOT assay, performed as previously described [18 (link)]. In brief, PBMCs from P. vivax-infected subjects were cultured with R848 and recombinant human IL-2 (Mabtech, Nacka Strand, Sweden) at 37°C in a 5% CO2 incubator for 96 h. Then, the ELISPOT assay was performed by coating with 15 μg/mL of anti-human IgG MT91/145 (Mabtech), 5 μg/mL of DBL-TH and Sal I antigens, or 1 μg/mL of tetanus toxoid (TT) antigen (Merck Millipore, Billerica, MA, USA) onto Multi-Screen IP plates (Merck Millipore). Cells were seeded in duplicate to yield 5 × 10⁴ cells per anti-human IgG-coated well and 1 × 10⁶ cells per antigen-coated well. After overnight incubation, 1 μg/mL of detection antibodies MT78/145 (Mabtech) was added and the mixture incubated for 2 h at RT. After washing, streptavidin-HRP at 1:1000 dilution (Mabtech) was added; plates were incubated for 1 h, then TMB substrate added. Plates were then rinsed with deionized water and analyzed with a Bioreader 5000 ELISPOT Reader. A positive ELISPOT response was defined as detectable spots in duplicate wells with the total spots in the specific antigen-coated wells being at least twice the number of spots detected with the negative control samples. The antigen-uncoated wells were used as negative controls for DBL-TH-specific ASC analysis.

PBMC were stimulated with 1 µg/mL R848 and 10 ng/mL IL2 (MabTech) at 2x10⁶ cells/mL for 5d at 37°C. ELISpot plates (Merck) were activated and coated ON at 4°C with 2 µg/mL HBsAg (Roche Diagnostics), washed 1x with PBS, and blocked with RPMI for 2h at RT. Stimulated PBMC were added to ELISpot plates at 1-2x10⁵/well in RPMI and incubated for 18h at 37°C. B cells were subsequently removed, and plates were washed 5x with PBS. 1 µg/ml detection antibody MT78/145 (MabTech) was added in PBS+0.5%BSA for 2h at RT. After washing, Streptavidin-HRP (MabTech) in PBS+0.5%BSA was added for 1h at RT, followed by TMB (MabTech). Spot development was stopped by rinsing with H₂O, and spot numbers were quantified with ImmunoSpot Reader (CTL Europe).