Bcip nbt phosphatase substrate

The Enzyme-Linked ImmunoSpot assay (ELISpot) was conducted to quantify antigen-specific antibody-secreting B cells (ASC). The PVDF membranes of ELISpot plate were activated by incubating in 35% ethanol for 10 min at room temperature. The plate was washed thrice with PBS buffer to remove residue ethanol, and 500 ng/well of bacterial antigen (bCOE) added, in PBS buffer at 4°C overnight. Following washing, 200 µl of complete RPMI containing 10% FBS was added and plates incubated at 37°C, 5% CO_2, for 1 h, to prevent non-specific binding. Splenocytes were isolated from mice 10 days after final immunization using Percoll density gradients. Viable splenocytes were counted by trypan blue method (Thermo Fisher Scientific). Cells were diluted in complete RPMI medium to 1 × 10⁶, 5 × 10⁵, and 1 × 10⁵ cells/well and then cultured at 37 °C, 5% CO₂ for 2 days. After removal of unbound cells by washing, anti-mouse IgG alkaline phosphatase (Sigma) was added and following 2 h incubation at 37°C, the cells were washed three times as before. ASC were visualized by addition of BCIP/NBT phosphatase substrate (Sigma) and colour spots counted under microscope and computer.

Multiscreen_HTS filter Elispot plates (Mabtech, Stockholm, Sweden) were coated with 15 μg/mL of human IFNγ antibody (1-DIK) overnight at 4 °C. A total of 100,000 PBMCs were placed in each well and stimulated with 5 μg/mL of pooled and/or individual STIP1 peptides from the aforementioned peptide library for 18 h with 5 IU/mL of IL-2. The CD3/CD28/CD2 T cell activator (STEMCELL Technologies, Vancouver, BC, Canada) was used as the positive control. The plates were then developed using the 1:1000 human biotinylated IFNγ detection antibody (7-B6–1), followed by streptavidin ALP and BCIP/NBT phosphatase substrate (Sigma Aldrich). The number of spot forming units (SFU) were quantified using the Mabtech Iris^TM FluoroSpot/ELISpot reader system equipped with Spot reader software version 1.1.9 and included in the analysis. Out of the 15 individuals with high STIP1 autoantibodies, seven individuals returned for the follow-up and were recruited for the characterisation of STIP-specific T cells.