Mahas4510

Short term ex-vivo ELISPOTs were used to determine the frequencies of antigen-specific IgG secreting plasmablasts in peripheral blood of TB patients and healthy controls. Ninety-six (96) ELISPOT- well filter plates (MAHA S4510, Millipore) were coated overnight with either 100 µl of PBS containing mycobacterium-specific recombinant proteins (6 µg/ml PPD, 10 µg/ml ESAT-6, Ag85A, Ag85B, and CFP-10). Tetanus toxoid (2 µg/ml) was used as positive control while PBS-coated wells served as negative controls. Plates were washed and blocked with RPMI containing 1% BSA at 37°C for 2 hours. Purified PBMCs (2x10⁵/ml) were added into each antigen-coated well and the plates were incubated for 5 hours at 37°C in a 5% CO₂ incubator. Plates were washed with PBS and 0.05% PBS-Tween and developed with biotin-SP-conjugated AffiniPure fragment donkey anti-human IgG (Jackson ImmunoResearch), strepavidin- AKP (BD biosciences) and its substrate (AP-conjugate substrate kit, Biorad, USA). Developed plates were air-dried and the number of resultant spots enumerated on an AID ELISPOT reader. Frequencies of antigen-specific PBs were calculated and later presented as spot forming units per million PBMCs.

Antibody secreting cells (ASCs) were assessed using ELISPOTs. Spleen (1 × 10⁶ cells and 0.5 × 10⁶ cells) or bone marrow cells (1 × 10⁶ cells and 0.5 × 10⁶ cells) were added to 96-well cellulose ester membrane plates (MAHAS4510; Millipore) coated with NP₁₃-BSA (10 μg/mL) or GPI₈-BSA (10 μg/mL) and incubated at 37°C and 5% CO₂ for 8 h. To measure total IgG, wells were coated with sheep anti-mouse IgG (#AC111; Millipore) at 10 μg/mL. Concentration of cells to measure total IgG ASCs were 1 × 10⁵, 0.5 × 10⁵, and 0.1 × 10⁵ per well. Anti-NP IgG, anti-GPI IgG or total IgG ASCs were detected using goat anti-mouse IgG conjugated to ALP (#3310-4; MabTek) and visualized using the BCIP/NBT system (# 50-81-00; KPL, USA). Spots were counted using an automated reader (AID ELISPOT Reader System, software version 7) and calculated as ASCs/1 × 10⁶.
Blood was collected and serum separated at times indicated for ELISA. 96-well MaxiSorp plates were coated with GPI-BSA (10 ng/well) and titrated serum was incubated for at least 24 h at 4°C. Anti-GPI IgG was detected using goat anti-mouse IgG-HRP (SouthernBiotech) and visualized with 3,3′,5,5′- tetramethylbenzidine (TMB) substrate (#T8665, Sigma Aldrich). Antibody concentrations were quantitated using standard curves of known IgG protein concentrations.

PBMCs from patients were isolated at defined time points and analyzed by modified enzyme-linked immunospot (ELISPOT) assay in duplicates. IgM, IgG and IgA ELISPOT assays in our study were performed as described previously [16 (link),17 (link)]. In brief, 96-well plates (MAHA S4510, Millipore, Burlington, MA, USA) were coated overnight at 4 °C with antibodies against IgM, IgA or IgG (Sigma Aldrich). Plates were washed twice with PBS and blocked for 2 h at 37 °C with PBS/1% BSA before cells incubated overnight at 37 °C, 5% CO₂ in humidified atmosphere. Plates were then washed four times with PBS/0.25% Tween-20. Detection solution (detection antibody diluted 1:2000 in PBS/1% BSA) was added for 2 h. Plates were washed four times with PBS/1% BSA prior adding streptavidin (1:400) for 1 h. Plates were washed twice with PBS/0.25% Tween-20 and twice with PBS. Spots were developed adding 100 µL/well of 3-amino-9-ethylcarbazole at 0.3 mg/mL diluted in 0.1 M sodium acetate pH 5.0 and 0.03% hydrogen peroxide for 5 min in the dark. After several washing steps with distilled water and overnight drying spots were counted with the ImmunoSpot Series 3B Analyzer and ImmunoSpot 3.0 software (CTL, Cleveland, OH, USA). To exclude unspecific binding a human IgM/IgG Double-Color ELISPOT (hIgMIgG-DCE-1M/2, ImmunoSpot^®, Cleveland, OH, USA) assay kit was used according to the manufacturer’s instructions.

Nitrocellulose-backed 96-well MAHAS4510 plates (Millipore) were coated overnight at 4 °C with either Tat or NY-ESO-1 (1 μg/mL each) in 50 mM sodium bicarbonate buffer (pH 9.6). Plates were washed and blocked for 2 h at 37 °C with 10 % fetal calf serum in RPMI. PBMCs were seeded at 3 X 10⁵ cells/well and incubated for 18 h at 37 °C. Spot-forming cells (SFCs) were then detected using 1 μg/mL of biotinylated anti-human IgG (Jackson Immunoresearch Laboratories) for 1.5 h at room temperature. Ab binding was evaluated by the addition of streptavidin, alkaline phosphatase conjugate and a chromogenic alkaline phosphatase substrate (Pierce).