405 select plate washer

Immulon 2HB 96-well plates were coated with 100 ng/well of purified B. miyamotoi recombinant BmaA diluted in carbonate bicarbonate coating buffer (90 mM NaHCO₃, 60 mM Na₂CO₃; pH 9.6) and incubated overnight at 4 ° C. The plate wells were subjected to five washes with Tris-buffered saline–Tween 20 [TBS-T; 20 mM Tris, 140 mM NaCl, 2.7 mM KCl, 0.05 % Tween 20 (pH 7.4)] using a BioTek 405 Select plate washer (BioTek, Winooski, VT), followed by addition of 300 μL blocking buffer (3 % fetal bovine serum in TBS-T) for 60 min at room temperature. Post-block, plates were washed, and sera in blocking buffer (1:100) was added to individual wells in duplicate and incubated for 45 min at room temperature. Plates were washed followed by addition of HRP-conjugated goat anti-human IgG Fc, F(ab’)2 fragment (1:5000) (Life Technologies, Carlsbad, CA) and incubated for 45 min. Plates were washed and developed by addition of KPL SureBlue TMB Microwell Peroxidase substrate (100 μL) (Seracare, Milford, MA) for 10 min. Reaction was terminated with 1 N HCL and samples read at 450 nm using an ELx808IU Ultra microplate reader (Biotek, Winooski, VT). Cutoff values to determine positive samples were calculated by 3 standard deviations above the mean optical density for all healthy control samples. Patient samples were assayed in duplicate and replicated 2 times.