Anti human igg h l hrp

A competition ELISA, using an antibody to BAFF that recognizes a conformationally-dependent epitope, was used to determine the level of BAFF in the native conformation on the memBAFF cells. Recombinant human soluble BAFF was coated on microtiter plates (Greiner Bio-One, Monroe, NC, USA), 3.6 ng/well in PBS, overnight at 4°C. The ELISA plate was then washed (TBS, 0.1% Tween 20) and blocked (Casein/PBS; Thermo Fisher Scientific, Waltham, MA, USA). In a polypropylene plate, serially diluted soluble BAFF, cells expressing memBAFF, or vector control cells were incubated with 4.5 ng of anti-BAFF (clone 6B2.1) for 1 hour at RT with shaking. After the incubation, 50 μL of the BAFF/antibody mixture was added to the ELISA plate and shaken for 12 minutes at RT. The plate was washed and the plate-bound anti-BAFF was developed using donkey, anti-Human IgG (H + L) HRP (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA) followed by OPD substrate. The plate was read on a Molecular Devices plate reader and the data analyzed using SoftMaxPro 3.1.2 software (Molecular Devices, Sunnyvale, CA, USA) using a 4 parameter curve fit. The soluble BAFF titration was used to establish the standard curve to quantify the number of memBAFF molecules on the cells.