Anti human fviii igg2a

IgG subclass of anti-FVIII antibodies was assessed using an in-house established ELISA assay. Briefly, 96-well plates (Corning) were coated overnight with 1 µg/mL rhFVIII in PBS at room temperature. Plates were blocked using 1% bovine serum albumin (Sigma-Aldrich) in PBS. Standards or plasma samples were diluted in 1% bovine serum albumin in PBS. Anti-FVIII IgG isotype-specific antibodies were detected by anti-mouse horseradish peroxidase–conjugated IgG1, IgG2a (both from Southern Biotech, Birmingham, AL), IgG2b, or IgG2c (both from Abcam) and 3,3',5,5'-tetramethylbenzidine substrate solution (Perbio Science). Absorbance was detected at 450 nm using an ELISA reader (Tecan). The concentration of anti-FVIII isotype–specific antibodies was estimated from a standard curve obtained using serial dilutions of mouse anti-human FVIII IgG1 (Merck Millipore), anti-human FVIII IgG2a (Thermo Fisher Scientific) starting at 50 ng/mL, or mouse anti-FVIII reference plasma starting at 50 arbitrary units per milliliter.

Ninety-six well half-area plates (Corning) were coated overnight with 1 µg/mL rhFVIII in carbonate coating buffer at 4°C. Plates were blocked with 10% fetal bovine serum (Life Technologies, Carlsbad, CA) in PBS. Plasma samples were loaded as serial dilutions in 10% fetal bovine serum/PBS. Anti-FVIII immunoglobulin G (IgG) antibodies were detected using horseradish peroxidase–conjugated anti-mouse IgG (Abcam, Cambridge, UK), followed by 3,3',5,5'-tetramethylbenzidine substrate solution (Thermo Fisher Scientific, Waltham, MA). Absorbance was read at 450 nm using an ELISA reader (Tecan, Maennedorf, Switzerland). Antibody titer was expressed as the highest dilution of plasma sample showing a positive result (optical density > cutoff point). The cutoff point was set at 10× average of blank controls analyzed in 1 assay. Alternatively, the concentration of anti-FVIII–specific antibodies was estimated from a standard curve obtained using serial dilutions of anti-human FVIII IgG2a (Thermo Fisher Scientific), starting at 50 ng/mL.