Biotin conjugated anti human igg

Briefly, 96-well ELISA plates (Costar, Corning) were coated with goat anti–human Ig (SouthernBiotech) in PBS at 4°C overnight, then blocked with 1% BSA blocking buffer at room temperature for 1 hour. The culture supernatants and serially diluted standards were then added and incubated at room temperature for 1 hour. After washing, biotin-conjugated anti–human IgG (SouthernBiotech) Ab was added and incubated at room temperature for 1 hour. After washing, avidin-HRP (BioLegend) was added to the plates, incubated for 30 minutes, and visualized with TMB substrate reagents (Invitrogen, Thermo Fisher Scientific). OD₄₅₀–OD₅₇₀ was measured by a spectrophotometer (Bio-Rad). The amount of Ig in duplicate wells was calculated based on the standard.

IgM and IgG levels in the culture supernatants were measured by standard ELISA as described (17 (link)). Briefly, 96-well ELISA plates (Costar) were coated with goat anti-human Ig (Cat#2010-01, Southern Biotech), blocked with 1% BSA in blocking buffer and incubated with serially diluted culture supernatants and standards. After washing, the biotin-conjugated anti-human IgG (Cat#2040-08, SouthernBiotech) or anti-human IgM biotin (Cat#2020-08, Southern Biotech) was added, followed by Avidin-HRP (Lot: B152022, Biolegend). The HRP-activity was visualized with TMB substrate reagents (Invitrogen) and OD₄₅₀-OD₅₇₀ was measured by spectrophotometer (Bio-Rad). The amount of Ig was calculated according to the standard curve.