Goat anti human igg fc

SDS-PAGE was performed on 4%–20% gradient gels under reducing and non-reducing conditions, and resolved proteins were transferred to polyvinylidene fluoride (PVDF) membranes. Goat anti-human IgG-Fc (abcam; cat# ab97221) and goat anti-human C_κ (Thermo Fisher Scientific; cat# 31129) were used to detect human IgG-Fc and κ LC, respectively. A rabbit anti-goat IgG-HRP Ab was used to detect the bound primary Abs. GAPDH was detected by probing the membrane with primary mouse anti-GAPDH (Santa Cruz Biotechnology; cat# sc-322330) Ab, followed by horse anti-mouse IgG-horseradish peroxidase (Cell Signaling; cat# 7076). Immunoreactive proteins were visualized using an ECL Kit (GE Healthcare, Cat. No. RPN2106).

Concentration of mAb in crude and purified samples were calculated by quantitative sandwich ELISA. A 96-well, half-area microtiter plate (Corning, New York, USA) was coated with goat anti-human IgG Fc (Abcam, Cambridge, UK) overnight at 1:1,000 dilution in 1×PBS. Plate washing was with 1×PBS-T (1×PBS with 0.05% Tween 20), and blocking was with 5% (w/v) skim milk in 1×PBS. Serially diluted plant samples and human IgG1 kappa isotype antibody standard (Abcam, Cambridge, UK) were incubated on the coated plates for 2 h at 37 °C, followed by detection with goat anti-human Kappa-HRP (SouthernBiotech, Alabama, USA) at 1:3,000 in 1×PBS. Finally, TMB one solution substrate (Promega, Wisconsin, USA) was added and the reaction was quenched with 1M H₂SO₄. The absorbance was measured at 450 nm on a NS-100 Nano Scan microplate reader (Hercuvan, Shah Alam. Malaysia).