Hrp goat anti human igg fc secondary antibody

For ELISA assays, antigen protein was coated on a 96-well plate (Costar) at 50 ng/well in PBS overnight at 4°C. For the soluble VH binding assay, horseradish peroxidase (HRP)-conjugated mouse anti-FLAG tag antibody (A8592, Sigma-Aldrich) was used to detect VH binding. For detection of human Fc protein, HRP-goat anti-human IgG Fc secondary antibody (A18817, Thermo Fisher Scientific) was used. For the competition ELISA, 200 nM of VH-Fc 1-16 was incubated with serially diluted VH 1-16-3 proteins, and the mixtures were added to antigen-coated wells. After washing, competition was detected by HRP-goat anti-human IgG Fc secondary antibody (A18817, Thermo Fisher Scientific). All colors were developed by 3,3′,5,5′-tetramethylbenzidine (TMB, Sigma) and stopped by 1 M H₂SO₄ followed by recording absorbance at 450 nm by iMark™ Microplate Absorbance Reader (Bio-Rad Laboratories). Experiments were performed in duplicate and the error bars denote ± 1 SD.