Goat anti human igg h l conjugated with hrp

Wild-type FGF23 or its alanine mutants were coated onto 96-well Maxisorp plates (Nunc) at a concentration of 2 μg/mL and incubated at 4 °C overnight. Plates were washed with PBS with 0.05% tween (PBST) prior to incubation with 200 μL of 1% BSA in PBST for 1 h. at room temperature, and followed by washing with PBST. Wild-type Burosumab or its alanine mutants were three-fold serially diluted in PBS starting at 9 µg/mL prior to adding into plates and incubating at room temperature for 2 h. Plates were washed again with PBST, and then 100 μL of goat anti-human IgG H&L conjugated with HRP (1:10,000, Abcam) was added to plates and incubated at room temperature for 1 h. After washing with PBST, the plate was incubated with 100 μL of TMB substrate (KPL) at room temperature for 3 min, and quenched with 100 μL of 1 N H₂SO₄. The absorbance was measured at 450 nm using a Spectramax S5 plate reader (Molecular Devices). Note that spot-check ELISA was performed in the same manner except that only two concentrations (0.04 and 1.2 µg/mL) of Burosumab were applied.

ELISA protocol was adapted from Quinlan et al.^{52 (link)}. Briefly, 100 μL of the wild-type GP in PBS was adsorbed to clear 96-well Maxisorp plates (Nunc) at a concentration of 1 μg/mL and left at 4 °C overnight. Plates were washed with PBS with 0.05% Tween 20 (PBST) and incubated with 100 μL of 2% BSA in PBST for 1 h at room temperature. Plates were washed with PBST then threefold serially diluted of anti-Nipah mAb were added to wells followed by incubation for 2 h. After washing with PBST, 100 μL of secondary antibody, goat anti-human IgG H&L conjugated with HRP (1:10,000, Abcam) were incubated for 1 h. Finally, plates were washed, and incubated with 100 μL of TMB substrate (KPL) for 5 min and quenched with 100 μL of 1 N H₂SO₄. Plates were read using a Tecan Infinite N200 Pro plate reader at 450 nm.