Horseradish peroxidase hrp conjugated goat anti mouse igg

Immunolon 4BX ELISA plates (Thermo Life Sciences, Philadelphia, PA) were coated overnight at 4°C with 100 ng/well of rHA or anti-mouse IgG (γ-chain specific)–peroxidase antibody produced in goat in carbonate buffer (pH 9.4) with 250 ng/mL bovine serum albumin (BSA). The plates were blocked for 2 h at 37°C with blocking buffer (2% BSA, 1% gelatin in PBS/0.05% Tween 20). Serum samples were initially diluted 1:100 and then further serially diluted in 1:2 in blocking buffer. Serially diluted samples were added to the assay plate in duplicate and incubated overnight at 4°C. Plates were washed three times with wash buffer (PBS with 0.05% Tween 20) and horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (Southern Biotech, Birmingham, AL, USA) at a 1:3000 dilution and incubated for 2 h at 37°C. Plates were washed 7 times with the wash buffer prior to development with 100 μL of 0.1% 2,2ʹ-azino-bis(3-ethylbenzothiaozoline-6 – sulfonic acid; ABTS) solution with 0.05% H₂O₂ for 40 min at 37°C. The reaction was terminated with 1% (w/v) sodium dodecyl sulfate (SDS). Colorimetric absorbance at 414 nm was measured using a PowerWaveXS (Biotek, Winooski, VT, USA) plate reader. The magnitude of IgG responses among groups was analyzed by area under the curve (AUC) analysis using Prism (GraphPad Software).^{14 (link)}