Anti human igg horseradish peroxidase hrp conjugate

Plates were sensitized with 50 μl of Pgp3 antigen (500 ng/ml in 0.1M NaHCO₃, pH 9.6) overnight at 4°C. Antigen was decanted, plates washed 2x with PBS with 0.3% Tween-20 (Sigma Aldrich, St. Louis, MO, PBST2), and 50 μl of serum samples diluted 1:50 in 250 μl PBST2 in 5% milk buffer solution, was added to wells in duplicate. Positive and negative control samples were added to each plate. After shaking at room temperature (RT) for 2 hours, samples were decanted and plate washed 4x with PBST2. Fifty μl of anti-human IgG-horseradish peroxidase (HRP) conjugate (1:10,000 dilution in PBST, Southern Biotech, Birmingham AL) was added to each well. After shaking at RT for 1h, antibody was decanted, plates washed 4x with PBST, and 50 μl 3, 3, 5, 5-tetramethlybenzidine (TMB) was added to each well for 6 minutes. The reaction was stopped with 50 μl of H₂SO₄ and the optical density (OD) was read at 450 nm using a SpectraMax M5 Microplate Reader (Molecular Devices, LLC, Sunnyvale, CA). All data were normalized to the 200 unit (U) control at which point an OD of >0.582 was considered positive for antibodies to Pgp3 based on ROC analysis of a panel of previously classified positive and negative sera.