Quantifying Anti-PTM Antibodies Using Modified ELISA

Anti-PTM antibodies were detected using an in-house enzyme-linked immunosorbent assay (ELISA), based on modified fetal calf serum (FCS) as described previously (10 (link)). Briefly, modified and non-modified FCS were coated to a Nunc Maxisorp ELISA plate (430,341, Thermofisher). In between each sequential step plates were washed three times using Phosphate Buffered Saline (PBS)/0.05%Tween (Sigma, P1379). After blocking [PBS/1% Bovine Serum Albumin (BSA)] for 6 h at 4°C, plates were incubated overnight at 4°C with 1/50, 1/100 or 1/1000 diluted serum. Each plate contained a standard of anti-PTM antibody positive serum to calculate arbitrary units. After incubation, IgG levels were detected using horseradish peroxidase (HRP) labeled Rabbit-anti-Human IgG (Dako, P0214). Plates were developed by incubating with 2,2′-azino-bis[3-ethylbenzothiazoline-6-sulfonic acid] (ABTS)/0.015% H₂O₂ (A1888 and 7,722-84-1, both from Merck) and absorbance at 415 nm was measured using a microplate reader (Bio-Rad iMark). The cut-off for positivity was set as the mean arbitrary units plus two times the standard deviation of 100 HCs, excluding values higher than 10x the mean.