Enzyme-Linked Immunosorbent Assay for Anti-AFB1 Antibodies

Detection and titration of specific anti-AFB₁ Abs were carried out by ELISA, as previously described [15] (link). Briefly, serially ten-fold diluted (from 1∶4 and 1∶10 to 1∶400,000) immune serum or control (pre-immune serum) was added to each well of polystyrene microtiter plates coated with AFB₁-BSA conjugate (Sigma-Aldrich) or BSA control protein. After incubation, reaction was detected by adding rabbit anti-bovine IgG (whole molecule) peroxidase conjugate Abs (Sigma-Aldrich, product number A7414) and chromogen/substrate solution. The optical density (OD) at 450 nm was read by using a Multiskan Ascent spectrophotometer (Labsystems, Helsinki, Finland), and the titer of each immune serum was defined as the inverse of the highest dilution that gave 0.1 OD above the pre-immune serum at the same dilution. To compensate for between-plate variability, individual plates were normalized to the mean of the appropriate positive control. For statistic evaluation the logarithms of anti-AFB₁ Ab titers were considered.