The Determination of AFM1 has been based on an Enzyme-Linked Immunoassay (ELISA) using the RIDASCREEN FAST test kit (R-biopharm, Germany, Product No: R1121). This method is quick, reliable and cost effective for the estimation of AFM1 and has been included in the official collection of test procedures by the German Federal Board of Health. This test kit is sufficient for 96 determinations (including the calibration curve). The basis of the test is the antigen–antibody reaction.
10 g of infant formula powder was added to a flask, which was filled up to 100 ml with distilled water. Mixture was then homogenized and warmed in a water bath (50C) for 30 min. Samples were centrifuged 10 min/3500 rpm/10C. The skimmed part was used directly in the test (100 µl per well).
100 µL of antibody was added to the bottom of wells, mixed and incubated for 15 min at room temperature. Then, the liquid was poured out of the wells and the wells were filled with 250 µL washing buffer and poured out the liquid again. This washing step was repeated twice.
Standards and sample solutions of 100 µL were added to the wells to occupy the binding sites proportionately then mixed gently and incubated at room temperature (20–25C) in the dark. Then, the liquid was poured out of the wells and the wells were filled with 250 µL washing buffer and poured out the liquid again. This washing step was repeated twice.
In the next stage, 100 µL of enzyme conjugate were added to occupy the remaining free binding sites and in washing step, 250 µL of washing buffer washed any unbound enzyme conjugates.
Then, 100 µL of enzyme substrate/chromogen were added to the wells and incubated for 30 minutes at room temperature in the dark. Bond enzyme conjugate converted the chromogen into a blue product, and then 100 µL of stop solution was added to each well, which leads to yellow discoloration of chromogen. The measurement of AFM1 was done photometrically at 450 nm. Then the AFM1 concentration in ng/L was read from the calibration curve.