Immunoaffinity pre-concentration combined with on-column visual detection as a tool for rapid aflatoxin M1 screening in milk

A non-instrumental rapid test was developed for the screening of aflatoxin M1 (AfM1) in milk at the 40 ng/L level. The method combines on one immunoaffinity gel layer, AfM1 pre-concentration and direct competitive immunoassay detection with visual evaluation. Aflatoxin B1-horse radish peroxidase (AfB1-HRP) and Sepharose 4B-immobilized anti-aflatoxin B1 monoclonal antibody with a 79% cross-reaction for AN I were used. The assay was performed in a standard column for solid phase extraction using a milk sample volume of 10 mL, Raw milk, pasteurized milk, milk powder, kefir and yogurt were analyzed with the developed test. It was shown that pasteurized milk could be analyzed without any sample preparation. The other types of milk were analyzed after centrifugation. For the assay procedure an aliquot of milk was flowed through the immunoaffinity gel layer in the tube by pushing the plunger of a syringe. Further, AfB1-HRP was applied and bound to free antibody binding sites. After addition of the chromogenic Substrate the results were visually evaluated. Blue colour developed in case of negative milk samples while no colour appeared for positive samples. This method showed a false negative rate of only 2% and a high throughput (20-35 min for six samples).