Simultaneous non-instrumental detection of aflatoxin B1 and ochratoxin A using a clean-up tandem immunoassay column

A set-up and simple method based on the clean-up tandem immunoassay approach was developed for the visual detection of two analytes. The method was based on a 1 mL column with one clean-up layer and two detection immunolayers. As detection immunolayers CNBr-activated Sepharose 4B with coupled secondary rabbit anti-mouse antibodies was used. Different specific antibodies were coupled to each detection immunolayer. The analysis was realised in a competitive ELISA format with visual detection of the developed colour for each detection immunolayer and took 20 min for six sample extracts. The described method was applied to the simultaneous detection of aflatoxin B1 and ochratoxin A in spices with cut-off levels at 5 and 10 μg kg−1, respectively. Results were confirmed by LC–MS/MS with immunoaffinity column clean-up.