Flow-through membrane-based enzyme immunoassay for rapid detection of ochratoxin A in wheat

A flow-through membrane-based enzyme immunoassay for the rapid detection of ochratoxin A in wheat was developed (patent pending). An Immunodyne ABC membrane was coated with rabbit anti-mouse immunoglobulins and free protein binding sites were blocked. After these antibody-coated membranes were placed on an absorbent layer in a plastic test device, a sequential competitive enzyme immunoassay was performed. The following reactants were successively dropped onto the membrane: wash solution, a dilution of monoclonal anti-ochratoxin A immunoglobulins, wash solution, ochratoxin A standard solution or sample extract solution, a dilution of ochratoxin A-horseradish peroxidase conjugate, and wash solution. Finally, substrate solution (H2O2-3,3',5,5'-tetramethylbenzidine) was added for color reaction. The dot color intensity on the membrane was visually compared with that of the negative control, which showed the most intense blue color because of the inverse relationship between toxin concentration and color development. A portable colorimeter was used to confirm and quantify the visual observations. An ochratoxin A concentration of 0.4 ng/ml in buffer solution suppressed the color development completely. With the use of a simple sample preparation procedure it was possible to eliminate matrix interference. A wheat sample spiked with 4 microg/kg resulted in a complete color suppression. With coated membranes, the immunoassay could be performed in less than 15 min.