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 me mbrane was coated with rabbit anti-mouse immunoglobulins and free protein b inding sites were blocked. After these antibody-coated membranes were place d 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-oc hratoxin A immunoglobulins, wash solution, ochratoxin A standard solution o r sample extract solution, a dilution of ochratoxin A-horseradish peroxidas e conjugate, and wash solution. Finally, substrate solution (H2O2-3,3',5,5' -tetramethylbenzidine) was added for color reaction. The dot color intensit y on the membrane was visually compared with that of the negative control, which showed the most intense blue color because of the inverse relationshi p 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 developm ent completely. With the use of a simple sample preparation procedure it wa s possible to eliminate matrix interference. A wheat sample spiked with 4 m u g/kg resulted in a complete color suppression. With coated membranes, the immunoassay could be performed in less than 15 min.