One-step immunoassay based on switching peptides for analyzing ochratoxin A in wines

Abstract

A one-step immunoassay is presented for the detection of ochratoxin A (OTA) using an antibody complex with switching peptides. Because the switching peptides (fluorescence-labeled) were able to bind the frame region of antibodies (IgGs), they were dissociated from antibodies immediately when target analytes were bound to the binding pockets of antibodies. From the fluorescence signal measurements of switching peptides, a quantitative analysis of target analytes, via a one-step immunoassay without any washing steps, could be performed. As the first step, the binding constant (K-D) of OTA to the antibodies was estimated under the continuous flow conditions of a surface plasmon resonance biosensor. Then, the optimal switching peptide, among four types of switching peptides, and the reaction condition for complex formation with the switching peptide were determined for the one-step immunoassay for OTA analysis. Additionally, the selectivity test of one-step immunoassay for OTA was carried out in comparison with phenylalanine and zearalenone. For the application to the one-step immunoassay to detect OTA in wines, two types of sample pre-treatment methods were compared: (1) a liquid extraction was carried out using chloroform as a solvent with subsequent resuspension in phosphate-buffered saline (total analysis time < 1 h); (2) direct dilution of the wine sample (total analysis time < 0.5 h). Finally, the direct dilution method was found to be effective for the one-step immunoassay based on the switching peptide assay for OTA in wines with a markedly improved total analysis time (< 0.5 h). Additionally, the assay results were compared with commercial lateral flow immunoassay.