Rapid Detection of Ochratoxin A in Malt by Cytometric Bead Array Based on Indirect Competition Principle

Abstract

Abstract A cytometric bead array (CBA) method based on indirect competition principle was developed for the sensitive and rapid detection of ochratoxin A (OTA) in malt. The malt samples were extracted by 60% methanol/PBS and the extracts were diluted five times with 20% methanol/PBS. After centrifugation, the supernatant was collected to prepare sample solution for analysis. The fluorescence-encoded microsphere surface was labeled with bovine serum albumin OTA (BSA-OTA) to compete with OTA in samples for anti-OTA monoclonal antibody (mAb). Then, FITC-IgG was added to bind with the captured mAb on the microsphere. After centrifugation and washing, the mean fluorescence intensity from FITC on the surface of microsphere was detected by a BD FACSCalibur analyzer for accurately qualitative and quantitative analysis of OTA. The results showed that the half inhibitory concentration (IC 50 ) was 1.20 ng mL −1 with the correlation coefficient ( R 2 ) of 0.989, and the limit of detection (LOD) for OTA was 0.12 ng mL −1 . The average recovery rates in malt samples were 93.9%–97.4% with relative standard deviations (RSDs) less than 3.6% at three spiking levels. Sixteen malt samples were analyzed and OTA was found in two samples with the contents less than 3.83 μg kg −1 which was lower than the maximum permitted residue level (5 µg kg −1 ) proposed by the European Union. All the positive samples were confirmed by LC-MS/MS. In this study, the CBA technique based on indirect competition principle was developed for the first time for successful detection of OTA in malt samples. The method was easy, rapid, sensitive and reliable with high potential for the qualitative and quantitative of multiple mycotoxins in other complex matrices.