Immunoassays For Detection Of Four Major Mycotoxins In Agro-products

Mycotoxins are toxic secondary metabolites produced by various fungi such as Aspergillus, Fusarium and Penicillium which grow on grains of corn, wheat, other legumes and cereal during harvest, storage, transportation or processing of such agricultural produce. More than 400 types of mycotoxins have been reported globally with majority of them having carcinogenic, teratogenic and mutagenic effects. Previous studies have shown that co-occurrence of multiple mycotoxins in a single commodity has a synergistic detrimental effect as compared to presence of a single mycotoxin. Mycotoxins pose severe threat to human and animal health via contamination of grains, feeds or processed foods along the food chain. Detection and control of mycotoxins in foods have become areas of emphasis in food safety research. Currently available detection methods of mycotoxins include bioassays, chemical methods or instrumental analyses. These are either non-specific, not sensitive or of low precision. Instrumental analyses such as HPLC require expensive equipment and technically demanding and thus are not suitable in local laboratories, though they provide quantitative data and have high sensitivity. However, immunoassay, unlike other assay, is simple, selective and robust for rapid and high throughput screening of different mycotoxins in feeds and food samples. This research describes five immunoassays based on immune-chromatographic principle, electrochemical immune-sensor and protein array technology for detection of four major types of mycotoxins, namely aflatoxin B1 (AFB1), ochratoxin A (OTA), fumonisin B1 (FB1) and zearalenone (ZEN). Major research achievement are summarized as follows:1. Competitive indirect ELISA (ic-ELISA) for quantitative detection of ochratoxin AThe monoclonal antibody 2D8 against OTA was identified as IgG1 isotype and has a titer of 1:1.024×106 with affinity constant of 3.75×109 L/M. This mAB 2D8 had low cross-reactivity with ochratoxin B (OTB) and none with aflatoxin B1 (AFB1), fumonisin B1 (FB1), zeralenone (ZEN), citrinin (CIT), patulin (PAT) and deoxynivalenol (DON). A ci-ELISA was then developed for quantitative determination of OTA. Under the optimized conditions, the assay has a detection limit of 0.08 ng/mL, working range from 0.07 ng/mL to 2.41 ng/mL and a recovery rate between 91.2% and 110.3%(with<15%CV) in spiked samples. This ic-ELISA can be used for rapid and quantitative detection of OTA in food and feed samples.2. Magnetic nanoparticle and biotin-streptavidin-based ci-ELISA for quantitative detection of zearalenoneIn order to minimize the detection time while maximizing the efficiency, precision and accuracy, a novel ELISA based on magnetic nanoparticles and biotin/streptavidin-HRP (MNP-bsELISA) was integrated and tested for detection of ZEN. Under the optimized conditions, the working range of this assay was 0.07-2.41 ng/mL with a detection limit of 0.04 ng/mL. The recovery rate ranged between 92.8% and 111.9%(with<10% CV) in spiked samples. Parallel analysis of cereals and feed samples revealed high correlation between MNP-bsELISA and liquid chromatography-tandem mass spectrometry.3. Magnetic nanoparticle-based electrochemical detection of ochratoxin AIn order to develop a test with high sensitivity towards some mycotoxins, such as OTA, magnetic nanoparticles-based electrochemical immuno-sensing assay (MNEIA) was tested. The assay had mininal detection limit of 0.007 ng/mL, working range from 0.01 ng/mL to 0.82 ng/mL and a recovery rate between 78.7% and 91.6%(with <15% SD and CV) in spiked samples. This method has a good stability and enable the detection even at trace levels.4. Dual flow immune-chromatographic assay for simultaneous detection of ochratoxin A and zearalenoneFor rapid screening and improved performance in detection output, we developed a single-step dual flow immuno-chromatographic assay (DICGA) that allows qualitative and quantitative detection of OTA and ZEN in corn, wheat and other feed samples. With qualitative DICGA, the detection limit was 0.625 ng/mL for OTA and 1.25 ng/mL for ZEN. With quantitative DICGA, the detection limit was 0.25 ng/mL and 0.5 ng/mL for OTA and ZEN, respectively. The corresponding detection range was between 0.32-12.16 ng/mL and 0.58-39.72 ng/mL. The recovery rate of both toxins in corn, wheat and feed samples ranged between 79.6-105.2%(with<15% CV). Results of naturally contaminated samples those were analyzed using both DICGA and liquid chromatography-tandem mass spectrometry revealed good correlation. Hence, this DICGA can be used for rapid qualitative or quantitative analysis of OTA and ZEN in cereal and feed samples.5. Antibody-based chip assay for high-throughsut simultaneous detection of four mycotoxinsSince simultaneous contamination of several mytoxins in food or feed is common and would have increased toxicity, we developed an antibody chip assay for high-throughput and simultaneous quantization of four mycotoxins (AFB1, OTA, FB1 and ZEN) using the nitrocellulose membrane as carrier. The detection limit of AFB1, OTA, FB1 and ZEN were 0.21,0.19,0.24 and 0.09 ng/mL, respectively. The corresponding detection range for the above mycotoxins were 0.47-55.69 ng/mL; 0.48-127.11 ng/mL; 0.56-92.57 ng/mL, and 0.22-31.36 ng/mL. The recovery rate in spiked samples ranged between 79.2-113.4%(with< 10% CV). This work offers a high-throughput tool for simultaneous quantitative detection of multiple targets and has a broad application prospects in analysing contamination of foods or feeds with mixed mycotoxins.To summarize, we attempted to integrate the specific monoclonal antibodies with magnetic nanoparticles, biotin-streptavidin, electrochemical and chip technologies for signal amplification and high-throughput detection of mycotoxins. The above assay systems (ci-ELISA, MNP-bsELISA, MNEIA, DICGA and antibody chip) can satisfy the demands for convenient, precise, accurate and efficient detection of mycotoxins in feeds and foods as part of the surveillance programs. The strategies used in this study provide important insights for the development of novel methods targeting other small molecules.