| Mycotoxins are the secondary metabolites of fungi, which may occur in the farm cultivation, harvest,storage, transportation and processing of agro-products. Then the agro-products, food and feedstuffsmay be contaminated by mycotoxins, which directly affect the quality and safety of agro-products. Themain mycotoxins in the agro-products are aflatoxins, zearalenone, ochratoxins, deoxynivalenol andsome others. The toxicities of myxotoxins mainly are carcinogenesis, teratogenesis, genetic toxicity,immunotoxicity and cytotoxicity. Myxotoxins could damage the liver, kidney, nervous tissue,hemopoietic and skin tissue of human and animals, and then induce the acute and chronic intoxicationto the organism. The mycotoxin contamination has been one of the difficult problems of general interestin the agro-product quality and safety and has received great attention from all works of life. Thisproblem has been utilized as one of the technical trade barriers by the developed countries.China territory is vast and the climate is in large difference and changes, which enable one kind offarm crops contaminated by multiple kinds of fungi. Therefore, the farm crops and agro-productscultivated in the natural environment may be contaminated by multiple kinds of mycotoxins.Mycotoxins may enter the body of human and animals when they intake the contaminated food. Thesynergy health hazard of several mycotoxins on humans and animals is much more serious than theharm alone. In recent years, the multiple mycotoxin contamination has aroused more and more attention.Developing sensitive, rapid and high throughput detection technology is the important strateges tomonitor the multiple mycotoxins contamination and gurantee the agro-product quality and safety.Nowadays, the main technology for high throughput detection of multiple mycotoxins is multifuctionalpurification column linked high performance liquid chromatography tandem mass spectrometry andimmune rapid analysis method. While, this former method may be affected by the sample matrix and isnot suitable for on-site and rapid detection. Most of immunoassay methods can just detect one kind ofmycotoxin, which is hard to meet the request of high throughput detection of multiple mycotoxins.To solve above urgent problems, main research contents and novelties of this work are as follows:1. The highly sensitive monoclonal hybriboma cell lines were screened for ochratoxin A andzearalenone. And series of sensitive monoclonal antibodies were prepared for mycotoxins, whichprovided the self-developed key regents for the detection of mycotoxin composite contamination.A sensitive and specific monoclonal antibody against ochratoxin A was prepared. With the animalimmunization, cell fusion and semisolid medium culture-gradient screening, three monoclonal cell linesagainst ochratoxin A were obtained, named1H2,1F8and3E2. The antibody produced by1H2has thehighest titer and best sensitivity. The subtype of1H2antibody is IgG1, and the affinity constant is1.03×109L/mol, which means1H2antibody is of high affinity.1H2has good specificity for ochratoxinA, and has no obvious cross-reactivity with the analog ochratoxin B. It also has no obviouscross-reactivity with other mycotoxins, such as aflatoxin B1, G1, zearalenone, deoxynivalenol, and thecross-reactivity to these mycotoxins are below0.3%. The sensitivity (IC50value) of1H2against ochratoxin A is0.058ng/mL. Comparing with the reported anti-ochratoxin A monoclonal antibodies, thisone is of the best specificity and the highest sensitivity.A highly sensitive monoclonal antibody against zearalenone was prepared. Five monoclonal celllines for zearalenone were obtained, and2D3performed the highest titer and the best sensitivity. Afterthe optimization of the ELISA parameters, including the coating concentration of antigen, concentrationof antibody, coating buffer and pH value, the sensitivity (IC50value) of2D3antibody for zearalenonewas0.02ng/mL. Comparing with the reported monoclonal antibody,2D3has the best sensitivity.Indirect competitive ELISA based on the immunoaffinity column (IAC) was developed for detectionof ochratoxin A and zearalenone. Based on the1H2and2D3antibodies, the IAC was developed forsample preparation, and the indirect competitive ELISA with IAC (IAC-icELISA) for ochratoxin A andzearalenone were developed, respectively. After the purification with IAC, the effect of sample matrixhas been effectively elimilated. The developed IAC-icELISA was utilized to detect ochratoxin A andzearalenone in rice, wheat, soybean and peanut samples, which show good performance in accuracy andrecovery. The results of IAC-icELISA were in good agreement with those from high performance liquidchromatography determination. It means that this IAC-icELISA could be utilized for ochratoxin A andzearalenone detection in agro-products.2. The immunochromatographic technology for simultaneous detection of multiple mycotoxinscontamination was developed. And the immunochromatographic strip was successfully developed formultiple mycotoxins contamination.One kind of immunochromatographic strip for simultaneous detection of three mycotoxins wasfirstly developed. There were three test lines on the strip. Three mycotoxin antigens were separatelyimmobilized on each test line, respectively. Three mycotoxin antibodies were labeled on the colloidgold to prepare the mixed probe. This strip was of good specificity and there was no cross reactivitybetween the determination of these three mycotoxins. The visible detection limit of thisimmunochromatographic strip for aflatoxin B1, ochratoxin A, zearalenone was0.25,0.5,1ng/mL,respectively. This strip was of good stability and could be stored in the room temperature anddesiccative environment for more than six months. This strip was utilized to detect multiple mycotoxinsin maize, rice and peanut, and the results were in good agreement with the results of ELISA detection. Itmeant that this immunochromatographic strip was of good accuracy and reproducibility, and hadapplication prospect in the simultaneous and rapid evaluation of mycotoxin composite contamination inagro-products.3. The microarray immunochip was developed for simultaneous detection of multiple mycotoxinscontamination. And a polymer brush microarray immunochip was successfully developed forsimultaneous detection of three mycotoxins.A non-fouling polymer brush microarray immunochip was developed for simultaneous detection ofthree kinds of mycotoxins. The three-dimensional polymer brush can enlarge the specific surface area ofthe slide and increase the ability of combining the proteins. This biochip with non-fouling polymerbrush is weak in nonspecific adsorption. After the immobilization of the antigen, it can be directly used to perform detection and needs no coating process, which is unlike the ELISA method. And thedetection time could be shortened. The concentration of the immobilized antigen and the antibodysolutions were optimized. This method is of high sensitivity and the limits of detection for aflatoxin B1,ochratoxin A, zearalenone are4,4,3pg/mL, respectively. The linear ranges of this method for aflatoxinB1, ochratoxin A, zearalenone are4-330,12-3000,3-2000pg/mL. This high sensitive competitiveantigen microarray was utilized to detect three mycotoxins in the spiked peanut samples. Thistechnology was of high accuracy and the recovery was between85.9%and109.2%. This work offers apowerful high-throughput tool for fast screening of multiple contaminants in food quality monitoring.4. An immunoaffinity column linked high performance liquid chromatography tandem massspectrometry was developed. The immunoaffinity column for simultaneous purification andconcentration of four mycotoxins was successfully developed.The IAC for simultaneous capture of aflatoxin B1, ochratoxin A, zearalenone and T-2toxin wasdeveloped and the capacities for aflatoxin B1, ochratoxin A, zearalenone and T-2toxin were127.4ng,165.1ng,146.85ng,245.2ng, respectively. The preparation method for simultaneous extraction of thesefour mycotoxins was optimized. Combing with the IAC and the optimized extraction method for samplepreparation, the spiked and natural maize samples were evaluated with HPLC-MS/MS, which gave highrecoveries (between87%-102%) for these four mycotoxins. This self-developed IAC has wideapplication prospect in the conform test of detection of mycotoxins composite contamination. |
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