基于免疫分析的农产品真菌毒素混合污染同步检测技术研究
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摘要
真菌毒素是由真菌产生的次级代谢产物，由于真菌在农产品生长、收获、储运、加工过程中都会发生，因此真菌毒素极易污染农产品、食品和饲料等，直接影响农产品及其制品的质量安全。农产品中的真菌毒素主要为黄曲霉毒素、玉米赤霉烯酮、赭曲霉毒素、呕吐毒素等。真菌毒素对人、畜往往具有很强的致癌作用、致畸作用、遗传毒性、免疫毒性、细胞毒性等，可损害人和动物的肝脏、肾脏、神经组织、造血组织及皮肤组织等，从而引起对机体的急性或慢性中毒，严重威胁人、畜健康。真菌毒素污染已成为农产品质量安全的重要热点、难点问题，受到了社会各界的广泛关注，并被发达国家作为技术贸易壁垒手段之一。
我国地域辽阔、气候差异及变化较大，一种农作物被多种真菌侵染的情况较为常见。因此，一种自然条件生长的农产品可能受多种真菌毒素的混合污染。人和动物摄入受真菌毒素混合污染的农产品时，真菌毒素也会随之进入人体，几种真菌毒素协同作用对人和动物的健康危害作用可能比真菌毒素单独作用的危害大。近年来，真菌毒素混合污染受到了越来越多的关注，建立灵敏、快速、高检测通量的检测技术是真菌毒素混合污染监管和保障农产品质量安全的重要措施。目前，用于真菌毒素混合污染多组分检测的技术主要为多功能净化柱-高效液相与质谱联用方法和免疫快速检测方法等，然而多功能净化柱-高效液相与质谱联用方法受样品基质干扰大，特别是无法满足现场快速检测需求；免疫快速检测方法中主要针对单一真菌毒素污染，难以满足多种真菌毒素混合污染快速检测需求。
针对上述问题，本论文的研究内容和创新点如下：
1.选育出赭曲霉毒素A和玉米赤霉烯酮的高灵敏单克隆杂交瘤细胞株，并研制出系列真菌毒素高灵敏单克隆抗体，为多种真菌毒素混合污染检测研究提供自主核心试剂。
研制出一种灵敏度高、特异性好的抗赭曲霉毒素A单克隆抗体。本研究采用动物免疫、细胞融合、半固体培养-梯度筛选法获得3株能产生抗赭曲霉毒素A抗体的单克隆细胞株1H2、1F8、3E2，其中1H2细胞株效价和灵敏度最高。1H2抗体亚型为IgG1型，对赭曲霉毒素A的亲和力常数为1.03×109L/mol，属于高亲和力抗体。1H2抗体对赭曲霉毒素A的反应特异性好，与赭曲霉毒素A结构类似物赭曲霉毒素B交叉反应不明显，与其他种类的真菌毒素黄曲霉毒素B1、G1、玉米赤霉烯酮、呕吐毒素的交叉反应不明显，交叉反应率均小于0.3%。1H2抗体对OTA的灵敏度（IC50值）为0.058ng/mL。该抗体是迄今研究报道的对赭曲霉毒素A特异性最好、灵敏度最高的单克隆抗体。
研制出一种高灵敏度的抗玉米赤霉烯酮单克隆抗体。本研究筛选获得5株能产生抗玉米赤霉烯酮抗体的单克隆杂交瘤细胞株，其中2D3抗体的效价和灵敏度最高。通过对ELISA反应条件的优化，包括对抗原包被浓度，抗体使用浓度，封闭液，反应体系pH值，盐离子浓度的优化，2D3抗体对玉米赤霉烯酮的灵敏度（IC50值）为0.02ng/mL，是迄今报道的对玉米赤霉烯酮灵敏度最高的单克隆抗体。
建立了基于免疫亲和柱前处理的间接竞争ELISA检测赭曲霉毒素A和玉米赤霉烯酮的方法。以1H2抗体和2D3抗体为基础，分别建立了基于免疫亲和柱前处理的间接竞争ELISA检测赭曲霉毒素A和玉米赤霉烯酮的方法。经免疫亲和柱处理后，样品基质对免疫竞争反应的干扰得到有效抑制。将建立的ELISA方法用于大米、小麦、大豆、花生样品中赭曲霉毒素A和玉米赤霉烯酮的检测，回收率高，检测结果与标准方法高效液相色谱法的一致性较好，说明该基于免疫亲和前处理的ELISA方法能用于农产品中赭曲霉毒素A和玉米赤霉烯酮的检测分析。
2.研究建立了农产品多种真菌毒素混合污染免疫层析同步检测技术，并成功研制出多种真菌毒素混合污染同步检测的免疫层析试纸条。
研制出一种能同步检测三种真菌毒素的免疫层析试纸条。试纸条具有三条检测线，每条检测线上固定一种真菌毒素的完全抗原，将三种真菌毒素抗体分别标记到胶体金上，基于竞争性免疫层析反应原理，可同步检测黄曲霉毒素B1、赭曲霉毒素A、玉米赤霉烯酮三种真菌毒素。该免疫层析试纸条特异性好，三种真菌毒素之间无交叉反应，对黄曲霉毒素B1、赭曲霉毒素A、玉米赤霉烯酮的可视化检测限分别为0.25、0.5、1ng/mL。检测试纸条的稳定性好，在室温干燥的环境下有效使用期达6个月以上。试纸条的准确性和重现性好，对玉米、大米和花生样品进行检测时，检测结果与ELISA方法的一致性较好，表明研制的免疫层析试纸条在农产品真菌毒素混合污染同步、快速检测中具有应用前景。
3.研究建立了多种真菌毒素混合污染微阵列免疫芯片同步检测技术，并成功研制出一种能同步检测三种真菌毒素的聚合物刷微阵列免疫芯片。
研制出一种能同步检测三种真菌毒素的免疫芯片。基于聚合物刷的微阵列免疫芯片，三维结构的聚合物刷扩大玻片比表面积，提高结合蛋白质分子的能力。且该刷状聚合物修饰的玻片非特异性吸附能力弱，在固定抗原后，不需像ELISA方法一样进行封闭，直接就可用于检测分析，大大缩短了检测时间。本实验通过对固定抗原浓度、抗体使用浓度等条件进行摸索和优化，对黄曲霉毒素B1和赭曲霉毒素A的检出限为4pg/mL，对玉米赤霉烯酮的检出限为3pg/mL。该方法对三种真菌毒素的线性范围分别为：黄曲霉毒素B14-330pg/mL；赭曲霉毒素A12-3000pg/mL；玉米赤霉烯酮3-2000pg/mL。该方法对添加三种真菌毒素的花生样品进行检测分析的准确性高，回收率在85.9-109.2%，该技术为农产品质量安全监测中多种污染物的高通量检测提供了技术支撑。
4.研究建立了多种真菌毒素混合污染免疫亲和-高效液相色谱-质谱联用检测技术，并成功研制出多种真菌毒素同步净化、富集的免疫亲和柱。
基于免疫亲和原理研制了能同步净化、富集黄曲霉毒素B1、赭曲霉毒素A、玉米赤霉烯酮和T-2毒素的免疫亲和柱，对四种真菌毒素的柱容量分别为127.4ng、165.1ng、146.85ng、245.2ng。并对多种真菌毒素同步提取的前处理方法进行了优化，将优化的前处理提取方法与免疫亲合柱结合，用于HPLC-MS/MS检测中的样品前处理，对四种真菌毒素同步检测的回收率介于87%-102%之间，研制的免疫亲和柱在真菌毒素混合污染的确证性检测中具有广阔的应用前景。
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.
我国地域辽阔、气候差异及变化较大，一种农作物被多种真菌侵染的情况较为常见。因此，一种自然条件生长的农产品可能受多种真菌毒素的混合污染。人和动物摄入受真菌毒素混合污染的农产品时，真菌毒素也会随之进入人体，几种真菌毒素协同作用对人和动物的健康危害作用可能比真菌毒素单独作用的危害大。近年来，真菌毒素混合污染受到了越来越多的关注，建立灵敏、快速、高检测通量的检测技术是真菌毒素混合污染监管和保障农产品质量安全的重要措施。目前，用于真菌毒素混合污染多组分检测的技术主要为多功能净化柱-高效液相与质谱联用方法和免疫快速检测方法等，然而多功能净化柱-高效液相与质谱联用方法受样品基质干扰大，特别是无法满足现场快速检测需求；免疫快速检测方法中主要针对单一真菌毒素污染，难以满足多种真菌毒素混合污染快速检测需求。
针对上述问题，本论文的研究内容和创新点如下：
1.选育出赭曲霉毒素A和玉米赤霉烯酮的高灵敏单克隆杂交瘤细胞株，并研制出系列真菌毒素高灵敏单克隆抗体，为多种真菌毒素混合污染检测研究提供自主核心试剂。
研制出一种灵敏度高、特异性好的抗赭曲霉毒素A单克隆抗体。本研究采用动物免疫、细胞融合、半固体培养-梯度筛选法获得3株能产生抗赭曲霉毒素A抗体的单克隆细胞株1H2、1F8、3E2，其中1H2细胞株效价和灵敏度最高。1H2抗体亚型为IgG1型，对赭曲霉毒素A的亲和力常数为1.03×109L/mol，属于高亲和力抗体。1H2抗体对赭曲霉毒素A的反应特异性好，与赭曲霉毒素A结构类似物赭曲霉毒素B交叉反应不明显，与其他种类的真菌毒素黄曲霉毒素B1、G1、玉米赤霉烯酮、呕吐毒素的交叉反应不明显，交叉反应率均小于0.3%。1H2抗体对OTA的灵敏度（IC50值）为0.058ng/mL。该抗体是迄今研究报道的对赭曲霉毒素A特异性最好、灵敏度最高的单克隆抗体。
研制出一种高灵敏度的抗玉米赤霉烯酮单克隆抗体。本研究筛选获得5株能产生抗玉米赤霉烯酮抗体的单克隆杂交瘤细胞株，其中2D3抗体的效价和灵敏度最高。通过对ELISA反应条件的优化，包括对抗原包被浓度，抗体使用浓度，封闭液，反应体系pH值，盐离子浓度的优化，2D3抗体对玉米赤霉烯酮的灵敏度（IC50值）为0.02ng/mL，是迄今报道的对玉米赤霉烯酮灵敏度最高的单克隆抗体。
建立了基于免疫亲和柱前处理的间接竞争ELISA检测赭曲霉毒素A和玉米赤霉烯酮的方法。以1H2抗体和2D3抗体为基础，分别建立了基于免疫亲和柱前处理的间接竞争ELISA检测赭曲霉毒素A和玉米赤霉烯酮的方法。经免疫亲和柱处理后，样品基质对免疫竞争反应的干扰得到有效抑制。将建立的ELISA方法用于大米、小麦、大豆、花生样品中赭曲霉毒素A和玉米赤霉烯酮的检测，回收率高，检测结果与标准方法高效液相色谱法的一致性较好，说明该基于免疫亲和前处理的ELISA方法能用于农产品中赭曲霉毒素A和玉米赤霉烯酮的检测分析。
2.研究建立了农产品多种真菌毒素混合污染免疫层析同步检测技术，并成功研制出多种真菌毒素混合污染同步检测的免疫层析试纸条。
研制出一种能同步检测三种真菌毒素的免疫层析试纸条。试纸条具有三条检测线，每条检测线上固定一种真菌毒素的完全抗原，将三种真菌毒素抗体分别标记到胶体金上，基于竞争性免疫层析反应原理，可同步检测黄曲霉毒素B1、赭曲霉毒素A、玉米赤霉烯酮三种真菌毒素。该免疫层析试纸条特异性好，三种真菌毒素之间无交叉反应，对黄曲霉毒素B1、赭曲霉毒素A、玉米赤霉烯酮的可视化检测限分别为0.25、0.5、1ng/mL。检测试纸条的稳定性好，在室温干燥的环境下有效使用期达6个月以上。试纸条的准确性和重现性好，对玉米、大米和花生样品进行检测时，检测结果与ELISA方法的一致性较好，表明研制的免疫层析试纸条在农产品真菌毒素混合污染同步、快速检测中具有应用前景。
3.研究建立了多种真菌毒素混合污染微阵列免疫芯片同步检测技术，并成功研制出一种能同步检测三种真菌毒素的聚合物刷微阵列免疫芯片。
研制出一种能同步检测三种真菌毒素的免疫芯片。基于聚合物刷的微阵列免疫芯片，三维结构的聚合物刷扩大玻片比表面积，提高结合蛋白质分子的能力。且该刷状聚合物修饰的玻片非特异性吸附能力弱，在固定抗原后，不需像ELISA方法一样进行封闭，直接就可用于检测分析，大大缩短了检测时间。本实验通过对固定抗原浓度、抗体使用浓度等条件进行摸索和优化，对黄曲霉毒素B1和赭曲霉毒素A的检出限为4pg/mL，对玉米赤霉烯酮的检出限为3pg/mL。该方法对三种真菌毒素的线性范围分别为：黄曲霉毒素B14-330pg/mL；赭曲霉毒素A12-3000pg/mL；玉米赤霉烯酮3-2000pg/mL。该方法对添加三种真菌毒素的花生样品进行检测分析的准确性高，回收率在85.9-109.2%，该技术为农产品质量安全监测中多种污染物的高通量检测提供了技术支撑。
4.研究建立了多种真菌毒素混合污染免疫亲和-高效液相色谱-质谱联用检测技术，并成功研制出多种真菌毒素同步净化、富集的免疫亲和柱。
基于免疫亲和原理研制了能同步净化、富集黄曲霉毒素B1、赭曲霉毒素A、玉米赤霉烯酮和T-2毒素的免疫亲和柱，对四种真菌毒素的柱容量分别为127.4ng、165.1ng、146.85ng、245.2ng。并对多种真菌毒素同步提取的前处理方法进行了优化，将优化的前处理提取方法与免疫亲合柱结合，用于HPLC-MS/MS检测中的样品前处理，对四种真菌毒素同步检测的回收率介于87%-102%之间，研制的免疫亲和柱在真菌毒素混合污染的确证性检测中具有广阔的应用前景。
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.
引文
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