黄曲霉毒素替代物绿色免疫分析研究
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摘要
黄曲霉毒素（Aflatoxin）是一类真菌毒素，主要由黄曲霉（Aspergilus flavus）、寄生曲霉（Aspergilus parasiticus）和集峰曲霉（Aspergillus nomius）产生，广泛污染花生、玉米、大豆、大米、高粱等大宗农作物。黄曲霉毒素是目前发现的致癌能力最强的真菌毒素，严重危害人畜健康，世界各国对食品、农产品中黄曲霉毒素的含量都有严格限定，一些发达国家借此对我国出口农产品设置贸易壁垒，致使我国出口农产品常因黄曲霉毒素超标发生贸易问题，造成巨大的经济损失。由于产毒真菌在各级食物链中广泛分布，在一定环境条件下便会侵染作物并产生黄曲霉毒素，因此，黄曲霉毒素与农兽药不同，无法通过药源管理实现有效控制。因此，为了保障消费者安全，减少贸易壁垒给我国带来的经济损失，保障社会可持续发展，实现让消费者吃上放心食品的中国梦，需要强大的检测技术作为支撑，防止有毒有害食品流入市场，走入餐桌。
目前，黄曲霉毒素的检测方法有很多，包括薄层层析法、免疫亲和柱净化-荧光光度法、高效液相色谱法等。免疫分析法是众多检测方法的一个重要的补充，它具有检测灵敏度高、选择性好、样品前处理简便、成本低等优势。黄曲霉毒素是小分子化合物，需要采用竞争免疫分析模式进行检测，即通过包被抗原与待测化合物共同竞争结合抗体，以待测化合物浓度为横坐标，相应的B/B0值为纵坐标建立标准曲线，从而对样品中的分析物进行定量。在黄曲霉毒素的免疫分析中，包被抗原为偶联有大分子蛋白（如牛血清白蛋白）的黄曲霉毒素，抗原的合成过程需要大量黄曲霉毒素标准物，在方法的建立与应用中，同样需要使用黄曲霉毒素标准物建立标准曲线，不仅价格昂贵，而且直接威胁操作人员身体健康，如果对检测后含有剧毒黄曲霉毒素标准物的废液处理不当，极易造成实验室及环境的严重二次污染问题。
本研究针对以上问题，研制出了两类黄曲霉毒素替代抗原，一种黄曲霉毒素替代标准物，并以此为基础，研究建立了粮油产品黄曲霉毒素绿色免疫分析技术，为粮油等种植业大宗农产品中黄曲霉毒素污染的监测提供了灵敏、快速、简便、成本低、环保的新方法。本论文研究的主要内容和创新点如下：
1.研制出黄曲霉毒素噬菌体八肽替代抗原，将噬菌体肽用作黄曲霉毒素替代抗原建立免疫分析技术，开辟了黄曲霉毒素绿色免疫检测的新途径。
研究构建了噬菌体展示随机八肽库，以抗黄曲霉毒素单克隆抗体1C11为基础，筛选出五种噬菌体肽可用作黄曲霉毒素替代抗原，通过对包被抗体浓度、噬菌体肽浓度、pH值、盐离子浓度、甲醇浓度等理化参数的优化，分别构建基于五种噬菌体肽的免疫竞争曲线，其中CM4用作黄曲霉毒素替代抗原建立的ELISA法灵敏度最高，检测黄曲霉毒素B1的IC50值为0.290ng/mL；研究了花生、玉米、大米三种代表粮油产品的基质效应对该检测方法的影响，不同样品基质对ELISA标准曲线和最大OD值均有一定程度的影响，经过多次试验，发现在样品稀释液中添加一定浓度的牛血清白蛋白（BSA）（2%-4%）可以使得基质效应减少到可接受范围；对花生、玉米、大米三种空白样品添加不同浓度的AFB1和混合黄曲霉毒素，样品检测回收率均在60-120%之间，标准偏差在10%以内；分别采用传统ELISA与基于噬菌体肽替代抗原的ELISA检测天然污染黄曲霉毒素的样品，检测结果具有很好的相关性（R2=0.931）。与国内外相关噬菌体肽替代抗原相比发现，本研究中建立的基于噬菌体肽替代抗原的ELISA法检测灵敏度最高，且对黄曲霉毒素B1、B2、G1、G2的通用性最强。
2.首次研制出纳米抗体用作黄曲霉毒素替代抗原，研究建立了基于纳米抗体替代抗原的免疫分析技术，为黄曲霉毒素等剧毒污染物绿色免疫分析研究提供了新的技术平台。
以抗黄曲霉毒素单克隆抗体1C11作为免疫原免疫羊驼，提取外周血RNA，采用重链抗体铰链区特异性引物，克隆获得两类不同亚型的重链抗体IgG2a及IgG3的重链可变区片段（VHH），大小在400-600bp之间。再将扩增得到的VHH片段连接到噬菌粒载体pComb3X上，电转化大肠杆菌ER2738，通过M13KO7辅助噬菌体拯救获得库容量为1.1109pfu的纳米抗体库。经过三轮生物亲和淘选，从中获得三种能够与抗体1C11可变区特异性结合的噬菌体展示纳米抗体，通过对包被抗体浓度、噬菌体浓度、pH值、盐离子浓度、甲醇浓度等参数的优化，建立了基于三种噬菌体展示纳米抗体用作替代竞争抗原的ELISA法，其中基于phage2-5的ELISA法具有最高的检测灵敏度，对四种黄曲霉毒素（AFB1、AFB2、AFG1、AFG2）检测的IC50值分别为0.054ng/mL、0.140ng/mL、0.077ng/mL、0.373ng/mL；对三种纳米抗体进行表达纯化，直接用作包被抗原，对包被浓度、抗体1C11浓度、反应体系pH值、盐离子浓度、甲醇浓度等各条件进行优化，建立了基于纳米抗体的竞争抑制曲线，其中VHH2-5作为包被抗原建立的ELISA法检测灵敏度最高，对黄曲霉毒素B1的IC50值为0.160ng/mL，反应对四种黄曲霉毒素（AFB1、AFB2、AFG1、AFG2）的交叉反应率分别是100%、54.4%、90.4%和37.7%；以花生、大米、玉米三种空白样品作为基质进行AFB1和黄曲霉毒素总量的添加回收实验，AFB1的样品添加回收率均在70-120%之间；分别采用基于纳米抗体的ELISA法与常规ELISA法对天然污染黄曲霉毒素的样品进行检测，两种方法检测结果一致，表明本研究建立的基于纳米抗体能够很好的应用于黄曲霉毒素的污染监测中。
3.首次将纳米抗体用作黄曲霉毒素替代标准物，研究建立了基于纳米抗体替代标准物的快速检测技术，开辟了黄曲霉毒素绿色免疫检测的新途径。
将纳米抗体VHH2-5用作黄曲霉毒素替代标准物，以纳米抗体VHH2-5的浓度作为横坐标，以ELISA检测的B/B0值作为纵坐标，建立标准曲线，在相同的B/B0下，替代标准物浓度与AFB1浓度呈良好的线性（R2=0.998），以VHH2-5浓度为横坐标，AFB1浓度为纵坐标，建立线性相关曲线。采用两步法计算样品中黄曲霉毒素含量，首先根据显色值计算替代标准物浓度，然后通过VHH2-5与AFB1的线性相关方程，转换为黄曲霉毒素含量。以花生、玉米和大米作为代表样品，分别构建三种基质的转换方程，测定AFB1的添加回收率，结果均在70-110%；组间和组内标准偏差分别在0.7-6.2%和8.3-18.7%之间。选取了20份天然污染黄曲霉毒素的样品，采用替代标准物ELISA法进行检测，与高效液相色谱法检测结果进行比对，相关系数R2值为0.988，对比结果高度一致；此外，对纳米抗体VHH2-5进行耐热性能测定，并与实验室制备的黄曲霉毒素M1抗独特型多克隆抗体进行比较，结果表明纳米抗体比传统抗体具有更好的温度稳定性。因此，本研究研制的纳米抗体替代标准物能够很好的应用于实际样品中黄曲霉毒素污染的检测，并且具有良好的稳定性。
The aflatoxins are mycotoxins produced mainly by the fungal species Aspergillus flavus，Aspergillus parasiticus and Aspergillus nomius, which occur on a wide range of agriculturalcommodities and food matrices, such as peanuts, corns, soybeans, rice and sorghum, etc. Aflatoxin is upto now one of the most potent carcinogens, which is harmful to human and livestock. Every country inthe world has set up strict regulations towards aflatoxins in food and agro-products. Some developedcountries set up trade barrier on aflatoxins which cause serious economic loss to our export ofagro-products due to exceeding concentration of aflatoixns. Mycotoxin-producing fungi can occur inany food chain and will produce aflatoxins under some circumstances. As a result, the occurrence ofaflatoxin is unavoidable and difficult to be elimited by regulation. In order to protect the health ofcustomers, eliminate the economic loss, guarantee the sustainable development of society and realizethe Chinese Dream of ‘eat safe’, powerful detection techniques are required to avoid harmful foodentering into the market and onto the table.
There are a variety of analytical methods for aflatoxins, including the original thin-layerchromatographic (TLC) method, immunoaffinity column purification coupled with fluorescencedetection, high performance liquid chromatography (HPLC), etc. Immunoassays are importantsupplement to those methods, which have advantages such as high sensitivity, selectivity, rapid samplepreparation, and cost effective, etc. Aflatoxins are small moleculars, so it is necessary to use anaflatoxin-protein conjugate as coating antigen, competing combination to antibody with free aflatoxins,so called competitive immunoassay. In the case of aflatoxins, the coating antigen is always synthesizedby conjugating protein, such as bovine serum albumin to aflatoxin molecular. Huge amount of aflatoxinstandard is needed during synthesis, as well as in the development of an immunoassay, which is notonly quite expensive, but also causes potential hazardous to analysts and the environment.
In our research, two types of aflatoxin substitute antigen and one surrogate standard were obtained.Green immunoassays based on surrogate antigen or standard were developed in this research, whichprovide a sensitive, fast, simple, cost effective and environmental friendly method to regulate aflatoxinsin agro-products. The main contents and innovations are as follow:
1. Phage-displayed peptides that mimic aflatoxin antigen were developed in this research. Greenimmunoassay towards aflatoxin was constructed based on those mimotopes, which provide technicalsupport to green analysis of aflatoxin.
A random-8-peptide phage-displayed library was constructed and used as a source of peptides thatmimic aflatoxins. Five mimotope peptides were obtained by panning-elution from the library. Afteroptimization of the concentration of coating antibody and phage, pH value, ion strength, and methanolconcentration, etc, competitive ELISAs were constructed based on five phage-displayed peptides.ELISA based on phage CM4has the highest sensitivity with IC50value of0.290ng/mL. Matrix effect was studied by using peanut, corn and rice as representative samples. By adding2-4%BSA in extractdilution buffer, matrix effect was successfully eliminated to an acceptable range. Further validationindicated relatively good recovery (60-120%) with RSD less than10%. Natural contaminated sampleswere analyzed for aflatoxin concentration by both conventional ELISA and phage ELISA. The resultsshowed good correlation (R2=0.931). It can be concluded that the mimotope preparation is an effectivesubstitute for the aflatoxin based coating antigen in ELISA and can be used in real sample analysis.
2. This is the first application of nanobody technique in the development of antigen mimotope,which provides a new technical platform for green analysis of aflatoxin.
An alpaca was immunized by anti-aflatoxin monoclonal antibody1C11. Total RNA was extractedfrom alpaca’s blood and VHH genes (400-600bp) were cloned by PCR using two pairs of hingespecified primers. The phage displayed VHH library was constructed by ligating amplified VHH geneswith plasmid pComb3X and electrotransformated to E. coli ER2738. The achieved library has a size of1.1109pfu. After3rounds of biopanning, three anti-idiotype nanobodies were selected. Phage ELISAwas developed after a series of optimization of coating antibody concentration, phage workingconcentration, pH value, ionic strength and methanol concentration, etc. ELISA based on phage2-5hasthe best sensitivity with IC50value of0.054ng/mL,0.140ng/mL,0.077ng/mL and0.373ng/mLtowards AFB1, AFB2, AFG1and AFG2, respectively. Three anti-idiotype nanobodies were expressed,purified and used directly as substitute coating antigen. After optimization of coating concentration,antibody concentration, pH value, ionic strength and methanol concentration, VHH ELISAs weredeveloped with three nanobodies. The best immunoassay developed with VHH2-5shows an IC50of0.160ng/mL towards aflatoxin B1and cross-reactivity toward aflatoxin B2, G1and G2of54.4%,90.4%,and37.7%, respectively. Good recoveries (70-120%) of aflatoxin B1were achieved from peanuts, riceand corn. A good correlation was found between the data obtained from the conventional ELISA andELISA based on a VHH coating antigen for the analysis of aflatoxins in peanuts and feedstuff. Theseresults indicated that the developed ELISA could well meet the specific detection of AFB1in realsamples.
3. This is the first research using nanobody as aflatoxin surrogate standard, which exploits a newway to develop immunoreagent.
Nanobody VHH2-5was applied as surrogate standard in competitive ELISA towards aflatoxin. Byusing the concentration of VHH2-5as x axis, the value of B/B0as y axis, a standard curve wasdeveloped. The concentration of VHH2-5showed excellent linear relationship with aflatoxinconcentration at the same inhibition value (R2=0.998). The concentration of aflatoxin was calculated bya two-step calculation: the concentration of VHH2-5was first achieved by a four-parameter logisticregression from the detected OD450value, and then converted to aflatoxin concentration by a linearequation. The average recoveries of AFB1from spiked peanut, rice and corn samples were in the rangeof70-110%. The inter-and intra-laboratory reproducibility was in the range of0.7-6.2%and8.3-18.7%, respectively. The assay was compared to a high-performance liquid chromatographic (HPLC) methodfor determination of20naturally contaminated peanut samples, displaying a good correlation(R2=0.988). We also tested the thermostability of VHH2-5, by comparison with anti-idiotypepolyclonal antibody towards aflatoxin M1developed in our laboratory, VHH2-5showed better stabilityunder high temperature. In conclusion, VHH2-5could be used as surrogate standard and applied in thecontamination monitoring of aflatoxins in real samples.
目前，黄曲霉毒素的检测方法有很多，包括薄层层析法、免疫亲和柱净化-荧光光度法、高效液相色谱法等。免疫分析法是众多检测方法的一个重要的补充，它具有检测灵敏度高、选择性好、样品前处理简便、成本低等优势。黄曲霉毒素是小分子化合物，需要采用竞争免疫分析模式进行检测，即通过包被抗原与待测化合物共同竞争结合抗体，以待测化合物浓度为横坐标，相应的B/B0值为纵坐标建立标准曲线，从而对样品中的分析物进行定量。在黄曲霉毒素的免疫分析中，包被抗原为偶联有大分子蛋白（如牛血清白蛋白）的黄曲霉毒素，抗原的合成过程需要大量黄曲霉毒素标准物，在方法的建立与应用中，同样需要使用黄曲霉毒素标准物建立标准曲线，不仅价格昂贵，而且直接威胁操作人员身体健康，如果对检测后含有剧毒黄曲霉毒素标准物的废液处理不当，极易造成实验室及环境的严重二次污染问题。
本研究针对以上问题，研制出了两类黄曲霉毒素替代抗原，一种黄曲霉毒素替代标准物，并以此为基础，研究建立了粮油产品黄曲霉毒素绿色免疫分析技术，为粮油等种植业大宗农产品中黄曲霉毒素污染的监测提供了灵敏、快速、简便、成本低、环保的新方法。本论文研究的主要内容和创新点如下：
1.研制出黄曲霉毒素噬菌体八肽替代抗原，将噬菌体肽用作黄曲霉毒素替代抗原建立免疫分析技术，开辟了黄曲霉毒素绿色免疫检测的新途径。
研究构建了噬菌体展示随机八肽库，以抗黄曲霉毒素单克隆抗体1C11为基础，筛选出五种噬菌体肽可用作黄曲霉毒素替代抗原，通过对包被抗体浓度、噬菌体肽浓度、pH值、盐离子浓度、甲醇浓度等理化参数的优化，分别构建基于五种噬菌体肽的免疫竞争曲线，其中CM4用作黄曲霉毒素替代抗原建立的ELISA法灵敏度最高，检测黄曲霉毒素B1的IC50值为0.290ng/mL；研究了花生、玉米、大米三种代表粮油产品的基质效应对该检测方法的影响，不同样品基质对ELISA标准曲线和最大OD值均有一定程度的影响，经过多次试验，发现在样品稀释液中添加一定浓度的牛血清白蛋白（BSA）（2%-4%）可以使得基质效应减少到可接受范围；对花生、玉米、大米三种空白样品添加不同浓度的AFB1和混合黄曲霉毒素，样品检测回收率均在60-120%之间，标准偏差在10%以内；分别采用传统ELISA与基于噬菌体肽替代抗原的ELISA检测天然污染黄曲霉毒素的样品，检测结果具有很好的相关性（R2=0.931）。与国内外相关噬菌体肽替代抗原相比发现，本研究中建立的基于噬菌体肽替代抗原的ELISA法检测灵敏度最高，且对黄曲霉毒素B1、B2、G1、G2的通用性最强。
2.首次研制出纳米抗体用作黄曲霉毒素替代抗原，研究建立了基于纳米抗体替代抗原的免疫分析技术，为黄曲霉毒素等剧毒污染物绿色免疫分析研究提供了新的技术平台。
以抗黄曲霉毒素单克隆抗体1C11作为免疫原免疫羊驼，提取外周血RNA，采用重链抗体铰链区特异性引物，克隆获得两类不同亚型的重链抗体IgG2a及IgG3的重链可变区片段（VHH），大小在400-600bp之间。再将扩增得到的VHH片段连接到噬菌粒载体pComb3X上，电转化大肠杆菌ER2738，通过M13KO7辅助噬菌体拯救获得库容量为1.1109pfu的纳米抗体库。经过三轮生物亲和淘选，从中获得三种能够与抗体1C11可变区特异性结合的噬菌体展示纳米抗体，通过对包被抗体浓度、噬菌体浓度、pH值、盐离子浓度、甲醇浓度等参数的优化，建立了基于三种噬菌体展示纳米抗体用作替代竞争抗原的ELISA法，其中基于phage2-5的ELISA法具有最高的检测灵敏度，对四种黄曲霉毒素（AFB1、AFB2、AFG1、AFG2）检测的IC50值分别为0.054ng/mL、0.140ng/mL、0.077ng/mL、0.373ng/mL；对三种纳米抗体进行表达纯化，直接用作包被抗原，对包被浓度、抗体1C11浓度、反应体系pH值、盐离子浓度、甲醇浓度等各条件进行优化，建立了基于纳米抗体的竞争抑制曲线，其中VHH2-5作为包被抗原建立的ELISA法检测灵敏度最高，对黄曲霉毒素B1的IC50值为0.160ng/mL，反应对四种黄曲霉毒素（AFB1、AFB2、AFG1、AFG2）的交叉反应率分别是100%、54.4%、90.4%和37.7%；以花生、大米、玉米三种空白样品作为基质进行AFB1和黄曲霉毒素总量的添加回收实验，AFB1的样品添加回收率均在70-120%之间；分别采用基于纳米抗体的ELISA法与常规ELISA法对天然污染黄曲霉毒素的样品进行检测，两种方法检测结果一致，表明本研究建立的基于纳米抗体能够很好的应用于黄曲霉毒素的污染监测中。
3.首次将纳米抗体用作黄曲霉毒素替代标准物，研究建立了基于纳米抗体替代标准物的快速检测技术，开辟了黄曲霉毒素绿色免疫检测的新途径。
将纳米抗体VHH2-5用作黄曲霉毒素替代标准物，以纳米抗体VHH2-5的浓度作为横坐标，以ELISA检测的B/B0值作为纵坐标，建立标准曲线，在相同的B/B0下，替代标准物浓度与AFB1浓度呈良好的线性（R2=0.998），以VHH2-5浓度为横坐标，AFB1浓度为纵坐标，建立线性相关曲线。采用两步法计算样品中黄曲霉毒素含量，首先根据显色值计算替代标准物浓度，然后通过VHH2-5与AFB1的线性相关方程，转换为黄曲霉毒素含量。以花生、玉米和大米作为代表样品，分别构建三种基质的转换方程，测定AFB1的添加回收率，结果均在70-110%；组间和组内标准偏差分别在0.7-6.2%和8.3-18.7%之间。选取了20份天然污染黄曲霉毒素的样品，采用替代标准物ELISA法进行检测，与高效液相色谱法检测结果进行比对，相关系数R2值为0.988，对比结果高度一致；此外，对纳米抗体VHH2-5进行耐热性能测定，并与实验室制备的黄曲霉毒素M1抗独特型多克隆抗体进行比较，结果表明纳米抗体比传统抗体具有更好的温度稳定性。因此，本研究研制的纳米抗体替代标准物能够很好的应用于实际样品中黄曲霉毒素污染的检测，并且具有良好的稳定性。
The aflatoxins are mycotoxins produced mainly by the fungal species Aspergillus flavus，Aspergillus parasiticus and Aspergillus nomius, which occur on a wide range of agriculturalcommodities and food matrices, such as peanuts, corns, soybeans, rice and sorghum, etc. Aflatoxin is upto now one of the most potent carcinogens, which is harmful to human and livestock. Every country inthe world has set up strict regulations towards aflatoxins in food and agro-products. Some developedcountries set up trade barrier on aflatoxins which cause serious economic loss to our export ofagro-products due to exceeding concentration of aflatoixns. Mycotoxin-producing fungi can occur inany food chain and will produce aflatoxins under some circumstances. As a result, the occurrence ofaflatoxin is unavoidable and difficult to be elimited by regulation. In order to protect the health ofcustomers, eliminate the economic loss, guarantee the sustainable development of society and realizethe Chinese Dream of ‘eat safe’, powerful detection techniques are required to avoid harmful foodentering into the market and onto the table.
There are a variety of analytical methods for aflatoxins, including the original thin-layerchromatographic (TLC) method, immunoaffinity column purification coupled with fluorescencedetection, high performance liquid chromatography (HPLC), etc. Immunoassays are importantsupplement to those methods, which have advantages such as high sensitivity, selectivity, rapid samplepreparation, and cost effective, etc. Aflatoxins are small moleculars, so it is necessary to use anaflatoxin-protein conjugate as coating antigen, competing combination to antibody with free aflatoxins,so called competitive immunoassay. In the case of aflatoxins, the coating antigen is always synthesizedby conjugating protein, such as bovine serum albumin to aflatoxin molecular. Huge amount of aflatoxinstandard is needed during synthesis, as well as in the development of an immunoassay, which is notonly quite expensive, but also causes potential hazardous to analysts and the environment.
In our research, two types of aflatoxin substitute antigen and one surrogate standard were obtained.Green immunoassays based on surrogate antigen or standard were developed in this research, whichprovide a sensitive, fast, simple, cost effective and environmental friendly method to regulate aflatoxinsin agro-products. The main contents and innovations are as follow:
1. Phage-displayed peptides that mimic aflatoxin antigen were developed in this research. Greenimmunoassay towards aflatoxin was constructed based on those mimotopes, which provide technicalsupport to green analysis of aflatoxin.
A random-8-peptide phage-displayed library was constructed and used as a source of peptides thatmimic aflatoxins. Five mimotope peptides were obtained by panning-elution from the library. Afteroptimization of the concentration of coating antibody and phage, pH value, ion strength, and methanolconcentration, etc, competitive ELISAs were constructed based on five phage-displayed peptides.ELISA based on phage CM4has the highest sensitivity with IC50value of0.290ng/mL. Matrix effect was studied by using peanut, corn and rice as representative samples. By adding2-4%BSA in extractdilution buffer, matrix effect was successfully eliminated to an acceptable range. Further validationindicated relatively good recovery (60-120%) with RSD less than10%. Natural contaminated sampleswere analyzed for aflatoxin concentration by both conventional ELISA and phage ELISA. The resultsshowed good correlation (R2=0.931). It can be concluded that the mimotope preparation is an effectivesubstitute for the aflatoxin based coating antigen in ELISA and can be used in real sample analysis.
2. This is the first application of nanobody technique in the development of antigen mimotope,which provides a new technical platform for green analysis of aflatoxin.
An alpaca was immunized by anti-aflatoxin monoclonal antibody1C11. Total RNA was extractedfrom alpaca’s blood and VHH genes (400-600bp) were cloned by PCR using two pairs of hingespecified primers. The phage displayed VHH library was constructed by ligating amplified VHH geneswith plasmid pComb3X and electrotransformated to E. coli ER2738. The achieved library has a size of1.1109pfu. After3rounds of biopanning, three anti-idiotype nanobodies were selected. Phage ELISAwas developed after a series of optimization of coating antibody concentration, phage workingconcentration, pH value, ionic strength and methanol concentration, etc. ELISA based on phage2-5hasthe best sensitivity with IC50value of0.054ng/mL,0.140ng/mL,0.077ng/mL and0.373ng/mLtowards AFB1, AFB2, AFG1and AFG2, respectively. Three anti-idiotype nanobodies were expressed,purified and used directly as substitute coating antigen. After optimization of coating concentration,antibody concentration, pH value, ionic strength and methanol concentration, VHH ELISAs weredeveloped with three nanobodies. The best immunoassay developed with VHH2-5shows an IC50of0.160ng/mL towards aflatoxin B1and cross-reactivity toward aflatoxin B2, G1and G2of54.4%,90.4%,and37.7%, respectively. Good recoveries (70-120%) of aflatoxin B1were achieved from peanuts, riceand corn. A good correlation was found between the data obtained from the conventional ELISA andELISA based on a VHH coating antigen for the analysis of aflatoxins in peanuts and feedstuff. Theseresults indicated that the developed ELISA could well meet the specific detection of AFB1in realsamples.
3. This is the first research using nanobody as aflatoxin surrogate standard, which exploits a newway to develop immunoreagent.
Nanobody VHH2-5was applied as surrogate standard in competitive ELISA towards aflatoxin. Byusing the concentration of VHH2-5as x axis, the value of B/B0as y axis, a standard curve wasdeveloped. The concentration of VHH2-5showed excellent linear relationship with aflatoxinconcentration at the same inhibition value (R2=0.998). The concentration of aflatoxin was calculated bya two-step calculation: the concentration of VHH2-5was first achieved by a four-parameter logisticregression from the detected OD450value, and then converted to aflatoxin concentration by a linearequation. The average recoveries of AFB1from spiked peanut, rice and corn samples were in the rangeof70-110%. The inter-and intra-laboratory reproducibility was in the range of0.7-6.2%and8.3-18.7%, respectively. The assay was compared to a high-performance liquid chromatographic (HPLC) methodfor determination of20naturally contaminated peanut samples, displaying a good correlation(R2=0.988). We also tested the thermostability of VHH2-5, by comparison with anti-idiotypepolyclonal antibody towards aflatoxin M1developed in our laboratory, VHH2-5showed better stabilityunder high temperature. In conclusion, VHH2-5could be used as surrogate standard and applied in thecontamination monitoring of aflatoxins in real samples.
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