【作者】 赵军；

【导师】 王爱萍；

【作者基本信息】 郑州大学 ， 细胞生物学， 2018， 硕士

【摘要】 真菌在农产品中大量滋生,它的代谢物存在于各种农业原材料和商品中。真菌毒素具有400多种不同的化学结构,其中赭曲霉毒素A（Ochratoxin A,OTA）是其主要的组成部分。OTA具有肾毒性、免疫毒性和致畸作用,被国际癌症研究机构（IARC）归类为致癌物质。现如今,最常用的检测方法为仪器分析法和免疫分析法。仪器分析法往往需要昂贵的仪器、操作繁琐且需要专业的技术人员。免疫分析法具有操作简单、灵敏度高、特异性强的优点,可以快速检测大量的待测样品。本实验制备了OTA单克隆抗体,研制出直接竞争ELISA试剂盒,从而建立了快速、灵敏的OTA的检测方法。本研究把OTA和牛血清白蛋白（Bovine serum albumin,BSA）偶联合成免疫原OTA-BSA,把OTA和鸡卵清蛋白（Ovalbumin,OVA）偶联合成检测原OTA-OVA。采用聚丙烯酰胺凝胶电泳和紫外扫描的方法,鉴定合成的抗原,结果显示合成成功。OTA-BSA和OTA-OVA偶联比分别为7.5:1与5:1。免疫结果表明OTA-BSA免疫的小鼠,产生了免疫应答。用OTA-OVA包板进行检测,其中1号小鼠免疫效果最好,其多抗血清效价最高可达1:51200,半数抑制浓度IC₅₀为7.98 ng/mL。选取免疫效价较高的1号小鼠,取其脾脏与骨髓瘤细胞（SP2/0）进行融合。取杂交瘤细胞上清液,采用间接ELISA法测其效价,用间接竞争ELISA法测其抑制,从而筛选阳性克隆。通过3轮亚克隆,共得到6株阳性克隆细胞株,分别命名为1F5、2C5、2D7、2G3、3D7和3F6。选择阳性值最高的2G3制备腹水,之后对腹水进行纯化。采用SDS-PAGE鉴定,纯化后单克隆抗体只含有2条清晰的条带,分子量分别为50 kDa和25 kDa,说明得到的单克隆抗体纯度高。通过ELISA鉴定,2G3纯化前后的效价都达到1:8.192×10⁷,IC₅₀为1.22 ng/mL,亲和力常数为2.11×10¹¹ mol/L。用亚型鉴定试剂盒检测2G3,其亚型为IgG1、κ轻链型。该细胞株特异性高,敏感性好,可以用来研制试剂盒。采用过碘酸钠法,将2G3连接到HRP上,制备得到酶标抗体。根据酶联免疫吸附试验的原理,应用酶标抗体,研制出直接竞争ELISA试剂盒,从而快速检测OTA残留。经条件优化后,OTA浓度与结合率呈良好的线性关系（y=-32.94x+61.00,R²=0.981）,IC₅₀为2.16 ng/mL,加标样品回收率范围为80.3%¹08%。该试剂盒与赭曲霉毒素B、C的交叉反应率分别为3.5%和0.28%,与其他生物毒素均无交叉反应。板间和板内平均变异系数分别为6.41%和7.07%。玉米、小麦和花生样品OTA的检测限分别为0.134、0.203和0.292 ng/mL。本研究制备的直接竞争ELISA（cd-ELISA）试剂盒,可以用于快速定量检测食品以及饲料中的OTA。更多还原

【Abstract】 Ochratoxins are toxic metabolites which are secreted by fungi such as Aspergillus and Penicillium.Fungi are abundant in agricultural products,and its metabolites are found in various agricultural raw materials and commodities.Mycotoxins have more than 400 different chemical structures,of which OTA is the main component.OTA has nephrotoxicity,immunotoxicity and teratogenic effects and IARC categorizes it as a carcinogen.Instrumental analysis and immunoassay are the most commonly used methods.Instrumental analysis methods often require expensive instruments,cumbersome operations,and require specialized technicians.Immunoassays have the advantages of simple operation,high sensitivity and strong specificity.It can quickly detect a large number of samples to be tested.In this experiment,OTA monoclonal antibodies were prepared and a competitive direct ELISA kit was developed,thereby establishing a rapid,sensitive detection method for OTA.In this study,OTA was coupled to BSA to synthesize the immunogen OTA-BSA,and OTA was coupled OVA to synthesize detecting antigen OTA-OVA.The synthesized antigens were identified by polyacrylamide gel electrophoresis and UV scanning spectrum.The results showed that the synthesis was successful.The OTA-BSA and OTA-OVA coupling ratios were 7.5:1 and 5:1,respectively.The immunization results showed that mice immunized with OTA-BSA produced an immune response.The OTA-OVA was used as coating antigen for detection.Among them,mouse 1 had the best immune effect.The titer of multi-antibody serum was up to 1:51200,and the value of IC50 was 7.98 ng/mL.Mouse 1 spleen cells fused with mouse myeloma cells SP2/0.After fusion,the supernatant of the hybridoma cells was taken and the potency was measured by indirect ELISA.The inhibition was measured by indirect competitive ELISA and the positive clones were screened.After three rounds of subcloning,six positive clone cell lines named 1F5,2C5,2D7,2G3,3D7 and 3F6 were obtained.The 2G3 with the highest positive value was selected to prepare ascites,after which ascites was purified.Polyacrylamide gel electrophoresis was used for identification.The purified monoclonal antibody contained only two clear bands with molecular weights of 50kDa and 25 kDa,indicating that the monoclonal antibody had higher purity.By ELISA,the titer before and after 2G3 purification reached 1:8.192×10⁷.The IC₅₀ was1.22 ng/mL and the affinity constant was 2.11×10¹¹ mol/L.2G3 was detected using a subtype identification kit,and its subtypes were IgG1 andκlight chain type.The cell line has high specificity and sensitivity and can be used to develop a kit.Using sodium periodate method,2G3 was attached to HRP to prepare enzyme-labeled antibody.According to the principle of enzyme-linked immunosorbent assay,the enzyme-labeled antibody was used to develop a competitive direct ELISA kit to rapidly detect OTA residues.After optimizing the conditions,the OTA concentration and the binding rate showed a good linear relationship（y=-32.94x+61.00,R²=0.981）.The IC₅₀ was 2.16 ng/mL and the sample recovery rate range was 80.3%¹08%.The cross-reactivity rates of the kit with ochratoxin B and C were 3.5%and 0.28%,respectively,and there was no cross-reaction with other biotoxins.The inter-panel and intra-board average coefficients of variation were 6.41%and 7.07%,respectively.The detection limits of OTA in corn,wheat and peanut were 0.134,0.203 and 0.292 ng/mL,respectively.It can be concluded that the competitive direct ELISA kit prepared in this study can be used for rapid and quantitative detection of OTA in food and feed.更多还原