【作者】 孟辉；

【导师】 沈建忠； 王战辉；

【作者基本信息】 中国农业大学 ， 基础兽医学， 2013， 博士

【摘要】 赭曲霉毒素A (Ochratoxin A, OTA)是由赭曲霉和疣孢青霉等青霉菌产生的次级代谢产物,具有高毒活性,广泛存在于食品和饲料中,严重危害人和动物的健康。本文制备了OTA的单克隆抗体、单链抗体和免疫亲和色谱柱(Immunoaffinity Chromatography, IAC),建立了可以检测OTA残留的酶联免疫检测法(Enzyme linked Immunosorbent Assay, ELISA)、免疫亲和色谱柱-超高效液相色谱串联质谱法(IAC-UPLC-MS/MS)和化学发光酶联免疫检测法(Chemiluminescence Enzyme Immunoassay, CLEIA),为OTA的残留检测提供了快速筛选和定量分析的技术手段。制备出了单克隆抗体4H10,抗体效价为1：1.28×106,亲和力常数Ka=5.31×109mol L-1,重链为IgG1型,轻链为κ型,与OTA、赭曲霉毒素B (Ochratoxin B, OTB)和赭曲霉毒素C(Ochratoxin C,OTC)的交叉反应率分别为100%、26.98%、137.23%,IC50值分别为1.29、4.78、0.94ng mL-1,与黄曲霉毒素(B1、B2、G1、G2、M1、M2,)、伏马菌素B1、玉米赤霉烯酮、呕吐毒素、T-2毒素均无交叉反应。同时以杂交瘤细胞为基因来源,制备出了抗OTA的单链抗体,抗体对OTA的IC50值是8.12ng mL-1,与OTA、OTB和OTC的交叉反应率分别为100%、15.25%、124.34%,与黄曲霉毒素(B1、B2、G、G2、M1、M2)、伏马菌素B1、玉米赤霉烯酮、呕吐毒素、T-2毒素均无交叉反应。以单抗4H10作为抗体材料,建立了玉米、大豆、小麦和猪饲料中OTA的间接竞争酶联免疫检测法(ciELISA),方法线性范围是0.3～3.0μg kg-1,玉米、大豆、小麦的检测限为0.15μgkg-1,猪饲料的检测限为0.60μg kg-1。在1.0～100μg kg-1的添加水平,回收率为80.5～99.1%,变异系数为6.6～13.6%。ciELISA方法的灵敏度、回收率和精密度均符合我国和欧盟相关规定,为玉米、大豆、小麦和猪饲料快速筛选提供了技术手段。以单抗4H10作为抗体材料,制备出了IAC柱。IAC柱动态柱容量为5.59μg mL-1(13.85nmol mL-1gel),绝对柱容量为0.978μg mL-1gel (2.4nmol mg-1Ab). IAC柱重复使用20次后,柱容量(2.25μg mL-1gel)仍能达到初始柱容量的41.3%,回收率和样品净化效果无明显变化,具有良好的重复使用性。以IAC柱作为净化手段建立了谷物(大米、燕麦、大豆、玉米、小麦、荞麦、小米)和猪饲料中OTA的IAC-UPLC-MS/MS定量分析和确证方法,检测限为0.2～0.3μgkg-1,定量限为0.5～1.0pg kg-1,在四个添加水平(0.5～100μg kg-1),回收率为74.1%～104.8%,日内变异系数为4.1～11.3%,日间变异系数为4.7～15.8%,方法准确可靠,为谷物和猪饲料的确证和定量分析提供了技术手段。玉米、大豆、小麦和猪饲料样品中OTA的ciELISA检测方法与IAC-UPLC-MS/MS方法相关系数R2=0.9604,具有良好的相关性。以单抗4H10作为抗体材料,建立了检测牛奶中OTA的ciELISA方法,方法线性范围是0.3～3.0μgL-1,检测限为0.15μg L-1,在0.5～5.0μg L-1的添加水平,回收率为97.1～103.2%,变异系数为7.9～13.8%。同时建立了检测牛奶中OTA的CLEIA检测方法,方法线性范围是0.064～0.67μgL-1,检测限为0.03μgL-1,在0.1～5.0μgL-1的添加水平,回收率为98.3～104.1%,变异系数为11.2～15.4%。本研究建立的牛奶样品的ciELISA和CLEIA检测方法的回收率和精密度均符合我国和欧盟相关规定,为牛奶样品的快速筛选和定量分析提供了技术手段。更多还原

【Abstract】 Ochratoxin A (OTA) are mainly produced by several species of Aspergillus Ochraceus and Penicillium Verrucosum moulds, providing high toxicology which threatens the health of human and animal. In this study, a monoclonal antibody (mAb) and a single chain antibody (scFv) were produced. Based on the mAb, enzyme linked immunosorbent assay (ELISA) and chemiluminescence enzyme immunosorbent assay (CLEIA) were developed. An immunoaffinity chromatography (1AC) for clean-up OTA from matrices was developed. Moreover, an IAC-UPLC-MS/MS method was developed. These methods were used for determination of OTA in samples.In this study, a mAb was produced from a stable hybridoma cell line (4H10), which belongs to the immunoglobulin G1(κ-light chain) isotype. The titer of mAb was1:1.28×106and the affinity constant was5.31×109mol L-1of the mAb. A competitive indirect enzyme-linked immunosorbent assay (ciELISA) was used to characterize the mAb. The concentrations causing50%inhibition (IC50) of binding of mAb to OTA-ovalbumin (OTA-OVA) by free OTA, ochratoxin B (OTB), and ochratoxin C (OTC) were1.29,4.78, and0.94ng mL-1,respectively. Aflatoxin (AF) B1, B2, G1, G2, M,, M2, fumonisin B1(FB1, zearalenone (ZEN), deoxynivalenol (DON) and T-2toxin did not inhibit the binding of mAb to the marker antigen in ciELISA. The relative cross-reactivities of mAb to OTA, OTB and OTC were calculated to be100%,26.98%and137.23%, respectively. Based on the hybridoma cell line, a scFv was produced. The relative cross-reactivities of scFv to OTA, OTB and OTC were calculated to be100%,15.25%and124.34%, respectively. AFB,, B2, G1, G2, M1, M2, FB1, ZEN, DON, and T-2toxin did not inhibit the binding of scFv to the marker antigen in ciELISA.The ciELISA method using the mAb for determination of OTA in maize, soybean, wheat and pigfeed were developed. The linear range of ciELISA was0.3～3.0μg kg-1. The limit of detection (LOD) of the optimized ciELISA method was0.15μg kg-1in maize, soybean and wheat. Meanwhile, the LOD in pigfeed was0.6μg kg-1. At1.0～100μg kg-1fortified levels in maize, soybean, wheat and pigfeed, the mean recoveries of OTA ranged from80.5%to99.1%with coefficients of variations (CVs)6.6-13.6%. The accuracy and precision of the method at this level fall within the EU and China regulatory limits. This method can be used for determination of OTA in maize, soybean, wheat and pigfeed.Based on the mAb, an IAC for clean-up OTA from matrices was developed. The maximum OTA-binding capacity of the IAC was approximately5592ng of OTA per milliliter of gel (13.85nmol mL-1gel) which was equivalent to about978ng of OTA per milligram of antibody (2.4nmol mg-1Ab). An IAC-UPLC-MS/MS method using IAC for determination of OTA in cereals and pigfeeds was developed. This methodology has been validated in eight different matrices (millet, maize, soybean, wheat, rice, oat, buckwheat and pigfeed) with highly satisfactory results. The IAC-UPLC-MS/MS method offers a limit of quantification (LOQ, S/N=10) ranging from0.5μg kg-1to1.0μg kg-1and a limit of detection (LOD, S/N=3) ranging from0.2μg kg-1to0.3μg kg-1in cereals and pigfeeds. The IAC-UPLC-MS/MS method offers a good LOQ and LOD. The accuracy and precision at this level fall within the EU and China regulatory limits. The IAC-UPLC-MS/MS method can be used for determination and confirmation of OTA in cereal and pigfeed.A ELISA and a high sensitive chemiluminescence enzyme immunoassay (CLEIA) method using the4H10mAb for determination of OTA in milk was developed. The linear range of ELISA was0.3～3.0μg L-1and the LOD of ELISA was0.15μg L-1. At0.5～5.0μg L-1fortified levels in milk, mean recoveries ranged from97.1～103.2%with coefficients of variations (CVs)7.9-13.8%. The linear range of CLElA was0.064～0.67μg L-1and the LOD of CLEIA was0.03μg L-1. At0.1-5.0μg L-1fortified levels in milk, mean recoveries ranged from98.3～104.1%with coefficients of variations (CVs)11.2～15.4%. The accuracy and precision at this level fall within the EU and China regulatory limits. The ELISA and CLEIA methods can be used for determination of OTA in milk.更多还原