真菌毒素类高灵敏高通量快速检测方法研究
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摘要
真菌毒素是一些真菌在生长过程中产生的致病性、致死性的次级代谢产物，在粮食生产、贮存、加工和流通环节都有可能因为保存条件不当污染真菌造成污染。由于大多数真菌素素的毒性剧烈，并具有致癌、致畸和致突变的“三致”作用，因此各种检测方法被开发用于检测粮食谷物中的各种真菌毒素。免疫分析法具有高灵敏、快速、高通量、多残留和现场检测的优势，现已发展成为真菌毒素检测快速筛查和检测的重要手段之一。本课题采用免疫学原理，将真菌毒素半抗原偶联到蛋白载体，通过免疫和细胞筛选，制备了特异性单克隆抗体的基础上，建立起黄曲霉毒素B1、黄曲霉毒素M1、赭曲霉毒素A、玉米赤霉烯酮、脱氧雪腐镰刀菌烯醇和T-2毒素六种物质的快速检测方法，主要研究内容如下：
（1）将真菌毒素分子衍生后制备的半抗原，通过紫外、质谱等进行鉴定后，通过活性酯法或者CDI法与载体蛋白（血蓝蛋白、牛血清白蛋白和卵清蛋白）偶联，制备了六种真菌毒素半抗原的完全抗原；通过免疫BALB/c小鼠、细胞融合和亚克隆，总共获得8株针对六种真菌毒素特异性或群选型单克隆抗体。
（2）对筛选得到的8株单克隆抗体的性质进行了鉴定，其亲和常数分别为黄曲霉毒素B14.2×109L/mol(11A9)和2.29×109L/mol(5C3)；黄曲霉毒素M11.28×109L/mol(G3D9)和2.29×109L/mol(H1)；赭曲霉毒素A1.09×109L/mol；玉米赤霉烯酮2.26×109L/mol；脱氧雪腐镰刀菌烯醇1.6×108L/mol；T-2毒素1.35×108L/mol。并测定了其抗体亚型和交叉反应率。
（3）试验优化了包被抗原和一抗使用浓度、标准品稀释液的缓冲液的pH、离子强度、甲醇含量、一抗和二抗的反应时间、显色时间等ELISA条件。在最适ELISA条件下，测得抗体的IC50分别为：黄曲霉毒素B10.043±0.003ng/mL（11A9）和0.025±0.005ng/mL（5C3）；黄曲霉毒素M10.032±0.006ng/mL（G3D9）和0.035±0.005ng/mL（H1）；赭曲霉毒素A0.092±0.006ng/mL；玉米赤霉烯酮0.054±0.004ng/mL；脱氧雪腐镰刀菌烯醇31.60±2.89ng/mL；T-2毒素0.431±0.019ng/mL。
（4）在建立的8株单克隆抗体细胞株基础上，建立了六种真菌毒素的ELISA，并通过添加回收对方法进行了验证，其回收率均在80%～120%之间，批内批间差异均小于20%。
（5）通过优化酶解条件，制备了AFB1单抗的F(ab')2片段，通过免疫兔子，制备得到AFB1的抗独特型抗体，并对其进行了鉴定。用来开发黄曲霉毒素无毒试剂盒；通过实际样品添加回收试验，其回收率在110%～130%之间，批内批间变异系数均小于10%。
（6）制备了可用于快速现场检测的AFB1、AFM1、OTA、ZEN、DON和T-2生物毒素胶体金快速检测试纸条，其检测限LOD分别为0.2、0.1、0.2、1、50和5ng/mL，cut-off值分别为0.5、0.2、1.0、2.0、100和10ng/mL。
（7）制备了可以同时检测4种真菌毒素（ZEN、T-2、AFB1和OTA）的试纸条，其LOD分别为2、16、1和2ng/mL，cut-off值分别为为4、32、2和4ng/mL。
Mycotoxins are pathogenic and fatal secondary metabolites which are produced by somefungi during growing. Mycotoxins could be fabricated during the production, storage,processing and circulation of cereal in case of improper conditions. Because of the acutetoxicity as well as the ability of carcinogenicity, teratogenicity and mutagenicity, all kinds ofdetection methods were developed. With advantages in high sensitivity, rapid, highthroughput and more residues and field test, immunoassay has been one of the importantmeans of rapid screening and detection mycotoxin detection. In this research completeantigens were prepared by conjugating the haptens of mycotoxins to the proteins carrier, andthen were used to immunize mice. The cell lines of monoclonal antibodies (mAbs) thatagainst AFB1, AFM1, OTA, ZEN, DON and T-2toxin were obtained through cell-screening.Rapid assay methods for the six mycotoxins were developed based on the mAbs. The maincontents are as follows.
(1) The haptens were obtained by deriving the mycotoxins, and confirmed byUV-spectroscopy and mass. Subsequently complete antigens were obtained by conjugatingthe haptens with protein carriers. Finally eight cell lines were obtained through immunization,cell fusion and subclone.
(2) Characteristics of mAbs were identified, the affinity constant was AFB14.2×109L/mol(11A9) and2.29×109L/mol (5C3);AFM11.28×109L/mol (G3D9) and2.29×109L/mol(H1);OTA1.09×109L/mol;ZEN2.26×109L/mol; DON1.6×108L/mol; T-2toxin1.35×108L/mol, respectively. Antibody subtypes and cross reaction were also measured.
(3) Some conditions were optimized, such as the concentrations of coating antigens andantibodies, the pH, ion strength and methanol concentration of the standard solutions, reactiontime of primary antibodies, Secondary antibodies and colour developing. Under the bestconditions, the IC50were0.043±0.003ng/mL(AFB1,11A9)and0.025±0.005ng/mL(AFB1,5C3),0.032±0.006ng/mL(AFM1, G3D9) and0.035±0.005ng/mL(AFM1, H1),0.092±0.006ng/mL (OTA),0.054±0.004(ZEN) ng/mL,31.60±2.89ng/mL(DON),0.431±0.019ng/mL(T-2toxin), respectively.
(4) The ELISA methods for six mycotoxins were developed on the base of obtained eightmAbs. The ELISA methods were confirmed by spiked assay. The recoveries were between80%~120%, and the differences between group and within group were less than20%.
(5) F(ab')2fragment of AFB1mAbs were prepared by optimizing the enzymatichydrolysis conditions of antibodies. Anti-idiotype antibody was obtained by immunizingrabbits, and was used to develop nontoxic ELISA kit. The kit was confirmed by spiked assay.The recoveries were between110%~130%, and the differences between group and withingroup were less than10%.
(6) The rapid field-test strips for detecting AFB1, AFM1, OTA, ZEN, DON andT-2toxinwere developed. The LOD is0.2,0.1,0.2,1,50,5ng/mL and cut-off level were0.5,0.2,1.0,2.0,100,10ng/mL, respectively.
(7) The muitiple rapid field-test strips for simultaneously detecting ZEN、T-2、AFB1andOTA was developed. The LOD is2,16,1,2ng/mL and cut-off level were4,32,2,4ng/mL,respectively.
（1）将真菌毒素分子衍生后制备的半抗原，通过紫外、质谱等进行鉴定后，通过活性酯法或者CDI法与载体蛋白（血蓝蛋白、牛血清白蛋白和卵清蛋白）偶联，制备了六种真菌毒素半抗原的完全抗原；通过免疫BALB/c小鼠、细胞融合和亚克隆，总共获得8株针对六种真菌毒素特异性或群选型单克隆抗体。
（2）对筛选得到的8株单克隆抗体的性质进行了鉴定，其亲和常数分别为黄曲霉毒素B14.2×109L/mol(11A9)和2.29×109L/mol(5C3)；黄曲霉毒素M11.28×109L/mol(G3D9)和2.29×109L/mol(H1)；赭曲霉毒素A1.09×109L/mol；玉米赤霉烯酮2.26×109L/mol；脱氧雪腐镰刀菌烯醇1.6×108L/mol；T-2毒素1.35×108L/mol。并测定了其抗体亚型和交叉反应率。
（3）试验优化了包被抗原和一抗使用浓度、标准品稀释液的缓冲液的pH、离子强度、甲醇含量、一抗和二抗的反应时间、显色时间等ELISA条件。在最适ELISA条件下，测得抗体的IC50分别为：黄曲霉毒素B10.043±0.003ng/mL（11A9）和0.025±0.005ng/mL（5C3）；黄曲霉毒素M10.032±0.006ng/mL（G3D9）和0.035±0.005ng/mL（H1）；赭曲霉毒素A0.092±0.006ng/mL；玉米赤霉烯酮0.054±0.004ng/mL；脱氧雪腐镰刀菌烯醇31.60±2.89ng/mL；T-2毒素0.431±0.019ng/mL。
（4）在建立的8株单克隆抗体细胞株基础上，建立了六种真菌毒素的ELISA，并通过添加回收对方法进行了验证，其回收率均在80%～120%之间，批内批间差异均小于20%。
（5）通过优化酶解条件，制备了AFB1单抗的F(ab')2片段，通过免疫兔子，制备得到AFB1的抗独特型抗体，并对其进行了鉴定。用来开发黄曲霉毒素无毒试剂盒；通过实际样品添加回收试验，其回收率在110%～130%之间，批内批间变异系数均小于10%。
（6）制备了可用于快速现场检测的AFB1、AFM1、OTA、ZEN、DON和T-2生物毒素胶体金快速检测试纸条，其检测限LOD分别为0.2、0.1、0.2、1、50和5ng/mL，cut-off值分别为0.5、0.2、1.0、2.0、100和10ng/mL。
（7）制备了可以同时检测4种真菌毒素（ZEN、T-2、AFB1和OTA）的试纸条，其LOD分别为2、16、1和2ng/mL，cut-off值分别为为4、32、2和4ng/mL。
Mycotoxins are pathogenic and fatal secondary metabolites which are produced by somefungi during growing. Mycotoxins could be fabricated during the production, storage,processing and circulation of cereal in case of improper conditions. Because of the acutetoxicity as well as the ability of carcinogenicity, teratogenicity and mutagenicity, all kinds ofdetection methods were developed. With advantages in high sensitivity, rapid, highthroughput and more residues and field test, immunoassay has been one of the importantmeans of rapid screening and detection mycotoxin detection. In this research completeantigens were prepared by conjugating the haptens of mycotoxins to the proteins carrier, andthen were used to immunize mice. The cell lines of monoclonal antibodies (mAbs) thatagainst AFB1, AFM1, OTA, ZEN, DON and T-2toxin were obtained through cell-screening.Rapid assay methods for the six mycotoxins were developed based on the mAbs. The maincontents are as follows.
(1) The haptens were obtained by deriving the mycotoxins, and confirmed byUV-spectroscopy and mass. Subsequently complete antigens were obtained by conjugatingthe haptens with protein carriers. Finally eight cell lines were obtained through immunization,cell fusion and subclone.
(2) Characteristics of mAbs were identified, the affinity constant was AFB14.2×109L/mol(11A9) and2.29×109L/mol (5C3);AFM11.28×109L/mol (G3D9) and2.29×109L/mol(H1);OTA1.09×109L/mol;ZEN2.26×109L/mol; DON1.6×108L/mol; T-2toxin1.35×108L/mol, respectively. Antibody subtypes and cross reaction were also measured.
(3) Some conditions were optimized, such as the concentrations of coating antigens andantibodies, the pH, ion strength and methanol concentration of the standard solutions, reactiontime of primary antibodies, Secondary antibodies and colour developing. Under the bestconditions, the IC50were0.043±0.003ng/mL(AFB1,11A9)and0.025±0.005ng/mL(AFB1,5C3),0.032±0.006ng/mL(AFM1, G3D9) and0.035±0.005ng/mL(AFM1, H1),0.092±0.006ng/mL (OTA),0.054±0.004(ZEN) ng/mL,31.60±2.89ng/mL(DON),0.431±0.019ng/mL(T-2toxin), respectively.
(4) The ELISA methods for six mycotoxins were developed on the base of obtained eightmAbs. The ELISA methods were confirmed by spiked assay. The recoveries were between80%~120%, and the differences between group and within group were less than20%.
(5) F(ab')2fragment of AFB1mAbs were prepared by optimizing the enzymatichydrolysis conditions of antibodies. Anti-idiotype antibody was obtained by immunizingrabbits, and was used to develop nontoxic ELISA kit. The kit was confirmed by spiked assay.The recoveries were between110%~130%, and the differences between group and withingroup were less than10%.
(6) The rapid field-test strips for detecting AFB1, AFM1, OTA, ZEN, DON andT-2toxinwere developed. The LOD is0.2,0.1,0.2,1,50,5ng/mL and cut-off level were0.5,0.2,1.0,2.0,100,10ng/mL, respectively.
(7) The muitiple rapid field-test strips for simultaneously detecting ZEN、T-2、AFB1andOTA was developed. The LOD is2,16,1,2ng/mL and cut-off level were4,32,2,4ng/mL,respectively.
引文
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