【作者】 王鹏；

【导师】 匡华；

【作者基本信息】 江南大学 ， 食品科学与工程， 2023， 博士

【摘要】 近些年来,食品安全问题频发,尤其以柑橘、茶叶、大豆与玉米为代表的大宗食品经常被检出残留有多种有毒化学物质,其中异噁草酮、丙炔氟草胺、除草醚、甲羧除草醚、硝磺草酮及其代谢物、磺草酮及代谢物、麦草畏、草甘膦、唑螨酯与喹螨醚等13种化学残留物多次被检出。而随着我国食品安全检测体系的完善与检测范围的扩大,抽检的食品样本数量逐年递增,传统的仪器检测方法逐渐无法满足基层样本的抽检需求,亟需研发更加高效与便捷的检测方法。因此本研究以上述化学残留物为研究对象,通过计算模拟技术辅助设计筛选半抗原,合成人工抗原,研发单克隆抗体,建立了可以检测柑橘、茶叶、大豆与玉米中上述化学残留物的胶体金快速检测方法。1、半抗原设计、筛选与合成化学残留物结构简单、分子量过低无法引起免疫应答,需要经过化学改造以获得可与载体蛋白偶联的活性基团。通过RDkit计算,选择活性基团后与靶标分子功能结构相似度>50%的半抗原进行量子计算分析,通过Gaussian计算与Discovery Studio定位分析后得到分子在电子密度为0.001 a.u.平面上的电势分布图。（1）以筛选高特异性抗体为目的,选择分子整体空间结构与靶标分子重合,特征基团上原子的电性与靶标分子相同,分子表面静电势与靶标分子一致的半抗原进行合成,包括:异噁草酮、丙炔氟草胺、麦草畏与草甘膦、唑螨酯、喹螨醚的半抗原。（2）以筛选宽谱识别性抗体为目的,选择母核结构与靶标分子重合,改造位点不影响母核结构上原子的电性与分子表面静电势分布的半抗原进行合成,包括:除草醚、甲羧除草醚、硝磺草酮及其代谢物、磺草酮及其代谢物的半抗原。2、单克隆抗体的制备。以牛血清白蛋白（BSA）、卵清蛋白（OVA）为载体蛋白,偶联半抗原制备完全抗原。以BSA偶联物免疫小鼠,采用杂交瘤细胞技术筛选分泌高性能抗体的细胞株,采用诱生腹水方式制备单克隆抗体。制备高特异性抗体（与靶标化学残留物的结构类似物交叉率<5%）:CLO-m Ab 4C4、FLU-m Ab 1A7、DIC-m Ab 1B5、GLY-m Ab 1B9、FEP-m Ab 2G9与FEA-m Ab 3F10,分别对异噁草酮、丙炔氟草胺、麦草畏、草甘膦、唑螨酯与喹螨醚的IC₅₀值分别为2.47 ng/m L、0.59 ng/m L、39.41 ng/m L、1160 ng/m L、206.88 ng/m L、103.61 ng/m L。此外还筛选了宽谱识别性抗体NIT-m Ab 5G7,可以同时识别除草醚(IC₅₀值为1.54 ng/m L)、甲羧除草醚(IC₅₀值为2.20 ng/m L);抗体BIF-m Ab 3E3也可以同时识别除草醚(IC₅₀值为73.14ng/m L)、甲羧除草醚(IC₅₀值为5.76 ng/m L);抗体MES-m Ab 4G7可以同时识别硝磺草酮(IC₅₀值为4.21 ng/m L)及其代谢物4-甲砜基-2-硝基苯甲酸(IC₅₀值为9.11 ng/m L)与2-氨基-4-甲砜基苯甲酸(IC₅₀值为9.47 ng/m L)、磺草酮(IC₅₀值为8.14 ng/m L)及其代谢物2-氯-4-甲磺酰基苯甲酸(IC₅₀值为12.5 ng/m L);抗体MES-m Ab 3H7可以同时识别硝磺草酮(IC₅₀值为3.76 ng/m L)及其代谢物4-甲砜基-2-硝基苯甲酸(IC₅₀值为10.81ng/m L)与2-氨基-4-甲砜基苯甲酸(IC₅₀值为7.36 ng/m L)。所有抗体亲和力常数均大于1×10⁷L/mo L,属于高亲和力抗体。3、食品中化学残留物的胶体金试纸条检测方法开发优化金标抗体的标记条件、抗原抗体使用量与金标抗体稀释液种类,制备了试纸条,并应用于柑橘、茶叶、大豆与玉米的检测。除草甘膦外的化学残留物采用乙腈/乙酸的混合溶剂进行提取,提取液离心后取上清液浓缩,残留物用含10%乙腈的0.01 M磷酸盐缓冲液（p H 7.4）重悬后检测。含草甘膦的样本需分别将提取液与重悬液更改为酸性超纯水与含10%乙腈的0.01 M硼酸盐缓冲液（p H 8.8）。柑橘中5种化学残留物的视觉检测限（v LOD）为1～100 ng/g;茶叶中4种化学残留物的v LOD为2～200ng/g;大豆中9种化学残留物的v LOD分别为2～500 ng/g;玉米中6种化学残留物的v LOD为10～20ng/g,均能完全满足国家标准的限量要求。经添加回收实验验证,该方法回收率在70%～120%之间。与仪器检测方法相比较,检测结果一致,且极大的缩短了检测时间,20～40min内即可得到检测结果。同时在单一污染物试纸条检测方法的基础上研发了多重胶体金试纸条免疫层析检测法,可以同时检测大豆中硝磺草酮、异噁草酮与丙炔氟草胺的含量,检测结果与单一试纸条检测方法一致,可以满足国家标准的限量要求。综上所述,本课题创新了小分子半抗原识别特异性调控方法,研发了可识别13种食品中典型化学残留物的高灵敏度单克隆抗体,建立了食品中化学残留物的胶体金快速检测方法,能完全满足国家标准的限量要求与基层现场快速检测需求。更多还原

【Abstract】 In recent years,food safety problems have occurred frequently.In particular,a variety of toxic chemical residues often were detected in bulk foods of citrus,tea,soybeans,and corn,including clomazone,flumioxazin,nitrofen,bifenox,mesotrione and its metabolites,sultrione and its metabolites,dicamba,glyphosate,fenpyroximate and fenazaquin.With the improvement of my country’s food safety testing system and the expansion of testing scope,the number of food samples sampled is increasing year by year.Traditional instrument testing methods are gradually unable to meet the sampling needs of grassroots samples.There is an urgent need to develop more efficient and convenient testing methods.Therefore,in this study,the haptens of the 13 chemical residues were designed and screened by computational simulation technology,and the antigens were synthesized,10 monoclonal antibodies developed.Finally a colloidal gold rapid detection method was established.1.The designed,screened and synthesized of haptensThe chemical residues structure is too simple to cause an immune response.To coupled to carrier proteins,they need to be modified with obtain active groups.The haptens with a functional structural similarity of>50%to the target molecule is analyzed by quantum computing after selecting the active group with RDkit calculation.After Gaussian calculation and Discovery Studio positioning analysis,the potential distribution diagram of the molecule was obtained on the plane with an electron density of 0.001 a.u..（1）For the purpose of screening highly specific antibodies,the haptens were screened and synthesized that were similar to target molecule of overall spatial structure,atoms electrical properties and characteristic groups,including the haptens of clomazone,flumioxazin,dicamba,glyphosate,fenpyroximate and fenazaquin.（2）For the purpose of screening group-selective antibodies,the haptens were screened and synthesized that were similar to target molecule of parent core structure,including the haptens of nitrofen,bifenox,mesotrione and its metabolites,sultrione and its metabolites.2.Preparation of monoclonal antibodies.Bovine serum albumin（BSA）and ovalbumin（OVA）was conjugated with hapten.Mice were immunized with BSA conjugates,hybridoma cell technology was used to screen cell lines secreting high-performance antibodies,and monoclonal antibodies were prepared by inducing ascites.Preparation of highly specific antibodies（crossover rate<5%with structural analogs of the target chemical residue）:CLO-m Ab 4C4,FLU-m Ab 1A7,DIC-m Ab 1B5,GLY-m Ab 1B9,FEP-m Ab 2G9 and FEA-m Ab 3F10,the IC₅₀ values for clomazone,flumioxazin,dicamba,glyphosate,fenpyroximate and fenazaquin are 2.47 ng/m L,0.59 ng/m L,39.41 ng/m L,and1160 ng/m L,206.88 ng/m L,103.61 ng/m L respectively.In addition,the group-selective antibody NIT-m Ab 5G7 was also screened,which can simultaneously recognize nitrofen(IC₅₀value is 1.54 ng/m L)and bifenox(IC₅₀ value is 2.20 ng/m L);the antibody BIF-m Ab 3E3 can also simultaneously recognize nitrofen(IC₅₀ value:73.14 ng/m L),bifenox(IC₅₀ value:5.76ng/m L);the antibody MES-m Ab 4G7 can simultaneously recognize mesotrione(IC₅₀ value:4.21 ng/m L)and its metabolites 4-methylsulfonyl-2-nitrobenzoic acid(IC₅₀ value is 9.11ng/m L)and 2-amino-4-methylsulfonylbenzoic acid(IC₅₀ value is 9.47 ng/m L),sulfotrione(IC₅₀ value is 8.14 ng/m L)and its metabolite 2-chloro-4-methanesulfonylbenzoic acid(IC₅₀value is 12.5 ng/m L);the antibody MES-m Ab 3H7 can simultaneously recognize mesotrione(IC₅₀ value is 3.76 ng/m L)and its metabolites 4-methylsulfonyl-2-nitrobenzoic acid(IC₅₀value:10.81 ng/m L)and 2-amino-4-methylsulfon-ylbenzoic acid（IC50 value:7.36 ng/m L）.All antibody affinity constants are greater than 1×107L/mo L and are high-affinity antibodies.3.Development of colloidal gold test strip detection method for chemical residues in foods.The p H of the colloidal gold-labeled antibody,the amount of antigen and antibody used,and the type of gold-labeled antibody diluent were optimized,and test strips were prepared and applied to the detection of citrus,tea,soybeans,and corn.Chemical residues other than glyphosate were extracted using a mixed solvent of acetonitrile/acetic acid.The extract was centrifuged and the supernatant was concentrated.The residue was resuspended in 0.01 M phosphate buffer（p H 7.4）containing 10%acetonitrile and then detected.For samples containing glyphosate,the extraction solution and resuspension solution need to be changed to acidic ultrapure water and 0.01 M borate buffer（p H 8.8）containing 10%acetonitrile.The visual detection limit（v LOD）of 5 chemical residues in citrus is 1～100 ng/g;the v LOD of 4chemical residues in tea is 2～200ng/g;the v LOD of 9 chemical residues in soybeans is 2respectively～500 ng/g;the v LOD of 6 chemical residues in corn is 10～20 ng/g,all of which can fully meet the limit requirements of national standards.Verified by addition recovery experiments,the recovery rate of this method is between 70%and 120%.Compared with the instrument detection method,the detection results are consistent and the detection time is greatly shortened.The detection results can be obtained within 20～40min.At the same time,a multiple colloidal gold test strip immunochromatographic detection method was developed based on the single contaminant test strip detection method,which can simultaneously detect the contents of mesotrione,clomazone and propargyl flufenate in soybeans.The test results are consistent with the single test strip test method and can meet the limit requirements of national standards.To sum up,this project has innovated the specific regulation method of small molecule hapten recognition,developed a highly sensitive monoclonal antibody that can recognize 13typical chemical residues in food,and established a colloidal gold rapid detection of chemical residues in food.This method can fully meet the limited requirements of national standards and the needs of rapid on-site testing at the grassroots level.更多还原