动物源性食品中违禁促生长激素的免疫快速检测技术研究
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摘要
食品安全问题屡禁不止,随着监管力度的加强,对配套检测技术的性能也提出了更高的要求,快速、准确、高灵敏和高通量成为检测技术发展的趋势,而免疫检测技术作为目前最常用的快速筛查手段,势必要跟上国家对监管技术的需求,本研究以食品安全问题较为严重的莱克多巴胺、沙丁胺醇、睾酮、甲基睾酮和去甲基睾酮这些促生长激素类药物为研究对象,从抗原的设计与合成入手,制备高灵敏和高亲和力的单克隆抗体,并研发相应的免疫快速检测技术。
(1)本研究首先设计合成了新型莱克多巴胺半抗原OAA,并获得了较高纯度的半抗原产物,通过紫外和电泳鉴定,成功合成了莱克多巴胺免疫原OAA-KLH和包被抗原OAA-OVAo通过免疫检测,确认了免疫原具有较好的免疫效果,通过细胞融合和筛选,并在常规细胞筛选阶段,直接使用猪尿样品进行抗基质干扰细胞株的同时筛选,获得莱克多巴胺的单克隆抗体3F11和9C2。其中单克隆抗体9C2的亲和常数为9.69×109L/mol,为高亲和力抗体,抗体亚型为IgG2b型。同时将该筛选方法应用于大分子乳铁蛋白单抗的筛选,获得了高特异性的乳铁蛋白抗体,证明了筛选方法的可行性。
(2)建立了莱克多巴胺间接竞争ic-ELISA检测方法,该ic-ELISA方法的最适条件为：包被抗原浓度0.25μg/mL;单克隆抗体浓度为0.025μg/mL;包被溶液为0.05M pH9.6CB缓冲溶液,标准品稀释液为0.01M pH7.4PBS溶液,其中NaCl含量为1.6%,甲醇含量为10%。在优化条件下,ic-ELISA方法的ICso值为0.229ng/mL,最低检测限(IClo)为0.012ng/mL,定量检测线性范围(IC20～IC80)为0.037ng/mL-1.412ng/mL。在不同水平的平均回收率在60%-100%之间,变异系数均小于10%。
建立了莱克多巴胺胶体金试纸条检测方法,确定金标记抗体的条件为0.1mol/L K2CO32μL/mL胶体金溶液；抗体用量2.4μg/mL胶体金溶液；NC膜上包被抗原浓度为0.8mg/mL。制备的试纸条对猪尿添加样品用比色法和消线法判断的检测限分别为2ng/mL和5ng/mL,用T线的灰度值建立的半定量曲线的检测限为0.1ng/mL,完全满足现场快速检测的需求。
(3)合成了两种沙丁胺醇半抗原,分别从沙丁胺醇分子结构两端暴露出抗原决定簇,并筛选了通用型和高特异性的沙丁胺醇高灵敏单克隆抗体。分别利用通用型抗体1E1和特异性3G6抗体建立了沙丁胺醇间接竞争ic-ELISA检测方法。
对于1E1抗体的ic-ELISA方法的最适条件为：包被抗原浓度0.25μg/mL;单克隆抗体浓度为0.12μg/mL;包被溶液为0.05M pH9.6CB缓冲溶液,标准品稀释液为0.01MpH7.4PBS溶液,其中NaCl含量为1.6%,甲醇含量为10%。在优化条件下,ICso为0.2ng/mL, Amax为1.685, Amax/IC50为8.425,最低检测限(IClo)为0.02ng/mL,定量检测线性范围(IC20～IC80)为0.047ng/mL-0.856ng/mLo在不同水平的平均回收率在80%-130%之间。
对于3G6抗体的的ic-ELISA方法的最适条件为：包被抗原浓度0.25μg/mL;单克隆抗体浓度为0.0625μg/mL;包被溶液为0.05M pH9.6CB缓冲溶液,标准品稀释液为0.01M pH7.4PBS溶液,其中NaCl含量为1.6%,甲醇含量为10%。在优化条件下,IC50为0.185ng/mL, Amax为1.756, Amax/IC50为9.492,最低检测限(IClo)为0.018ng/mL,定量检测线性范围(IC20～IC80)为0.0367ng/mL～0.935ng/mL。检测猪尿样本时无需稀释可以直接检测,在不同水平的平均回收率在90%-115%之间。
(4)针对睾酮的结构特点,设计了能够暴露睾酮特异性五元环的免疫原,制备了单-特异性识别睾酮的抗体。所制备的睾酮3G7抗体的IC50为0.11ng/mL, LOD检测限(IC1o计算)为0.016ng/mL,线性范围为0.034-0.394ng/mL。对其他的激素类似物交叉反应率都小于0.7%,组织样本的添加回收率在85%-110%。
(5)制备了能够特异性识别甲基睾酮的抗体(1E12)。跟甲基睾酮类似物交叉很低。所建立的甲基睾酮间接竞争ELISA的IC50为0.22ng/mL, LOD检测限(IC1o)为0.037ng/mL,线性范围为0.067-0.738ng/mL。对水产品的添加回收率为90%-110%。
(6)制备了通用型的的19-去甲基睾酮抗体(8F5),能够识别睾酮,雌二醇,甲基睾酮和炔雌醇类似物。所建立的19-去甲基睾酮ELISA的IC50为0.12ng/mL, LOD检测限(IC1o计算)为0.014ng/mL,线性范围为0.03-0.448ng/mL。所开发的间接竞争ELISA检测猪尿样品无需稀释,直接检测,回收率大于75%,极大地方便了现场样品的筛查。
With the rising of food safety issues, more demands are required on detection technology.Fast, accurate, highly sensitive and high-throughput become the trends. Immunoassays, whichhas been widely used in food safety, must meet the demands of country. Ractopamine,salbutamol, testosterone, methyltestosterone and19-nortestosterone, the drug of abuse, waschosen for this research, with the aim of development of rapid immunological detectiontechniques by design and synthesis of antigens to prepare sensitivity and high affinitymonoclonal antibodies.
This research was started from design and synthesis of a new type of ractopamine haptenOAA. High purity product hapten was obtained, and immunogen OAA-KLH and coatingantigen OAA-OVA was successful synthesized by UV-Vis and electrophoresis identification.The OAA-KLH showed good immune effect. By using pig urine samples in the cell line screenstage, ractopamine monoclonal antibody3F11and9C2with some anti-matrix effect wasobtained. The affinity constant of monoclonal antibody9C2is9.69u109L/mol, and theantibody subtype is IgG2b. Meanwhile, the screening method is applied to screening ofmacromolecules monoclonal antibody for lactoferrin, and high specificity antibody ofactoferrin was obatined, proving the feasibility of this screening methods.
An indirect competition ELISA(ic-ELISA) was established for ractopamine. The optimumconditions of the ic-ELISA method are as follows: coating concentration of antigen is0.25Pg/mL; monoclonal antibody concentration was0.025Pg/mL; coating buffer is0.05M pH9.6CB, standard dilute solution is0.01M pH7.4PBS with1.6%NaCl and10%methanol. And theIC50is0.229ng/mL, the detection limit (IC10) is0.012ng/mL, the linear quantitative detectionrange (IC20~IC80) is0.037ng/mL~1.412ng/mL. The average recoveries at different spikelevels are between60%-100%, and the coefficient of variation is less than10%.
Rapid colloidal gold test strip for ractopamine was developed. The optimum conditions fortest strip are as follows:2PL0.1mol/L K2CO3+1mL colloidal gold solution;2.4Pg/mL antibody+1mL colloidal gold solution×The coating antigen concentration on NC membrane is0.8mg/mL. The detection limits for ractopamine in swine urine is2ng/mL by color comparisonmethod,5ng/mL by T line disappearance. And a semi-quantitative detection limit0.1ng/mLwas obtained by acquiring the T line gray values.
Two salbutamol haptens with different epitopes was designed and synthesized. A sensitivitybroad-specific (1E1) and a sensitivity high specific (3G6) salbutamol monoclonal antibodywere obtained respectively. And an indirect competition ELISA(ic-ELISA) was established for1E1and3G6monoclonal antibody.
The optimum conditions of the ic-ELISA method for1E1are as follows: coatingconcentration of antigen is0.25Pg/mL; monoclonal antibody concentration was0.125Pg/mL;coating buffer is0.05M pH9.6CB, standard dilute solution is0.01M pH7.4PBS with1.6%NaCl and10%methanol. And the IC50is0.2ng/mL, the detection limit (IC10) is0.02ng/mL, thelinear quantitative detection range (IC20~IC80) is0.047ng/mL~0.856ng/mL. The averagerecoveries at different spike levels are between80%-130%, and the coefficient of variation is less than10%.
The optimum conditions of the ic-ELISA method for3G6are as follows: coatingconcentration of antigen is0.25Pg/mL; monoclonal antibody concentration was0.0625Pg/mL;coating buffer is0.05M pH9.6CB, standard dilute solution is0.01M pH7.4PBS with1.6%NaCl and10%methanol. And the IC50is0.185ng/mL, the detection limit (IC10) is0.018ng/mL,the linear quantitative detection range (IC20~IC80) is0.0367ng/mL~0.935ng/mL. And therecovery in spiked swine urine is between90%-115%.
A testosterone hapten with the five-membered ring as epitope was designed andsynthesized. And the high specific monoclonal antibody (3G7) for testosterone was obtained.The IC50of icELISA after optimization is0.11ng/mL èthe detection limit (IC10) is0.016ng/mL,the linear quantitative detection range is0.034ng/mL~0.394ng/mL. The cross-reactivity of thisantibody is less than0.7%with other hormone analogs. And the recovery in spiked tissuesample is between85%~110%.
A methyltestosterone hapten was designed and synthesized. And the high specificmonoclonal antibody (1E12) for methyltestosterone was obtained. The IC50of icELISA afteroptimization is0.22ng/mL èthe detection limit (IC10) is0.037ng/mL, the linear quantitativedetection range is0.067ng/mL~0.738ng/mL. And the recovery in spiked aquatic sample isbetween90%~110%.
A broad-sepcific monoclonal antibody (8F5) was obtained using19-nortestosterone hapten.The monoclonal antibody (8F5) can recognize testosterone, estradiol, and methyltestosteroneand ethinyl estradiol. The IC50of icELISA after optimization is0.12ng/mL for19-nortestosterone èthe detection limit (IC10) is0.014ng/mL, the linear quantitative detection rangeis0.03ng/mL~0.448ng/mL. And the recovery in spiked swine urine sample is greater than75%which is acceptable in field detection.
(1)本研究首先设计合成了新型莱克多巴胺半抗原OAA,并获得了较高纯度的半抗原产物,通过紫外和电泳鉴定,成功合成了莱克多巴胺免疫原OAA-KLH和包被抗原OAA-OVAo通过免疫检测,确认了免疫原具有较好的免疫效果,通过细胞融合和筛选,并在常规细胞筛选阶段,直接使用猪尿样品进行抗基质干扰细胞株的同时筛选,获得莱克多巴胺的单克隆抗体3F11和9C2。其中单克隆抗体9C2的亲和常数为9.69×109L/mol,为高亲和力抗体,抗体亚型为IgG2b型。同时将该筛选方法应用于大分子乳铁蛋白单抗的筛选,获得了高特异性的乳铁蛋白抗体,证明了筛选方法的可行性。
(2)建立了莱克多巴胺间接竞争ic-ELISA检测方法,该ic-ELISA方法的最适条件为：包被抗原浓度0.25μg/mL;单克隆抗体浓度为0.025μg/mL;包被溶液为0.05M pH9.6CB缓冲溶液,标准品稀释液为0.01M pH7.4PBS溶液,其中NaCl含量为1.6%,甲醇含量为10%。在优化条件下,ic-ELISA方法的ICso值为0.229ng/mL,最低检测限(IClo)为0.012ng/mL,定量检测线性范围(IC20～IC80)为0.037ng/mL-1.412ng/mL。在不同水平的平均回收率在60%-100%之间,变异系数均小于10%。
建立了莱克多巴胺胶体金试纸条检测方法,确定金标记抗体的条件为0.1mol/L K2CO32μL/mL胶体金溶液；抗体用量2.4μg/mL胶体金溶液；NC膜上包被抗原浓度为0.8mg/mL。制备的试纸条对猪尿添加样品用比色法和消线法判断的检测限分别为2ng/mL和5ng/mL,用T线的灰度值建立的半定量曲线的检测限为0.1ng/mL,完全满足现场快速检测的需求。
(3)合成了两种沙丁胺醇半抗原,分别从沙丁胺醇分子结构两端暴露出抗原决定簇,并筛选了通用型和高特异性的沙丁胺醇高灵敏单克隆抗体。分别利用通用型抗体1E1和特异性3G6抗体建立了沙丁胺醇间接竞争ic-ELISA检测方法。
对于1E1抗体的ic-ELISA方法的最适条件为：包被抗原浓度0.25μg/mL;单克隆抗体浓度为0.12μg/mL;包被溶液为0.05M pH9.6CB缓冲溶液,标准品稀释液为0.01MpH7.4PBS溶液,其中NaCl含量为1.6%,甲醇含量为10%。在优化条件下,ICso为0.2ng/mL, Amax为1.685, Amax/IC50为8.425,最低检测限(IClo)为0.02ng/mL,定量检测线性范围(IC20～IC80)为0.047ng/mL-0.856ng/mLo在不同水平的平均回收率在80%-130%之间。
对于3G6抗体的的ic-ELISA方法的最适条件为：包被抗原浓度0.25μg/mL;单克隆抗体浓度为0.0625μg/mL;包被溶液为0.05M pH9.6CB缓冲溶液,标准品稀释液为0.01M pH7.4PBS溶液,其中NaCl含量为1.6%,甲醇含量为10%。在优化条件下,IC50为0.185ng/mL, Amax为1.756, Amax/IC50为9.492,最低检测限(IClo)为0.018ng/mL,定量检测线性范围(IC20～IC80)为0.0367ng/mL～0.935ng/mL。检测猪尿样本时无需稀释可以直接检测,在不同水平的平均回收率在90%-115%之间。
(4)针对睾酮的结构特点,设计了能够暴露睾酮特异性五元环的免疫原,制备了单-特异性识别睾酮的抗体。所制备的睾酮3G7抗体的IC50为0.11ng/mL, LOD检测限(IC1o计算)为0.016ng/mL,线性范围为0.034-0.394ng/mL。对其他的激素类似物交叉反应率都小于0.7%,组织样本的添加回收率在85%-110%。
(5)制备了能够特异性识别甲基睾酮的抗体(1E12)。跟甲基睾酮类似物交叉很低。所建立的甲基睾酮间接竞争ELISA的IC50为0.22ng/mL, LOD检测限(IC1o)为0.037ng/mL,线性范围为0.067-0.738ng/mL。对水产品的添加回收率为90%-110%。
(6)制备了通用型的的19-去甲基睾酮抗体(8F5),能够识别睾酮,雌二醇,甲基睾酮和炔雌醇类似物。所建立的19-去甲基睾酮ELISA的IC50为0.12ng/mL, LOD检测限(IC1o计算)为0.014ng/mL,线性范围为0.03-0.448ng/mL。所开发的间接竞争ELISA检测猪尿样品无需稀释,直接检测,回收率大于75%,极大地方便了现场样品的筛查。
With the rising of food safety issues, more demands are required on detection technology.Fast, accurate, highly sensitive and high-throughput become the trends. Immunoassays, whichhas been widely used in food safety, must meet the demands of country. Ractopamine,salbutamol, testosterone, methyltestosterone and19-nortestosterone, the drug of abuse, waschosen for this research, with the aim of development of rapid immunological detectiontechniques by design and synthesis of antigens to prepare sensitivity and high affinitymonoclonal antibodies.
This research was started from design and synthesis of a new type of ractopamine haptenOAA. High purity product hapten was obtained, and immunogen OAA-KLH and coatingantigen OAA-OVA was successful synthesized by UV-Vis and electrophoresis identification.The OAA-KLH showed good immune effect. By using pig urine samples in the cell line screenstage, ractopamine monoclonal antibody3F11and9C2with some anti-matrix effect wasobtained. The affinity constant of monoclonal antibody9C2is9.69u109L/mol, and theantibody subtype is IgG2b. Meanwhile, the screening method is applied to screening ofmacromolecules monoclonal antibody for lactoferrin, and high specificity antibody ofactoferrin was obatined, proving the feasibility of this screening methods.
An indirect competition ELISA(ic-ELISA) was established for ractopamine. The optimumconditions of the ic-ELISA method are as follows: coating concentration of antigen is0.25Pg/mL; monoclonal antibody concentration was0.025Pg/mL; coating buffer is0.05M pH9.6CB, standard dilute solution is0.01M pH7.4PBS with1.6%NaCl and10%methanol. And theIC50is0.229ng/mL, the detection limit (IC10) is0.012ng/mL, the linear quantitative detectionrange (IC20~IC80) is0.037ng/mL~1.412ng/mL. The average recoveries at different spikelevels are between60%-100%, and the coefficient of variation is less than10%.
Rapid colloidal gold test strip for ractopamine was developed. The optimum conditions fortest strip are as follows:2PL0.1mol/L K2CO3+1mL colloidal gold solution;2.4Pg/mL antibody+1mL colloidal gold solution×The coating antigen concentration on NC membrane is0.8mg/mL. The detection limits for ractopamine in swine urine is2ng/mL by color comparisonmethod,5ng/mL by T line disappearance. And a semi-quantitative detection limit0.1ng/mLwas obtained by acquiring the T line gray values.
Two salbutamol haptens with different epitopes was designed and synthesized. A sensitivitybroad-specific (1E1) and a sensitivity high specific (3G6) salbutamol monoclonal antibodywere obtained respectively. And an indirect competition ELISA(ic-ELISA) was established for1E1and3G6monoclonal antibody.
The optimum conditions of the ic-ELISA method for1E1are as follows: coatingconcentration of antigen is0.25Pg/mL; monoclonal antibody concentration was0.125Pg/mL;coating buffer is0.05M pH9.6CB, standard dilute solution is0.01M pH7.4PBS with1.6%NaCl and10%methanol. And the IC50is0.2ng/mL, the detection limit (IC10) is0.02ng/mL, thelinear quantitative detection range (IC20~IC80) is0.047ng/mL~0.856ng/mL. The averagerecoveries at different spike levels are between80%-130%, and the coefficient of variation is less than10%.
The optimum conditions of the ic-ELISA method for3G6are as follows: coatingconcentration of antigen is0.25Pg/mL; monoclonal antibody concentration was0.0625Pg/mL;coating buffer is0.05M pH9.6CB, standard dilute solution is0.01M pH7.4PBS with1.6%NaCl and10%methanol. And the IC50is0.185ng/mL, the detection limit (IC10) is0.018ng/mL,the linear quantitative detection range (IC20~IC80) is0.0367ng/mL~0.935ng/mL. And therecovery in spiked swine urine is between90%-115%.
A testosterone hapten with the five-membered ring as epitope was designed andsynthesized. And the high specific monoclonal antibody (3G7) for testosterone was obtained.The IC50of icELISA after optimization is0.11ng/mL èthe detection limit (IC10) is0.016ng/mL,the linear quantitative detection range is0.034ng/mL~0.394ng/mL. The cross-reactivity of thisantibody is less than0.7%with other hormone analogs. And the recovery in spiked tissuesample is between85%~110%.
A methyltestosterone hapten was designed and synthesized. And the high specificmonoclonal antibody (1E12) for methyltestosterone was obtained. The IC50of icELISA afteroptimization is0.22ng/mL èthe detection limit (IC10) is0.037ng/mL, the linear quantitativedetection range is0.067ng/mL~0.738ng/mL. And the recovery in spiked aquatic sample isbetween90%~110%.
A broad-sepcific monoclonal antibody (8F5) was obtained using19-nortestosterone hapten.The monoclonal antibody (8F5) can recognize testosterone, estradiol, and methyltestosteroneand ethinyl estradiol. The IC50of icELISA after optimization is0.12ng/mL for19-nortestosterone èthe detection limit (IC10) is0.014ng/mL, the linear quantitative detection rangeis0.03ng/mL~0.448ng/mL. And the recovery in spiked swine urine sample is greater than75%which is acceptable in field detection.
引文
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