三聚氰胺单克隆抗体制备及其高灵敏快速检测技术研究
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摘要
三聚氰胺奶粉污染事件使我国乳制品行业遭到重创,国内外纷纷设置了在各种食品中三聚氰胺的允许残留限量。在我国,三聚氰胺已被列入生乳收购及乳制品产品出厂必检项目。为满足庞大的检测业务要求,亟需开发高通量、快速、高灵敏的三聚氰胺筛查手段应用于三聚氰胺的日常监控。鉴于此,本论文开展了以下研究:
本文首先设计合成并鉴定了三种三聚氰胺半抗原:MEL-MCA,MEL-ACA和MEL-PABA;采用活性酯法制备并鉴定了三种免疫原MEL-MCA-BSA,MEL-ACA-BSA和MEL-PABA-BSA;采用混合酸酐法制备并鉴定了三种包被原MEL-MCA-OVA,MEL-ACA-OVA和MEL-PABA-OVA;通过动物免疫实验,研究不同半抗原结构对免疫效果的影响。结果表明三种免疫原均具有免疫原性,其中以免疫原MEL-ACA-BSA和包被原MEL-PABA-OVA的异源免疫分析组合效果最好。
将制备的三种免疫原免疫BALB/C小鼠以制备抗三聚氰胺单克隆抗体,共获得4株可稳定分泌抗三聚氰胺单克隆抗体的杂交瘤细胞株,制备腹水并进行免疫特性分析,其抗体亚型为IgG1型,抗体亲和常数为2.67×109 L/mol,该抗体除与灭蝇胺交叉反应率为132%外,与其他三聚氰胺结构类似物无交叉反应,为目前报道的免疫特性最好的抗三聚氰胺单克隆抗体。
利用获得的单克隆抗体建立了三聚氰胺间接竞争ELISA检测方法并制备了试剂盒。通过对ELISA影响各因素的优化,最终确定其IC50为6±0.55ng/ml,奶粉最低检测限为100μg/kg,液态奶最低检测限为20ng/ml。添加回收实验结果表明,奶粉样品和液态奶样品的添加回收率分别为93%-101.9%和83.6%-96.9%,变异系数均小于10%;用国家标准方法GB/T 22388-2008 LC-MS/MS法进行确证,表明本研究所制备的ELISA试剂盒检测结果准确可靠,可用于乳及乳制品中三聚氰胺的检测,且试剂盒在4°C可保存1年,完全满足三聚氰胺残留检测的实际要求。
利用单克隆抗体制备了特异性识别、净化三聚氰胺的IAC柱,并建立了奶粉和液态奶中三聚氰胺的IAC-LC-MS/MS检测方法,IAC柱动态柱容量为24.14 n mol/ml gel,绝对柱容量为3.36 n mol/mg抗体,IAC柱最佳重复使用次数为8次;建立的IAC-LC-MS/MS检测方法的检测限为10ng/ml,定量限为20ng/ml,实际样品添加回收率为79.4-91.8%,变异系数小于10%,符合国际规定的残留分析方法要求,可用于实际样品中三聚氰胺残留检测。
采用柠檬酸钠法制备金纳米粒子,利用抗三聚氰胺单克隆抗体制备胶体金标记物,建立了基于胶体金免疫层析技术的乳及乳制品中三聚氰胺残留的高灵敏快速检测方法。通过优化确定胶体金标记抗体的最佳pH值为8.0,胶体金标记最佳抗体用量为8μg/ml,包被原最佳包被浓度和最佳二抗包被浓度为0.5mg/ml制备出三聚氰胺胶体金免疫层析试纸条。结果表明试纸条对三聚氰胺标准品的检测限为12.5ng/ml,实际奶粉样品的检测限为37.5μg/kg,对三聚氰胺结构类似物无交叉反应性,可满足三聚氰胺实际检测需求。
利用冠醚对金纳米粒子进行修饰制备功能化金纳米粒子,建立基于分子识别的三聚氰胺高灵敏快速检测比色法。通过优化确定三聚氰胺的检测线性范围为10-500ng/ml,检测限为6ng/ml,奶粉样品添加回收率在98.4%-105.6%之间,变异系数小于10%,可实现三聚氰胺简单、快速、高灵敏检测。
The melamine contamination event of milk powder greatly impacted China’s dairy industry. And the maximum residue limits of melamine in food have been set up both in domestic and abroad. In China, melamine has been one of the conventional detection items for milk and related products. Therefore, high-throμghput, rapid, and highly sensitive screening methods are urgently needed to satisfy the needs for melamine detection. In view of this, this dissertation carried out the following studies:
In this dissertation, three haptens (MEL-MCA, MEL-ACA and MEL-PABA) were designed, synthesized and identified. Three immunogens (MEL-MCA-BSA, MEL-ACA-BSA and MEL-PABA-BSA) and three coating antigens (MEL-MCA-OVA, MEL-ACA-OVA and MEL-PABA-OVA) were conjμgated by the active ester method and mixed anhydride method, respectively. The effects of hapten structure for immunity were investigated by animal immunization experiment. Results showed that the synthesis of these three immunogens was successful, and revealed that the MEL-ACA-BSA antibody/MEL-PABA-OVA coating conjμgate combination was the best for heterologous ELISA system.
Three immunogens was applied to immunize BALB/c mice to produce anti-melamine monoclonal antibody. And four monoclonal cell lines which could steadily secrete anti-melamine monoclonal antibody were obtained successfully. The subtype of antibody was identified as IgG1 by the commercial Kit. And the antibody affinity constant was calculated to be 2.67×109 L/mol. The most important property of the prepared antibody was that there was no cross-reaction to other MEL analogues except with cyromazine. The antibody achieved in this study was the best high-quality antibody among the currently reported monoclonal antibody against MEL.
Based on the obtained monoclonal antibody, an indirect competitive ELISA detection method of MEL was established and a relative ELISA Kit was assembled. Factors that may affect the performances of ELISA were optimized systematically. The IC50 value was 6±0.55ng/ml and the limit of detection (LOD) was 100μg/kg in powder and 20ng/ml in liquid milk, respectively. Recovery results showed that the recoveries ranged from 93 to 101.9% in milk powder samples and from 83.6 to 96.9% for liquid milk, respectively and variation coefficients were less than 10%. Real samples were determined throμgh the developed ELISA Kit of our laboratory. Detection results were also futher compared with results of by LC-MS/MS method according to national standard GB/T22388-2008, which showed highly consistency of the two methods. The stability results of the ELISA Kit indicated that the Kit stored at 4°C for one year can still be useful, which satisfy the requirement of the determination of MEL residues.
Based on the obtained anti-melamine monoclonal antibody, we prepared the immunoaffinity chromatography column to specifically purify MEL and developed the IAC-LC-MS/MS method of measure MEL in powder and liquid milk. The results showed that the dynamic column capacities of IAC was 24.14 n mol/ml gel,and the specific column capacity of IAC was 3.36 n mol/mg antibody, respectively. The best regeneration cycle of IAC was 8 cycles. The limit of detection of IAC-LC-MS/MS method was 10ng/ml,and the limit of quantification was 20ng/ml, respectively. The recoveries of melamine from spiked milk and milk products samples ranged from 79.4 to 91.8%, and the variation coefficients were less than 10%, and which satisfied the requirement of the determination of MEL residues.
In this dissertation, colloidal gold was also prepared via sodium citrate reduction method. Based on the anti-melamine antibody, we developed a high-sensitivity and fast detection method for the MEL by the colloidal gold immunochromatographic assay. Several factors that affected assay performance were optimized. Finally, the optimal pH of gold-labeled antibodies was 8.0, the optimal antibody amount was 8μg/ml, and the optimal concentrations of coating and second antibody were 0.5mg/ml. The test strip for MEL had a visual detection limit of 12.5ng/ml in stand solution and 37.5μg/kg in powder, and had no cross-reactivity to MEL analogues, which could meet the demand of practical MEL testing.
Beside, we futher developed a sensitive and rapid visually and spectroscopic method for MEL detection by the molecular recognition between crown ether functionalized gold nanoparticles (GNPs) and amino group in MEL. Under the optimal conditions, MEL could be selectively detected in a concentration range from 10ng/ml to 500ng/ml with a limit of detection as 6ng/ml. Recovery results showed that the recoveries ranged from 98.4 to 105.6% in milk powder samples, and variation coefficients were less than 10%, which could be applicable for simple, rapid and sensitivity MEL detection.
本文首先设计合成并鉴定了三种三聚氰胺半抗原:MEL-MCA,MEL-ACA和MEL-PABA;采用活性酯法制备并鉴定了三种免疫原MEL-MCA-BSA,MEL-ACA-BSA和MEL-PABA-BSA;采用混合酸酐法制备并鉴定了三种包被原MEL-MCA-OVA,MEL-ACA-OVA和MEL-PABA-OVA;通过动物免疫实验,研究不同半抗原结构对免疫效果的影响。结果表明三种免疫原均具有免疫原性,其中以免疫原MEL-ACA-BSA和包被原MEL-PABA-OVA的异源免疫分析组合效果最好。
将制备的三种免疫原免疫BALB/C小鼠以制备抗三聚氰胺单克隆抗体,共获得4株可稳定分泌抗三聚氰胺单克隆抗体的杂交瘤细胞株,制备腹水并进行免疫特性分析,其抗体亚型为IgG1型,抗体亲和常数为2.67×109 L/mol,该抗体除与灭蝇胺交叉反应率为132%外,与其他三聚氰胺结构类似物无交叉反应,为目前报道的免疫特性最好的抗三聚氰胺单克隆抗体。
利用获得的单克隆抗体建立了三聚氰胺间接竞争ELISA检测方法并制备了试剂盒。通过对ELISA影响各因素的优化,最终确定其IC50为6±0.55ng/ml,奶粉最低检测限为100μg/kg,液态奶最低检测限为20ng/ml。添加回收实验结果表明,奶粉样品和液态奶样品的添加回收率分别为93%-101.9%和83.6%-96.9%,变异系数均小于10%;用国家标准方法GB/T 22388-2008 LC-MS/MS法进行确证,表明本研究所制备的ELISA试剂盒检测结果准确可靠,可用于乳及乳制品中三聚氰胺的检测,且试剂盒在4°C可保存1年,完全满足三聚氰胺残留检测的实际要求。
利用单克隆抗体制备了特异性识别、净化三聚氰胺的IAC柱,并建立了奶粉和液态奶中三聚氰胺的IAC-LC-MS/MS检测方法,IAC柱动态柱容量为24.14 n mol/ml gel,绝对柱容量为3.36 n mol/mg抗体,IAC柱最佳重复使用次数为8次;建立的IAC-LC-MS/MS检测方法的检测限为10ng/ml,定量限为20ng/ml,实际样品添加回收率为79.4-91.8%,变异系数小于10%,符合国际规定的残留分析方法要求,可用于实际样品中三聚氰胺残留检测。
采用柠檬酸钠法制备金纳米粒子,利用抗三聚氰胺单克隆抗体制备胶体金标记物,建立了基于胶体金免疫层析技术的乳及乳制品中三聚氰胺残留的高灵敏快速检测方法。通过优化确定胶体金标记抗体的最佳pH值为8.0,胶体金标记最佳抗体用量为8μg/ml,包被原最佳包被浓度和最佳二抗包被浓度为0.5mg/ml制备出三聚氰胺胶体金免疫层析试纸条。结果表明试纸条对三聚氰胺标准品的检测限为12.5ng/ml,实际奶粉样品的检测限为37.5μg/kg,对三聚氰胺结构类似物无交叉反应性,可满足三聚氰胺实际检测需求。
利用冠醚对金纳米粒子进行修饰制备功能化金纳米粒子,建立基于分子识别的三聚氰胺高灵敏快速检测比色法。通过优化确定三聚氰胺的检测线性范围为10-500ng/ml,检测限为6ng/ml,奶粉样品添加回收率在98.4%-105.6%之间,变异系数小于10%,可实现三聚氰胺简单、快速、高灵敏检测。
The melamine contamination event of milk powder greatly impacted China’s dairy industry. And the maximum residue limits of melamine in food have been set up both in domestic and abroad. In China, melamine has been one of the conventional detection items for milk and related products. Therefore, high-throμghput, rapid, and highly sensitive screening methods are urgently needed to satisfy the needs for melamine detection. In view of this, this dissertation carried out the following studies:
In this dissertation, three haptens (MEL-MCA, MEL-ACA and MEL-PABA) were designed, synthesized and identified. Three immunogens (MEL-MCA-BSA, MEL-ACA-BSA and MEL-PABA-BSA) and three coating antigens (MEL-MCA-OVA, MEL-ACA-OVA and MEL-PABA-OVA) were conjμgated by the active ester method and mixed anhydride method, respectively. The effects of hapten structure for immunity were investigated by animal immunization experiment. Results showed that the synthesis of these three immunogens was successful, and revealed that the MEL-ACA-BSA antibody/MEL-PABA-OVA coating conjμgate combination was the best for heterologous ELISA system.
Three immunogens was applied to immunize BALB/c mice to produce anti-melamine monoclonal antibody. And four monoclonal cell lines which could steadily secrete anti-melamine monoclonal antibody were obtained successfully. The subtype of antibody was identified as IgG1 by the commercial Kit. And the antibody affinity constant was calculated to be 2.67×109 L/mol. The most important property of the prepared antibody was that there was no cross-reaction to other MEL analogues except with cyromazine. The antibody achieved in this study was the best high-quality antibody among the currently reported monoclonal antibody against MEL.
Based on the obtained monoclonal antibody, an indirect competitive ELISA detection method of MEL was established and a relative ELISA Kit was assembled. Factors that may affect the performances of ELISA were optimized systematically. The IC50 value was 6±0.55ng/ml and the limit of detection (LOD) was 100μg/kg in powder and 20ng/ml in liquid milk, respectively. Recovery results showed that the recoveries ranged from 93 to 101.9% in milk powder samples and from 83.6 to 96.9% for liquid milk, respectively and variation coefficients were less than 10%. Real samples were determined throμgh the developed ELISA Kit of our laboratory. Detection results were also futher compared with results of by LC-MS/MS method according to national standard GB/T22388-2008, which showed highly consistency of the two methods. The stability results of the ELISA Kit indicated that the Kit stored at 4°C for one year can still be useful, which satisfy the requirement of the determination of MEL residues.
Based on the obtained anti-melamine monoclonal antibody, we prepared the immunoaffinity chromatography column to specifically purify MEL and developed the IAC-LC-MS/MS method of measure MEL in powder and liquid milk. The results showed that the dynamic column capacities of IAC was 24.14 n mol/ml gel,and the specific column capacity of IAC was 3.36 n mol/mg antibody, respectively. The best regeneration cycle of IAC was 8 cycles. The limit of detection of IAC-LC-MS/MS method was 10ng/ml,and the limit of quantification was 20ng/ml, respectively. The recoveries of melamine from spiked milk and milk products samples ranged from 79.4 to 91.8%, and the variation coefficients were less than 10%, and which satisfied the requirement of the determination of MEL residues.
In this dissertation, colloidal gold was also prepared via sodium citrate reduction method. Based on the anti-melamine antibody, we developed a high-sensitivity and fast detection method for the MEL by the colloidal gold immunochromatographic assay. Several factors that affected assay performance were optimized. Finally, the optimal pH of gold-labeled antibodies was 8.0, the optimal antibody amount was 8μg/ml, and the optimal concentrations of coating and second antibody were 0.5mg/ml. The test strip for MEL had a visual detection limit of 12.5ng/ml in stand solution and 37.5μg/kg in powder, and had no cross-reactivity to MEL analogues, which could meet the demand of practical MEL testing.
Beside, we futher developed a sensitive and rapid visually and spectroscopic method for MEL detection by the molecular recognition between crown ether functionalized gold nanoparticles (GNPs) and amino group in MEL. Under the optimal conditions, MEL could be selectively detected in a concentration range from 10ng/ml to 500ng/ml with a limit of detection as 6ng/ml. Recovery results showed that the recoveries ranged from 98.4 to 105.6% in milk powder samples, and variation coefficients were less than 10%, which could be applicable for simple, rapid and sensitivity MEL detection.
引文
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