抗克百威—三唑磷双特异性单克隆抗体研究
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摘要
本论文以杀虫剂克百威和三唑磷为研究对象,合成了一种克百威半抗原4—[[(2,3-二氢-2,2-二甲基-7-苯并呋喃基氧)羰基]氨基]丁酸(BFNB)和两种三唑磷半抗原O-乙基,O-[1-苯基-(H-1,2,4-三唑-3-基),N-(3-羧基-丙基)]硫代磷酸胺(THBu)、O-乙基,O-[1-苯基-(H-1,2,4-三唑-3-基),N-(5-羧基-丙基)]硫代磷酸胺(THHe),然后制备人工抗原,免疫小鼠,通过杂交瘤技术进行细胞融合,获得各自的杂交瘤细胞,并制备了相应的单克隆抗体,腹水效价在10~6以上。在此基础上,采用细胞酶缺陷技术和显微操作技术进行四体杂交瘤细胞的试验研究,获得的单克隆抗体具有双特异性功能和反应性强的特点。
试验对单克隆抗体及其ELISA方法进行了优化:使用Coming公司的COASTAR酶标板、以PBS(0.05M,pH8.0)为抗体包被缓冲溶液、CBS(0.05M,pH9.6)为抗原包被缓冲溶液,37℃包被2h、封闭溶液以2%脱脂牛奶、抗克百威单抗以0.05M PBS缓冲溶液的离子浓度(pH值为7.0)、而抗三唑磷单抗以0.02MPBS(pH值为7.4)为佳。研究建立了克百威和三唑磷直接竞争的ELISA方法(包被抗体模式),结果表明,克百威ELISA方法的检测灵敏度120达到了1.89ng/mL(线性范围为1.11～45.95ng/mE),三唑磷ELISA方法的检测灵敏度120达到了0.15ng/mL(线性范围为0.07～3.90 ng/mL),经过对环境样品、水果和蔬菜等样品进行添加回收率试验,结果表明,除了米和面粉的回收率有所偏高外,其他样品的回收率结果均在75%～130%范围内波动,表明所建立的ELISA方法是可靠的。
对分泌克百威的杂交瘤细胞采用8-氮鸟嘌呤筛选培养,使细胞HGPRT酶缺失,对分泌三唑磷的杂交瘤细胞采用6-氯嘌呤筛选培养,使细胞TK酶缺失,然后进行细胞融合、筛选、检测,获得了一株能分泌抗克百威和三唑磷的双特异性单克隆抗体的四体杂交瘤细胞(12C1-2H12),制备腹水并纯化抗体,通过一系列的反应缓冲体系的筛选和优化,建立了相应ELISA方法。从灵敏度结果比较可知,双特异性单克隆抗体对克百威和三唑磷的检测灵敏度(120)分别为4.46ng/mL和0.36 ng/mL,与单克隆抗体相比略有降低(I_(20)升高)。交叉反应率实验结果表明所制备的双特异性单克隆抗体与克百威和三唑磷相应的结构类似物均无明显交叉反应。结果表明所制备的双特异性单克隆抗体具有很高的双特异性和较高的检测灵敏度。采用双特异性单抗ELISA方法,进行了土壤和水质样品、蔬菜和水果样品中克百威和三唑磷添加回收率试验,水样的检测结果略有偏高,尤其是自来水样;土壤、蔬菜和水果样品的回收率结果均在70～130%范围内,检测结果表明所制备的双特异性单克隆抗体能适用与环境样品、干粉样品,水果和蔬菜样品的检测,所建立的免疫分析方法准确可靠。
通过以上试验研究,本文初步提出了较为系统的基于杂交瘤技术的双特异性单克隆抗体制备技术体系、以及相应的ELISA方法和评价技术,并对双特异性单克隆抗体方法原理进行了初步的研究与探讨。
One hapten of insecticide carbofuran 2,2-dimethyl-2,3-dihydrobenzofuranyl, 7-N-(3- carboxypropyl) carbamate (BFNB) and two haptens of pesticide triazophos O-ethyl, O-(1-phenyl-1H-1,2,4-triazol-3- yl) N-(3-carboxypropyl)phosphoramidothioate (THBu) and [O-ethyl, O-(1-phenyl-1H-1,2,4-triazol-3-yl) N-(5-carboxyamyl) phosphoramidothioate (THHe) were synthesized and then covalently coupled with carrier protein BSA and OVA, respectively. The mouse were immunized with artificial antigens, then mouse spleen lymphocytes were fused with myeloma cells by cross-tumour technique. The hybridoma cells were selected and cloned. The monoclonal antibodies were obtained with high titres, which of ascitic fluids up to 1×10~(-6) by indirect ELISA. The tetradoma cell was selected based on the technique of lacking functional enzyme and the technique of microscopic operation, then the bi-specific monoclonal antibodyby were obtained with the characteristics of bi-specificity and high reactivity.
The conditions of ELISA for those insecticides above were optimized as follows. Costar Immuno plate from corning corporation was selected as the coating plate in the experiment. The optimized coating buffer were PBS(0.05 M, pH8.0) and CBS( 0.05 M, pH9.6) for AC format(antibodies-coated) and CC format (conjugate-coated), respectively. The plate was incubated at 37°C for 2 hours and blocked by 2% defatted milk powder. The optimized buffers were 0.05M PBS (pH7.0) and 0.02M PBS (pH7.4) for carbofuran and triazophos, respectively. Based on the optimized ELISA conditions, the direct competitive ELISA (antibody coating format) were adopted for the determination of carbofuran and triazophos. The limits of detection(I_(20)) of the selected ELISA were 1.89 ng/mL and 0.15 ng/mL for carbofuran and triazophos, respectively. And the linear concentrations were ranged from 1.11 to 45.95 ng/mL and 0.07 to 3.90 ng/mL for carbofuran and triazophos, respectively. Different kinds of samples such as water and soil samples, vegetable and fruit samples were spiked with carbofuran and triazophos. The results showed that the recoveries of most of the samples ranged from 75% to 130%, except for rice and wheat powder samples. However, it was proved that the selected ELISA was reliable for detecting carbofuran and triazophos.
In order to get the hybrid-hybridoma(tetradoma) cells, the hybridoma producing monoclonal antibody to carbofuran was selected by 8-N-Ag which make the cells lacking of hypoxanthibe- guanine phosphoribosyltransferase (HGPRT). And the hybridoma producing monoclonal antibody to triazophos was selected by 6-chloropurine which made the cells lacking of thymidine kinase(TK). Then the mouse hybrid hybridomas (tetradoma) was fused with the selected hybridomas and cloned by the technique of microscopic operation. One tetradoma line (12C1-2H12) secreted immunoglobulin manifesting parental and bi-specific binding characteristics was established. The bi-specific monoclone was produced by ascite and purified. Then the optimal conditions for the ELISA were established based on the optimization of buffer condition. From the result, the detection limits I_(20) of the ELISA based on bi-specific monoclonal antibody were 4.46 and 0.36 ng/mL for carbofuran and triazophos, respectively. The value of I_(20) was higher than that of the monoclonal antibody. The bi-specific monoclonal antibodies were obtained with high specificity to the analytes, and with very low cross-reactivity to carbofuran and triazophos related compounds. There were no obvious difference of cross-reactivity between the bi-McAb and McAb. It was testified that those bi-McAbs obtained was successful and could be used for detecting carbofuran and triazophos, respectively. The optimized ELISA based on Bi-McAb was applied in environmental samples, vegetable samples and fruit samples. From the results, the recoveries for water samples showed an abnormal tendency, especially from the sample of tap water, which had the highest tendency in tested water samples. The recoveries for soil, vegetable and fruit samples were ranged from 75% to 130%. Therefore, the Bi-McAb was suitable to detect the analyte residue in the soil samples, vegetable samples and fruit samples.
In this study, the technique system for producing bi-specific monoclonal antibody based on hybrid-hybridoma and its ELISA method was esteblished successfully. And some of principle for bi-McAb to pesticide was discussed.
试验对单克隆抗体及其ELISA方法进行了优化:使用Coming公司的COASTAR酶标板、以PBS(0.05M,pH8.0)为抗体包被缓冲溶液、CBS(0.05M,pH9.6)为抗原包被缓冲溶液,37℃包被2h、封闭溶液以2%脱脂牛奶、抗克百威单抗以0.05M PBS缓冲溶液的离子浓度(pH值为7.0)、而抗三唑磷单抗以0.02MPBS(pH值为7.4)为佳。研究建立了克百威和三唑磷直接竞争的ELISA方法(包被抗体模式),结果表明,克百威ELISA方法的检测灵敏度120达到了1.89ng/mL(线性范围为1.11～45.95ng/mE),三唑磷ELISA方法的检测灵敏度120达到了0.15ng/mL(线性范围为0.07～3.90 ng/mL),经过对环境样品、水果和蔬菜等样品进行添加回收率试验,结果表明,除了米和面粉的回收率有所偏高外,其他样品的回收率结果均在75%～130%范围内波动,表明所建立的ELISA方法是可靠的。
对分泌克百威的杂交瘤细胞采用8-氮鸟嘌呤筛选培养,使细胞HGPRT酶缺失,对分泌三唑磷的杂交瘤细胞采用6-氯嘌呤筛选培养,使细胞TK酶缺失,然后进行细胞融合、筛选、检测,获得了一株能分泌抗克百威和三唑磷的双特异性单克隆抗体的四体杂交瘤细胞(12C1-2H12),制备腹水并纯化抗体,通过一系列的反应缓冲体系的筛选和优化,建立了相应ELISA方法。从灵敏度结果比较可知,双特异性单克隆抗体对克百威和三唑磷的检测灵敏度(120)分别为4.46ng/mL和0.36 ng/mL,与单克隆抗体相比略有降低(I_(20)升高)。交叉反应率实验结果表明所制备的双特异性单克隆抗体与克百威和三唑磷相应的结构类似物均无明显交叉反应。结果表明所制备的双特异性单克隆抗体具有很高的双特异性和较高的检测灵敏度。采用双特异性单抗ELISA方法,进行了土壤和水质样品、蔬菜和水果样品中克百威和三唑磷添加回收率试验,水样的检测结果略有偏高,尤其是自来水样;土壤、蔬菜和水果样品的回收率结果均在70～130%范围内,检测结果表明所制备的双特异性单克隆抗体能适用与环境样品、干粉样品,水果和蔬菜样品的检测,所建立的免疫分析方法准确可靠。
通过以上试验研究,本文初步提出了较为系统的基于杂交瘤技术的双特异性单克隆抗体制备技术体系、以及相应的ELISA方法和评价技术,并对双特异性单克隆抗体方法原理进行了初步的研究与探讨。
One hapten of insecticide carbofuran 2,2-dimethyl-2,3-dihydrobenzofuranyl, 7-N-(3- carboxypropyl) carbamate (BFNB) and two haptens of pesticide triazophos O-ethyl, O-(1-phenyl-1H-1,2,4-triazol-3- yl) N-(3-carboxypropyl)phosphoramidothioate (THBu) and [O-ethyl, O-(1-phenyl-1H-1,2,4-triazol-3-yl) N-(5-carboxyamyl) phosphoramidothioate (THHe) were synthesized and then covalently coupled with carrier protein BSA and OVA, respectively. The mouse were immunized with artificial antigens, then mouse spleen lymphocytes were fused with myeloma cells by cross-tumour technique. The hybridoma cells were selected and cloned. The monoclonal antibodies were obtained with high titres, which of ascitic fluids up to 1×10~(-6) by indirect ELISA. The tetradoma cell was selected based on the technique of lacking functional enzyme and the technique of microscopic operation, then the bi-specific monoclonal antibodyby were obtained with the characteristics of bi-specificity and high reactivity.
The conditions of ELISA for those insecticides above were optimized as follows. Costar Immuno plate from corning corporation was selected as the coating plate in the experiment. The optimized coating buffer were PBS(0.05 M, pH8.0) and CBS( 0.05 M, pH9.6) for AC format(antibodies-coated) and CC format (conjugate-coated), respectively. The plate was incubated at 37°C for 2 hours and blocked by 2% defatted milk powder. The optimized buffers were 0.05M PBS (pH7.0) and 0.02M PBS (pH7.4) for carbofuran and triazophos, respectively. Based on the optimized ELISA conditions, the direct competitive ELISA (antibody coating format) were adopted for the determination of carbofuran and triazophos. The limits of detection(I_(20)) of the selected ELISA were 1.89 ng/mL and 0.15 ng/mL for carbofuran and triazophos, respectively. And the linear concentrations were ranged from 1.11 to 45.95 ng/mL and 0.07 to 3.90 ng/mL for carbofuran and triazophos, respectively. Different kinds of samples such as water and soil samples, vegetable and fruit samples were spiked with carbofuran and triazophos. The results showed that the recoveries of most of the samples ranged from 75% to 130%, except for rice and wheat powder samples. However, it was proved that the selected ELISA was reliable for detecting carbofuran and triazophos.
In order to get the hybrid-hybridoma(tetradoma) cells, the hybridoma producing monoclonal antibody to carbofuran was selected by 8-N-Ag which make the cells lacking of hypoxanthibe- guanine phosphoribosyltransferase (HGPRT). And the hybridoma producing monoclonal antibody to triazophos was selected by 6-chloropurine which made the cells lacking of thymidine kinase(TK). Then the mouse hybrid hybridomas (tetradoma) was fused with the selected hybridomas and cloned by the technique of microscopic operation. One tetradoma line (12C1-2H12) secreted immunoglobulin manifesting parental and bi-specific binding characteristics was established. The bi-specific monoclone was produced by ascite and purified. Then the optimal conditions for the ELISA were established based on the optimization of buffer condition. From the result, the detection limits I_(20) of the ELISA based on bi-specific monoclonal antibody were 4.46 and 0.36 ng/mL for carbofuran and triazophos, respectively. The value of I_(20) was higher than that of the monoclonal antibody. The bi-specific monoclonal antibodies were obtained with high specificity to the analytes, and with very low cross-reactivity to carbofuran and triazophos related compounds. There were no obvious difference of cross-reactivity between the bi-McAb and McAb. It was testified that those bi-McAbs obtained was successful and could be used for detecting carbofuran and triazophos, respectively. The optimized ELISA based on Bi-McAb was applied in environmental samples, vegetable samples and fruit samples. From the results, the recoveries for water samples showed an abnormal tendency, especially from the sample of tap water, which had the highest tendency in tested water samples. The recoveries for soil, vegetable and fruit samples were ranged from 75% to 130%. Therefore, the Bi-McAb was suitable to detect the analyte residue in the soil samples, vegetable samples and fruit samples.
In this study, the technique system for producing bi-specific monoclonal antibody based on hybrid-hybridoma and its ELISA method was esteblished successfully. And some of principle for bi-McAb to pesticide was discussed.
引文
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