三种重金属单抗的制备与抗体基因序列分析及其免疫检测方法的建立
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摘要
重金属污染事件层出不穷,愈演愈烈,畜禽产品重金属残留不同程度超标,威胁人畜健康。快速、廉价地监测生境中重金属是减小其对人类及动物危害的先决条件。建立更快速、更经济的免疫分析法检测重金属是生产及经济发展的需要。本研究旨在建立能够稳定分泌高亲和力和特异性抗重金属汞、铜和锌的单克隆抗体的杂交瘤细胞株,克隆抗重金属汞、铜和锌单克隆抗体的重链和轻链可变区基因,建立其免疫分析方法并得到初步应用。
本研究将重金属(汞、铜和锌)通过双功能螯合剂与血蓝蛋白偶联制备了免疫抗原,与牛血清白蛋白偶联制备了检测抗原。通过BCA法、紫外分光光度法、SDS-PAGE电泳和石墨炉原子吸收光谱法鉴定重金属汞、铜和锌螯合物人工抗原。用合成的免疫抗原免疫BALB/c小鼠,采用杂交瘤技术建立能稳定分泌抗重金属汞、铜和锌单克隆抗体的杂交瘤细胞株,分析杂交瘤细胞的染色体,鉴定。通过抗体亚型鉴定试剂盒鉴定单克隆抗体的类和亚类,通过体内诱导法大量制备并纯化抗体,通过间接酶联免疫吸附测定(ELISA)法检测抗体效价、亲和力和细胞株分泌抗体的稳定性,间接竞争ELISA(IC-ELISA)法测定抗体的特异性。通过提取杂交瘤总RNA、RT-PCR,扩增抗体可变区基因,克隆测序分析该基因。采用矩阵实验确定抗原抗体的最佳组合,通过对封闭物、pH值、离子强度等影响因子的研究,确定IC-ELISA的最佳工作参数。
研究结果表明:成功制备了汞、铜和锌的免疫抗原和检测抗原,并建立了能稳定分泌特异性抗重金属汞、铜和锌单克隆抗体的杂交瘤细胞株分别为3G4-G5、9D11-F3和Z1A5,抗体均为IgM亚类,轻链为kappa型,亲和力达10~9L/mol～10~(10)L/mol,半数抑制浓度(IC50)分别为1.15μM、0.257μg/mL和0.628μg/mL,与其他金属的交叉反应低。从杂交瘤细胞3G4-G5、9D11-F3和Z1A5中克隆得到了抗重金属汞、铜和锌单克隆抗体的重链和轻链可变区基因。在IC-ELISA最佳工作参数确定的情况下,建立定量测定金属离子汞、铜和锌的方法,检测范围(IC20～IC80)分别为0.15μM～9.0μM,0.055μg/mL～1.198μg/mL和0.062μg/mL～6.341μg/mL,最低检测限分别为3.94×10~(-5)mM,0.0227μg/mL和0.015μg/mL。水样中的汞、铜和锌添加回收率为分别为88.9%～107.3%,87.4%～106.8%和88.6%～109.4%。
本研究成功制备了重金属汞、铜和锌抗原,建立了3株能稳定分泌高亲和力、高特异性抗重金属汞、铜和锌单克隆抗体的杂交瘤细胞,且克隆得到了抗重金属汞、铜和锌单克隆抗体的重链和轻链可变区基因,为下一步通过基因工程改造抗体奠定了基础。建立重金属汞、铜和锌残留IC-ELISA检测法,并能有效地用于实验室水样的重金属汞、铜和锌残留检测,为重金属汞、铜和锌残留检测试剂盒的研发奠定了基础,为农畜产品及环境安全检测提供了重要的工具,对提高风险评估工作的效率和质量,保障食品安全有重要现实意义。
Heavy metal pollution incidents have been emerging endlessly and increasing fiercely. The poultry products existed heavy metal in different degree are in excess of the standards, which are jeopardizing the health of human and animals. Monitoring heavy metals in the environment rapidly and inexpensively is a prerequisite to minimize harm for human and animals. The development of immunoassays detecting heavy metals residues has been a promising trend because of its speediness and cheapness. The aim of this study was to establish hybridoma cell lines that can steadily secrete high affinity and specificity monoclonal antibodies (McAbs) against mercury, copper and zinc respectively, and clone the variable region genes of the McAb to develop and apply indirect competitive enzyme linked i m m u n o s o r b e n t a s s a y ( I C - E L I S A ) f o r m e r c u r y, c o p p e r a n d z i n c .
In this study, heavy metal (mercury, copper and zinc) was coupled to carrier proteins such as keyhole limpet hemocyanin (KLH) and bovine serum albumin (BSA) via bifunctional chelator. Carrier protein of immunizing antigen and detection antigen was KLH and BSA respectively. The artificial antigens of mercury, copper and zinc were identified with the methods of bicinchoninic acid (BCA), ultraviolet spectrophotometry, sodium-dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), and graphite furnace atomic absorption spectrometry. BALB/c mice were immunized with immunizing antigen and the hybridoma cell lines that can steadily secrete high affinity and specificity monoclonal antibodies against mercury, copper and zinc respectively were established by hybridoma technique. And the chromosomes of hybridoma cell were analyzed. Additionally, the class and subclass of McAbs were determined by Mouse Monoclonal Antibody Isotyping Kit. Purified McAbs were prepared after ascites fluid was produced in BALB/c mice. The titer and affinity of McAbs and the stability of the hybridoma cell lines secreting McAbs were determined by indirect ELISA, the specificity of McAbs was determined by competitive inhibition ELISA. The total RNA of hybridoma cell lines was isolated, and the variable region genes were amplified by RT-PCR to analyze the variable region genes. The optimal working parameter of IC-ELISA was determined through the research of working concentration of antigen and antibody, blocking agent, pH and ionic strength. The results showed that the synthesis of immune and detection antigen of mercury, copper and zinc was successful. Three hybridoma cell lines producing stably specific MAbs against mercury, copper and zinc were obtained, which were named G4-G5, 9D11-F3 and Z1A5 respectively. The class and subclass of all McAbs were IgM and kappa respectively. The affinity constant of antibodies was 10~9L/mol~10~(10)L/mol. The IC50 values of 3G4-G5, 9D11-F3 and Z1A5 was 1.15μM, 0.257μg/mL and 0.628μg/mL respectively. The cross-reactivities were lower than other metal ions. The variable region genes of the McAbs against mercury, copper and zinc respectively were amplified from 3G4-G5, 9D11-F3 and Z1A5. With the optimization of working parameter, the standard curves of IC-ELISA for mercury, copper and zinc were established. The detection range (IC20~IC80) was 0.15μM~9.0μM, 0.055μg/mL~1.198μg/mL and 0.062μg/mL~6.341μg/mL respectively. The lowest detection limit was 3.94×10~(-5)mM, 0.0227μg/mL and 0.015μg/mL respectively. Recoveries of mercury, copper and zinc from the analyte-fortified into tap water and ultrapure water were in the range of 88.9%~107.3%, 87.4%~106.8% and 88.6%~109.4% respectively.
The synthesis of antigen of mercury,copper and zinc was successful. Three hybridoma cell lines producing stably MAbs with high affinity and specificity against mercury, copper and zinc were obtained. The variable region genes of the McAbs that are against mercury, copper and zinc respectively were amplified, which laid a foundation for transformation antibody with genetic engineering. The IC-ELISA for mercury, copper and zinc was established. The immunoassays method set the stage of the development of immunoassay test kits for determining residues of mercury, copper and zinc, which could provide an important tool for farm products as well as environmental safety test, so it would have the realistic significance to improve the efficiency and quality of risk assessment.
本研究将重金属(汞、铜和锌)通过双功能螯合剂与血蓝蛋白偶联制备了免疫抗原,与牛血清白蛋白偶联制备了检测抗原。通过BCA法、紫外分光光度法、SDS-PAGE电泳和石墨炉原子吸收光谱法鉴定重金属汞、铜和锌螯合物人工抗原。用合成的免疫抗原免疫BALB/c小鼠,采用杂交瘤技术建立能稳定分泌抗重金属汞、铜和锌单克隆抗体的杂交瘤细胞株,分析杂交瘤细胞的染色体,鉴定。通过抗体亚型鉴定试剂盒鉴定单克隆抗体的类和亚类,通过体内诱导法大量制备并纯化抗体,通过间接酶联免疫吸附测定(ELISA)法检测抗体效价、亲和力和细胞株分泌抗体的稳定性,间接竞争ELISA(IC-ELISA)法测定抗体的特异性。通过提取杂交瘤总RNA、RT-PCR,扩增抗体可变区基因,克隆测序分析该基因。采用矩阵实验确定抗原抗体的最佳组合,通过对封闭物、pH值、离子强度等影响因子的研究,确定IC-ELISA的最佳工作参数。
研究结果表明:成功制备了汞、铜和锌的免疫抗原和检测抗原,并建立了能稳定分泌特异性抗重金属汞、铜和锌单克隆抗体的杂交瘤细胞株分别为3G4-G5、9D11-F3和Z1A5,抗体均为IgM亚类,轻链为kappa型,亲和力达10~9L/mol～10~(10)L/mol,半数抑制浓度(IC50)分别为1.15μM、0.257μg/mL和0.628μg/mL,与其他金属的交叉反应低。从杂交瘤细胞3G4-G5、9D11-F3和Z1A5中克隆得到了抗重金属汞、铜和锌单克隆抗体的重链和轻链可变区基因。在IC-ELISA最佳工作参数确定的情况下,建立定量测定金属离子汞、铜和锌的方法,检测范围(IC20～IC80)分别为0.15μM～9.0μM,0.055μg/mL～1.198μg/mL和0.062μg/mL～6.341μg/mL,最低检测限分别为3.94×10~(-5)mM,0.0227μg/mL和0.015μg/mL。水样中的汞、铜和锌添加回收率为分别为88.9%～107.3%,87.4%～106.8%和88.6%～109.4%。
本研究成功制备了重金属汞、铜和锌抗原,建立了3株能稳定分泌高亲和力、高特异性抗重金属汞、铜和锌单克隆抗体的杂交瘤细胞,且克隆得到了抗重金属汞、铜和锌单克隆抗体的重链和轻链可变区基因,为下一步通过基因工程改造抗体奠定了基础。建立重金属汞、铜和锌残留IC-ELISA检测法,并能有效地用于实验室水样的重金属汞、铜和锌残留检测,为重金属汞、铜和锌残留检测试剂盒的研发奠定了基础,为农畜产品及环境安全检测提供了重要的工具,对提高风险评估工作的效率和质量,保障食品安全有重要现实意义。
Heavy metal pollution incidents have been emerging endlessly and increasing fiercely. The poultry products existed heavy metal in different degree are in excess of the standards, which are jeopardizing the health of human and animals. Monitoring heavy metals in the environment rapidly and inexpensively is a prerequisite to minimize harm for human and animals. The development of immunoassays detecting heavy metals residues has been a promising trend because of its speediness and cheapness. The aim of this study was to establish hybridoma cell lines that can steadily secrete high affinity and specificity monoclonal antibodies (McAbs) against mercury, copper and zinc respectively, and clone the variable region genes of the McAb to develop and apply indirect competitive enzyme linked i m m u n o s o r b e n t a s s a y ( I C - E L I S A ) f o r m e r c u r y, c o p p e r a n d z i n c .
In this study, heavy metal (mercury, copper and zinc) was coupled to carrier proteins such as keyhole limpet hemocyanin (KLH) and bovine serum albumin (BSA) via bifunctional chelator. Carrier protein of immunizing antigen and detection antigen was KLH and BSA respectively. The artificial antigens of mercury, copper and zinc were identified with the methods of bicinchoninic acid (BCA), ultraviolet spectrophotometry, sodium-dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), and graphite furnace atomic absorption spectrometry. BALB/c mice were immunized with immunizing antigen and the hybridoma cell lines that can steadily secrete high affinity and specificity monoclonal antibodies against mercury, copper and zinc respectively were established by hybridoma technique. And the chromosomes of hybridoma cell were analyzed. Additionally, the class and subclass of McAbs were determined by Mouse Monoclonal Antibody Isotyping Kit. Purified McAbs were prepared after ascites fluid was produced in BALB/c mice. The titer and affinity of McAbs and the stability of the hybridoma cell lines secreting McAbs were determined by indirect ELISA, the specificity of McAbs was determined by competitive inhibition ELISA. The total RNA of hybridoma cell lines was isolated, and the variable region genes were amplified by RT-PCR to analyze the variable region genes. The optimal working parameter of IC-ELISA was determined through the research of working concentration of antigen and antibody, blocking agent, pH and ionic strength. The results showed that the synthesis of immune and detection antigen of mercury, copper and zinc was successful. Three hybridoma cell lines producing stably specific MAbs against mercury, copper and zinc were obtained, which were named G4-G5, 9D11-F3 and Z1A5 respectively. The class and subclass of all McAbs were IgM and kappa respectively. The affinity constant of antibodies was 10~9L/mol~10~(10)L/mol. The IC50 values of 3G4-G5, 9D11-F3 and Z1A5 was 1.15μM, 0.257μg/mL and 0.628μg/mL respectively. The cross-reactivities were lower than other metal ions. The variable region genes of the McAbs against mercury, copper and zinc respectively were amplified from 3G4-G5, 9D11-F3 and Z1A5. With the optimization of working parameter, the standard curves of IC-ELISA for mercury, copper and zinc were established. The detection range (IC20~IC80) was 0.15μM~9.0μM, 0.055μg/mL~1.198μg/mL and 0.062μg/mL~6.341μg/mL respectively. The lowest detection limit was 3.94×10~(-5)mM, 0.0227μg/mL and 0.015μg/mL respectively. Recoveries of mercury, copper and zinc from the analyte-fortified into tap water and ultrapure water were in the range of 88.9%~107.3%, 87.4%~106.8% and 88.6%~109.4% respectively.
The synthesis of antigen of mercury,copper and zinc was successful. Three hybridoma cell lines producing stably MAbs with high affinity and specificity against mercury, copper and zinc were obtained. The variable region genes of the McAbs that are against mercury, copper and zinc respectively were amplified, which laid a foundation for transformation antibody with genetic engineering. The IC-ELISA for mercury, copper and zinc was established. The immunoassays method set the stage of the development of immunoassay test kits for determining residues of mercury, copper and zinc, which could provide an important tool for farm products as well as environmental safety test, so it would have the realistic significance to improve the efficiency and quality of risk assessment.
引文
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