Zn-金属硫蛋白间接竞争型ELISA的建立与应用
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摘要
ELISA以其快速、准确、安全、简便的特点而成为定量测定生物体内微量有机物的理想方法。本实验的目的就是为了建立一套快速、有效、方便并能定量测定猪体组织中MT含量的ELISA法，为动物科学中的研究和应用提供MT的定量测定技术。
1．猪MT抗小鼠抗血清的制备：用戊二醛法将纯品Zn-MT与载体蛋白(BSA)偶联后，作为人工抗原，对昆明小鼠皮下注射。首次免疫时抗原中MT量分别为8ug/只、16ug/只、32ug/只、64ug/只、128ug/只；加强免疫时所用抗原量依次加倍。前3次免疫时采用的是弗氏完全佐剂乳化，后3次免疫采用弗氏不完全佐剂乳化。两批小鼠加强免疫的时间隔分别为10日和14日。用免疫双扩散方法测定抗血清效价。得出以下结论：当首次免疫抗原中MT的量为16-32ug/只，每次加强免疫时抗原量加倍，加强免疫间隔时间为10日时，其免疫效果较佳，可使大部分小鼠抗血清效价达到1：16以上。
2．抗体的纯化及鉴定：将效价达到1：16以上的MT小鼠抗血清用饱和硫酸铵盐析法浓缩出γ-球蛋白，然后用DEAE-Sephadex A50层析柱分离提纯IgG。实验中发现洗脱液pH值为7.4时洗脱第一峰即为纯化的IgG，用不连续缓冲系统聚丙烯酰胺凝胶浓度梯度电泳鉴定层析洗脱下的IgG达到电泳纯。对纯化后的IgG进行辣根过氧化物酶标记，用直接ELISA鉴定其为MT的特异性抗体。
3．ELISA的建立：运用固相抗原间接非竞争型ELISA和固相抗原间接竞争型ELISA检测猪体组织中部分样品MT的含量，结果表明：用间接非竞争型ELISA测定样品时其结果不够稳定，同一样品所对应的0D值相差较大，此方法仅适合于样品中MT定性检测；采用间接竞争型ELISA测定样品，0D值比较稳定。用方阵滴定法确定了间接竞争型ELISA的各项具体条件：抗原最佳包被浓度为6.25ug/ml，一抗(鼠抗猪)使用适宜浓度为50ug/ml，酶标抗体(山羊抗小鼠)的使用浓度为1/8000倍，反应时间为3hr。用标准MT，采用间接竞争型ELISA方法拟合的标准曲线回归方程为：y=10~5×2~((22.32880-0.39583x))，相关系数为：R=0.9592。本方法的灵敏度为4.1ng/ml，批间和批内变异系数为9.9760％-14.3858％，在实际应用中有较好的稳定性。说明本试验建立的间接竞争型ELISA具有特异性强、灵敏度较高、稳定性和重复性好、适合于大批量样品的检测等优点，不失为对MT进行定量分析的一种有效方法。
ELISA is becoming an optimal method that could determine the quantum of organic compound in organism because of its speediness, specificity and sensitivity. The purpose of our research was to establish a set of complete assay determined quantum of metallothionein in swine' s body.
1. the polyclonal antiserum in mice of MT which was distilled from swine was prepared : After the rarefied MT-II was coupled to carrier protein BSA by means of GD, the protein-hapten conjugates was acted as man-made antigen. Kun Ming mice were injected subcutaneous by the antigen with the carrier protein of BSA. And each group had the lever of MT in antigen were corresponding 8ug/mouse,16ug/mouse,32ug/mouse, 64ug/mouse,128ug/mouse in the first immunity. The quantum of MT in antigen is doubling in the latter boosting immunity in turn. The antigen had been emulsified with Freund' s complete adjuvant in the earlier 3 times and with Freund' s incomplete adjuvant in the latter 3 times. The two batches of mouse were boosted with the same condition except different time between two immunities, one was 10 days, and the other was 2 weeks. After the antiserum titters being determined by the double immunodiffeoion in two dimensions assay , we had these results: The level of MT in antigen was 16-32ug/mouse and the level of MT was doubling in progression, the time between two boosting immunities is 10 days, the impact is fine, which could make the antiserum titers of a majority of mice attach to 1 :16.
2.The antibody was purified and identified: The antiserum whose titer attach to 1 I 16 was condensed to get Y-globulin with ammonium sulphate precipitation assay, and then the y -globulin was purified to IgG on a DEAE-Sephadex A50 column. We found when the pH value of washing buffer was 7. 4, the first apex of curve corresponded the solution of purified IgG whose purification was identified with discontinuous on different concentration polyacrylamide electrophoresis. After being conjugated with HRP, the IgG which is the specifically antibody to MT was identified with direct ELISA.
3. Establishment of ELISA: There are two assays which were the indirect competitive ELISA and the indirect uncompetitive ELISA, and they were used to determined MT in the sample of swine .the result showed: the result on determined with the indirect uncompetitive ELTSA were erratic, and the same
sample of OD value is much discrepant, and this assay is fit for determining the nature of MT. However, the result on MT which was determined by the indirect competitive ELISA were steady and this assay is fit to determine the quantum of MT, and it is comparntively a fine method. Using the method of chessboard titration chose the proper reaction for the indirect competitive ELISA: the carrier antigen of concentration is 6. 25ug/ml; the first antibody (mouse-antiswine) of suitable concentration is 50ug/ml; and the immunoglobulin-HRP(goat-antimouse) of diluent concentration is 1/8000;the reaction of time is 3hr. After liner regression analysis, a linear equation : y=105x2(22.32880-0.39583x), coefficient of correlation: R=0. 9592, and the sensitivity of assay to detect the rabbit MT is 4. Ing/ml. Intra-assay coefficient of variation and Inter-assay coefficient of variation are 9. 9760%-14. 3858%, which showed that this assay has fine stability. It proved that the establishment in our experimentation of indirect competitive ELISA has highly specificity, sensitivity, stability and repetitiveness. And it is fit for determining a big batch samples, and it is an effective method for determining the quantum of MT.
1．猪MT抗小鼠抗血清的制备：用戊二醛法将纯品Zn-MT与载体蛋白(BSA)偶联后，作为人工抗原，对昆明小鼠皮下注射。首次免疫时抗原中MT量分别为8ug/只、16ug/只、32ug/只、64ug/只、128ug/只；加强免疫时所用抗原量依次加倍。前3次免疫时采用的是弗氏完全佐剂乳化，后3次免疫采用弗氏不完全佐剂乳化。两批小鼠加强免疫的时间隔分别为10日和14日。用免疫双扩散方法测定抗血清效价。得出以下结论：当首次免疫抗原中MT的量为16-32ug/只，每次加强免疫时抗原量加倍，加强免疫间隔时间为10日时，其免疫效果较佳，可使大部分小鼠抗血清效价达到1：16以上。
2．抗体的纯化及鉴定：将效价达到1：16以上的MT小鼠抗血清用饱和硫酸铵盐析法浓缩出γ-球蛋白，然后用DEAE-Sephadex A50层析柱分离提纯IgG。实验中发现洗脱液pH值为7.4时洗脱第一峰即为纯化的IgG，用不连续缓冲系统聚丙烯酰胺凝胶浓度梯度电泳鉴定层析洗脱下的IgG达到电泳纯。对纯化后的IgG进行辣根过氧化物酶标记，用直接ELISA鉴定其为MT的特异性抗体。
3．ELISA的建立：运用固相抗原间接非竞争型ELISA和固相抗原间接竞争型ELISA检测猪体组织中部分样品MT的含量，结果表明：用间接非竞争型ELISA测定样品时其结果不够稳定，同一样品所对应的0D值相差较大，此方法仅适合于样品中MT定性检测；采用间接竞争型ELISA测定样品，0D值比较稳定。用方阵滴定法确定了间接竞争型ELISA的各项具体条件：抗原最佳包被浓度为6.25ug/ml，一抗(鼠抗猪)使用适宜浓度为50ug/ml，酶标抗体(山羊抗小鼠)的使用浓度为1/8000倍，反应时间为3hr。用标准MT，采用间接竞争型ELISA方法拟合的标准曲线回归方程为：y=10~5×2~((22.32880-0.39583x))，相关系数为：R=0.9592。本方法的灵敏度为4.1ng/ml，批间和批内变异系数为9.9760％-14.3858％，在实际应用中有较好的稳定性。说明本试验建立的间接竞争型ELISA具有特异性强、灵敏度较高、稳定性和重复性好、适合于大批量样品的检测等优点，不失为对MT进行定量分析的一种有效方法。
ELISA is becoming an optimal method that could determine the quantum of organic compound in organism because of its speediness, specificity and sensitivity. The purpose of our research was to establish a set of complete assay determined quantum of metallothionein in swine' s body.
1. the polyclonal antiserum in mice of MT which was distilled from swine was prepared : After the rarefied MT-II was coupled to carrier protein BSA by means of GD, the protein-hapten conjugates was acted as man-made antigen. Kun Ming mice were injected subcutaneous by the antigen with the carrier protein of BSA. And each group had the lever of MT in antigen were corresponding 8ug/mouse,16ug/mouse,32ug/mouse, 64ug/mouse,128ug/mouse in the first immunity. The quantum of MT in antigen is doubling in the latter boosting immunity in turn. The antigen had been emulsified with Freund' s complete adjuvant in the earlier 3 times and with Freund' s incomplete adjuvant in the latter 3 times. The two batches of mouse were boosted with the same condition except different time between two immunities, one was 10 days, and the other was 2 weeks. After the antiserum titters being determined by the double immunodiffeoion in two dimensions assay , we had these results: The level of MT in antigen was 16-32ug/mouse and the level of MT was doubling in progression, the time between two boosting immunities is 10 days, the impact is fine, which could make the antiserum titers of a majority of mice attach to 1 :16.
2.The antibody was purified and identified: The antiserum whose titer attach to 1 I 16 was condensed to get Y-globulin with ammonium sulphate precipitation assay, and then the y -globulin was purified to IgG on a DEAE-Sephadex A50 column. We found when the pH value of washing buffer was 7. 4, the first apex of curve corresponded the solution of purified IgG whose purification was identified with discontinuous on different concentration polyacrylamide electrophoresis. After being conjugated with HRP, the IgG which is the specifically antibody to MT was identified with direct ELISA.
3. Establishment of ELISA: There are two assays which were the indirect competitive ELISA and the indirect uncompetitive ELISA, and they were used to determined MT in the sample of swine .the result showed: the result on determined with the indirect uncompetitive ELTSA were erratic, and the same
sample of OD value is much discrepant, and this assay is fit for determining the nature of MT. However, the result on MT which was determined by the indirect competitive ELISA were steady and this assay is fit to determine the quantum of MT, and it is comparntively a fine method. Using the method of chessboard titration chose the proper reaction for the indirect competitive ELISA: the carrier antigen of concentration is 6. 25ug/ml; the first antibody (mouse-antiswine) of suitable concentration is 50ug/ml; and the immunoglobulin-HRP(goat-antimouse) of diluent concentration is 1/8000;the reaction of time is 3hr. After liner regression analysis, a linear equation : y=105x2(22.32880-0.39583x), coefficient of correlation: R=0. 9592, and the sensitivity of assay to detect the rabbit MT is 4. Ing/ml. Intra-assay coefficient of variation and Inter-assay coefficient of variation are 9. 9760%-14. 3858%, which showed that this assay has fine stability. It proved that the establishment in our experimentation of indirect competitive ELISA has highly specificity, sensitivity, stability and repetitiveness. And it is fit for determining a big batch samples, and it is an effective method for determining the quantum of MT.
引文
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