基于均相化学发光法的肿瘤标记物CA125检测试剂和JAK1与相互作用蛋白分析试剂的研制
发布时间:2018-12-30 19:27
【摘要】:研究背景及目的采用均相化学发光免疫分析技术研制CA125的定量检测试剂,评价各项性能指标;同时,研制了基于该免疫分析技术细胞裂解液中分析JAK1和IL-2RB,JAK1和IL-10RA相互作用的核心原料和分析试剂。方法1研制CA125的均相化学发光定量免疫分析试剂1.1参考标准品的配制:用标准品稀释液将CA125抗原配置成1,12,34,118,310,670U/mL系列参考品溶液,每管1mL于-20 ℃保存,融化后于4 ℃保存,避免反复冻融。1.2抗体与发光微球的连接:CA125的配对标记抗体200 μg加入到超滤管中,以8,000 rpm离心6 min,用抗体与受体微球的连接缓冲液洗涤5次。CA125的包被抗体分别取30,50,70,100μg与1 mg发光微球连接,加入10 μL的抗体与受体微球连接溶液,1.25μL的10%Tween-20,37 ℃避光震荡48 h。加入10 μL的封闭液封闭结合位点。37 ℃避光孵育1h后离心洗涤。将连接抗体的微球溶液调整浓度为5 mg/mL。1.3生物素与抗体的连接:将CA125的配对包被抗体100μg加入到超滤管中,以8,000 rpm离心6 min,用生物素标记缓冲液洗涤5次,加入0.5 μL的22 mg/mL的活化生物素,用生物素标记缓冲液定容至200 μL,室温孵育2 h。将连接产物加入超滤管,用生物素保存液洗涤5次,最后收集超滤管中液体,并将浓度调整至5 mg/mL。1.4试剂性能指标的评价:包括标准曲线的绘制,灵敏度,特异性实验,回收率,干扰实验及与国外试剂盒的比较实验等。2 研制均相化学发光定量免疫分析JAK1和IL-2RB,JAK1和IL-10RA相互作用的核心原料和分析试剂2.1从Hela细胞中提取总RNA,通过RT-PCR逆转录cDNA,扩增IL-2RB,IL-10RA。经双酶切、连接、连接产物的转化、挑克隆及质粒提取,得到重组质粒 pENTER-IL-2RB-His 和 pENTER-IL-10RA-His。2.2 重组质粒 pENTER-IL-2RB-His 和 pENTER-IL-10RA-His 的 PCR、双酶切、测序鉴定。2.3重组质粒的转染、间接免疫荧光和免疫印迹检测IL-2RB和IL-10RA蛋白的真核表达。2.4免疫共沉淀检测JAK1和IL-2RB、JAK1和IL-10RA的相互作用。2.5均相化学发光免疫分析法检测JAK1-IL-2RB、JAK1-IL-10RA的蛋白相互作用。结果1自制的CA125均相化学发光免疫分析试剂的分析灵敏度为0.29 U/mL。分析内和分析间的变异系数分别为4.4%-6.8%,9.9%-12.7%。自制试剂临床血清样本值与化学发光法的测值具有良好的相关性。2 经PCR、双酶切鉴定IL-2RB和IL-10RA重组质粒为阳性克隆,并且在293T细胞中成功表达,免疫印迹可在对应的位置看到目的蛋白条带。3基于CA125均相化学发光免疫分析技术,当受体发光微球连接抗体为30 μg时,能够检测出JAK1-IL-2RB,JAK1-IL-10RA的相互作用。结论自制的CA125均相化学发光免疫分析试剂的各项指标均达到检测试剂的要求,为研制肿瘤标记物CA125试剂盒提供了核心原料,也为其他均相化学发光免疫分析试剂的研制提供了技术支持。成功构建了重组质粒pENTER-IL-2RB-His 和 pENTER-IL-10RA-His,并在 293T 细胞中成功表达。共转染JAK1和IL-2RB,JAK1和IL-10RA质粒的细胞裂解液中用免疫共沉淀和新型均相化学发光免疫分析技术也可以检测出其相互作用。这为进一步研究JAK1蛋白及通路蛋白相互作用奠定了基础。
[Abstract]:The background and purpose of the study were to develop a quantitative detection reagent for CA125 by homogeneous chemiluminescence immunoassay, and to evaluate the performance indexes. At the same time, the core raw materials and analytical reagents for the interaction of JAK1 and IL-2RB, JAK1 and IL-10RA in the cell lysis solution based on the immune analysis were developed. Method 1 The preparation of a homogeneous chemiluminescent immunoassay reagent for CA125 was developed: 1, 12, 34, 118, 310, 670U/ mL series of reference solutions were prepared by standard dilution, and 1mL per tube was stored at-20.degree. C. and then stored at 4 鈩,
本文编号:2396032
[Abstract]:The background and purpose of the study were to develop a quantitative detection reagent for CA125 by homogeneous chemiluminescence immunoassay, and to evaluate the performance indexes. At the same time, the core raw materials and analytical reagents for the interaction of JAK1 and IL-2RB, JAK1 and IL-10RA in the cell lysis solution based on the immune analysis were developed. Method 1 The preparation of a homogeneous chemiluminescent immunoassay reagent for CA125 was developed: 1, 12, 34, 118, 310, 670U/ mL series of reference solutions were prepared by standard dilution, and 1mL per tube was stored at-20.degree. C. and then stored at 4 鈩,
本文编号:2396032
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