肿瘤筛查用（热休克蛋白90α）快速检测试剂的研制

发布时间：2018-01-17 03:13

本文关键词：肿瘤筛查用（热休克蛋白90α）快速检测试剂的研制　出处：《郑州大学》2017年硕士论文　论文类型：学位论文

【摘要】：目的:热休克蛋白90α(HSP 90α)是细胞受应激原刺激后诱导产生的一种应激蛋白,在肿瘤组织中HSP90α表达量达正常组织表达水平的2-10倍。其在血液中的含量与肿瘤恶性程度呈正相关。HSP90α作为一种全新肿瘤标志物,用于肺癌、胃癌、肝癌、乳腺癌、结直肠癌等多个肿瘤的临床检测。患者血浆中HSP90α含量水平对应患者病情变化,可实时、较准确地反映治疗效果,在临床上可为医生提供诊断、治疗、预后的客观依据。本实验针对此蛋白进行系统研究,建立人血清中热休克蛋白90α(HSP90α)的荧光免疫层析定量检测方法。方法:1、采用人HSP90α融合蛋白,进行抗原纯度、浓度和生物活性的鉴定,免疫小鼠、利用间接酶联免疫吸附法(ELISA)效价鉴定、细胞融合、单克隆抗体制备、单抗纯度和亚型鉴定、蛋白纯化等实验操作步骤制备纯度较高的HSP90α单克隆抗体和多克隆抗体。2、研制用于定量检测的荧光免疫层析检测试纸条,通过采用荧光免疫层析技术,对荧光微球制备工艺进行优化,并对最佳反应时间、线性范围、精密性、回收率、临床检测等性能评价方面进行系统研究。初步建立了热休克蛋白90α荧光免疫层析检测方法。结果:1、共筛选出16株单克隆细胞株,经过亚型筛选以后共选出8株阳性细胞株,编号分别为3B2、4E3、4F4、5E2、7D1、7E4、8F2、8G4。2、经过8株单抗和多抗配对,挑选出8F2和多抗成为最优的一对配对抗体。确定了各反应条件,最佳反应时间为5min。线性范围0.39ng/m L-100ng/m L;灵敏度为0.0578ng/ml;精密性质控品高值(30ng/m L)CV=7.46%;低值(10ng/m L)CV=8.39%,高、中、低值血清的回收率分别为100.56%、99.76%、94.1%;与国外HSP90αELISA检测试剂盒平行检测40份临床患者血清,结果显示两者相关系数为0.9685。结论:1、通过单克隆抗体制备的实验过程,成功筛选出8株阳性细胞株,并挑选出一对配对抗体用于后续实验的研究。2、超声波技术在荧光微球活化过程中可降低聚集现象。3、初步建立的方法具有良好的精密性和线性,临床符合率能满足临床检测技术要求,有望完善后报批用于临床。
[Abstract]:Objective: heat shock protein 90 伪 (HSP90 伪) is a kind of stress protein induced by stressors. The expression of HSP90 伪 in tumor tissues was 2-10 times higher than that in normal tissues. The expression of HSP90 伪 in blood was positively correlated with the malignancy of tumor. HSP90 伪 was regarded as a new tumor marker. It can be used for clinical detection of lung cancer, gastric cancer, liver cancer, breast cancer, colorectal cancer and so on. The plasma HSP90 伪 level can reflect the effect of treatment in real time and accurately. It can provide the objective basis of diagnosis, treatment and prognosis for doctors in clinic. A fluorescence immunochromatographic method for quantitative detection of heat shock protein 90 伪 (HSP90 伪) in human serum was established. Assays of concentration and biological activity, immunizing mice, titer identification by indirect enzyme-linked immunosorbent assay (Elisa), cell fusion, monoclonal antibody preparation, monoclonal antibody purity and subtype identification. HSP90 伪 monoclonal antibody and polyclonal antibody. 2 were prepared in the process of protein purification, and a fluorescent immunochromatographic test strip was developed for quantitative detection. The preparation process of fluorescent microspheres was optimized by using fluorescence immunochromatography, and the optimum reaction time, linear range, precision and recovery rate were obtained. The method of heat shock protein 90 伪 fluorescence immunochromatography was established. Results: 1. A total of 16 monoclonal cell lines were screened. After subtype selection, 8 positive cell lines were selected, numbered 3B2O4E3E3E3F4F4F4E2O7E4O7E4F2O8G4.2and matched by 8 McAbs and polyclonal antibodies, respectively. 8F2 and polyclonal antibodies were selected as the best pair of paired antibodies. The optimum reaction conditions were determined. The optimum reaction time was 5 min. The linear range was 0.39 ng / m L-100ng / mL; The sensitivity is 0.0578ng / ml; Precision property control with high value of 30ng / m CVC 7.46; The recoveries of high, middle and low value serum were 100.56 and 99.76 ~ 94.1, respectively. 40 serum samples of clinical patients were detected in parallel with HSP90 伪 ELISA test kit. The correlation coefficient was 0.9668 5. Conclusion: 1. Through the preparation of monoclonal antibodies, 8 positive cell lines were successfully screened, and a pair of paired antibodies were selected for further study. 2. Ultrasonic technology can reduce the aggregation of fluorescent microspheres in the process of activation. The preliminary method has good precision and linearity, and the clinical coincidence rate can meet the technical requirements of clinical detection. It is expected to be perfect and approved for clinical use.
【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R730.43

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