發(fā)布時(shí)間：2018-01-17 03:13

  本文關(guān)鍵詞：腫瘤篩查用（熱休克蛋白90α）快速檢測(cè)試劑的研制　出處：《鄭州大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 單克隆抗體制備 熒光免疫檢層析法 熱休克蛋白90α 熒光微球活化

【摘要】：目的:熱休克蛋白90α(HSP 90α)是細(xì)胞受應(yīng)激原刺激后誘導(dǎo)產(chǎn)生的一種應(yīng)激蛋白,在腫瘤組織中HSP90α表達(dá)量達(dá)正常組織表達(dá)水平的2-10倍。其在血液中的含量與腫瘤惡性程度呈正相關(guān)。HSP90α作為一種全新腫瘤標(biāo)志物,用于肺癌、胃癌、肝癌、乳腺癌、結(jié)直腸癌等多個(gè)腫瘤的臨床檢測(cè)�；颊哐獫{中HSP90α含量水平對(duì)應(yīng)患者病情變化,可實(shí)時(shí)、較準(zhǔn)確地反映治療效果,在臨床上可為醫(yī)生提供診斷、治療、預(yù)后的客觀依據(jù)。本實(shí)驗(yàn)針對(duì)此蛋白進(jìn)行系統(tǒng)研究,建立人血清中熱休克蛋白90α(HSP90α)的熒光免疫層析定量檢測(cè)方法。方法:1、采用人HSP90α融合蛋白,進(jìn)行抗原純度、濃度和生物活性的鑒定,免疫小鼠、利用間接酶聯(lián)免疫吸附法(ELISA)效價(jià)鑒定、細(xì)胞融合、單克隆抗體制備、單抗純度和亞型鑒定、蛋白純化等實(shí)驗(yàn)操作步驟制備純度較高的HSP90α單克隆抗體和多克隆抗體。2、研制用于定量檢測(cè)的熒光免疫層析檢測(cè)試紙條,通過(guò)采用熒光免疫層析技術(shù),對(duì)熒光微球制備工藝進(jìn)行優(yōu)化,并對(duì)最佳反應(yīng)時(shí)間、線性范圍、精密性、回收率、臨床檢測(cè)等性能評(píng)價(jià)方面進(jìn)行系統(tǒng)研究。初步建立了熱休克蛋白90α熒光免疫層析檢測(cè)方法。結(jié)果:1、共篩選出16株單克隆細(xì)胞株,經(jīng)過(guò)亞型篩選以后共選出8株陽(yáng)性細(xì)胞株,編號(hào)分別為3B2、4E3、4F4、5E2、7D1、7E4、8F2、8G4。2、經(jīng)過(guò)8株單抗和多抗配對(duì),挑選出8F2和多抗成為最優(yōu)的一對(duì)配對(duì)抗體。確定了各反應(yīng)條件,最佳反應(yīng)時(shí)間為5min。線性范圍0.39ng/m L-100ng/m L;靈敏度為0.0578ng/ml;精密性質(zhì)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別為100.56%、99.76%、94.1%;與國(guó)外HSP90αELISA檢測(cè)試劑盒平行檢測(cè)40份臨床患者血清,結(jié)果顯示兩者相關(guān)系數(shù)為0.9685。結(jié)論:1、通過(guò)單克隆抗體制備的實(shí)驗(yàn)過(guò)程,成功篩選出8株陽(yáng)性細(xì)胞株,并挑選出一對(duì)配對(duì)抗體用于后續(xù)實(shí)驗(yàn)的研究。2、超聲波技術(shù)在熒光微球活化過(guò)程中可降低聚集現(xiàn)象。3、初步建立的方法具有良好的精密性和線性,臨床符合率能滿足臨床檢測(cè)技術(shù)要求,有望完善后報(bào)批用于臨床。
[Abstract]:Objective: heat shock protein 90 偽 (HSP90 偽) is a kind of stress protein induced by stressors. The expression of HSP90 偽 in tumor tissues was 2-10 times higher than that in normal tissues. The expression of HSP90 偽 in blood was positively correlated with the malignancy of tumor. HSP90 偽 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 偽 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 偽 (HSP90 偽) 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 偽 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 偽 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 偽 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.
【學(xué)位授予單位】：鄭州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R730.43

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