摘要
[目的]以合成抗原EGFRvⅢ-BSA作为被检测物,建立检测抗EGFRvⅢ单链抗体的间接ELISA检测方法并优化其反应条件。[方法]利用N-马来酰亚胺甲基环己烷-1-羧酸琥珀酰亚胺酯(SMCC)将EGFRvⅢ与BSA偶联制备抗原。利用方阵滴定法对间接ELISA法的抗原包被浓度,单链抗体的稀释度、包被时间和温度、包被液、抗His-tag多抗稀释度和酶标抗体的稀释度进行优化,并对建立的间接ELISA方法的方法学进行验证。[结果]通过SDS-PAGE电泳和全波长紫外扫面显示EGFRvⅢ-BSA合成成功。用PBS于37℃包被1.25μg/m L的EGFRvⅢ-BSA 2 h,加入19.044μg/L的ChiMR1、1∶8 000的兔抗His-tag多抗和1∶10 000酶标抗体可获得最佳ELISA检测结果。应用优化后的条件检测4.761~152.35μg/L范围内的ChiMR1显示线性良好。线性方程为y=(A-D)/[1+(x/C)^B]+D(A:1.99706;B:-1.09760;C:35.54885;D:0.01558),R2=0.99923;60μg/L和20μg/L的质控品的检测精密度分别为7.142%和4.828%;准确度分别为91.8%和82.90%,准确度和精密度良好。[结论]建立的间接ELISA方法的线性、准确度和精密度良好,为临床疾病的检测奠定良好基础。
[Objective]The synthetic antigen EGFRvⅢ-BSA was detected as a test object. By optimizing the reaction conditions to establish of the indirect ELISA method of single-chain antibody fragment（ Sc Fv） against EGFRvⅢ. [Methods]EGFRvⅢ antigen was conjugated with BSA by SMCC to prepare antigen. By using the matrix titration method of indirect ELISA method for antigen coating concentration,antibody dilution,coating time and temperature,coating solution,anti His-tag polyclonal antibody and enzyme labeled antibody dilution was optimizated,and the established indirect ELISA methodology for verification. [Results]EGFRvⅢ-BSA was successfully synthesized by SDS-PAGE electrophoresis and full wavelength ultraviolet scanning. EGFRvⅢ-BSA（ 1. 25 g/m L） was coated with PBS at 37 ℃ for 2 h,added Chi MR1（ 19. 044 g/L）,added polyclonal antibody against His-tag（ 1 ∶ 8 000） and enzyme labeled antibody（ 1 ∶ 10 000） to obtain the best results of ELISA.Chi MR1 was used to detect the optimized conditions of 4. 761-152. 35 g/L within the scope of the show good Linearity. Linearequation is y =（ A-D）/[1（ x/C） ^B]D（ A： 1. 99706; B： 1. 09760; C： 35. 54885; D： 0. 01558） and R2 is 0. 99923. The precision of the quality control products of 60 μg/L and 20μg/L are 7. 142% and 4. 828% respectively. The accuracy is 91. 80%and 82. 90% respectively and the accuracy and precision are good. [Conclusion]The linearity,accuracy and precision of establishing indirect ELISA method is good and can be used for the detection of clinical diseases.
出处
《生物技术》
北大核心
2017年第4期342-348,共7页
Biotechnology
基金
国家自然科学基金项目(“细胞色素P450 2A13在非小细胞肺癌中高表达的表观遗传调控机制研究”,No.81603189)
贵州省自然科学基金项目(“HDAC抑制剂-OFA抗体偶联药物的制备及其抗肿瘤活性的研究”,No.黔科合J字[2014]2018号)
贵阳市科技计划(联合基金)项目(“细胞色素p450 2A13在肺癌组织中异常表达的表观遗传调控机制”,No.[20161001]03)
贵州省科学技术厅人才团队项目(“贵州省高层次创新性人才培养计划-“百”层次人才”,No.黔科合平台人才[2016]5677
“贵州省民族药药效物质基础研究科技创新人才团队”,No.黔科合平台人才[2016]5613)
贵州省教育厅项目(“现代药物研究开发协同创新中心”,No.黔教合协同创新字[2013]04)
