摘要
目的：研发人血清及尿液可溶性胎盘生长因子（sPLGF）的ELISA检测试剂盒并进行性能测试。方法：以抗人PLGF单克隆抗体包被微孔板，加入重组sPLGF和生物素标记的检测抗体，形成抗原抗体复合体，加人链亲合素辣根过氧化物酶和底物TMB实现比色反应；从线性范围、准确度、精密度、样品基质及样品前处理方法等指标进行性能评价，并与市售进口同类产品进行比较。结果：捕获抗体的最适包被量为400ng／well，包被条件为4℃孵育16～20h，检测抗体的最适工作浓度为1μg／ml，样品最适反应条件为37℃孵育2h，适合的封闭液及样品稀释液组合为3％BSA—PBS和0．1％BSA—PBS；验证该试剂盒的有效检测范围为15．6～2000μg／ml，准确度为85％～115％；批次内变异系数cV≤10％，批次间变异系数cv≤15oA；其检测范围、准确度、精密度、灵敏度及样品适用范围等与市售进口试剂盒基本一致。结论：本项目开发的人sPLGF检测试剂盒实现了人血清和尿液中的内源sPLGF的定量检测，其检测性能稳定、可靠，具有比进口试剂盒检测范围更广、价格更低的优势，可用于临床检测血液及尿液中的内源性sPLGF水平。
Objective： To develop an ELISA kit for detecting soluble placental growth factor （sPLGF） in human serum and urine, and analyze its performance. Methods： A specific monoclonal antibody to human sPLGF was pre-coated on- to a microplate. Recombinant sPLGF and biotin-conjugated detection antibody were added to form a complex. A strep- tavidin HRP was covalently linked to the detection antibody, allowing for a colorimetric reaction in the presence of sub- strate TMB. The performance index of the kit, including line range, accuracy, intra-assay precision, inter-assay preci- sion, sensitivity, and sample substrate and preprocessing were detected and compared with an imported commercial kit （R＆D）. Results： The optimal coating amount of capture antibody was 400 ng/well of the ELISA kit. The coating con- ditions were as following., the wells were pre-coated at 4℃ for 16-20 hours the optimum working concentration of the detection antibody was 1μg/mlthe samples were incubated at 37℃ for 2 hours and the best blocking buffer and assay diluents were 3 M BSA/PBS or 0.1% BSA/PBS buffer. The detection and quantification of human sPLGF was verified within the range of 15.6-2000 pg/ml. The linearity of the assay was 85%-115%. The intra-assay and inter-assay varia- bility with coefficients of variation were - 10% and -15%, respectively. The performance was comparable to the commercial kit （R＆D） with a good consistency in most aspects as line range, recovery, precision and sensitivity and sample scope of application. Conclusion.. The developed human sPLGF ELISA kit realizes the quantitative measure- ment of endogenous sP1GF level in human serum and urine samples. This quantitative immunoassay kit is stable andreliable with advantages of wider testing range and lower cost compared to the imported kit, which can be used clinical- ly to determine human sPLGF in serum and urinary.
出处
《中国计划生育学杂志》
2016年第11期772-777,789,共7页
Chinese Journal of Family Planning
基金
四川省科研院所科技成果转化资金项目(13010137)
