定量检测组织型纤溶酶原激活剂夹心ELISA方法的建立

Establishment of a Sandwich ELISA for Quantitative Measurement of Tissue-type Plasminogen Activator

目的:建立灵敏、特异的组织型纤溶酶原激活剂(t-PA)定量测定方法,为血栓性疾病和肿瘤性疾病的早期诊断及疗效评估提供辅助手段。方法:采用抗t-PA多克隆抗体包被酶联板、HRP标记抗t-PA单克隆抗体为标记抗体、重组t-PA为标准品,建立定量测定t-PA的夹心ELISA双抗体法。以t-PA测定的特异性、灵敏性和重复性评价夹心ELISA测定法。结果:夹心ELISA测定法可检测t-PA浓度为0.5ng/mL的样品,不同样品的组内和组间的变异系数分别为4.7%和8.4%。采用夹心ELISA法测定40份正常人血浆,t-PA的平均含量为(4±2.1)ng/mL。结论:夹心ELISA测定法具有灵敏性高、特异性强的特点,可用于人血浆中t-PA水平的定量测定。

Objective： To develop a specific and sensitive method for quantitative gen activator （t-PA）. Methods： Anti t-PA polyclonal antibodies and HRP labelled used as coating antibody and labelled antibody, respectively. Sandwich ELISA for the measurement of tissue type plasmino- anti t-PA monoclonal antibodies were measurement of tissue t-PA was performed in microtiter plates using purified recombinant t-PA as standard and was judged by the specificity, sensitivity and reproducibility. Results： The lower limit of sensitivity of the assay of t-PA was 0.5 ng/mL. The coefficients of variation of the assay were 4.7% within assay and 8.4 % between assays, respectively. 40 samples from normal human plasma were assayed resulted in a mean value of （4＋2.1）ng/mL of t-PA, which was consistent with the clinical reported value. Conclusion： The developed sandwich ELISA was a specific and sensitive method for quantitative measurement of t-PA and can be used for quantitative detection of t-PA levels in human plasma in clinical.