抗人NGAL单克隆抗体的制备及其定量检测方法的建立

Preparation of monoclonal antibody against NGAL and establishment of an quantitative immunological detection methods for NGAL

目的获得抗人中性粒细胞明胶酶相关脂质运载蛋白(neutrophil gelatinase-associated lipocalin,NGAL)特异性单克隆抗体并建立双抗体夹心ELISA免疫定量检测方法。方法通过杂交瘤技术制备了抗人NGAL高亲和力高特异性单克隆抗体,筛选能够结合天然NGAL的单克隆抗体并建立双抗体夹心ELISA系统。以重组人NGAL为检测对象,对系统线性范围、灵敏度、精密度和准确度(回收试验)进行全面分析和评价。通过对肾病患病组和对照组尿液标本检测初步研究其在临床上的分析性能。结果获得了8株能稳定分泌抗人NGAL单克隆抗体的杂交瘤细胞株,筛选得到F2和G2两株单克隆抗体可用于双抗夹心ELISA免疫检测。本方法线性范围为0～40 ng/ml,灵敏度为4.8 ng/ml,批内变异CV≤10.04%,批间变异CV≤9.76%,平均回收率为101.9%。临床标本检测显示肾病组与对照组有显著差异(P<0.01)。结论本研究制备了高亲和力特异性单克隆抗体并建立了双抗体夹心ELISA方法,该方法灵敏度、精密度和准确性良好,能够检测体液标本中的天然NGAL蛋白,检测结果与临床基本符合,为其临床应用和推广奠定了基础。

In this study, with the self-made high specific monoclonal antibodies as raw material, a sandwich ELISA quantitative immunological detection method for protein neutrophil gelatinase-associated lipocalin （NGAL） was established. Using recombinant human NGAL as detection target, linear range, sensitivity, precision and accuracy of this ELISA system were measured and evaluated. Then we preliminarily studied on clinical application of NGAL through detection of NGAL in urine of the renal disease group and the control group. The liner range of the method was 0-40 ng/ml, the functional sensitivity of the method was 4.8 ng/ml; the intra-assay imprecision （CV） was lower than 10.04% and extra-assay imprecision （CV） was less than 9.76%; the recovery value of this system was between 97.4% and 106.4%. The method illustrated significant difference in urine NGAL level between renal disease group and control group （P 〈 0.01）. We concluded that the method is good in liner range, sensitivity, precision and accuracy, which may provide the foundation for the further application of NGAL on clinical detection.