NGAL单体分子检测急性肾損伤的特异性及其上转磷光定量POCT检测技术的研究

There are still no effective early biomarker and rapid detection method for the diagnosis of acute kidney injury (AKI). Most recent studies have confirmed that the neutrophil gelatinase-associated lipocalin (NGAL) related to neutrophil gelatin enzyme, is specifically overexpressed in the renal tubule epithelium and secreted into urine in AKI, which is positively correlated to the damage degree of AKI, and a potential early biomarker for the detection of AKI. But current detections by means of antibodies originated from NGAL homologous dimer as antigen and its ELISA test, have shown unstable results due to the multiple pathologic status and the lack of quick quantitative analysis. We have successfully prepared immunogen with different methods for NGAL and obtained the specific monoclonal antibodies recognizing renal tubules epithelium and the UCP labeling materials with good ability of transferring light at the (Y.Yb.Er) F3 site by chemical co-precipitation technique. Based on our unique monoclonal antibodies specific for monomer NGAL , This proposal will explore the specificity of monomer NGAL determination for clinical setting AKI and develop a novel quantitative urine POCT assay for the monomer NGAL via UCP technique . This study will improve significantly the diagnosis and treatment of AKI in the near future.

急性肾损伤（AKI）目前尚缺乏有效的早期检测指标及快速检测方法。新近研究发现中性粒细胞明胶酶相关脂质运载蛋白(NGAL)在AKI时特异性地高表达于肾小管上皮细胞并分泌入尿液，其水平与AKI损伤程度正相关，是AKI理想的早期诊断标志物。目前临床研究应用来源于中性粒细胞的NGAL同源二聚体为免疫原制备的抗体及ELISA检测方法，其测定值因不同的病理状态存在较大的差异，且不能作到快速定量检测。我们设计并制备不同形式的NGAL分子为免疫原，筛选获得了能针对肾上皮NGAL单体分子的特异性单抗;并通过化学共沉淀技术制备了以(Y.Yb.Er)F3为基础的且有良好上转发光性能的标记材料。本项目拟在此工作基础上，以人肾上皮单体型NGAL检测抗体为突破口；用临床标本研究单体型NGAL分子检测对AKI早期诊断的特异性；并建立基于UCP的尿液NGAL无创定量 POCT检测方法，对提高AKI的诊治水平具有重要意义。

中性粒细胞明胶酶相关脂质运载蛋白（neutrophil gelatinase-associated lipocalin，NGAL）是中性粒细胞中的小分子脂质运载蛋白，与肾脏功能关系密切，临床应用前景广阔。上转发光免疫层析技术具有特异性高、灵敏度高、稳定性好、安全、操作简便等特点，是POCT非常具有潜力的研究方向之一。本研究制备了可配对检测的抗NGAL单克隆抗体1-F2、1-G2；制备了上转发光颗粒(Y.Yb.Er) NaF4@SiO2，装配了NGAL上转发光检测卡并优化检测体系。NGAL上转发光检测方法的标准曲线方程为y=0.9278x+1.9029（r2=0.9947），其中y轴为lg NGAL浓度，x轴为lgT/C值；检出限为4.05 ng/ml；分析测量范围为5.6~1006.42ng/ml；批内、批间和日间变异系数均<5%，总变异系数<6%；回收实验总体回收率为101.65%；不与转铁蛋白、人血清白蛋白、胆红素、葡萄糖和肌酐发生交叉反应。说明NGAL上转发光检测卡精密度、正确度和特异性良好，可以满足临床检测需要。临床研究中收集正常成年人尿液701例，得到男性尿液NGAL浓度正常参考区间为0-69.11ng/ml，女性正常参考区间为0-63.92ng/ml，总体参考区间为0-67.46ng/ml。收集尿常规异常的体检病人尿液181例，发现尿红细胞阳性样本的NGAL浓度与正常人群没有显著差异（p>0.05），尿白细胞阳性样本、尿蛋白阳性样本的NGAL浓度与正常人群相比显著升高（p<0.01）。收集慢性肾脏病病人的尿液、血清样本27例，NGAL浓度与正常人群相比显著升高（p＜0.01），SCr平均浓度与正常人群有统计学差异（p＜0.05）。绘制ROC曲线，NGAL曲线下面积为0.981，cut-off值为76.09ng/ml，此时敏感度为96.3%，特异性为96.7%。