酶联免疫吸附法分析法检测中性粒细胞明胶酶相关脂质运载蛋白方法学的建立及临床应用评价

Development of a ELISA methodology for the determination of NGAL levels and the evaluation of clinical application

目的建立ELISA分析检测NGAL的方法并应用于临床。方法采用ELISA免疫分析及双抗体夹心法,对检测体系的标准曲线、最低检测量、稳定性、精密度、回收率、干扰实验等相关性能指标进行评价,通过检测健康体检人员及临床急性肾损伤患者尿液标本并与目前应用于临床的较成熟的ELISA方法进行比对,评价ELISA方法的临床应用可行性。结果该方法检测NGAL的标准曲线为y=4.720 8x-0.404 9,线性范围为0.0 ng/ml^40.0 ng/ml,相关系数(r)为0.999 4,最低检测限为0.729 ng/ml。批内、批间精密度均<10%。该方法的正常参考值为<11.04 ng/ml,ROC曲线的AUC为0.995,敏感度为91.88%,特异度为85.71%,准确度为90.95%,阳性预示值为97.31%,阴性预示值为65.22%。该方法与化学发光法呈正相关(r=0.993)。结论 ELISA免疫分析法检测NGAL符合临床诊断的相关要求,可应用于临床急性肾功能损伤患者的诊断及疾病监测。

Objective To establish a method to detect neutrophil gelatinase-associated lipocalin（ NGAL） by using enzyme-linked immuno sorbent assay（ ELISA） and apply to the clinic. Methods Assay performance characteristics,such as the standard curve,the minimum detectable level,the stability,precision,recovery were evaluated for ELISA. Healthy reference populations and clinical urine specimens were measured to evaluate the perspective of the clinical application. Results The standard curve was y = 4. 72 8x-0. 404 9,the linear range was 0. 0 ng/m-40. 0 ng/ml,the correlation coefficient（ r） was 0. 999 4,the lowest detection limit was 0. 729 ng/ml. Intra-batch and inter-batch precision was less than 10%. The normal reference range of this method was 11. 04 ng/ml,the AUC of ROC was 0. 995,the sensitivity was 91. 88%,the specificity was 85. 71%,the accuracy was 90. 95% and the positive predicted value was 97. 31% and the negative predicted value was 65. 22%. The method has a strong correlation with chemiluminescence method（ r = 0. 993）. Conclusion The ELISA method for detecting NGAL achieves clinical application standards and can be used for the diagnosis and surveillance of acute kidney injury patients.