尿液中性粒细胞明胶相关脂质运载蛋白时间分辨免疫荧光分析方法的建立及临床应用评价

Development and application of a time-resolved fluoroimmunoassay for urinary Neutrophil gelatinase-associated lipocalin

目的建立尿液中性粒细胞明胶相关脂质运载蛋白(NGAL)时间分辨免疫荧光分析方法并应用于临床检测。方法根据美国临床和实验室标准化协会(CLSI)制定的相关EP系列评价文件,使用本实验建立的时间分辨免疫荧光分析方法,对尿液NGAL的最低检测限、精密度、准确度、稳定性、分析测量范围、Hook效应、生物参考区间进行检测评价。通过检测健康体检人员及临床急性肾损伤患者尿液样本与Abbott ARCHITECT化学发光法进行比对,使用SPSS软件进行统计学分析。结果本实验所建立的方法最低检测限为1.5 ng/ml,批内不精密度与总不精密度均<6.0%,回收率为93%~107%,当NGAL浓度高达3×10~5ng/ml时,无Hook效应(5×10~5ng/ml亦不会出现假阴性),该方法与Abbott ARCHITECT化学发光具有很强的相关性(r=0.989),且37℃加速7 d荧光信号保留率均>80%。结论本实验室研究方法检测尿液NGAL各项性能指标均满足临床要求,可应用于急性肾损伤患者的诊断。

Objective To establish a method to detect urine neutrophil gelatinase-associated lipocalin（ NGAL） by using time-resolved fluroimmunoassay（ TRFIA） and to apply it to the clinic. Methods According to the correlative EP documents formulated by the CLSI,the limits of detection（ Lo D）,imprecision,accuracy,analytical measurement range（ AMR） and biological reference interval of NGAL in human urine were evaluated by the method of TRFIA kit,and then the results were compared with the chemiluminescent immunoassay（ CLIA） method（ Abbott ARCHITECT-）. The agreements between the assays were assessed by SPSS statistical analysis. Results The lowest limit of detection was 1. 5 ng/ml. The within-run imprecision and the total imprecision were both lower than 6. 0%. The spiking recovery was within 93%-107%. There was no Hook effect when the sample was at 3 × 10^5ng/ml NGAL（ There will be no false negatives even at 5 × 10^5ng/ml）. The correlation coefficient（ r） of clinical comparison test with Abbott ARCHITECT was greater than 0. 989. The thermal stability result showed that the retention of fluorescence signal was more than 80% at 37 ℃ for 7 days. Conclusion The time-resolved fluroimmunoassay（ TRFIA） method for detecting NGAL achieved corresponding clinical application standards and can be used for the diagnosis of patients with acute kidney injury（ AKI）.