PLA2R-IgG间接荧光定量免疫层析分析的构建与应用

Development and Application of an Indirect Fluorescent Immunochromatographic Assay for Quantitative Detection of PLA2R-IgG

目的 血清抗磷脂酶A2受体抗体IgG (PLA2R-IgG)水平是诊断和治疗特发性膜性肾病(IMN)的重要依据,而目前国内外常规检测手段主要是酶免法。为提升检测的便捷性,同时满足灵敏、宽量程分析需求,本研究构建了一种新的PLA2R-IgG检测技术。方法 采用包裹铕元素的微球示踪,对反应步骤进行选择,对微球制备液的p H、微球-抗体反应比例和反应时间优化,本文基于间接法构建了PLA2R-IgG的荧光定量免疫层析检测方法,并进行了初步临床评价。结果 本方法的灵敏度达0.7 RU/ml,标准曲线方程为y=0.771x-1.437,相关系数0.995,线性测量范围为0.7~1 500 RU/ml,回收率为86.27%~98.98%,平均批内变异系数为8.13%,交叉反应率均小于0.1%,试剂37℃储存10 d稳定。本方法与市售酶免试剂盒相关性为0.953,阴阳性判断一致,对IMN的检出率为76.9%。结论 采用两步法反应的PLA2R-IgG间接荧光定量免疫层析分析,快速、灵敏、准确,具有临床实用性。

Objective Serum anti-phospholipase A2 receptor antibody IgG(PLA2R-IgG)is an important basis for the diagnosis and treatment of idiopathic membranous nephropathy.By now,its conventional detection method is ELISA.To improve the reaction convenience,and obtain the assay sensitivity and wide detecting range,a novel immunoassay was established for PLA2R-IgG detection.Methods Here,based on europium fluorescent microspheres an indirect immunochromatographic assay(ICA)for PLA2R-IgG was developed and evaluated.The reaction process was firstly chosen,and the method parameters were assessed like p H of activating buffer for microspheres,the ratio of europium microspheres to antibodies,and reaction time.Results The performance metrics of the newly proposed assay were analyzed that the sensitivity was 0.7 RU/ml of PLA2R-IgG,the standard curve equation was y=0.771 x-1.437 with 0.995 of R,the linear working range was 0.7-1500 RU/ml,the recoveries were 86.27%-98.98%,the average variable coefficient of intra assay was 8.13%,the cross reaction ratio were above 0.1%and PLA2R-IgG-ICA reagents were stable for 10 d at 37℃.The correlation was 0.953 between developed method and commercial ELISA kits,the consistency was also high.The positive detection rate was 76.9%.Conclusion The established PLA2R-IgG-ICA using two-step dosing was rapid,sensitive,quantitative and accurate with potential clinical application.