检测β-CTX和N-MID水平的双标记时间分辨免疫荧光分析方法的建立和评价  

Establishment and evaluation of a double-labeled timeresolved immunofluorescence analysis method for detecting levels ofβ-CTX and N-MID

作　　者：毛骞[1] 陈翠翠 梁焕坤 刘鹏娥 钟树海 李来庆 MAO Qian;CHEN Cuicui;LIANG Huankun;LIU Penge;ZHONG Shuhai;LI Laiqing(Department of Endocrinology,Affiliated Hospital,Beihua University,Jilin 132001,China;Guangzhou Youdi Biotechnology Co.,Ltd.,Guangzhou 510663,China)

机构地区：[1]北华大学附属医院内分泌科,吉林吉林132001 [2]广州优迪生物科技股份有限公司,广东广州510663

出　　处：《吉林大学学报（医学版）》2021年第3期770-776,共7页Journal of Jilin University：Medicine Edition

基　　金：吉林省卫计委卫生技术创新项目(2017J076)。

摘　　要：目的:研制一种检测β胶联降解产物(β-CTX)和骨钙素N端中分子片段(N-MID)水平的双标记时间分辨免疫荧光分析方法(TRFIA)并评价其检测性能。方法:将抗β-CTX和N-MID的4E5和2B7单克隆抗体(MAb)作为包被抗体包被在96孔培养板,采用铕离子(Eu^(3+))和钐离子(Sm^(3+))分别标记2G6和5A3 MAb作为检测抗体,建立双抗体夹心TRFIA分析法并制备成试剂盒。通过试剂盒的灵敏度、准确度(稀释回收率)、特异性、精密度、稳定性和临床样本比对等实验评价其检测性能。结果:制备的双标记TRFIA试剂盒对β-CTX的检测灵敏度为0.025μg·L^(-1),线性范围为0.025~5.000μg·L^(-1),对N-MID的检测灵敏度为0.5μg·L^(-1),线性范围为0.5~200.0μg·L^(-1)。β-CTX平均稀释回收率为102.13%,N-MID平均稀释回收率为103.02%,与其他常见骨检测指标无明显交叉反应,特异性较强。β-CTX批内变异系数(CV)为5.81%~7.82%,批间CV为5.97%~8.02%;N-MID批内CV为6.05%~8.32%,批间CV为6.14%~8.56%;TRFIA试剂盒可在4℃稳定保存6个月,37℃稳定保存7 d。结论:建立的双标记TRFIA方法具有高灵敏度、高特异度、高准确率和方便快捷等优点,适用于大批量临床样品的检测。Objective:To develop a double-labeled time-resolved immunofluorescence analysis(TRFIA)method for the determination of the levels ofβ-cross-linked C-terminal telopeptide of typeⅠcollagen(β-CTX)and N-terminal middle molecular fragment of osteocalein(N-MID)of osteocalcin,and to evaluate its detection performance.Methods:The 4E5和2B7 monoclonal antibodies(MAb)ofβ-CTX and N-MID antigens were constructed,and then coated as coating antibodies in 96-well plates,and the antibodies were labeled with europium ion(Eu^(3+))and samarium ion(Sm^(3+)),respectively.A double antibody sandwich TRFIA method was established and prepared into a kit.The detection performance was evaluated by the sensitivity,accuracy(dilution recovery),specificity,precision,stability,and clinica sample alignment of the kit.Results:The prepared double-labeled TRFIA Kit had a detection sensitivity of0.025μg·L^(-1) forβ-CTX and a linear range of 0.025-5.000μg·L^(-1);the detection sensitivity for N-MID was 0.5μg·L^(-1),and the linear range was 0.5-200.0μg·L^(-1);the average dilution recovery rate ofβ-CTX was 102.13%,and the average dilution recovery rate of N-MID was 103.02%;there were no obvious cross-reaction with other common bone test indicators and the specificity was high.The intra-assay coefficient of variation(CV)ofβ-CTX was 5.81%-7.82%,and the inter-assay CV was 5.97%-8.02%;the intra-assay CV of N-MID was 6.05%-8.32%,and the inter-assay CV was 6.14%-8.56%;the TRFIA Kit could be stored stably at 4℃for half a year and stably stored at 37℃for 7 d.Conclusion:The establishedβ-CTX and N-MID double-labeled TRFIA method has the advantages of high sensitivity,high specificity,high accuracy,convenience and speed,and is suitable for the detection of largescale clinical samples.

关 键 词：β胶联降解产物 N端中分子片段 骨质疏松症 双抗体夹心 双标记时间分辨免疫荧光分析 

分 类 号：R331[医药卫生—人体生理学]