纳米颗粒增强透射免疫比浊法检测乳酸脱氢酶同工酶3方法的建立及性能评价

摘要

目的 建立纳米颗粒增强透射免疫比浊法(PETIA)测定乳酸脱氢酶同工酶3(LDH3)的分析方法 .方法 确定最适反应条件,包括抗体浓度、胶乳微球粒径、交联剂[1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(EDAC)/N-羟基琥珀酰亚胺(NHS)]浓度、缓冲液种类及pH值、反应时间,并建立检测血清LDH3的PETIA方法 .按美国临床实验室标准化委员会(NCCLS)EP5-A2文件要求,评价方法 的精密度、线性范围、回收率、抗干扰能力和分析灵敏度,并初步建立参考区间.结果 PETIA最适反应条件:抗体浓度为0.1 mg/mL、胶乳微球粒径为120 nm、交联剂(EDAC/NHS)浓度为10 mg/mL、缓冲液为磷酸盐缓冲液(PBS)、pH值为7.8时偶联率最佳,室温(25℃)和低温(4℃)放置24 h偶联率保持不变.采用建立的PETIA检测LDH3低值(16.5 U/L)、中值(65.0 U/L)和高值(155.0 U/L)的批内变异系数(CV)分别为5.77%、4.02%、3.77%,批间CV分别为6.59%、4.44%、3.93%;线性范围为3.6~200.0 U/L;平均回收率为102.1%;900 U/L类风湿因子、500μmol/L胆红素、9 mmol/L甘油三酯、5 g/L血红蛋白、5 g/L维生素C均无明显干扰;分析灵敏度为3.6 U/L;参考区间为13.5~75.9 U/L.结论 建立的PETIA测定LDH3具有方法 简单、快速、灵敏等优点,且结果 准确,可用于全自动生化分析仪,能够满足临床检验的要求.%Objective To establish nano particle-enhanced turbidimetric immunoassay(PETIA) for determining serum lactate dehydrogenase isoenzyme 3(LDH3).Methods The optimal reaction conditions [antibody concentration,latex particle size,1-ethyl-3-(3-dimethylaminopropyl)carbodiimide(EDAC)/ N-hydroxysuccinimido(NHS) concentration,the kind and pH value of buffer solution and reaction time] were determined. PETIA for determining serum LDH3 was established. According to the National Committee for Clinical Laboratory Standards EP5-A2,the precision,linear range,recovery rate,anti-interference ability and analytic sensitivity were evaluated,and the initial reference interval was established. Results The optimal reaction conditions were antibody concentration 0.1 mg/mL,latex particle size 120 nm,EDAC/ NHS concentration 10 mg/mL,phosphate buffered saline(PBS) with pH value 7.8 and coupling rate remaining unchanged under room temperature(25℃) and low temperature(4℃) for 24 h. For LDH3 with low value(16.5 U/L),median value(65.0 U/L) and high value(155.0 U/L),the within-run coefficients of variation(CV) were 5.77%,4.02% and 3.77%,and the between-run CV were 6.59%,4.44% and 3.93%. The linear range was 3.6-200.0 U/L. The average recovery rate was 102.1%. The factors of 900 U/L rheumatoid factor,500μmol/L bilirubin,9 mmol/L triglyceride,5 g/L hemoglobin,5 g/L vitamin C had no interference for PETIA in the determination of LDH3. The analytic sensitivity was 3.6 U/L. The reference interval was 13.5-75.9 U/L. Conclusions PETIA for the determination of LDH3 is simple,rapid,sensitive and accurate,and it can be used in automatic biochemical analyzers,which can meet the requirements of clinical determination.