人心肌肌钙蛋白I化学发光免疫分析方法的建立

Establishment of chemiluminescent immunoassay for determination of human cardiac troponin I (cTnI)

目的 建立检测人心肌肌钙蛋白I(cTnI)化学发光免疫分析方法。方法 用重组cTnI免疫新西兰兔获得抗体 ,经DEAE 2 3层析和 (NH4) 2 SO4沉淀纯化后 ,用碳化二亚胺 (EDC)法标记辣根过氧化物酶 ,SephadexG 2 0 0纯化。优化鲁米诺 H2 O2 肉桂酸发光体系 ,建立了cTnI化学发光免疫分析法。用该法检测 138例正常人及病人血清标本 ,确定正常值参考范围 ,并与ELISA法比较。结果 所得抗体的滴度为 1∶80 0 0。优化后的发光体系为 :鲁米诺 (4 .5mmol/L) ,H2 O2 (7.5mmol/L) ,四苯硼钠 (0 .8mmol/L) ,肉桂酸 (0 .4mmol/L) ,比单独用肉桂酸或四苯硼钠作增强剂灵敏度分别增加了 1.19和 1.0 5倍。本法检测灵敏度为0 .2ng/ml,线性范围为 0 .4～ 5 0ng/ml,批内CV平均 9.0 % ,批间CV平均 11.8% ,回收率为 10 4 .2 %。Hb浓度在 0～ 5 0nmol/L ,T Bil在 0～ 15 μmol/L ,TG在 0～ 2 .0mmol/L对测定结果无影响。与ELISA比较 ,两法结果一致 (P >0 0 5 ) ,相关系数r=0 .985 2(P <0 .0 1)。用BioCheck标准品为校正物 ,确定临床诊断参考范围为 <2 .12ng/ml,诊断符合率为 96 .2 %。结论 建立的cTnI化学发光免疫分析法可常规用于临床检验。

Objective To establish a method of chemiluminescent immunoassay for determination of cTnI.Method Antibodies were produced by immunizing New Zealand rabbits with recombinant cTnI and were purified by DEAE-23 chromatography and (NH4)2SO4 centrifugation.Conjugation of horseradish peroxidase to immunoglobulin G was performed by EDC method and purified by Sephadex-G200 chromatography.Chemiluminescent immunoassay was established by optimizing luminol-H 2O 2-coumaric acid-NaTPB luminescent system.Sensitivity,linear scope,precision,interference,and recovery for the method were evaluated.The reference range was defined by testing 138 serum samples from healthy subjects and compared to the results determined by ELISA.Result The antibody titer of ELISA was 1∶8 000.The optimized luminescent system was as follows:luminol:(4.5 mmol/L),H 2O 2(7.5 mmol/L),NaTPB(0.8 mmol/L) and coumaric acid(0.4 mmol/L).The sensitivity of this method was 0.2ng/ml with 1.19 and 1.05-fold increase compared to using coumaric acid and NaTPB respectively.Linear scope was 0.4～50 ng/ml.Average intra-assay and inter-assay precision was 9.0% and 11.8% respectively.Average recovery was 104.2%. The concentrations (for hemoglobin:0～50mmol/L,total bilirubin:0～15 μmol/L,triglyceride:0～2.0 mmol/L) do not influence the testing result.Compared with ELISA,relative coefficient was 0.9852 (P> 0.05).The reference range for clinical diagnosis was less than 2.12 ng/ml by using Biocheck standard reference as rectifying sample.The coincident rate for diagnosis was 96.15%.Conclusion Successful establishment of chemiluminescent immunoassay method for cTnI paved a way for clinical application.