组织因子途径抑制物-2的时间分辨荧光免疫测定方法的建立及评价

Establishment and evaluation of time-resolved fluoroimmunoassay for quantitative detection of tissue factor pathway inhibitor-2

目的建立测定血液中组织因子途径抑制物-2（tissue factor pathway-2,TFPI-2）含量的时间分辨荧光免疫测定方法（time-resolved fluorescence immunoassay,TRFIA）。方法以兔抗人TFPI-2多克隆抗体作为固相抗体,单克隆抗体3C8作为第一抗体,原核表达的人TFPI-2作为标准品,Eu3＋标记的羊抗鼠单克隆抗体作为检测抗体,采用双抗体夹心法建立TFPI-2 TRFIA分析系统,并与酶联免疫吸附法（ELISA）进行比较。结果自制TFPI-2 TRFIA试剂标准曲线的线性范围为0.1~50 ng/mL,灵敏度为0.048 ng/mL,批内和批间变异系数均≤8.39%。应用该方法测定56例冠状动脉粥样硬化性心脏病（coronary atherosclerotic heart disease,CHD）患者及24例正常体检者血浆中的TFPI-2分别为（10.58±7.12）和（8.53±2.32）ng/mL。结论我们建立的TFPI-2 TRFIA分析方法与ELISA相比灵敏度更好,定量检测结果能够敏感准确地反映血液内TFPI-2的浓度变化,特异性和稳定性均符合要求。

Objective To establish a new and high sensitive method of time-resolved fluorescence immunoassay（TRFIA） for the quantification of human tissue factor pathway inhibitor-2（TFPI-2）. Methods Polyclonal anti-human TFPI-2 rabbit IgG was used as the capture antibody,monoclonal antibody 3C8 as the first antibody,hTFPI-2 expressed by E.coli as the standards,and Eu3＋-Iabelled goat anti-mouse IgG as the second antibody. Results The linear range of standard curve of TFPI-2 TRFIA was 0.1-50 ng/mL,and the sensitivity was 0.049 ng/mL.The intra-and inter-coefficients of variation were both ≤8.39%.The concentration of hTFPI-2 in plasma of 56 patients with coronary atherosclerotic heart disease（CHD） and 24 health controls were（10.58±7.12） and（8.53±2.32） ng/mL,respectively. Conclusions The TFPI-2 TRFIA kit we developed is more sensitive than ELISA kit,and it can be used to detect TFPI-2 in circulation.