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

摘要

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 antihuman 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 atherosclerofic 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.%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.