用多克隆抗体构建的夹心免疫传感器检测心肌肌钙蛋白I

Measurement of Cardiac Troponin I by Sandwich Immunobiosensor Prepared with the Polyclonal Antibody

用表面等离子体子共振生物传感器构建对心肌肌钙蛋白 I特异性的免疫传感器检测心肌肌钙蛋白 I,并建立两种检测方法 :直接法的最低检测限为 2 .5 μg/L,基于传感膜上的夹心免疫法的灵敏度为 0 .5 μg/L,检测范围为 0 .5～ 2 0 μg/L,批内及批间精密度分别为 3 .5 %～ 4.9% ,6.1 %～ 7.4% ;用夹心法及国外试剂盒对 40名健康献血者和 2 0例急性心肌梗死患者血清心肌肌钙蛋白 I水平进行检测 ,两者符合率为 95 % .

Cardiac and skeletal muscle troponin I(cTnI, sTnI) were purified from human left ventricular tissue and skeletal muscle tissue by affinity chromatographic method, respectively. The specific polyclonal antibody to cTnI was prepared by immuned pure New Zealand rabbit with the purified cTnI and purified by nitrocellulose membrance filtration-affinity chromatography with sTnI. A specific immunobiosensor for cTnI was developed by using surface plasmon resonance(SPR) biosensor with biotin as an intermediate layer and with the biotinylated-specific polyclonal antibody as capture antibody and used in a direct detection of cTnI in sera with the lowest detection limit of 2.5 μg/L. A more sensitive sandwich immunobiosensor for cTnI was developed by using biotinylated-specific polyclonal antibody as capture antibody(the first antibody) and polyclonal antibody as the second antibody. The sandwich immunoassay showed an excellent sensitivity of 0^5 μg/L and the detection range of 0^5-20 μg/L and within-run variation of 3^5%-4^9% and between-run variation of 6^1%-7^4%. The cTnI′s in the sera of 40 healthy donors and 20 patients with acute myocardial infarction(AMI) were monitored by using the sandwich immunoassay and the Lifesign MI Troponin I test device, respectively and it was found that the coincidence of the two assays was 95%, indicating that the developed sandwich immunoassay with SPR biosensor is suitable in clinical determination.