电化学酶联免疫分析法测定人牙本质涎磷蛋白的研究

Detection of dentin sialophosphoprotein by electrochemical enzyme-linked immunoassay

目的探讨电化学酶联免疫分析法(ELISA)测定牙本质涎磷蛋白(DSPP)的可行性。方法选取牙本质涎磷蛋白标准品以辣根过氧化物酶(HRP)为标记酶、邻联茴香胺(ODA)为酶催化反应的底物,分别用电化学ELISA法及传统光学ELISA法检测酶催化产物。对比两种检测方法对DSPP检测的线性范围及检测限的差异。结果用电化学ELISA法检测酶催化产物,产物在-0.63 V(vs.Ag/AgCl)有一个灵敏的还原峰,进而可以用于游离HRP的检测,其线性范围为0.04～1.0 ng/mL,检测限为0.01 ng/mL。应用于DSPP标准品的检测,线性范围为2.5～200.0 pg/mL,检出限为2.5 pg/mL,灵敏度显著高于传统光度ELISA检测法。结论电化学ELISA法可以作为检测痕量DSPP的一个新方法。

Objective To investigate the sensitivity of electrochemical enzyme-linked immunoassay for the detection of dentin sialophosphoprotein （DSPP）. Methods Standard DSPP were detected by electrochemical enzyme linked immunoassay and traditional spectroscopic enzyme-linked immunosorbent assay （ELISA） respectively. The sensitivity of the two methods was compared and analyzed. Results HRP could catalyze the oxidation of ODA to produce an electroactive product, which exhibited a reduction peak at -0. 63 V （vs. Ag/AgC1）. Free HRP was detected in the concentration range from 0. 04 to 1.0 ng/mL, with the detection limit of 0. 01 ng/mL. DSPP, in combination with the sandwich ELISA procedure, could be further detected in the concentration range from 2. 5 pg/mL to 200. 0 pg/mL with the detection limit of 2. 5 pg/mL. The sensitivity of electrochemical enzyme-linked immunoassay was significantly higher than that of traditional spectroscopic ELISA. Conclusion Electrochemical enzyme-linked immunoassay is a new way of detecting DSPP with more sensitivity.