液体芯片技术定量测定人体血清CEA、AFP、NSE和tPSA

Quantitative determination of carcinoembryonic antigen(CEA), alpha fetoprotein(AFP),neuron specific enolase(NSE) and total prostate specific antigen(tPSA) by multiplexed bead-based immunoassay

目的应用液体芯片技术,联合定量测定人体血清癌胚抗原(CEA)、甲胎蛋白(AFP)、神经元特异性烯醇化酶(NSE)和前列腺特异性抗原(tPSA),并对其临床应用进行评价。方法制备交联微球以及生物素标记抗体,利用双抗体夹心法对60个血清样本进行测定,并将其结果与化学发光免疫分析法(CLIA)作比较。结果同时检测CEA、AFP、tPSA、NSE的线性范围分别为0.078～200 ng/mL、0.030～30.3 ng/mL、0.007～7.5 ng/mL、0.146～75 ng/mL;最低检测限为26.0 pg/mL、19.7 pg/mL、4.9 pg/mL、73.2 pg/mL;批内精密度CV<9.0%,批间精密度CV<13.2%;检测结果与CLIA测值的相关系数r分别为0.986、0.979、0.964、0.958(P<0.001)。结论液体芯片技术是一种具有极大优势的新型检测技术。该技术打破传统测定技术每次只能测定一个指标的限制,并且具有高通量、高灵敏度、检测时间短、样本用量少等特点。

Objective To measure the levels of carcinoembryonic antigen （CEA）, alpha fetoprotein （AFP）, neuron specific enolase （NSE） and total prostate specific antigen （tPSA） in human serum using multiplexed bead-based immunoassay, finally carry out valuation on its clinical practice. Methods Antibody-conjugated microspheres and biotinylated detection antibodies were prepared. The levels of CEA, AFP, NSE and tPSA in clinical serum samples were also measured using a sandwich immunoassay, and compared the performance of this method with chemiluminescence immunoassay（CLIA）. Results Dynamic range were 0.039~400 ng/mL, 0.030~121 ng/mL, 0.007~30 ng/mL, 0.146-300 ng/mL, and lower limits of detection were 26.0 pg/mL, 19.7 pg/mL, 4.9 pg/mL, 73.2 pg/mL for CEA, AFP, tPSA and NSE, respectively. Intra-assay variances were less than 9%, and inter-assay variances were less than 14%. Correlation coefficients（r） were 0.986, 0.979（P〈0.001, n=64）, 0.964（P〈0.001, n=60）, 0.958（P〈0.001, n=64） compared with CLIA for CEA, AFP, tPSA and NSE, respectively. Result obtained from multiplexed bead-based immunoassay was compatible with chemiluminescence immunoassay（CLIA）. However, only 1 microtiter of serum and 2 to 3 hours were required for the multiplexed bead-based immunoassay. Conclusion The multiplexed bead-based immunoassay is a sensitive, reproducible and a high-throughput method. This method may further be developed for clinical application.