磁微粒化学发光酶免疫分析法检测血清fPSA的方法学建立.docVIP

磁微粒化学发光酶免疫分析法检测血清fPSA的方法学建立 　　摘要:本研究建立了一种定量分析人血清中游离前列腺特异性抗原(f-PSA)的高灵敏度磁微粒化学发光酶免疫分析方法。采用了一步双抗体夹心法,将单克隆抗体连接在磁微粒表面,以辣根过氧化物酶(Horseradish Peroxidase, HRP)为标记酶,结合鲁米诺(luminol)高灵敏化学发光反应为检测体系进行深入研究。建立检测试剂的主要指标:检测范围0.02～50ng/ml;最低检出限0.02ng/ml,分析内变异4.7%;回收率在90%～110%之间。对510例样本进行测定临床研究,与Roche检测结果符合程度高,相关系数达到0.995。结果表明,该检测方法稳定,可靠,在临床分析人血清中f-PSA的辅助诊断前列腺癌方面具有很高的应用价值。 　　关键词:游离前列腺特异抗原,磁微粒化学发光酶免疫分析法,肿瘤标志物 　　 　　Key words: prostate specific antigen,microbeads-based chemiluminescence immunoassay. Tumor marker 　　Gao wei 　　Kaifeng City Maternity Hospital 　　[Abstract]A sensitive chemiluminescence enzyme immunoassay based on magnetic micro-beads was established to detect the free prostate specific antigen (f-PSA) in human serum. One antigen was coated on the magnetic micro-beads and the other was labled with horseradish peroxidase(HRP), with the highly sensitive Luminol chemiluminescence reaction as the detection system. The linear range of this method for PSA was 0.02~50ng/ml with the limit detection of 0.02 ng. The intra-assay coefficients of variation is lower than 4.7% and the percent recovery was in the range of 90%~110%. F-PSA in 510 clinical samples were detected with this method. Compared with Roche chemiluminescence immunoassays, the correlation coefficient of 0.995 was obtained. The results demonstrated that this method is reliable and shows proposed potential application in the clinical analysis of f-PSA for prostate cancer diagnosis. 　　 　　前列腺特异性抗原(Prostate Specific Antigen, PSA)是人体前列腺上皮组织细胞分泌的一种分子量为30～33 kDa的单链糖蛋白分子[1～3]。前列腺癌(Prostate Cancer, PCa)病人血清中PSA浓度升高;良性前列腺增生(Benign Prostate Hyperplasia, BPH)病人血清中,PSA浓度也会升高;随着年龄的增加,血清中PSA浓度也有不同程度的升高,在4～10 ng/ml的浓度范围就形成了诊断灰区[4~6]。PCa病人血清中f-PSA浓度水平与BPH病人以及正常人相比,明显偏低,游离前列腺特异抗原百分率对PCa的特异性更高,可降低临床检验筛查的假阳性率。建立特异性强、检测灵敏度高的新型f-PSA检测试剂将会对前列腺相关疾病的检测具有较高的临床应用价值。 　　1.材料及方法 　　1.1试剂: PSA、fPSA单克隆抗体购自美国Seradyn公司;辣根过氧化物酶、鲁米诺购自sigma公司;f-PSA化学发光免疫分析(CLIA)试剂盒购自Roche公司。 　　1.2仪器:LUMO微孔板化学发光检测仪、IWO型洗板机(郑州安图生物工程有限公司);罗氏ELESYS2010电化学发光仪。 　　1.3临床样本: