微孔板化学发光酶免疫分析法分析人血清中游离前列腺特异性抗原

Microplate Chemiluminescence Enzyme Immunoassay for the Quantitative Analysis of Free Prostate-specific Antigen in Human Serum

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）的高灵敏度微孑L板化学发光酶免疫分析方法，以碱性磷酸酶（alkalinephosphatase，ALP）为标记酶，4-甲氧基-4-（3″-磷酰氧基苯）-螺旋-（1，2-二氧杂环丁烷-3，2′-金刚烷）（4-methoxy-4-（3″-phosphate-phenyl）-spiro-（1，2-dioxetane-3，2′-adamantane），AMPPD）-ALP高灵敏的化学发光反应为检测体系，通过检测发光强度对人血清中f-PSA进行定量。对几种物理化学参数如温育时间、免疫反应步骤以及检测时间等进行了优化，采用了一步双抗体夹心法，37％恒温静置温育2h，洗涤后加入50μL AMPPD，30～80min内检测。该方法线性范围为0．15～20μg/L，相关系数大于0．998；检出限可达0．01μd/L，批内和批间相对标准偏差（C．V．）均小于7％；回收率在88％～108％之间。对人体血清中共存的6种常见肿瘤标志物CA50、CA125、CA15-3、CA242、CEA和AFP的偶联反应进行考察，特异性良好。为了验证该方法用于商业试剂盒的可行性，在4℃和37％条件下分别进行了3d，5d，7d的稳定性考察，其线性相关系数仍均大于0．998，相对标准偏差小于6％。对98例实际血样进行测定，并与进口试剂盒（Monobind．USA）作临床比对，相关性良好。这些结果均表明，该分析体系稳定，可靠，可以用于商业化试剂盒的开发，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。

A microplate chemiluminescence enzyme immonoassay with high sensitivity for the quantitative analysis of free prostate specific antigen （f-PSA） in human serum was established. The immunoassay utilized alkaline phosphatase （ALP） as the labeled enzyme, and the highly sensitive ALP-AMPPD （4-methoxy-4-（ 3″-phosphate-phenyl）-spiro-（1,2-dioxetane-3,2′-adamantane） ） chemiluminescence reaction as the detection system. Several physieochemieal parameters such as incubation time, immunoreaetion sequence, and detection time were studied and optimized. A one-step sandwiched reaction mode was applied with incubation at 37℃ for 2 h and chemilumineseent detection with 50 μL AMPPD for 30 - 80 min. The linear range of the proposed method for f-PSA was 0.15-20 μg/L with r =0. 998. The detection limit was 0.01 μg/L. Inter-assay and intra-assay RSD were both below 7%. The recovery was 88% - 108%. The cross-reactivity of six normal tumor markers in human serum, including CA50, CA125, CA15-3, CA242, CEA and AFP, was studied. It was shown that this method exhibited high specificity to f-PSA. The stability of the method was evaluated by identifying its analytical performance after stored at 4℃ and 37℃ for 3, 5, 7 days with RSD lower than 6% and correlation coefficient higher than 0. 998, which showed satisfied reliability and stability. Compared with CLIA kit purchased from Monobind. Inc. USA, there was a good correlation between the two methods. The proposed method has shown great potential in the clinical analysis of f-PSA for prostate cancer diagnosis, as well as in the commercial kits development.