快速检测游离前列腺特异抗原ELISA方法的建立

Development of an ELISA for rapid detection of free prostate specific antigen

目的 建立快速检测人血清中游离前列腺特异抗原(f-PSA)的ELISA方法.方法 利用抗f-PSA的单克隆抗体(单抗)杂交瘤细胞株,一株单抗2D1用于固相包被,另一株单抗2E4进行辣根过氧化物酶标记,采用二步法,建立定量测定人血清f-PSA的双抗体夹心ELISA法.对该法的敏感度、精密度、准确性、特异性进行了分析.应用该法与瑞典CanAg公司的f-PSA ELISA试剂盒同时检测了18例前列腺癌和25例前列腺增生患者血清标本的f-PSA含量,进行了两种方法检测结果的相关性分析.结果 以双抗体夹心ELISA法检测f-PSA,在f-PSA 0～20μg/L范围内线性良好;敏感度为0.025 μg/L;批内变异系数为4.5%～6.2%,批间变异系数为3.9%～7.2%;回收率为94.3%～111.1%;与α1抗糜蛋白酶结合的结合型PSA交叉率为0.7%;检测43份临床标本f-PSA含量的结果与瑞典CanAg公司f-PSA试剂盒检测结果的相关系数为0.995.结论 所建立的双抗体夹心ELISA方法是一种特异、敏感、简便实用的f-PSA快速检测方法,有助于在PSA低水平升高的重叠范围内(4～10μg/L)鉴别前列腺增生与前列腺癌.

Objective To develop an ELISA for rapid and sensitive detection of free prostate specific antigen(f-PSA). Methods A sandwich ELISA was established by using two kinds of antibodies against f-PSA. McAb 2D1 was coated on a microtiter plate;and 2E4 was labeled with horseradish peroxidase(HRP). The specificity,sensitivity,precision,and accuracy of the ELISA were analyzed. The concentration of f-PSA in 43 serum samples collected from 18 patients with prostate cancer and 25 patients with benign prostate hypertrophies were detected by using the sandwich ELISA and CanAg Free PSA EIA kit(CanAg Diagnostics Inc,Sweden),respectively. The results were compared. Results The detection limit of the ELISA was 0.025 μg/L within an acceptable limit between 0 and 20 μg/L. The recovery rate of f-PSA ranged from 94.3% to 111.1%. The within-run and between-day coefficient of variation(CV) varied from 4.5% to 6.2% and 3.9% to 7.2%,respectively. It exhibited a cross-reactivity of 0.7% with PSA bound to α-1 antichymotrypsin(PSA-ACT). There was a good correlation between the results of f-PSA detection in 45 serum samples measured by the ELISA and CanAg Free PSA EIA kit(r = 0.995). Conclusions This sandwich ELISA is a rapid,sensitive and precise method in detecting f-PSA. In comparison with the commercial test kit of f-PSA,the ELISA was satisfying for its clinical applications. This method can be used to discriminate between benign and malignant prostate disease with a serum PSA level of 4-10 μg/L.