前列腺小体外泌蛋白ELISA检测方法的建立和初步评价

Establishment of ELISA detection method for prostatic exosomal protein,and its primary evaluation

目的建立前列腺小体外泌蛋白ELISA检测方法并评价其作为慢性前列腺炎辅助诊断的可行性。方法从慢性前列腺炎患者尿液中提取并纯化前列腺小体外泌蛋白,以纯化的前列腺小体外泌蛋白为抗原免疫实验BALB/c小鼠,得到单克隆抗体经过纯化后,建立ELISA检测方法。并对笔者所在医院的140例慢性前列腺炎患者尿液及正常人60例尿液中的前列腺小体外泌蛋白含量进行检测和评价。通过正交试验确定特异性抗体和酶标抗体的最佳工作浓度,并对试剂盒进行了4℃,12个月保存实验。确定该检测方法的临界值。结果慢性前列腺炎症患者尿液中前列腺小体外泌蛋白含量明显高于正常人尿液中含量,有显著性差异(Z=10.74,P<0.05)。实验室临界值为1.24 ng/ml。在140份慢性前列腺炎患者尿样中,阳性率为88.5%;特异性为90%。将前列腺小体外泌蛋白试剂盒测定结果与临床金标准比较[Kappa=0.75(95%CI=0.652 to 0.848),P<0.01],该检测试剂盒与临床金标准有高度一致性。试剂盒各个成分在低温保存12个月可以保持稳定。结论前列腺小体外泌蛋白的检测试剂盒灵敏度高,特异性强,稳定性合格,可以为临床前列腺炎诊断提供可靠的依据。

Objective To shape ELISA detection method for prostatic exosomal protein [PESP] and to evaluate the method of applying indirect ELISA for the assistant diagnosis of chronic prostatitis in its feasibility. Methods PEP was isolated from the urines of chronic prostatitis patients and used as the immunogen for generating mouse monoclonal antibodies ;the purified antibodies were used to establish an indirect ELISA detection method to study 140 urine samples of chronic prostatitis patients and 60 normal control＇s urine samples collected from the department of urology in Jinan Military Greneral Hospital. The optimal dilution of specific antibody and secondary antibody were surveyed through the orthogonal trial. The kits stored at 4℃ for 12 months were tested. Differences between two groups and critical value were evaluated by using SPSS 16.0 software. Results The content of PESP in chronic prostatitis sample was much higher than the normal control samples,that had significant differences between the two groups（Z=10.74,P〈0.05）. The critical value in the laboratory is 1.24ng/ml. There were perfectly consistent compared with the results of PESP testing kits and clinical gold standard （Kappa= 0.75 [95%CI=0.652 to 0.848],P〈0.01）. The components of the prostasomes kits can be stabilized storing at 4℃ for 12 months. Conclusion The prostasomes ELISA kits is specific,sensitive,stable,and may provide a simple rapidly and reliable method for clinic prostatitis diagnosis.