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...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。展开更多
Based on the preparation of an anti-diethylstilbestrol(DES) monoclonal antibody,a simple and convenient indirect competitive enzyme-linked immunosorbent assay(ELISA) method for DES detection has been developed.The mon...Based on the preparation of an anti-diethylstilbestrol(DES) monoclonal antibody,a simple and convenient indirect competitive enzyme-linked immunosorbent assay(ELISA) method for DES detection has been developed.The monoclonal antibody demonstrated high sensitivity to DES with an IC50 value of 275 pg mL-1 and detection limit(LOD) of 90 pg mL-1.The specificity of the assay was studied by measuring cross-reactivity of the antibody with structurally related compounds of ethinyl estradiol(<7%),estrone(<0.1%),estriol(<0.1%),and diethylstilbestrol benzoate(<0.1%).Chicken,fish,shrimp,urine and bile spiked with different concentration of DES were detected by the developed method,and the recovery rates were greater than 79.5%.Intra-and inter-assay variations were about 6%.This method exhibited high stability with a coefficient of variation less than 10% in buffer and in real samples.The LODs in fish/shrimp,liver,feed and urine spiked with DES were 600,600,4800 and 600 pg mL-1,respectively.These results confirmed that the antibody to DES was successfully produced and could be used to establish ELISA methods for DES detection in food producing animals.展开更多
文摘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。
基金supported by the Shandong Natural Science Foundation (Y2008B31)the National High-Tech Research and Development Program of China (07AA10Z435, 2007AA06A407)+1 种基金the National Natural Science Foundation of China (20675048)the Fundamental Research Funds for the Central Universities (65011121)
文摘Based on the preparation of an anti-diethylstilbestrol(DES) monoclonal antibody,a simple and convenient indirect competitive enzyme-linked immunosorbent assay(ELISA) method for DES detection has been developed.The monoclonal antibody demonstrated high sensitivity to DES with an IC50 value of 275 pg mL-1 and detection limit(LOD) of 90 pg mL-1.The specificity of the assay was studied by measuring cross-reactivity of the antibody with structurally related compounds of ethinyl estradiol(<7%),estrone(<0.1%),estriol(<0.1%),and diethylstilbestrol benzoate(<0.1%).Chicken,fish,shrimp,urine and bile spiked with different concentration of DES were detected by the developed method,and the recovery rates were greater than 79.5%.Intra-and inter-assay variations were about 6%.This method exhibited high stability with a coefficient of variation less than 10% in buffer and in real samples.The LODs in fish/shrimp,liver,feed and urine spiked with DES were 600,600,4800 and 600 pg mL-1,respectively.These results confirmed that the antibody to DES was successfully produced and could be used to establish ELISA methods for DES detection in food producing animals.
