磷酸化Y盒结合蛋白1的抗体制备及其辅助诊断原发性肝癌合并肺转移的临床价值

摘要

Objective To prepare antibodies (pAbs) against phosphorylated Y-box binding protein 1 (pYB-1),perform qualitative detection of the ascites/pYB-1 ratio in patients with hepatocellular carcinoma with pulmonary metastasis (HCC-PM),and assess the clinical significance of the ascites/pYB-1 ratio as a diagnostic biomarker for HCC-PM.Methods Bioinformatic prediction and chemical synthesis was used to identify and generate the YB-1 polypeptide with phosphorylation at serine position 102 (KYLRSVGDG).Rabbits were immunized with the YB-1 polypeptide coupled to a carrier protein.Protein A affinity chromatography was used to prepare highly-purified pAbs.ELISA and SDS-PAGE were used to determine concentration and purity of the pAbs.A total of 109 ascites specimens were collected from patients (36 cases of HCC,44 cases HCC-PM,and 29 cases of liver cirrhosis) and concentrated to obtain the pYB-1.Western blotting was used to qualitatively detect pYB-1 in ascites.Regression analysis and receiver operating characteristic (ROC) curve analysis were used to assess the qualitative data.Results The prepared pAbs had a concentration of ≥ 1:1 × 106 and high purity.The pAbs/YB-1S102 specifically recognized endogenous pYB-1S102.The pYB-1S102 detected in ascites specimens from patients with HCC and HCC-PM,and the positive rate of detection was 30.6％ and 77.3％ respectively (P ＜ 0.01).The pYB-1S102 showed sensitivity of 77.3％ and a accuracy rate of 73.8％ for diagnosis of HCC-PM.Conclusion Detection of pYB-1S102 in ascites could be a useful biomarker for diagnosis and metastasis monitoring in patients with HCC.%目的 制备Y盒结合蛋白1磷酸化抗体(pAb/YB-1S102);探讨腹水中磷酸化YB-1 (pYB-1)作为标志物辅助诊断原发性肝癌合并肺转移的临床价值. 方法 通过生物信息学预测并化学合成102位丝氨酸磷酸化YB-1多肽,耦联载体蛋白后免疫家兔;protein A亲和层析法纯化抗血清pAb/YB-1S102,并验证其特异性.收集109例患者腹水并富集腹水中pYB-1,其中原发性肝细胞癌(HCC) 36例,HCC合并肺转移44例,肝硬化29例;采用Western blot定性检测腹水中pYB-1,并对定性结果进行回归判别与受试者工作特征(ROC)曲线分析.计数资料采用x2检验.结果 酶联免疫吸附法与十二烷基硫酸钠-聚丙烯酰胺凝胶电泳结果显示制备的pAb/YB-1S102效价≥1∶1×106,且纯度较高.Western blot结果证实pAb/YB-1S102能特异性识别内源性pYB-1S102.肝硬化患者腹水中未检出pYB-1S102,HCC合并肺转移患者腹水pYB-1S102阳性率(77.3％)显著高于HCC(30.6％,x2=11.69,P＜0.01);回归分析与ROC曲线结果表明pYB-1S102辅助诊断HCC合并肺转移的灵敏度为77.3％,符合率为73.8％(x2=17.56,P＜0.01). 结论 成功制备可识别内源性pYB-1的抗体pAb/YB-1102;通过定性检测腹水pYB-1,可鉴别诊断HCC合并肺转移.