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

Preparation of antibodies against phosphorylated YB-I and investigation of the clinical value as a biomarker to monitor hepatocdlular carcinoma with pulmonary metastasis

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l采用Westemblot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-1渤观效价≥1：1×106，且纯度较高。Westernblot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-1勖02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-1乳衄辅助诊断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-1S102通过定性检测腹水pYB-1，可鉴别诊断HCC合并肺转移。

[Abstract] 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-puritied 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 aseites. Regression analysis and receiver operating characteristic （ROC） curve analysis were used to assess the qualitative data. Resulis The prepared pAbs had a concentration of ≥ 1：1 x 106 and high purity. The pAbs/YB-1S102 specifically recotmired 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 ofpYB-1S102 in ascites could be a useful biomarker for diagnosis and metastasis monitoring in patients with HCC.