Identifications of diagnostic biomarkers specific binding to soluble proteins of adenocarcinoma A 549 cell lines by an autoantibodiomics

2001年，Hanash教授和他的同事首次报道，通过蛋白质组学分析，抗Annexin-I和Annexin-II的自身抗体经常(37-40%)存在于肺腺癌的血浆中。此后，许多研究人员利用相同的技术在癌症患者血浆中鉴定出针对来自癌细胞的靶蛋白的自身抗体。我们还应用了新的基于蛋白质组学的分析来确定诊断肺腺癌的候选生物标志物。与基因分析方法相比，抗癌/肿瘤相关抗原自身抗体的鉴定具有简便、易行的优点。这项被称为自身抗体组学的技术有望应用于血浆中癌症诊断生物标记物的筛选。[方法]在增溶缓冲液中处理培养的人肺癌细胞株(A549)，并将其可溶性部分应用于样品溶液。用双向电泳法分离可溶性蛋白质，然后转移到PVDF膜上。封闭PVDF膜后，以血浆(500倍稀释度)为一抗，与膜上分离蛋白反应，用ECLplus试剂盒检测阳性斑点。结果与结论肺腺癌患者血浆中含有抗α-lace、伴侣蛋白、过氧化还蛋白-6自身抗体等8种生物标志物。我们认为这些自身抗体是诊断肺腺癌的候选生物标志物。

At 2001, Prof. Hanash and his colleagues first reported that the autoantibodies against Annexin-I ＆ II frequently (37-40%) were existed in plasma of lung adenocarcinoma using proteomics-based analysis. Thereafter many researchers have been identified autoantibody in cancer patients' plasma against targeted proteins derived from cancer cells using the same technique. We also applied the novel proteomics-based analysis to identify the candidate biomarkers for the diagnosis of lung adenocarcinoma. The advantages of the identification of autoantibodies against cancer/tumor related antigens were more easy and simple than the gene analytical method. This technique, so-called autoantibodiomics, was expected to apply the screening of cancer diagnostic biomarkers in plasma. In this project we identified candidate new lung cancer diagnosis markers.[Method] We treated a cultured human lung cancer cell strain (A549) in solubilization buffer solution and soluble fractions were applied a sample solution. The soluble proteins were separated with a two-dimensional electrophoresis and then transferred on a PVDF membrane. After blocking the PVDF membrane, plasma (500 times dilution) as a primary antibody was reacted with separated proteins on membrane, detected a positive spots using ECLplus reagent Kit. On the other hand, silver-staining gel was cut-off a positive spots corresponding to the ECL positive spots and analyzed them by a time-of-flight mass spectrometer (UltraFlex) or an ion trap type mass spectrometer ( LCQDECA) and a database searched a provided result and identified target proteins.[Results and conclusion] We found eight kinds of biomarkers, anti α-enlace, chaperonin, peroxiredoxin-6 autoantibodies, in patients' plasma of lung adenocarcinoma. We concluded that these autoantibodies were candidate biomarker to diagnose lung adenocarcinoma.