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

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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.