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Identification of Hepatocellular-Carcinoma-Associated Antigens and Autoantibodies by Serological Proteome Analysis Combined with Protein Microarray

Supporting Info

Lan Li^†^‡^§, Su-hong Chen^†^§, Chao-hui Yu^‡, You-ming Li*^‡ and Sheng-qi Wang*^†

Beijing Institute of Radiation Medicine, Number 27 Taiping Road, Beijing 100850, , and Department of Gastroenterology, The First Affiliated Hospital, Medical College, Zhejiang University, Number 79 Qingchun Road, Hangzhou 310003,

J. Proteome Res., 2008, 7 (2), pp 611–620

DOI: 10.1021/pr070525r

Publication Date (Web): December 28, 2007

†

Beijing Institute of Radiation Medicine.

‡

Zhejiang University.

These authors contributed equally to this work.

* To whom correspondence should be addressed: Beijing Institute of Radiation Medicine, Number 27 Taiping Road, Beijing 100850, People

s Republic of China. Telephone: +86-10-68210077X932211 . Fax: +86-10-68210077X932211. E-mail: sqwang@bmi.ac.cn (S.-q.W.); The First Affiliated Hospital, Medical College, Zhejiang University, Number 79 Qingchun Road, Hangzhou 310003, People

s Republic of China. Telephone: +86-571-87236603 . Fax: +86-571-87236611. E-mail: liyouming@dna.net.cn (Y.-m.L.).

Abstract

To comprehensively study autoantibodies in patients with hepatocellular carcinoma (HCC), we used an approach-based serology and proteomics technologies. Total proteins extracted from HepG2 cells and HepG2.2.15 cells were separated by two-dimensional gel electrophoresis (2DE) and then transferred onto polyvinylidene difluoride (PVDF) membranes, which were subsequently incubated with sera from HCC patients or from normal controls. As a result, 13 HCC-associated antigens were identified. Antigenicity of eight proteins was further confirmed using recombinant proteins by Western blotting (WB) and protein microarray. The results of antigen microarray analysis showed strong signals of keratin 8 and lamin A/C in chronic hepatitis controls; therefore, the autoantibodies to keratin 8 and lamin A/C may not be HCC-specific. These two antigens were removed from subsequent analyses. The frequencies of positive reactions to DEAD (Asp-Glu-Ala-Asp) box polypeptide 3, eukaryotic translation elongation factor 2 (eEF2), apoptosis-inducing factor (AIF), heterogeneous nuclear ribonucleoprotein A2 (hnRNP A2), prostatic binding protein, and triosephosphate isomerase (TIM) were significantly higher in HCC than in chronic hepatitis and normal individuals. Positive reactions to DEAD box polypeptide 3, eEF2, AIF, and prostatic binding protein were significantly more frequent in HCC than in any other cancer. The sensitivity of any individual antigen in HCC at stage I ranged from 50 to 85%. When the combinations of six antigens were analyzed, the sensitivity increased to 90%. We conclude that the detection of autoantibodies against the six antigens may have value on early diagnosis of HCC.

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