Autoantibodies to tumor-associated antigens combined with abnormal alpha-fetoprotein enhance immunodiagnosis of hepatocellular carcinoma

Abstract

The identification and characterization of tumor-associated antigens (TAAs) and their use in antigen mini-arrays for cancer immunodiagnosis has been of interest recently as an approach to cancer detection. In this study, autoantibodies in sera from a patient with HCC were used as probes to immunoscreen a HepG2 cDNA expression library for the identification of TAAs involved in malignant liver transformation. Recombinant proteins from two genes identified in this manner, Sui1 and RalA were expressed, purified and used as antigens in immunoassays to detect the presence of antibodies in sera from 77 patients with HCC, 30 with chronic hepatitis (CH), 30 with liver cirrhosis (LC) and 82 normal human sera (NHS). The prevalence of antibody to Sui1 and RalA in HCC were 11.7% (9/77) and 19.5% (15/77), respectively, which were significantly higher than prevalence in liver cirrhosis (3.3% and 3.3%), chronic hepatitis (0% and 0%) and normal human sera (0% and 0%). When Sui1 and RalA were added to a panel of eight other TAAs used in a previous study, the final cumulative prevalence of anti-TAA antibodies in HCC to the 10 TAA array was raised to 66.2% (51/77). The specificity for HCC compared with LC, CH and NHS, was 66.7%, 80.0%, and 87.8%, respectively. When anti-TAA was added to abnormal serum AFP as combined diagnostic markers, it raised the diagnostic sensitivity from 66.2% to 88.7%. AFP and anti-TAA were independent markers and the simultaneous use of these two markers significantly resulted in the increased sensitivity of HCC detection.

Fig. 1

Titer of autoantibodies to EP-HCC-1 (Sui1) and EP-HCC-13 (RalA) in HCC, liver cirrhosis (LC), chronic hepatitis (CH) and NHS. The range of antibody titers to Sui1 and RalA is expressed as optical density (OD) obtained from ELISA. The mean + 2SD and + 3SD of 82 normal human sera are shown in relationship to all serum samples. Panel A: Antibody titer to EP-HCC-1 (Sui1). Panel B: Antibody titer to EP-HCC-13 (RalA). Lane 1, CH; lane 2: LC; lane 3: HCC; lane 4: NHS.

Fig. 2

Representative Western Blot showing reactivity of ELISA positive HCC sera with recombinant proteins Sui1 (A) or RalA (B). To confirm the specificity of antibodies in positive sera for ELISA, all the positive sera were subjected to antibody absorption with recombinant protein Sui1 or RalA, followed by ELISA to evaluate the reduction of OD values (C, D). Panels A and C: The lane with “+” indicates anti-Sui1 polyclonal antibody used as positive control; lanes 1–3, three HCC positive sera; lanes 4 and 5, two normal human sera used as negative control. Panels B and D: The lane with “+” indicates anti-RalA monoclonal antibody used as positive control; lanes 1–3: three HCC positive sera; lane 4 and 5, two normal human sera used as negative control. The residual OD value after absorption with purified antigens is comparable to the “background” OD value shown by normal sera.