Objective: To analyse antigens transiently expressed on hepatoma cells during apoptotic response.
Methods: Hepatoma HCC9204 cells were treated with 6% ethanol for 6 hr to induce apoptosis. Apoptotic HCC9204 cells were isolated and homogenized. After centrifugation at 14,000 rpm, the supernatant was dialyzed against water with molecular weight cutoff at 12,000. The dialysate was used as hepatoma apoptotic cell-associated antigen (HACAA). Subtractive immunization technique was used to prepare polyclonal antibodies against apoptotic cell-associated antigens on hepatoma. The antibody response to HACAA was determined by ELISA and immunoblot assays. Localization of HACAA on hepatoma apoptotic cells was demonstrated immunocytochemically.
Results: This polyclonal antibody reacted more strongly with HACAA than with antigen likewise prepared from non-apoptotic hepatoma cell. The antigenic determinants were sensitive to trypsin and mainly located on the cytoplasmic membrane.
Conclusion: Epitopes are expressed on hepatoma apoptotic cells which can be used to examine an apoptotic response. The antibody reported in this paper is useful to screen novel apoptosis associated genes.