AIM:To elucidate the expression of the apoptosis-associatedmolecules in human primary hepatocellular carcinoma(HCC)cells,and prepare the monoclonal antibodies(mAb)againstthe apoptosis-associated antigens of HCC cells.METHODS:Human HCC cell line HCC-9204 cells wereinduced apoptosis with 60 mL·L~(-1)ethanol for 6 h and theirmorphological changes were observed by transmissionelectron microscope.The cell DNA fragmentations weredetected by Terminal Deoxynucleotidyl transferase-mediateddUTP nick end labeling(TUNEL)assay,and the cell DNAcontents by flow cytometry.Ten mice were immunized withethanol-induced apoptotic HCC-9204 cells with the methodof subtractive immunization,while the other 10 mice usedas the control were immunized by the routine procedures.The tail blood of all the mice were prepared after the lastimmunization,and the produced antibodies were determinedby the immunocytochemical ABC staining.The splenic cellsof the mice whose tail blood sera-HCC-9204 cells serumreactions were most different between the apoptotic andthe non-apoptotic were prepared and fused with the mousemyeloma cell line SP2/0 cells.The positive antibodies wereselected by ELISA assay.The fusion rates of hybridoma ceilsand the producing rates of antibodies were calculated.Thefused cells that secreted candidate objective antibody werecloned continually with the of limited dilution method,andthen selected and analyzed further by theimmunocytochemical ABC staining.The chromosomes of thecloned hybridoma cells that secreted objective mAb and themAb immunoglobulin(Ig)subtype of the prepared mAb werealso determined.The molecular mass of the mAb associatedantigen was analyzed by Western blot assay.RESULTS:HCC-9204 cells treated with 60 mL·L~(-1)ethanolfor 6 h,manifested obvious apoptotic morphological changes,the majority of the cells were TUNEL-positive,and the sub-G1 apoptotic peak was evident.There were 2 mice in theexperimental group whose tail blood serum reacted stronglywith the apoptotic HCC-9204 cells,but weakly with theirnon-apoptotic counterparts.In the fusion rates of hybridomacells as well as the producing rates of the antibody deseribedabove,there did not show significant difference betweenthe experimental and the control group,but weakly with non-apoptotic HCC-9204.However,the total producing rateof antibodies in the experimental group was significantlylower compared with the control(P<0.01),and so was theproducing rate of the antibodies which reacted strongly withboth apoptotic and non-apoptotic HCC-9204 cells(P<0.01).After cloned continually for several times the cell that producemAb which reacted strongly with the nuclei of ethanol-induced apoptotic HCC-9204 cells,but very weakly with thatof non-apoptotic cells was selected out.Chromosome analysisrevealed that the selected cell was with the universalcharacteristics of the monoclonal hybridoma cells whichsecreted mAb,and the Ig subtype of the prepared mAb wasIgG1.The molecular mass of this mAb associated antigenof was about 75 ku.CONCLUSION: Subtractive immunization is a useful method to prepare the mAb against the apoptosis-associated antigens of cells. The expression of some molecules increases to some extent in HCC-9204 cells in the process of apoptosis induced by low-concentration ethanol. The mAb that may be against ethanol-induced apoptosis-associated antigens of HCC cells was successfully prepared and primarily identified. AIM: To elucidate the expression of the apoptosis-associated molecules in human primary hepatocellular carcinoma (HCC) cells, and prepare the monoclonal antibodies (mAb) against the apoptosis-associated antigens of HCC cells. METHODS: Human HCC cell line HCC-9204 cells were induced of apoptosis with 60 mL·L -1 ethanol for 6 h and their morphological changes were observed by transmission electron microscope.The cell DNA fragments weredetected by Terminal Deoxynucleotidyl transferase-mediateddUTP nick end labeling (TUNEL) assay, and the cell DNAcontents by flow cytometry. Ten mice were immunized withethanol-induced apoptotic HCC-9204 cells with the method of subtractive immunization, while the other 10 mice usedas the control were immunized by the routine procedures. The tail blood of all the mice were prepared after the lastimmunization, and the the creatures antibodies were determined by the immunocytochemical ABC staining. The splenic cells of the mice whose tail blood sera-HCC-9204 cells serumreactions were most different between the apoptotic and the non-apoptotic were prepared and fused with the mouse myeloma cell line SP2 / 0 cells. The positive antibodies were selected by ELISA assay. The fusion rates of hybridoma ceils and the producing rates of antibodies were. Thefused cells that secreted candidate objective antibody were cloned continually with the of limited dilution method, and at selected and further enhanced by the immunocytochemical ABC staining. chromosomes of thecloned hybridoma cells that secreted objective mAb and themAb immunoglobulin (Ig) subtype of the prepared mAb were determined. The molecular mass of the mAb associatedantigen was analyzed by Western blot assay.RESULTS: HCC-9204 cells treated with 60 mL·L -1 ethanolfor 6 h, manifested a significant apoptotic morphological changes, the majority of the cells were TUNEL-positive, and the sub- G1 apoptotic peak was evident. There were 2 mice in the experimental group whose tail blood body highly strongly associated with the apoptotic HCC -9204 cells, but weakly with their non-apoptotic counterparts. In the fusion rates of hybridoma cells as well as the producing rates of the antibody deseribed above, there did not show significant difference betweenthe experimental and the control group, but weakly with non-apoptotic HCC-9204 .Wever, the total producing rate of antibodies in the experimental group was significantlylower compared with the control (P <0.01), and so was theproducing rate of the antibodies that arrested strongly withboth apoptotic andnon-apoptotic HCC-9204cells (P <0.01) . After cloned continually for several times the cell that producemAbs who caught strongly with the nuclei of ethanol-induced apoptotic HCC-9204 cells, but very weakly with that of non-apoptotic cells was selected out. Chromosome analysis wasvealed that the selected cells was with the universal characteristics of the monoclonal hybridoma cells which created mAb, and the Ig subtype of the prepared mAb was IgGl. The molecular mass of this mAb associated antigenof was about 75 ku. CONCLUSION: Subtractive immunization is a useful method to prepare the mAb against the apoptosis-associated antigens of cells. The expression of some molecules increases to some extent in HCC-9204 cells in the process of apoptosis induced by low-concentration ethanol. The mAb that may be against ethanol-induced apoptosis-associated antigens of HCC cells was successfully prepared andrized identified.