Purpose: To elucidate the expression of the apoptosis-associated molecules in human

Purpose: To elucidate the expression of the apoptosis-associated molecules in human main hepatocellular carcinoma (HCC) cells, and prepare the monoclonal antibodies (mAb) against the apoptosis-associated antigens of HCC cells. cells. The positive antibodies were selected by ELISA assay. The fusion rates of hybridoma cells and the generating rates of antibodies were calculated. The fused cells that secreted candidate objective antibody were cloned continually with the of limited dilution method, and then selected and analyzed further by the immunocytochemical ABC staining. The chromosomes of the cloned hybridoma cells that secreted objective mAb and the mAb immunoglobulin (Ig) subtype of the prepared mAb were also decided. The molecular mass of the mAb associated antigen was analyzed by Western blot assay. RESULTS: HCC-9204 cells treated with 60 mL?L-1 ethanol for 6 h, manifested obvious apoptotic morphological changes, SRT3109 the majority of the cells were TUNEL-positive, and the sub-G1 apoptotic peak was obvious. There were 2 mice in the experimental group whose tail blood serum reacted strongly with the apoptotic HCC-9204 cells, but weakly with their non-apoptotic counterparts. In the fusion rates of hybridoma cells as well as the generating rates of the antibody deseribed above, there did not show significant difference between the experimental and the control group, but weakly with non-apoptotic HCC-9204. However, the total generating rate of antibodies in the experimental group was significantly lower weighed against the control (< 0.01), therefore was the producing price from the antibodies which reacted strongly with both apoptotic and non-apoptotic HCC-9204 cells (< 0.01). After cloned constantly for several moments the cell that generate mAb which reacted highly using the nuclei of ethanol-induced apoptotic HCC-9204 cells, but extremely with this of non-apoptotic cells was selected away weakly. Chromosome analysis uncovered that the chosen cell was using the general characteristics from the monoclonal hybridoma cells which secreted mAb, as well as the Ig subtype from the ready mAb was IgG1. The molecular mass of the mAb linked antigen of was about 75 ku. Bottom line: Subtractive immunization is certainly a useful solution to prepare the mAb against the apoptosis-associated antigens of cells. The appearance of some substances increases somewhat in HCC-9204 cells along the way of apoptosis induced by low-concentration ethanol. The mAb which may be against ethanol-induced apoptosis-associated antigens of HCC cells was effectively ready and primarily discovered. INTRODUCTION Apoptosis is certainly an activity of active designed cell loss of life (PCD), which may be regulated by many types of biological factors encoded with a complete large amount of mammalian genes[1-16]. There isn't only the boost or loss of the appearance of some already-existed protein along the way of apoptosis, but also the creation and display of some brand-new apoptosis-associated substances that usually do not exhibit in non-apoptotic cells[17-20]. Currently, the knowledge about apoptosis-associated molecules is still limited. The conventional process to discover new apoptosis-associated molecules is usually to clone and sequence the apoptosis-associated genes of cells by the methods such as differentiated SRT3109 PCR and phage display first, and then performe the experiments to study the function of the expressed SRT3109 product of the candidate apoptosis-associated genes[21-25]. Preparing the antibodies against the associated antigens of apoptotic cells is also a very hopeful way to investigate apoptosis-associated molecules. There are already Rabbit polyclonal to ZNF264. some reports about the successful preparation of the polyclonal antibodies against apoptosis-associated molecules while their antigens are unclear and even monoclonal antibodies (mAb) at the condition that their associated antigens are specific[19,26,27]. However, there has been no statement until now about preparing the mAb against apoptosis-associated antigens at the condition that its associated antigens are still unspecific. In the present study, some mice were immunized by the method of.