Purification of Igm Monoclonal Antibody from Ascites Fluids by Using Fast Protein Liquid Chromatography

Hybridoma clone C3A8 was established as a result of fusion between the lymphocytes of Balb/c mice immunized with the MCF-7 breast carcinoma cell line and Sp2/0-Ag14 myeloma cells. The clone was secreted the monoclonal antibodies (Mab) either in culture supernatant or ascites fluid and still have a contaminants which need to be purify in order to get the desired antibody. The main objective of this study is to purify the Mab. The monoclonal antibodies were purified by using HiTrap IgM Purification column and Fast Protein Liquid Chromatography (FPLC). The flow rate for FPLC system was 1 ml/min and 0.3 bar pressures which successfully separated IgM in crude monoclonal antibodies. Before purification process, the recloning of hybridoma cells by limiting dilutions was carried out in this study and it showed the clone C3A8 secreted IgM monoclonal antibody with kappa light chain. The purified IgM was analyzed using sodium dodecyl sulphate polyalcrylamide gel electrophoresis (SDS-PAGE) indicated that purified IgM had 55 kDa of heavy chain and 27 kDa of light chain. Screening by cell-ELISA showed the purified Mab C3A8 reacted strongly with breast cancer cells (MCF7) and colon cancer cells (HT29). Through immunofluorescence staining, the antigen was detected to be located in the cytoplasm of MCF7 and HT29 cell lines but there were no positive staining detected on cervical cancer (HeLa) and fibroblast normal cells (3T3). The purified Mab was found to react specifically against a 55 kDa protein that was present in the extract of MCF7 and HT29 cell lines when immunoblotting was carried out. All the results mentioned above, suggest that the purified Mab C3A8 could be detected in breast and colon cancers cells.