Purification of igm monoclonal antibody from ascites fluids by using fast protein liquid chromatography
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| date | 2015-03-09 08:43:35 |
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| id | 11583 |
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| internalnotes | Ainul Fajariah A.Bakar, Noorjahan B. Alitheen, Muhajir Hamid, Siti Aishah M. Ali and Abdul Manaf Ali. 2007. A Mouse IgM Monoclonal Antibody Recognized Breast and Colon Cancer, http://mc.manuscriptcentral.com/hybridoma. Ali A.M., Ong, B.K., Yussof, K., Hamid, M., Ali, S.A. and Azimahtol H.L. 1996. Generation of a stable hybridoma clone secreting monoclonal antibody against breast cancer. Asia Pacific journal of molecular Biology and biotechnology. 4(2): 123-131. Amyx H.L. 1987. Control of animal pain and distress in antibody production and infectious diseases studies. J. Am. Vet. Med. Assoc. 191: 1287. Barbara S.H. and Patricia G.M. 2001. Breast cancer: hormones and other risk factors. Maturitas. 38: 108-116. Boucheix C., Perrot J.Y, Mirshahi M., Bernadou A. and Rosenfeld C. 1983. A Rapid Method for Detection of Membrane Antigens by Immunofluorescence and its Application to Screening Hybridoma Antibodies, Journal of Immunological Methods. 57: 145-150. Brodeur B.R., Tsang P. and Larose Y. 1985. Parameters Affecting Ascites Tumor Formation in Mice and Monoclonal Antibody Production. Journal of immunological Methods. 71: 265-272. Collinsa A.B, Smitha R.N and Stonea J.R. 2009. Classification of amyloid deposits in diagnostic cardiac specimens by immunofluorescence, Cardiovascular Pathology. 18: 205-216. Coons A.H., Creech H.J. and Jones R.N. 1941. Immunological properties of an antibody containing a fluorescent group. Proc. Soc. Exp. Biol Med. 47: 200. Elima J., Curado M.P, Ogunbiyi O., Oga E., Fabowale T., Igbinoba F., Osubor G., Out T., Kumai H., Koechlin A., Osinubi P., Dakuma P., Blattner W. and Adebamowo C.A. 2012. Cancer incidence in Nigeria: A report from population-based cancer registries. Cancer Epidemiology 36:271-278. Gathumbi J.K., Usleber E. and Martlbauer E. 2001. Production of ultrasensitive antibodies against aflatoxin B1. Lett. Appl. Microbiol. 32: 349-351. Gautam S. and Kai-Chee Loh 2010. Binding Characteristics of Polymeric Immunoglobulin Receptor-D1 Based Affinity Ligand for IgM Purification, Journal of Chromatography B. Mahana W. and Paraf A. 199). Mice Ascites as a Source of Polyclonal and Monoclonal Antibodies, Journal of Immunological Methods. 161: 187-192. Meng Q., Qi M., Chen D.Z., Yuan R., Goldberg I.D., Rosen E.M., Auborn K. and Fan S. 2000. Suppression of breast cancer invasion and migration by indole-3-carbinol: associated with up-regulation of BRCA1 and E-cadherin/catenin complexes. J Mol Med. 78: 155-165. Ministry of Health Malaysia. 2003. Second Report of The National Cancer Registry: Cancer Incidence In Malaysia 2003. National Cancer Registry, Malaysia ISSN 1675-8870. Price P.W., McKinney E.C., Wang Y., Sasser L.E., Kandasamy M.K., Matsuuchi L., Milcarek C., Deal R.B., Culver D.G. and Meagher R.B. 2009. Engineered cell surface expression of membrane immunoglobulin as a means to identify monoclonal antibody-secreting hybridomas. Journal of Immunological Methods. 343: 28-41. Raja Ghosh. 2001. Purification of lysozyme by microporous PVDF membrane-based chromatographic process, Biochemical Engineering Journal. 14: 109-116. Sood A.K., Bhatty R., Kamat A.A., Landen C.N., Han L., Thaker P.H., Li Y., Gershenson D.M., Lutgendorf S. and Cole S.W. 2006. Stress hormone-mediated invasion of ovarian cancer cells. Clin Cancer Res. 12: 369-375. Susan Ker-hwa Ou, Jenny Ming-chen Hwang and Paul H. Patterson. (1993). A modified method for obtaining large amounts of high titer polyclonal ascites fluid, Journal of Immunological Methods. 165: 75-80. Yuecheng Y., Hongmeng L. and Xiaoyan L. 2006. Clinical evaluation of E-cadherin expression andits regulation mechanism in epithelial ovarian cancer. ClinExp Metastasis. 23: 65-74. |
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| spelling | 11583 https://intelek.unisza.edu.my/intelek/pages/view.php?ref=11583 https://intelek.unisza.edu.my/intelek/pages/search.php?search=!collection407072 Restricted Document Article Journal UniSZA Unisza unisza image/jpeg inches 96 96 1419 78 78 779 2015-03-09 08:43:35 1419x779 5842-01-FH02-FBIM-15-02633.jpg UniSZA Private Access Purification of igm monoclonal antibody from ascites fluids by using fast protein liquid chromatography ARPN Journal of Engineering and Applied Sciences 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. 10 1 343-350 Ainul Fajariah A.Bakar, Noorjahan B. Alitheen, Muhajir Hamid, Siti Aishah M. Ali and Abdul Manaf Ali. 2007. A Mouse IgM Monoclonal Antibody Recognized Breast and Colon Cancer, http://mc.manuscriptcentral.com/hybridoma. Ali A.M., Ong, B.K., Yussof, K., Hamid, M., Ali, S.A. and Azimahtol H.L. 1996. Generation of a stable hybridoma clone secreting monoclonal antibody against breast cancer. Asia Pacific journal of molecular Biology and biotechnology. 4(2): 123-131. Amyx H.L. 1987. Control of animal pain and distress in antibody production and infectious diseases studies. J. Am. Vet. Med. Assoc. 191: 1287. Barbara S.H. and Patricia G.M. 2001. Breast cancer: hormones and other risk factors. Maturitas. 38: 108-116. Boucheix C., Perrot J.Y, Mirshahi M., Bernadou A. and Rosenfeld C. 1983. A Rapid Method for Detection of Membrane Antigens by Immunofluorescence and its Application to Screening Hybridoma Antibodies, Journal of Immunological Methods. 57: 145-150. Brodeur B.R., Tsang P. and Larose Y. 1985. Parameters Affecting Ascites Tumor Formation in Mice and Monoclonal Antibody Production. Journal of immunological Methods. 71: 265-272. Collinsa A.B, Smitha R.N and Stonea J.R. 2009. Classification of amyloid deposits in diagnostic cardiac specimens by immunofluorescence, Cardiovascular Pathology. 18: 205-216. Coons A.H., Creech H.J. and Jones R.N. 1941. Immunological properties of an antibody containing a fluorescent group. Proc. Soc. Exp. Biol Med. 47: 200. Elima J., Curado M.P, Ogunbiyi O., Oga E., Fabowale T., Igbinoba F., Osubor G., Out T., Kumai H., Koechlin A., Osinubi P., Dakuma P., Blattner W. and Adebamowo C.A. 2012. Cancer incidence in Nigeria: A report from population-based cancer registries. Cancer Epidemiology 36:271-278. Gathumbi J.K., Usleber E. and Martlbauer E. 2001. Production of ultrasensitive antibodies against aflatoxin B1. Lett. Appl. Microbiol. 32: 349-351. Gautam S. and Kai-Chee Loh 2010. Binding Characteristics of Polymeric Immunoglobulin Receptor-D1 Based Affinity Ligand for IgM Purification, Journal of Chromatography B. Mahana W. and Paraf A. 199). Mice Ascites as a Source of Polyclonal and Monoclonal Antibodies, Journal of Immunological Methods. 161: 187-192. Meng Q., Qi M., Chen D.Z., Yuan R., Goldberg I.D., Rosen E.M., Auborn K. and Fan S. 2000. Suppression of breast cancer invasion and migration by indole-3-carbinol: associated with up-regulation of BRCA1 and E-cadherin/catenin complexes. J Mol Med. 78: 155-165. Ministry of Health Malaysia. 2003. Second Report of The National Cancer Registry: Cancer Incidence In Malaysia 2003. National Cancer Registry, Malaysia ISSN 1675-8870. Price P.W., McKinney E.C., Wang Y., Sasser L.E., Kandasamy M.K., Matsuuchi L., Milcarek C., Deal R.B., Culver D.G. and Meagher R.B. 2009. Engineered cell surface expression of membrane immunoglobulin as a means to identify monoclonal antibody-secreting hybridomas. Journal of Immunological Methods. 343: 28-41. Raja Ghosh. 2001. Purification of lysozyme by microporous PVDF membrane-based chromatographic process, Biochemical Engineering Journal. 14: 109-116. Sood A.K., Bhatty R., Kamat A.A., Landen C.N., Han L., Thaker P.H., Li Y., Gershenson D.M., Lutgendorf S. and Cole S.W. 2006. Stress hormone-mediated invasion of ovarian cancer cells. Clin Cancer Res. 12: 369-375. Susan Ker-hwa Ou, Jenny Ming-chen Hwang and Paul H. Patterson. (1993). A modified method for obtaining large amounts of high titer polyclonal ascites fluid, Journal of Immunological Methods. 165: 75-80. Yuecheng Y., Hongmeng L. and Xiaoyan L. 2006. Clinical evaluation of E-cadherin expression andits regulation mechanism in epithelial ovarian cancer. ClinExp Metastasis. 23: 65-74. |
| spellingShingle | Purification of igm monoclonal antibody from ascites fluids by using fast protein liquid chromatography |
| summary | 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. |
| title | Purification of igm monoclonal antibody from ascites fluids by using fast protein liquid chromatography |
| title_full | Purification of igm monoclonal antibody from ascites fluids by using fast protein liquid chromatography |
| title_fullStr | Purification of igm monoclonal antibody from ascites fluids by using fast protein liquid chromatography |
| title_full_unstemmed | Purification of igm monoclonal antibody from ascites fluids by using fast protein liquid chromatography |
| title_short | Purification of igm monoclonal antibody from ascites fluids by using fast protein liquid chromatography |
| title_sort | purification of igm monoclonal antibody from ascites fluids by using fast protein liquid chromatography |