Purification and characterization of IgE produced by human myeloma cell line, U266 |
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| Authors: | S Ikeyama S Nakagawa M Arakawa H Sugino A Kakinuma |
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| Institution: | 1. Department of Pharmacy, Southwest Hospital, Third Military Medical University, Chongqing 400038, PR China;2. Department of Medical Oncology, Changzheng Hospital, Second Military Medical University, Shanghai 200070, PR China;3. School of Pharmacy, Second Military Medical University, Shanghai 200433, PR China;1. Department of Chemistry, Philipps-University Marburg, Hans-Meerwein-Straße 4, 35032 Marburg, Germany;2. Department of Neurology, Philipps-University Marburg, Baldingerstraße, 35043 Marburg, Germany;3. Institute for Medical Microbiology and Hospital Hygiene, University Hospital Giessen and Marburg, Hans-Meerwein-Straße, 35033 Marburg, Germany;4. Institute for Medical Microbiology, Justus-Liebig University, Biomedical Research Facility Seltersberg, Schubertstraße 81, 35392 Giessen, Germany;1. Department of Chemistry, University of Florida, P.O. Box 117200, Gainesville, FL 32611, USA;2. Université de Lyon, F-69622 Lyon, France;3. Université Lyon 1, Villeurbanne, France;4. Institut Lumière Matière, UMR5306 Université Lyon 1-CNRS, Université de Lyon, 69622 Villeurbanne Cedex, France;5. Institut Universitaire de France IUF, 103 Blvd St. Michel, 75005 Paris, France;6. Radboud University Nijmegen, Institute for Molecules and Materials, FELIX Laboratory, Toernooiveld 7, 6525ED Nijmegen, The Netherlands;7. van ‘t Hoff Institute for Molecular Sciences, University of Amsterdam, Science Park 904, 1098XH Amsterdam, The Netherlands;1. Life Sciences College of Nanjing Agricultural University, Nanjing 210095, Jiangsu, China;2. Key Laboratory of Animal Models and Human Disease Mechanisms of Chinese Academy of Sciences & Yunnan Province, Kunming Institute of Zoology, Kunming 650223, Yunnan, China |
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| Abstract: | Human IgE was isolated for the first time from the supernatant of the culture fluid of a human myeloma cell line, U266. The purification procedure consisted of salting out from the supernatant with ammonium sulfate, affinity chromatography on a lysine-Sepharose 4B column, ion exchange chromatography on a DEAE-Sephacel column, gel filtration and recycling chromatography on a Sephacryl S-300 column and removal of bovine proteins on an anti-bovine serum rabbit IgG-Sepharose 4B column. One hundred and twenty eight milligrams of IgE was recovered from 461 of culture fluid. The purification was extremely simplified by the introduction of immunoaffinity chromatography using the monoclonal antibody prepared by immunizing a mouse with an IgE preparation obtained by the above method: about 3.3 mg was recovered from 960 ml of culture fluid. The purified preparation was homogeneous as judged by the double-immunodiffusion test and end group analysis. The amino acid and carbohydrate compositions of the preparation coincided with those reported on other preparations obtained from the sera of myeloma patients. Our preparation, however, showed two bands with apparent mol. wts of 240,000 and 230,000 on sodium dodecyl sulfate polyacrylamide gel electrophoresis. When it was reduced with dithiothreitol and analyzed by electrophoresis, it gave two heavy chains and one light chain with apparent mol. wts of 80,000 and 76,000, and 28,000, respectively. On the other hand, the IgE molecule that was synthesized and secreted into the medium in the presence of tunicamycin (0.5 microgram/ml) gave only one heavy chain and one light chain with apparent mol. wts of 62,000 and 28,000, respectively. These results demonstrated that the two IgE molecular species contained in our preparation differed from each other in the carbohydrate moiety in their heavy chains. |
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