Purification and characterization of IgE produced by human myeloma cell line, U266.
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.
S
S
Ikeyama
S
S
Nakagawa
M
M
Arakawa
H
H
Sugino
A
A
Kakinuma
D000911:Antibodies, Monoclonal / Q000276:immunology; D002460:Cell Line; D055598:Chemical Phenomena; D011232:Chemical Precipitation; D002621:Chemistry; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007073:Immunoglobulin E / Q000302:isolation & purification*; D009194:Myeloma Proteins / Q000302:isolation & purification*
D000911:Antibodies, Monoclonal; D009194:Myeloma Proteins; D007073:Immunoglobulin E