Isolation and characterization of a monoclonal nonspecific suppressor factor (MNSF) produced by a T cell hybridoma.
By fusing Con A-activated BALB/c mice spleen cells with AKR thymoma BW5147 cells, we prepared a hybridoma producing a monoclonal nonspecific suppressor factor (MNSF). This factor inhibits a generation of LPS-induced immunoglobulin-secreting cells. We used ELISA for the bioassay of MNSF activity. With this method, a stable E17 hybridoma clone was selected, and its product in culture medium was isolated and characterized. MNSF fractionated on Sephadex G-100 in saline buffer shows a form with multiple m.w., but fractionated in 0.4 M pyridine-acetic buffer, it is limited to two species of approximately 24Kd and 16Kd. The MNSF was purified by hydroxyapatite chromatography, with marked effectiveness. MNSF activity was found exclusively in the 0.35 M sodium phosphate elution, and the content was further fractionated on subsequent gel filtration in the high ionic strength buffer described above. The purified factor exhibited two forms, of 24Kd and 16Kd, and showed peaks of pI 5.3 and 5.7, respectively, on isoelectric focusing. The MNSF preparation described here is stable at 56 degrees C and unaffected by 2-mercaptoethanol, but is unstable at pH 2.0 and is sensitive to tryptic proteolysis. We injected the hybridoma cells into the peritoneal cavity of pristane-primed F1 (AKR/J X BALB/c) mice, and a large amount of pure MNSF was obtained from the ascites, the characteristics of which were similar to those in the culture supernatant. Thus, the MNSF obtained from the E17 hybridoma consists of functionally identical but physicochemically different discrete proteins. This simple method of purification can serve as a probe for further characterization of MNSF and its application in in vivo experiments.
Nakamura
M
M
Ogawa
H
H
Tsunematsu
T
T
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001202:Ascitic Fluid / Q000378:metabolism; D003470:Culture Media; D004591:Electrophoresis, Polyacrylamide Gel; D000939:Epitopes / Q000276:immunology; D005260:Female; D006825:Hybridomas / Q000378:metabolism*; D051379:Mice; D008806:Mice, Inbred AKR; D008807:Mice, Inbred BALB C; D008970:Molecular Weight; D013491:Suppressor Factors, Immunologic / Q000032:analysis / Q000096:biosynthesis / Q000302:isolation & purification*; D013601:T-Lymphocytes / Q000378:metabolism*
D000596:Amino Acids; D003470:Culture Media; D000939:Epitopes; D013491:Suppressor Factors, Immunologic