The purification and characterization of rat gamma interferon by use of two monoclonal antibodies.
Two mouse monoclonal antibodies, designated DB-1 and DB-2, were isolated and used for the purification and characterization of recombinant rat interferon gamma (rRIF-gamma) derived from Chinese hamster ovary (CHO) cells. The two antibodies belong to different classes (DB-1 is an IgG1 and DB-2 an IgA) and display similar epitope specificities as shown in competition binding experiments. Both antibodies, raised against rRIF-gamma, exhibited high affinity for rat and mouse gamma interferon and efficiently neutralized the antiviral activity of both animal interferon species. Affinity chromatography analysis showed that a column with immobilized DB-1 was capable of complete binding of rat and mouse gamma interferon, both natural and recombinant DNA-derived. As visualized by SDS-polyacrylamide gel electrophoresis and Western blot analysis, the purified rRIF-gamma preparation consisted of at least seven molecular forms with Mr values ranging between 14 000 and 25 000, with a relative abundance of a 18 000 Mr protein. Gel permeation chromatography of crude rRIF-gamma gave coincident peaks of rRIF-gamma proteins (all different forms) and interferon activity corresponding to a Mr value of 45 000. The results suggest that the molecular heterogeneity was due to differential glycosylation and was not the consequence of a proteolytic degradation process.
P H
PH
van der Meide
M
M
Dubbeld
K
K
Vijverberg
T
T
Kos
H
H
Schellekens
D000818:Animals; D000911:Antibodies, Monoclonal; D000918:Antibody Specificity; D002846:Chromatography, Affinity; D000939:Epitopes; D006801:Humans; D007371:Interferon-gamma / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D051379:Mice; D008970:Molecular Weight; D009500:Neutralization Tests; D011499:Protein Processing, Post-Translational; D051381:Rats; D013045:Species Specificity
D000911:Antibodies, Monoclonal; D000939:Epitopes; D007371:Interferon-gamma