The purification and characterization of rat gamma interferon by use of two monoclonal antibodies
article
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-γ) 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-γ, 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-γ preparation consisted of at least seven molecular forms with M r values ranging between 14000 and 25000, with a relative abundance of a 18000 M r protein. Gel permeation chromatography of crude rRIF-γ gave coincident peaks of rRIF-γ proteins (all different forms) and interferon activity corresponding to a M r value of 45000. The results suggest that the molecular heterogeneity was due to differential glycosylation and was not the consequence of a proteolytic degradation process. Chemicals/CAS: gamma interferon, 82115-62-6; Antibodies, Monoclonal; Epitopes; Interferon Type II, 82115-62-6
Topics
gamma interferonmonoclonal antibodyaffinity chromatographyanimal cellanimal experimentblood and hemopoietic systemcharacterizationdrug analysisdrug bindingdrug identificationdrug interactiondrug isolationdrug purificationdrug screeningfemale genital systemgel filtrationglycosylationin vitro studypurificationspleenAnimalAntibodies, MonoclonalAntibody SpecificityChromatography, AffinityEpitopesHumanInterferon Type IIMiceMolecular WeightNeutralization TestsProtein Processing, Post-TranslationalRatsSpecies Specificity
TNO Identifier
230087
ISSN
00221317
Source
Journal of General Virology, 67(6), pp. 1059-1071.
Pages
1059-1071
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