Monoclonal antibodies to human thioredoxin reductase.
The thioredoxin system consisting of thioredoxin (Trx), thioredoxin reductase (TrxR), and NADPH is an electron donor for ribonucleotide reductase but has also been implicated in other cellular events, including secretion, growth promotion, regulation of transcription factors, protection against oxidative stress, and apoptosis. Mammalian TrxR is a dimeric flavoprotein with 58 kDa subunits each with a catalytically active selenocysteine residue. To study the function and expression of TrxR, we have produced and characterized, for the first time, monoclonal antibodies against human TrxR. Native placenta TrxR was used for immunization of BALB/c mice, followed by hybridization, cloning, and establishment of hybridomas producing specific antibodies against human TrxR. Three clones of IgG1, kappa subclass, termed anti-TrxR1, anti-TrxR2, and anti-TrxR3, were studied in detail. The isoelectric points (pIs) of the mAbs were 6.5, 6.0, and 6.5, respectively. The affinities (Ka) of the mAbs were 2 x 10(8) M-1. Inhibition ELISA using biotin-labeled versus nonconjugated mAb IgG revealed that all three mAbs recognized one immunodominant epitope. Western blot analysis showed that the antibodies specifically bound to a 58 kDa protein, representing the subunit of TrxR. A Trx-dependent insulin reduction assay was used for analysis of enzymatic activity and the antibodies neutralized the reductase activity.
Department of Biomedicine and Surgery, University of Linköping, Sweden. Anders.Rosen@mcb.liu.se
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Söderberg
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Sahaf
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Holmgren
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Rosén
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology* / Q000302:isolation & purification*; D000915:Antibody Affinity; D000918:Antibody Specificity; D005260:Female; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D013880:Thioredoxin-Disulfide Reductase / Q000276:immunology*
D000911:Antibodies, Monoclonal; D013880:Thioredoxin-Disulfide Reductase