The present invention concerns muteins of avidin and streptavidin with a reduced binding affinity for biotin as well as their use as interference elimination reagents in methods for the determination of an analyte e.g. in diagnostic tests such as for example immunoassays and nucleic acid hybridization assays. In addition the invention concerns the use of muteins of avidin and streptavidin as systems capable of regeneration for binding biotin e.g. for the analysis of biotinylated molecules, for examining receptor ligand interactions as well as for the affinity purification of biotinylated molecules.