Application of Four Anti-Human Interferon-α Monoclonal Antibodies for Immunoassay and Comparative Analysis of Natural Interferon-α Mixtures

Abstract

Four different mouse monoclonal antibodies to human interferon-α (IFN-α) were evaluated for application in quantitative and comparative analysis of natural IFN-α mixtures. Binding to IFN-α subtypes in solution revealed individual reactivity patterns. These patterns changed if the IFN-α molecules were immobilized either passively to a surface or bound by another antibody. Also, substitution of a single amino acid in IFN-α₂ affected the binding, apparently by altering the conformation. Isoelectric focusing of three natural IFN-α preparations from different sources, followed by immunoblotting, resulted in individual patterns with each of the four m Abs and also demonstrated variation in the composition of the IFN-α preparations. None of the mAbs was subtype specific, but by combining the different mAbs, and also applying polyclonal anti-human IFN-α antibodies, it was possible to design sensitive sandwich ELISAs with broad or more limited IFN-α subtype specificity.