Functional analysis of interferon-alpha subtypes using monoclonal antibodies to interferon-alpha 4a--subtype reactivity, neutralisation of biological activities and epitope analysis.

A panel of monoclonal antibodies (mAb) to a major human interferon-alpha (IFN-alpha) subtype, -alpha 4a, have been produced, characterised and used for studies of structure/function relationships of IFN-alpha subtypes. The mAb were tested for effects on receptor binding of IFN-alpha 4a, reactivity with other major subtypes -alpha 1, -alpha 2b and -alpha 14 by competitive ELISA and western immunoblotting, and for neutralisation of antiviral and antiproliferative activities of the four subtypes. The mAb could be grouped according to reactivity with IFN-alpha subtypes, group I (designated I-4-A) reacted with -alpha 4a and -alpha 2b, group II (I-4-C and I-4-F) reacted with -alpha 4a and -alpha 1, group III (I-4-D), I-4-G and I-4-H) reacted with -alpha 4a only, whereas group IV (I-4-I) reacted with -alpha 4a, -alpha 1 and -alpha 2b. No mAb reacted with IFN-alpha 14. Sequence comparisons of reactive and non-reactive IFN-alpha subtypes, and reactivity patterns with IFN-alpha fragments obtained by Lys-C digestion indicated that the epitopes were located in the N-terminal region (group I), in two regions of the middle of the molecule (group III and IV) and in the C-terminal region (group II). Binding of mAb to any of these four distinct epitopes neutralised the biological activities of IFN-alpha 4a, and in all cases, except I-4-A, inhibited receptor binding. Only the group III mAb bind to an epitope proposed to be in the vicinity of residues 30-40 which are implicated, from in vitro mutagenesis studies, in receptor binding. Binding of mAb to the other 3 epitopes neutralises biological activities by indirect mechanisms. These results emphasise the antigenic diversity between highly homologous IFN-alpha subtypes, which may have a wider functional significance. Individual mAb will have practical applications in the purification and detection of several IFN-alpha subtypes and so facilitate their further characterisation. By virtue of their different mechanisms of neutralisation, this panel of mAb will be useful in further studies of receptor interaction and signal transduction by IFN-alpha, and illustrate principles which are relevant to immunochemical studies of the receptor interactions of other cytokines.