IGE ANTIBODIES TO RECOMBINANT FORMS OF FEL-D-I - DICHOTOMY BETWEEN FLUID-PHASE AND SOLID-PHASE BINDING-STUDIES

Background: The major cat allergen Fel d I consists of two polypeptide chains linked by disulfide bonds, each of which has been expressed in bacteria. To investigate the antigenic structure of Fel d 1; antibody binding to the native molecule and to each recombinant chain were com pared. Methods: Polyclonal human IgE and IgG antibodies and monoclonal antibodies (mAbs) to Fel d I were compared for binding to Fel d I, ch ain 1, or chain 2 by fluid-phase inhibition radioimmunoassay, RAST, an d immunoabsorption. Results: In the fluid-phase assay, neither recombi nant chain significantly inhibited the binding of antibody to native F el d I at concentrations of up to 10 mu g/ml. partial inhibition was o bserved when chain 1 was used, which inhibited the binding of two mAbs by 40% and 75%. In contrast, when the solid-phase RAST assay was used , IgE antibodies bound both chains with high specificity, and there wa s a good quantitative correlation between IgE antibody binding to Fel d I and both chain 1 (r = 0.58, p < 0.01) and chain 2 (r = 0.47, p < 0 .01). Up to 70% of IgG or IgE anti-Fel d I antibodies could be absorbe d by either chain 1 or chain 5 and both chains in combination produced similar absorption values in response to native Fel d I. Four mAbs we re fully absorbed by chain I, but not chain 2, and three mAbs were nor absorbed by either chain. Conclusions: The results demonstrate a dich otomy between antibody binding to recombinant Fel d I chains, which ma y be explained by confirmational differences between the chains in the fluid phase or on solid supports. The results also suggest that chain I is art important site for mAb-defined B-cell epitopes on Fel d I.