Monoclonal antibodies to denatured ragweed pollen allergen Amb a I: characterization, specificity for the denatured allergen, and utilization for the isolation of immunogenic peptides of Amb a I

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The epitope structure of short ragweed allergen Amb a I (formerly antigen E or AgE) has been investigated by characterizing monoclonal antibodies (MAbs) from mice immunized with Amb a I. The MAbs to Amb a I that we previously produced and characterized (HaA1, HaB1 and HaC1) reacted with determinants which were conformationally dependent. Denaturation of Amb a I, which is a prerequisite for peptide isolation and hence epitope localization, destroyed these determinants. We report here the isolation of 15 MAbs produced against denatured Amb a I and present their reactivities with native Amb a I, denatured Amb a I, and peptide fragments of Amb a I. None of the 15 MAbs to denatured Amb a I bind to native Amb a I, as judged by their lack of binding to Amb a I in immune complex with HaA1 and HaC1. All 15 MAbs reacted with the extensively reduced and alkylated form of denatured Amb a I and 14 of the 15 were shown by Western blotting techniques and ELISA to have activity against the isolated alpha chain or beta chains of Amb a I, but not both. In addition, several MAbs react with tryptic fragments of Amb a I which allowed us to isolate and sequence two tryptic peptides of Amb a I. Antisera raised against KLH-coupled synthetic analogs of these peptides reacted well with intact, denatured Amb a I. Finally, during chromatographic purification of Amb a I from fresh pollen extracts, various MAbs began to react with Amb a I as a function of stage of isolation, suggesting that some proportion of Amb a I spontaneously denatures during purification, converting from the allergenic form possessing two major B cell epitopes to an allergenically inactive form.