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.
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.
Smith
J J
JJ
Department of Microbiology and Immunology, University of North Carolina, Chapel Hill 27514.
Olson
J R
JR
Klapper
D G
DG
D000485:Allergens; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology* / Q000302:isolation & purification; D000918:Antibody Specificity; D000937:Antigen-Antibody Reactions; D052179:Antigens, Plant; D004591:Electrophoresis, Polyacrylamide Gel; D000939:Epitopes / Q000032:analysis*; D051379:Mice; D008807:Mice, Inbred BALB C; D008970:Molecular Weight; D010446:Peptide Fragments / Q000276:immunology; D010940:Plant Proteins; D011058:Pollen / Q000276:immunology*; D011489:Protein Denaturation
D000485:Allergens; C026967:Amb a I protein, Ambrosia artemisiifolia; D000911:Antibodies, Monoclonal; D052179:Antigens, Plant; D000939:Epitopes; D010446:Peptide Fragments; D010940:Plant Proteins