PURIFICATION AND IMMUNOCHEMICAL CHARACTERIZATION OF RECOMBINANT AND NATIVE RAGWEED ALLERGEN AMB-A-II

The complete sequence of a cDNA encoding Amb a II and its relationship to the Amb a I family of allergens has recently been described [Roger s et al. (1991) J. Immun. 147, 2547-2552; Griffith et al. (1991a), Int . Archs Allergy appl. Immun. %, 296-304]. In this study, we present re sults generated with rabbit antipeptide antisera that recognize Amb a II or Amb a I, but not both. The specificity of two anti-Amb a II anti peptide sera, anti-RAE-50.K and anti-RAE-51.K, was verified on Western blots of recombinant Amb a II and Amb a I. 1. These two sera, directe d against separate regions of the Amb a II molecule, detected three in dividual 38-kDa Amb a II isoforms on 2D Western blots of aqueous ragwe ed pollen extract. These Amb a II isoforms have pI in the 5.5-5.85 ran ge and can be easily distinguished from Amb a I isoforms with pI in th e 4.5-5.2 range detected by an anti-Amb a I specific peptide antiserum . The Amb a II isoforms have also been individually purified from poll en, positively identified as Amb a II by amino acid sequencing, and vi sualized as separate bands on IEF gels. An analysis of Amb a II cDNA s equences generated by PCR led to the prediction of three Amb a II isof orms with pl of 5.74, 5.86 and 5.97 that are very similar to the pI de duced from 2D Western blot analysis. Recombinant Amb a I.1 and Amb a I I have been expressed in E. coli, purified in their denatured form, an d examined by ELISA for their capacity to bind pooled allergic human I gE. Purified native Amb a and Amb a II from pollen were shown to have very similar IgE-binding properties. In contrast, Amb a II had a marke dly reduced IgE-binding capacity as compared to Amb a I.1. These data suggest that recombinant Amb a I.1 and Amb a II, isolated in a denatur ed form, differ significantly in their IgE-binding properties whereas the native molecules isolated from pollen do not.