Mapping of Bet v I Epitopes by Using Murine Monoclonal Antibodies

Abstract

The different determinants of birch pollen extracts, as shown by SDS-PAGE analysis, range from 10 to 94 kDa. These determinants were then electrotransferred on nitrocellulose strips and allowed to react with human IgE Ab from sensitive patients in order to identify the allergenic determinants. Several minor (43, 35, 28 and 21 kDa) and the major (17 kDa) allergenic determinants were identified. Murine monoclonal antibodies (mAb) were then produced against the major allergenic determinant (Bet v I) and their specificity confirmed by immunoblot. One of them, mAb 3F10, was used to affinity-purify the Bet v I. The purity of this material was confirmed by SDS-PAGE analysis and its reactivity on immunoblot against human IgE ensured its biological activity. These mAb were then gathered on four families based on their pattern of reactivity with Bet v I. Indeed four different epitopes on the molecule were identified. Binding inhibition studies using two of them (mAb 5F9 and 8F12) suggested that the epitopes of Bet v I recognized by these mAb are not overlapping. On another hand, the binding of 8H7 and 3F10 was partially inhibited by 5F9 and the binding of 3F10, by 8F12. These data suggest that those two latter epitopes are somewhat overlapping. Finally, the mAb 5F9 could inhibit the binding of human IgE on the affinity-purified Bet v I up to 40% and then shares a common idiotope with human specific IgE Ab of allergic patients.

Characterization of a dominant antigenic determinant of Par o I encoded by recombinant DNA

“…Whereas T-cell epitopes consist of peptidic segments of antigens, peptides representing B-cell dominant epitopes have been rarely described, as antibodies usually recognize tridimensionai structures. As expected, B-ceil epitopes of major allergens from several sources have been reported to be conformational [2][3][4][5][6].…”

“…in frame with betagalactosidase is: gly thr ser scr cys arg leu val (pro). The focusing of antibody response to Par o I and Par J I on a linear epitope is unusual: allergens, as antigens, in general have conformational epitopes [4][5][6][7][8]. residues have been described in the response to viral and protozoan antigens [16][17][18].…”

“…Dominant epitopes have been attributed to the pollen allergens Amha 1 from Amhrosia[2\. Lolp 1. and Poa p 1 from grass [3.4], Sin a I from Siuapis alha [5] and Bet v 1 from birch [6]. These epitopes are defined by mouse MoAbs inhibiting a high proportion of human antibody binding to the respective allergeti.…”

Seasonal enhancement of IL‐4 induced IgE synthesis by peripheral blood mononuclear cells of atopic patients

“…The goat anti-human IgE antiserum (Atlantic Antibodies, Scarborough, ME, U.S.A.) was purified as previously described [28]. The anti-human IgE MoAbs (7H8; 3D 10) were prepared in our laboratory as reported in other studies [29,30]. These MoAbs were purified from mouse ascitic fiuids or culture supernatants by passage through a protein A-Sepharose CL4B column (Pharmacia, Piscataway, NJ, U.S.A.).…”

Monoclonal Antibodies Raised against the Major Apple Allergen, Mal d 1, are Useful Tools for Epitope Studies

“…Monoclonal antibodies (MAbs) are useful tools for the characterization of allergen extracts and for the purification of allergenic molecules (Marc-Series et al, 1990). In addition, they could be used for an epitopic definition (mapping) of the major allergen, which could lead to a better understanding of the molecular basis of allergenicity.…”