Allergenic and antigenic determinants of latex allergen Hev b 1: peptide mapping of epitopes recognized by human, murine and rabbit antibodies.

BACKGROUND

The rubber elongation factor in Hevea rubber (Hev b 1) is one of the most important latex allergen and is leading cause of latex type 1 hypersensitivity in children with spina bifida.

OBJECTIVE

The aim of this study was to define the allergenic and antigenic epitopes of Hev b 1.

METHODS

The immunoglobulin- (Ig)E and IgG antibody binding sites on Hev b 1 allergen were delineated by enzyme linked immunosorbent assay (ELISA) using synthetic overlapping peptides covering the whole Hev b 1 sequence. In order to improve the binding capacity and specificity all peptides were biotinylated at the N-terminal end via a 6-aminohexanoic acid as spacer and then adsorbed to streptavidin pre-coated microtitre plates. Fine mapping to define the essential amino acid residues for the antibody binding was achieved by using overlapping peptides with one amino acid offset.

RESULTS

It was demonstrated that the IgE epitopes were located in different regions of Hev b 1 including the C-terminal segment (121-137) and the segments with amino acid residues of 30-49 and 46-64. Two monoclonal antibodies (MoAbs) II2F3 and II4G9 raised against purified Hev b 1 recognized the C-terminal segment only. The results of epitope mapping with three rabbit antisera revealed that five positive peptides, including the epitope peptides 31-49, 46-64 and 121-137, were involved in the antibody-binding sites. Fine mapping on the segments 46-64 and 121-137 showed that the two MoAbs reacted with the peptide 125-134 in the C-terminal region, whereas the peptide with amino acids 124-134 was essential for recognition by human IgE antibodies. Epitopes to rabbit polyclonal IgG and human IgE were also found to be involved in the amino acid residues of 47-59.

CONCLUSION

Our results indicate that the most allergenic/antigenic portions of Hev b 1 allergen are the C-terminal region and the region with amino acid residues of 31-64. In both regions, the minimal IgE-binding epitope is almost identical with the IgG-binding epitope.