IgE recognition of multiple novel Api m 10 isoforms evaluated by protein array technology

Abstract

Background: Until now, two splice variants of the honeybee venom allergen Api m 10 have been described. Variant 2 exhibits IgE reactivity with approximately 50% of honeybee venom sensitized patients. Our proteomic analysis of honeybee venom gave an indication for the existence of additional Api m 10 isoforms. As such, further research was needed to identify these isoforms and investigate the impact of protein heterogeneity on IgE recognition. Method: Novel isoforms were searched by sequence analysis of cloned RT-PCR amplicons. All isoforms were produced as synthetic peptides or aglycosylated recombinant proteins and were spotted on nitrocellulose-coated glass slides. The colorimetric protein array technology was used to test IgE reactivity of the complete panel of isoforms using sera of 22 Api m 10 sensitized patients. Results: Nine additional Api m 10 isoforms were found, which derive from the same genomic locus by a complicated alternative splicing. Some truncated isoforms are produced by frameshifts which introduce an alternative stop codon. All 11 variants were obtained as synthetic peptides or purified bacterial recombinants. The colorimetric protein array showed differential IgE reactivity of different Api m 10 isoforms and allowed to predict an IgE epitope. Conclusion: Differential IgE reactivity of eleven Api m 10 isoforms was investigated using the colorimetric protein array technology. This approach shows some major benefits for testing IgE reactivity of a broad antigen panel. First, compared to many other technologies, only very low amounts of serum (25 µl) are needed. Second, in contrast to fluorescent arrays, the colorimetric signal detection allows the use of much cheaper scanners