Identification and expression of an allergen Asp f 13 from Aspergillus fumigatus and epitope mapping using human IgE antibodies and rabbit polyclonalantibodies

The Aspergillus genus of fungi is known to be one of the most prevalent aer oallergens. On two-dimensional immunoblotting using patients' sera containi ng IgE specific for Asp f 13, an allergen with a molecular mass of 33 kDa a nd a pI of 6.2 was identified. This allergen was also present in A. fumigat us culture filtrates. Furthermore, the sequence of the Asp f 13 cDNA was id entical to that for alkaline protease isolated from A. fumigatus and showed 42-49 %, identity of amino acids with two proteases from P. cyclopium and T. album and with the Pen c 1 allergen from P. citrinum. Asp f 13 coding se quences were expressed in Escherichia coli as a [His](6)-tagged fusion prot ein which was purified by Ni2+-chelate affinity chromatography. Recombinant Asp f 13 was recognized by rabbit polyclonal antibodies against Asp f 13 a nd by IgE antibodies from subject allergic to A. fumigatus. To identify and characterize the linear epitopes of this allergen, a combination of chemic al and enzymatic cleavage and immunoblotting techniques, with subsequent N- terminal sequencing and mass spectrometry, were performed. At least 13 diff erent linear epitopes reacting with the rabbit anti-hsp f 13 antiserum were identified, located throughout the entire molecule. In contrast, IgE from A. fumigatus-sensitive patients bound to three immunodominant epitopes at t he C-terminal of the protein.