Isolation, purification and characterization of a protein from indoor strains of Wallemia sebi that is antigenic to humans

The purpose of this project is to isolate and characterize a human allergen from the moderate xerophile Wallemia sebi. This obscure fungus turns out to be very common in some homes all over the world. It is known that it causes allergic responses in sensitized individuals, but until now, the identity of the major allergenic protein was unknown. The second purpose is to develop antibodies for its detection in house dust. The quantification of human allergen (antigen) levels can be helpful in studying allergic responses and performing exposure assessments. Human sera obtained from atopic patients showing fungal sensitization were screened against crude W. sebi extracellular protein extracts by Western Immunoblot and Enzyme-linked immunosorbant assays (ELISA). Based on its immunogenic response to human polyclonal antibodies, a 47kDa dimer was recognized as antigenic and chosen as the target protein for this work. This protein was purified by fast protein liquid chromatography (FPLC) and further characterized by liquid chromatography tandem mass spectrometry (LC-MS/MS), isoelectric focusing, and by a glycoprotein assay. The antigenicity of the 47kDa protein was confirmed by its ability to produce polyclonal antibodies in rabbits. Capture ELISA and Western immunoblot with the purified 47kDa protein, the Wallemia arthrospore protein extracts and the arthrospore-spiked fine dust confirmed that the polyclonal antibody was capable of detecting the targeted W. sebi antigen. Cross-reactivity Capture ELISA tests using spore protein extracts from various indoor fungi showed that the rabbit polyclonal antibodies produced were specific to the W. sebi target protein. Subsequent research will involve producing monoclonal antibodies to the selected W. sebi antigen to develop an immunoassay for W.sebi detection in the indoor environment.