Detection of Penicillium aurantiogriseum by ELISA utilizing antibodies produced against its exoantigens

An indirect competitive ELISA was developed using rabbit antiserum against the exoantigens (ExAgs) of Penicillium aurantiogriseum, and cross-reactivities we redetermined with 15 ExAgs from other species and genera of fungi and with the water-soluble extracts from four grains. The assay had good sensitivity to P. aurantiogriseum ExAgs (1 micrograms per ml serum), with generally low or no cross-reactivity with grain extracts and the other fungi, including five species of Penicillium, four species of Aspergillus, three species of Fusarium, two species of Mucor and one species of Alternaria. Immunoblotting analysis of the ExAgs from P. aurantiogriseum yielded at least 20 distinct antigenic bands, with about 3 or 4 being dark, 10-12 light and 5-8 faint in colour. Most of the bands had molecular masses of about 70-90 kDa. In contrast to P. aurantiogriseum, the ExAgs from the other fungi either did not react in the immunoblotting assay with the antiserum, or reacted only weakly with it. One antigenic band from P. roqueforti, however, reacted moderately strongly with the antiserum. The ELISA and immunoblotting analysis were used to detect P. aurantiogriseum in naturally contaminated wheat samples, and in wheat samples spiked with a second common storage fungus, Aspergillus ochraceus. The results demonstrated that the antiserum was able to detect P. aurantiogriseum in a background of other fungal species, and that these did not produce false positives. There was also a positive association between the concentration of P. aurantiogriseum ExAgs, as measured by ELISA, and the amount of mould as determined by the number of colony-forming units. Immunoblotting qualitatively confirmed the ELISA results obtained with fungi when cultured on liquid or on solid (wheat) media. The data suggest that the immunoassays developed for P. aurantiogriseum are useful for the detection and identification of P. aurantiogriseum on the basis of its ExAgs, with the advantages of being more efficient, simple and reliable than conventional techniques.