Use of enzyme immunoassays to quantify total fungi and possibly the genera Aspergillus, Penicillium and Fusarium in cereal samples

Abstract

A double antibody sandwich enzyme-linked immunosorbent assay (DAS ELISA) was constructed using an antiserum raised against Penicillium aurantiogriseum var. melanoconidium. The assay protocol was selected and preliminary optimisation carried out using a pure preparation of the homologous fungus (i.e. fungus against which antibodies (antisera) had been raised) as test material. Reproducibility was monitored. Intra-plate variation was acceptable but inter-plate variability was poor, suggesting that the method needs further improvement. Preliminary investigations into the sensitivity of the test for other fungi were carried out using mycelial preparations of fungi grown in synthetic liquid medium.

Results indicated greater and preferential detection of the three major groups of storage fungi (Aspergillus, Eurotium and Penicillium) over field fungi. The assay was, however, unable to detect spores grown in the laboratory on synthetic medium. Samples from spiked and unspiked barley were also used as test material. The assay was unable to detect the homologous fungus although present as both mycelium and spores from spiked barley. Further, the results showed that a high cross-reactivity was apparent with unspiked barley extracts.

The work carried out has allowed basic assay parameters and methodology to be set but clearly the assay requires further refinement in order to identify fungi from commodity. Further work is necessary to identify more suitable antibodies for utilisation in the DAS ELISA and/or to identify other assay designs which work better than the DAS ELISA.