Detection of Endophyte Toxins by ELISA Assay

Abstract

Endophytic fungi are associated with many common pasture grasses, in an association beneficial to both organisms, with endophyte-infected grasses demonstrating greater persistence under environmental stress, than endophyte-free grasses. However, this agronomic advantage is obtained at the expense of animal health. Perennial ryegrass staggers (RGS), common in New Zealand, and fescue toxicoses (FT), prevalent in the eastern USA, are the most widely recognised examples of such toxicoses. The compounds responsible for the toxicity of these grasses have been widely studied. A number of these toxins have been conjugated to protein, and used to raise antibodies capable of binding the toxin, and closely related structures, with high specificity for use in immunoassays such as ELISA for detection and quantitation of toxin. The ELISA, performed in 96-well PVC microtitre plates, typically requires only 50 μL sample per well, and has a detection limit in the pg/ml to ng/ml range. The ELISA is generally able to cope with crude extracts without extensive (and expensive) cleanup. The low cost and high throughput that this permits has allowed researchers to analyse samples from large-scale field trials. Immunological techniques have been used in the study of toxin accumulation in localised plant tissue, and for screening of endophytes for toxin production in vitro. They have also been used to probe plant material for compounds structurally related to the toxins to give an insight into the biosynthetic pathways of toxin synthesis. This paper details the production of antisera, and development and application of ELISA for endophyte toxins.