Development of immunoaffinity columns and immunoassays for the collection, partial purification and measurement of sporidesmin A and its metabolites
Permanent link to Research Commons versionhttps://hdl.handle.net/10289/15417
Monoclonal and polyclonal antibodies were produced for the development of competitive enzyme-linked immunosorbent assays (cELISA) for sporidesmin A, the mycotoxin that causes facial eczema in grazing livestock. Derivatives of sporidesmin conjugated to a number of proteins were used as immunising antigens and plate-coating antigens for cELISA. Two immunoassays, each detecting the presence of a different region of the sporidesmin A molecule have been developed. They allow the two major sporidesmins found in grass or in Pithomyces chartarum cultures to be distinguished. Organic solvent extraction of samples was avoided so that water-soluble metabolites could also be detected. To assist in the selection of animals resistant to facial eczema, the cELISAs were applied to a study of the metabolism of sporidesmin A in sheep. The cELISAs distinguish between sporidesmin A and its metabolites, as one antibody detects only sporidesmin A and the other detects sporidesmin A and metabolites. After sheep were dosed with sporidesmin A it was possible to monitor the excretion of the toxin in bile and its metabolite(s) in both bile and urine using the appropriate cELISA. There were no differences between resistant and susceptible sheep in the cumulative totals or excretion rates of immunoreactive metabolite in animals dosed with sporidesmin A. The detection of sporidesmin metabolites in sheep urine by cELISA was shown to have potential as a biomarker for measuring exposure to toxic pastures which could give early warning of the risk of facial eczema. An immunoaffinity chromatography matrix was prepared by coupling antibody to cyanogen bromide-activated Sepharose. The matrix bound sporidesmin A and immunoreactive metabolites from urine, and bound analytes were eluted with 40% methanol in water. The matrix capacity for sporidesmin A was 900 ng of sporidesmin A bound per mL of matrix, and for sporidesmin metabolites from urine it was approximately 500 ng of sporidesmin A immunoreactive equivalents per mL of matrix. A method was developed for the preparation of milk for immunoaffinity chromatography so that blocking of columns by milk fats was avoided. Sample concentration obtained using the immunoaffinity chromatography lowered the limit of quantification for the previously developed cELISA for sporidesmin A in milk by a factor of 100. A trial was carried out to determine whether sporidesmin residues appeared in milk following experimental ingestion of the toxin. Using the cELISA, sporidesmin metabolites were detected in milk samples collected after dosing. Further evidence for the presence of immunoreactive material was obtained by using immunoaffinity chromatography to recover the material from milk. Positive results in the sporidesmin A specific cELISA suggest the presence of free sporidesmin A at picogram levels. Purification from a much larger sample, and analyses by HPLC and mass spectrometry are needed to confirm this result. It should now be possible to use the anti-sporidesmin antibodies in immunochemical technologies and formats which are suitable for on-farm use. These could be used to help minimise exposure to sporidesmin A and provide an important advance in animal health.
The University of Waikato
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