Semiquantitative Determination of Ergot Alkaloids in Seed, Straw, and Digesta Samples Using a Competitive Enzyme-Linked Immunosorbent Assay

Abstract

Abstract. Ergot alkaloids present in endophyte-infected (Eϩ) tall fescue cause fescue toxicosis and other toxic effects in livestock that consume infected plant tissue, leading to significant financial losses in livestock production each year. The predominant method currently in use for quantifying ergot alkaloid content in plant tissue is through high-performance liquid chromatography (HPLC), which quantifies the amount of ergovaline, one of many ergot alkaloids in Eϩ plant tissue. The enzyme-linked immunosorbent assay (ELISA) method used in this study detects quantities of nonspecific ergot alkaloids and therefore accounts for greater amounts of the total ergot alkaloid content in Eϩ tissue than does HPLC. The ELISA can also be used to more expediently analyze a larger number of forage samples without sophisticated and costly analytical equipment and therefore could be more desirable in a diagnostic setting. The purpose of this study was to evaluate the between-day and within-run variability of the ELISA and to determine the binding efficiency of 6 ergot alkaloids to the 15F3.E5 antibody used in the competitive ELISA to ascertain its feasibility as a quick analysis tool for ergot alkaloids. Straw samples had an average coefficient of variation (CV) for concentration of 10.2% within runs and 18.4% between runs, and the seed samples had an average CV for concentration of 13.3% within runs and 24.5% between runs. The grass tissue-based lysergic acid standard curve calculated from the ELISA had an average r 2 of 0.99, with a CV of 2.1%. Ergocryptine, ergocristine, ergocornine, and ergotamine tartrate did not bind strongly to the 15F3.E5 antibody because of the presence of large side groups on these molecules, which block their binding to the antibody, whereas ergonovine and ergonovine maleate were bound much more efficiently because of their structural similarity to lysergic acid. Clarified rumen fluid was tested as an additional matrix for use in the ergot alkaloid competitive ELISA to determine whether future livestock metabolism experiments on the postingestion fate of ergot alkaloids in ruminants could utilize this assay as a quick screening tool for the presence of nonspecific ergot alkaloids in rumen fluid. HPLC and ELISA procedures were compared for their ability in determining ergot alkaloid toxicity based on the repeatability of the procedures and on the specific compounds they measure. The ratio of ELISA concentration to HPLC concentration (ergovaline) varied from 2.00 to 2.81 in seed samples and from 0.62 to 8.66 in straw samples, showing no consistent pattern between the 2 methods. Based on the lack of data at present for the identity of the toxin causing endophyte toxicosis and the lack of agreement between the ergovaline HPLC and ELISA analyses for ergot alkaloids, each method is equally valid as an indicator of toxicity and is the best means for determining the quantity of the specific toxin(s) they measure.Tall fescue (Festuca arundinacea) plants are often infected with the endophyte Neotyphodium c...

Analysis and Modification of Ergot Alkaloid Profiles in Fungi

“…In addition to the instrumental approaches described here, an immunological approach has been successful for detecting and quantifying ergot alkaloids in aggregate (e.g., Hiatt, Hill, Bouton, & Stuedemann, 1998;Hill, Thompson, Dawe, & Stuedemann, 1994;Schnitzius, Hill, Thompson, & Craig, 2001). In this case, the antibody was raised against lysergol coupled to human serum albumin and cross reacts with ergovaline and a variety of other ergot alkaloids.…”

BOARD-INVITED REVIEW: St. Anthony's Fire in livestock: Causes, mechanisms, and potential solutions1,2

“…This method has been successfully used to measure total ergot alkaloid concentrations in plant and fungal tissues Kelley, 1990, 1992;Hill and Agee, 1994;Schnitzius et al, 2001), digesta (Hill and Agee, 1994), urinary and biliary excretions (Stuedemann et al, 1998;Hill et al, 2000), and animal fat (Realini et al, 2005). However, the method suffers from a lack of selectivity in that it does not distinguish one ergot alkaloid from another.…”

“…The ELISA method Kelley, 1990, 1992;Hill and Agee, 1994;Schnitzius et al, 2001) excels in sensitive and rapid analysis for total ergot alkaloids. This method has been successfully used to measure total ergot alkaloid concentrations in plant and fungal tissues Kelley, 1990, 1992;Hill and Agee, 1994;Schnitzius et al, 2001), digesta (Hill and Agee, 1994), urinary and biliary excretions (Stuedemann et al, 1998;Hill et al, 2000), and animal fat (Realini et al, 2005).…”

Relationships among ergot alkaloids, cytochrome P450 activity, and beef steer growth

“…However, the ELISA assay lacks selectivity as it does not differentiate between ergot alkaloids which have necessitated the use of chromatographic methods to detect specific ergot alkaloids (Schultz et al, 2006; De Lorme et al, 2007; Foote et al, 2012; Wang et al, 2013). Schnitzius et al (2001), compared HPLC and ELISA detection of ergot alkaloids and found the methods were inconsistent in their identification of alkaloids, concluding that both had their advantages and disadvantages. Antigen (ergot alkaloids) detection is restricted to the specificity of the antibody used in the ELISA test.…”