Detection of Total Ergot Alkaloids in Cereal Flour and in Bread by a Generic Enzyme Immunoassay Method.

Four sets of polyclonal antibodies against ergot alkaloids ergometrine, ergotamine, α-ergocryptine, and ergocornine were produced and characterized in a competitive direct or indirect enzyme immunoassay (EIA). Standard curve LODs were 0.03 ng/mL (ergometrine EIA) to 2.0 ng/mL (ergocornine EIA). Three EIAs were highly specific, whereas the ergometrine EIA had a broad specificity pattern and reacted, albeit weakly, with all seven major ergot alkaloids and their epimeric forms. Using the ergometrine EIA, a generic test system was established in which total ergot alkaloids are quantified by a standard curve for a toxin mixture composed of three alkaloids that matched the ergot alkaloid composition in naturally contaminated rye and wheat products. Sample extraction with acetonitrile-phosphate-buffered saline at pH 6.0 without further cleanup was sufficient for EIA analysis. The LODs for total ergot alkaloids were 20 ng/g in rye and wheat flour and 14 ng/g in bread. Recoveries were 85-110% (RSDs of 0.1-11.7%) at a concentration range of 50-1000 ng/g. The total ergot alkaloid EIA was validated by comparison with HPLC-fluorescence detection. Although some under- and overestimation by the total ergot alkaloid EIA was observed, it was suitable for the reliable identification of positive samples at 10-20 ng/g and for the determination of total ergot alkaloids in a concentration range between 100 and 1000 ng/g.

Vaccination against ergot alkaloids and the effect of endophyte-infected fescue seed-based diets on rabbits.

Three sequential experiments were conducted with rabbits to 1) determine the effect of endophyte-infected (E+) tall fescue seed on rabbit performance and examine the effect of anti-ergot alkaloid immunization on rabbit performance and protectiveness against fescue toxicosis, 2) compare immunogens designed to elicit systemic anti-ergot alkaloid antibodies, and 3) select a superior adjuvant. In Exp. 1, rabbits (n = 6/treatment) fed E+ fescue seed diets (20%, 340 ppb total ergot alkaloids) had reduced (P < .05) intake and weight gain compared with endophyte-free (E-) controls, whereas apparent diet digestibility was not different between E+ and E-. Rabbits immunized against ergot alkaloids (E+ vac) with lysergol conjugated to human serum albumin (Ly-HSA) had greater (P < .05) intake than E+ rabbits during the wk 1 of a 3-wk dietary challenge. In Exp. 2, rabbits (n = 4/treatment) were immunized with Ly-HSA, with H100-B (ergot alkaloid hapten, H100-different protein carrier, B conjugate), or combinations of both with alum as adjuvant. Greatest (P < .001) anti-ergot alkaloid antibody (Ab) titer developed in the group immunized with H100-B. In Exp. 3, rabbits (n = 4/treatment) were immunized with the immunogen H100-B in conjunction with six adjuvants. Freund's incomplete adjuvant (FIA) in combination with DEAE-dextran and FIA alone gave highest anti-ergot titers. In summary, rabbit weight gain and intake were reduced by feeding E+ fescue seed diets, immunization against ergot alkaloids provided temporary improvement in intake, and H100-B conjugate with FIA or FIA + DEAE-dextran as adjuvants elicited a superior anti-ergot immune response. We believe that rabbits may serve as a model animal for fescue toxicosis research.

Antibody binding of circulating ergot alkaloids in cattle grazing tall fescue.

Direct evidence linking alkaloids found in endophyte-infected tall fescue forage with the livestock disorder known as fescue toxicosis is lacking. Physiologic effects of fescue toxicosis include reduced serum prolactin concentration in cattle. A monoclonal antibody specific to the lysergic moiety of ergot alkaloids was developed in mice after creating an immunogen by linking lysergol to human serum albumin. The antibody was specific to the lysergic moiety and, therefore, it cross-reacted with ergot alkaloids, lysergic acid, and lysergol. The antibody did not cross-react with alkaloid derivatives that had bromated or hydrogenated lysergic ring moieties. Fescue toxicosis conditions were elicited in yearling Angus steers by permitting them to graze endophyte-infected tall fescue containing > 650 micrograms/kg of ergovaline for 60 days. Passive immunization of steers by infusion of the monoclonal antibody increased serum prolactin concentration by 7 ng/ml, beginning immediately after infusion. Control steers did not respond to treatment with bovine serum albumin. Active immunization of yearling Angus heifers with immunogens containing lysergol or ergonovine linked to human serum albumin resulted in an antibody response.

[The effect of hydrogenated ergot alkaloids on immune functions. II. The primary antibody response and proliferative activity in mouse spleen cells in vitro]. / Vliv hydrogenovaných námelových alkaloidu na imunitní funkce. II. Primární protilátková odpoved' a proliferacní aktivita mysích slezinných bunek in vitro.

The present paper investigated the proliferative response of murine splenic cells in the tissue culture after stimulation with the polyclonal activators PHA, ConA and PWM. Proliferation of cells and DNA synthesis were measured by means of radioactively labelled thymidine. Furthermore, the primary antibody response of murine splenic cells in the tissue culture after activation with sheep red blood cells was examined. The response was measured by determining the number of plaque forming cells (PFC). The number of PFC's was determined by the plaque method of local haemolysis, in modification by the drop method. In these tests the ergot alkaloids dihydroergocornine, dihydroergocristine, dihydro-alpha-ergocryptine and dihydro-beta-ergocryptine were evaluated in two doses, 600 and 1200 micrograms/kg/day p. o. for the period of 14 days. Dihydroergocornine in these tests increased the primary antibody response in a statistically significant manner; on the other hand, after its administration in the higher dose decreased proliferation after PHA was found. The finding after dihydroergocristine administration was of interest. This ergot alkaloid slightly increased proliferation of cells after PHA and PWM as well as the primary antibody response of murine cells. After the administration of dihydro-alpha-ergocryptine increased proliferation after stimulation with PWM and increased primary antibody response, and after the administration of dihydro-beta-ergocryptine increased proliferation of cells after stimulation with PHA were found.