Determination of citrinin in barley by indirect and direct enzyme immunoassay

Polyclonal antibodies against the mycotoxin citrinin were raised in rabbits after immunization with citrinin conjugated to keyhole limpet hemocyanin. The antibodies were used in a competitive indirect enzyme immunoassay (EIA) with citrinin coupled to glucose oxidase as the solid-phase antigen for coating microtiter plates. Detection limits of this indirect EIA for citrinin ranged from 0.4 to 0.8 ng/mL for buffer solutions. Recoveries of citrinin added to ground barley at 100-2000 ng/g ranged from 105 to 112%, with coefficients of variation between 4.5 and 12%. A direct competitive EIA also was established, with citrinin coupled to horseradish peroxidase as the labeled antigen. Detection limits of this direct EIA for citrinin ranged from 2 to 4 ng/mL for buffer solutions. Recoveries of citrinin added to ground barley at 500-2000 ng/g ranged from 108 to 111%, with coefficients of variation between 8.4 and 26.9%. In naturally contaminated barley samples assayed with the indirect EIA, optimum extraction of citrinin was obtained in 30 min, and only one extraction was necessary to recover 72-76% of the analyte.