Development of a Chemiluminescent ELISA for quantification of Thiram in Honeybees

Here we describe the development of a chemiluminescent enzyme-linked immunosorbent assay (CL-ELISA) based on monoclonal antibodies (MAbs) for the detection and quantification of thiram in honeybees, that permits rapid and sensitive screening of a large number of samples. Thiram (tetramethylthiuram disulfide) [bis(dimethylthiocarbamyl)disulfide] is a wide spectrum nonsystemic dithiocarbamate fungicide, commonly applied in agriculture and horticulture as protectant on foliage and fruits, for seed treatment and as a vulcanizing agent in the rubber industry. Thiram is also present in the environment as an oxidation product of two other widely employed fungicides, ferbam and ziram [1], and it can persist in soil for several months. The use of pollinators, especially honeybees, as biological indicator is due to their capacity to reveal chemical impairment of the environment both by an higher mortality and retaining particles suspended in the air or on the plants. Various quantitative methods for thiram have been described, among them the ELISA [2] that has been the base of our research work. The assay was optimised and characterised by determining several parameters such as immunoreagents optimum concentrations, incubation time, tolerance to organic solvents and cross reactivity for thiram derivates and other. We estimated the sensitivity, as the IC(50) value and the detection limit (LOD) value. The assay was then applied to the analysis of honeybees samples fortified with thiram and of real honeybees samples collected in Italy and Russia. Two different extraction procedure were tested, a liquid-liquid extraction and a graphitized carbon-based solid-phase extraction. [1]. Lowen, WK. Determination of thiram in ferbam. J Assoc Off Anal Chem 1961; 44:584–589. [2]. Gueguen, F et al. Hapten synthesis for the development of a competitive inhibition enzyme-immunoassay for thiram. J Agric Food Chem 2000; 48:4492-4499.