Quantification of Imidacloprid in Honeybees: Development of a Chemiluminescent ELISA

A Chemiluminescente Enzyme-Linked Immuno-Sorbent Assay (CL-ELISA) for determination and quantification of the fungicide imidacloprid in honeybees was developed in an indirect competitive format. The assay was optimized by determining: the optimal coating conjugate concentration and anti-imidacloprid antiserum dilution, the effect of the incubation time on the competitive step, the tolerance to organic solvents. The IC50 and the limit of detection (LOD) values were 14.8 ng mL-1 and 0.11 ng mL -1, respectively, similar to those of colorimetric ELISA with a calibration range of 0.1 – 2600 ng mL-1. Cross reactivity of some related compounds such as three imidacloprid metabolites, 6-chloro nicotinic acid, 5-hydroxy-imidacloprid and imidacloprid olefin and one other chloronicotinoid insecticide, acetamiprid were tested. The assay was then applied to honeybee extracts obtained by using the liquid-liquid extraction. The calibration curves in honeybee extracts from liquid-liquid procedure gave an IC50 of 23.7 ng mL-1 and a LOD 1.6 ng mL-1. The average recovery value from honeybee extracts spiked with 100 and 1000 ng mL-1 of imidacloprid were 73% and 76% respectively. Finally, the assay was applied to honeybee samples collected during monitoring activities in Italy. Finally, the assay was applied to honeybee samples collected during monitoring activities in Italy and was founded that only five of the 27 samples were positives, with low concentrations of imidacloprid ranging between 1.2 and 15.4 ng g-1.