Development of an immunoassay (ELISA) for the quantification of thiram in lettuce.

Two competitive immunoassays, a laboratory assay based on microwell plates and a field test based on the use of polystyrene tubes, have been developed for the quantification of thiram in lettuces. Concerning the laboratory assay, the calibration curve for thiram had a linear range of 11 to 90 ng/mL and a detection limit of 5 ng/mL. Precision of the assay presented coefficient of variation values <9% and the recovery of thiram from lettuce averaged 89% across the range of the immunoassay method using 30 min extraction with water/acetone (50:50, v/v). The tube-based method was developed in order that an extract of lettuce, containing thiram at the MRL (8 ppm), would be found on the linear part of the standard curve. The calibration curve for thiram has a linear range of 100 to 800 ng/mL (1.39 to 11.1 ppm in lettuce) and a detection limit of 40 ng/mL.

Hapten synthesis for the development of a competitive inhibition enzyme-immunoassay for thiram.

An enzyme-linked immunosorbent assay (ELISA) was developed for the fungicide thiram. Two types of haptens were synthesized. The first type exhibits the two symmetrical N-alkyl dithiocarbamate patterns of thiram with a spacer arm linked to one of the N-methyl terminal group. The second type exhibits one of the two symmetrical N-alkyl dithiocarbamate patterns of thiram with a variable-length spacer arm linked to one sulfur atom. Polyclonal antibodies suitable for thiram detection were obtained from immunization with an hapten of the first type, while haptens of the second type were used as coating antigens to develop a competitive ELISA against thiram. The IC(50) value for thiram was estimated to be 0.24 microg/mL, with a detection limit of 0.03 microg/mL. The assay seems to be thiram-specific since no or little cross-reaction with other dithiocarbamates were observed.