Development of Immunoassay Based on Monoclonal Antibody Reacted with the Neonicotinoid Insecticides Clothianidin and Dinotefuran

Abstract

Enzyme-linked immunosorbent assay (ELISA) based on a monoclonal antibody (MoAb) was developed for the neonicotinoid insecticide clothianidin. A new clothianidin hapten (3-[5-(3-methyl-2-nitroguanidinomethyl)-1,3-thiazol-2-ylthio] propionic acid) was synthesized and conjugated to keyhole limpet hemocyanin, and was used for monoclonal antibody preparation. The resulting MoAb CTN-16A3-13 was characterized by a direct competitive ELISA (dc-ELISA). The 50% of inhibition concentration value with clothianidin was 4.4 ng/mL, and the working range was 1.5–15 ng/mL. The antibody showed high cross-reactivity (64%) to dinotefuran among the structurally related neonicotinoid insecticides. The recovery examinations of clothianidin for cucumber, tomato and apple showed highly agreement with the spiked concentrations; the recovery rate was between 104% and 124% and the coefficient of variation value was between 1.8% and 15%. Although the recovery rate of the dc-ELISA was slightly higher than that of HPLC analysis, the difference was small enough to accept the dc-ELISA as a useful method for residue analysis of clothianidin in garden crops.

Development of an Immunosensor Based on Surface Plasmon Resonance for Simultaneous Residue Analysis of Three Pesticides —Boscalid, Clothianidin, and Nitenpyram— in Vegetables

“…The applicability of the SPR immunosensor was confirmed by comparing the results obtained from the immunosensor with those of the individual dcELISAs, which, except for nitenpyram, had been evaluated by previous studies. 18,20 As shown in Fig. 5, the SPR immunosensor results correlated highly with those of the dcELISAs: R 2 = 0.98 for boscalid, R 2 = 1.00 for clothianidin, and R 2 = 0.98 for nitenpyram, with a slight bias of their slope: 0.77 for boscalid, 1.27 for clothianidin, and 1.12 for nitenpyram.…”

“…20 The anti-clothianidin MoAb and anti-nitenpyram MoAb were provided from Horiba Ltd. (Kyoto, Japan). 18 All other chemicals and reagents used were of analytical grade, and purchased from Wako Pure Chemical Industries, Ltd. or Nacalai Tesque, Inc. (Kyoto, Japan).…”

“…The haptens for boscalid and clothianidin were synthesized as described previously. 18,20 On the other hand, the synthesis method for nitenpyram is described in detail in Supporting Information 1: "Synthesis of nitenpyram hapten" with the scheme in Fig. S1.…”

“…30 Thus, we believed that the concept would be effective to develop a simultaneous SPR immunosensor. The clothianidin hapten was synthesized based on the published structure, 18 and then the linker for the nitenpyram hapten was introduced at the opposite side from clothianidin (Fig. 1).…”

“…Direct competitive enzyme-linked immunosorbent assays (dcELISAs) were developed for the monitoring of pesticide residues in vegetables as more simple and rapid methods compared to the above chromatography techniques, but of the single pesticide although simultaneous analysis is required in the field. [18][19][20] The development of simultaneous dcELISA for three pesticides was described previously. 21 Its reactivity was specific with each pesticide, but was difficult to put into practical use because optimization of the assay condition was complicated.…”