Development Of An Enzyme-linked Immunosorbent Assay For Pyrethroids

As a class of broad spectrum pesticides, pyrethroids were widely used in agriculture, and caused pyrethroids residues in plants and agricultural products affected human health. In this study, based an IC-ELISA established and optimized, an ELISA kit for deltamethrin residue determination was developed. Some researches was carried out on enzyme-linked immunoassay assay for pyrethroids muti-residues.An IC-ELISA for deltamethrin residue was developed. The optional working dilution of coated antigen and antibody were all 1:12800. When plates coated 37℃3 h or 4℃overnight after 37℃3 h and OPD reacted for 15 min, the sensitivity was best. Besides, the buffer solution used in ELISA with 10% methanol, pH 7.4,0.1 mol·L-1 NaCl were determined. Base on these reaction conditions, inhibition curver of deltamethrin was tested with linear range from 0.02μg·mL-1 to 100μg·mL-1. The formula was y=0.18x-0.1 483, R2=0.9848, IC50=4.00μg·mL-1.An ELISA kit for deltamethrin was developed. The technique parameters of the kit were tested with apple samples, as follows:IC50 ranged from 4.00μg·mL-1 to 7.71μg·mL-1, the limits of detection ranged from 0.01 mg·kg-1 to 0.02 mg·kg-1. When the spiked concentrations of deltamethrin were 0.1 mg·kg-1,0.5 mg·kg-1 and 1.0 mg·kg-1 in apple, the recovery of deltamethrin was 86.2～105.8% with coefficients of variation of 6.0-9.8%. There was low cross reactivity with other compounds structurally related to deltamethrin. It is indicated that the kit could be reserved for 6 months at 2-8℃.To develop the multi-residue analysis of pyrethroids, a new hapten was synthesized with 4-hydroxymethyl benzoic acid and 2,2-dimethyl-cyclopropane carboxylic acid. The hapten was covalently conjugated to carrier proteins BSA and OVA by the method of NHS. The conjugates were confirmed by UV scanning. Polyclonal antibody against hapten was successfully obtained by immunization of New Zealand white rabbits with artificial antigens Hapten-BSA. The highest titer of the antiserum was 51200. As coating antigen, Hapten-OVA was used to establish IC-ELISA. The dilution of the coating antigen and the antibody used in the ELISA were 1:6400 and 1: 12800 respectively. Furthermore, the buffer solution used in ELISA with 10% methanol, pH 7.4,0.1 mol·L-1 NaCl were also determined.Immunity-determination region of pyrethroids was proved with a series of specific experiments. The results showed that the hapten for pyrethroids must include benzene ring and ternary ring. In order to improve the sensitivity, adding cyanide ion, halogen and carbon chain are necessary, which in favor of ELISA for pyrethroids to be used in the actual production.