Development Of An Enzyme-Linked Immunoassay For Isoproturon

A competitive-indirect enzyme-linked immunosorbent assay (CI-ELISA) was developed for isoproturon.1. Two haptens of isoproturon were synthesized. One is hapten 4C [1-(3-carboxypropyl)-3-(4-iso–propylphenyl)-1-methylurea] and the other is hapten6C [1-(5-carboxypentyl)-3-(4-isopropylphenyl)-1-methylurea] synthesized. N-methyl-γ-aminobutyric acid was synthesized by hydrolysis of N-methylpyrrolidone, and reacted with p-isopropylphenylisocyanate to get hapten 4C. 6-methylaminohexanoic acid was synthesized by hydrolysis of N-methylcaprolactam, and reacted with p-isopropylphenylisocyanate to get hapten 6C. The two haptens were identified with TLC, IR and NMR. The hapten 4C was conjugated to ovalbumin (OVA) as the coating antigen. The hapten 6C was conjugated to bovine serum albumin (BSA) as the immunol antigen. The ratios of hapten 4C-OVA and hapten 6C-BSA were 36:1 and 46:1, respectively.2. The polyclonal antibody against isoproturon was preparated. Three New Zealand rabbits (A4746, A5051 and A5205) were immunized with immunol antigen. The results indicated that A4746, A5205 and A5051 elicited special antibodies to isoproturon. The titers are 160000, 20000, and 80000, respectively.3. The ELISA sensitivity of the two different coating antigens, hapten 4C-OVA and hapten 6C-OVA, were compared. The IC₅₀ of hapten 4C-OVA was 70 ng/mL and hapten6C-OVA was 500 ng/mL. Hapten 4C-OVA was used as the coating antigen.4. A CI-ELISA for isoproturon was developed. The linear working range of the assay was from 10 to 500 ng/mL. The regression equation was I=(22.888+ 0.043C^0.744)/(24.067+C^0.744). The IC₅₀ of the assay was 70 ng/mL. The limit of detection was 5 ng/mL. The cross reactivity for chlorbromuron, monolinuron, tebuthiuron, thidiazuron, neburon, and fluomethuron were all less than 0.1%.5. The developed ELISA method was applied to detect isoproturon in water and soil. The recovery rate in the water samples was 90%-115% and the coefficient of variation was 1.75%-7.02%. The recovery rate in the soil samples was 117%-160% and the coefficient of variation was 3.10%-6.65%.