Development Of Enzyme-liked Immunosorbent Assay (ELISA) For The Determination Of Phenylurea Herbicide Diuron

A direct competitive enzyme-linked immunosorbent assay (dcELIS A) was developed for the determination of phenylurea herbicide diuron. The content of this paper includes happen synthesis, preparation of polyclonal antibody against diuron, development of ELISA for the determination of diuron, recovery study, HPLC verification and real water sample analysis.Two haptens-hapten4C [1-(3-Carboxypropyl)-3-(3,4-dichlorophenyl)-1-methylurea] and hapten6C [l-(5-Carboxypentyl)-3-(3,4-dichlorophenyl)-1-methy-lurea] were prepared here. Methylene carbon chains can be received by hydrolysis of N-methyl pyrrolidone and N-methyl caprolactam. After that, methylene carbon chains were reacted with3,4-dichlorobenzene isocyanate to get hapten4C and hapten6C, respenctively. The hapten4C and hapten6C were conjugated to keyhole limpet hemocyanin (KLH) and bovine serum albumin (BSA) to get four immunogens, and conjugated to ovalbumin (OVA) and bovine serum albumin (BSA) to get coating antigens. The hapten4C conjugated to HRP was as the enzyme tracer. Then eight male rabbits were immunized with four immunogens. The titers of the antiserums were between10,000and100,000. Results indicated that rabbit immuned with hapten4C-KLH elicited special antiserum to diuron (100μg/L) showed85.5%inhibition. Antibody purification can be completed by protein A-sepharose4B immunoaffinity chromatography.In established icELLISA, the plate was coated with0.01μg per well of hapten6C-OVA, while the antibody was diluted in1:3000in PBS buffer (pH7.4). The sensitivity of the method (middle inhibition concentration) was IC50=0.48±0.09μg/L, and the detection limit was IC15=0.038±0.007μg/L. In established dcELISA, the amount of antibody-coated was0.25μg per well, while the dilution ratio of enzyme tracer was1:10000with PBS buffer (pH7.4). The sensitivity of this method was IC50=0.20±0.09μg/L, and the detection limit was IC15=0.015±0.007μg/L. The determination of12phenylurea herbicides with this established dcELISA suggested that5herbicides showed high cross-reactivity that the antibody can be used for the determination of this type of phenylurea herbicides. The recovery of dcELISA was89.04%-112.03%, and coefficient of variation was less than10%. The established dcELISA on quantitative determination of diuron was validated by HPLC. The dcELISA results were consistent well with HPLC results.17water samples, collected from Australia were analyzed on dcELISA. Among them,14samples showed positive, with0.07to3.10μg/L diuron residue. Then all the samples were sipked with2μg/L diuron, and the recovery were from96.6%to129.1%.