The Research On Enzyme-Linked Immunosorbent Assay For Diniconazole And Uniconazole

Enzyme-linked immunosorbent assay (ELISA) provide a highly sensitive, selective, simple and field-analytical method for the detection of pesticide residues. In this paper, the ELISA for the determination of diniconazole and uniconazole had been developed. The primary research results were described as below:Two haptens for diniconazole and uniconzole were synthesized. Hapten was conjugated to bovine serum albumin (BSA) for immunogen by carbodiimide method and to ovalbumin (OVA) for coating antigen by mixed anhydride method, respectively. The conjugates were confirmed by UV spectra, the conjugation molar ratio is 27:1 and 9:1 for the immunogen and coating antigen of diniconazole,17:1 and 5:1 for the immunogen and coating antigen of uniconazole. Polyclonal antibodies was generated by immunizing male New Zealand rabbits with the immunogen, the titers of the antibodies were 2.56×105 and 1.02×106, respectively.The effects of organic solvent, ionic intension and pH value on the affinity of diniconazole to antibody were evaluated. The resulted showed that 20% methanol, 0.4mol/L Na+, pH 7.5 were determined to optimum assay conditions. Under the optimized conditions, an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) was developed for detecting diniconazole, IC50 of which was 0.071±0.013mg/L and the limit of detection (LOD) was 1.28±0.8μg/L, the linear concentrations was kept well from 0.005mg/L to 5mg/L. CV of intra-plate and inter-plate for ic-ELISA was 1.82%-4.48% and 3.22%-8.74%, respectively. All the tested triazole fungicides (tebuconazole, hexaconazole, triadimefon, flutriafol, epoxiconazole, cyproconazole) did not show significant cross-reaction except for uniconazole. The ic-ELISA have been also established for uniconazole, under the optimized conditions (20% methanol,0.3mol/L Na+, pH 7.5), the linear concentrations ranged from 0.005-10mg/L, the LOD was 1.82±0.83μg/L, IC50 was 2.26±0.5mg/L for the determination of uniconazole. CV of intra-plate and inter-plate for ic-ELISA was 1.82%-5.05% and 1.93%-7.49%, respectively. Some triazole fungicides (tebuconazole, hexaconaz ole, triadimefon, flutriafol, epoxiconazole, cyproconazole) did not cross-react in this assay except diniconazole because of theirs structures.Recoveries of diniconazole into different samples such as water, soil, pear, grape, tomato and wheat flour were determined by ic-ELISA after sample matrix testing. These samples required simple cleanup procedure and the final remainder was diluted 1-4 times with buffer solution. At spiked different levels (0.005-0.5mg/L) recoveries of diniconazole were from 70%-120% with CV of 0.33%-10.42%. The average recoveries of uniconazole from fortified water and soil samples were in the range of 82.40%-105.92% with CV of 0.29%-8.52%. The method was satisfied with the requirements of the pesticide residue analysis, and the present study laid the foundation for production of the ic-ELISA kits.