Enzyme-Linked Immunosorbent Assay For The Determination Of Carbaryl Residues

An enzyme-linked immunosorbent assay (ELISA) based on polyclonal antibody and ELISA with enhanced chemiluminescent (ECL) detection of carbaryl in food samples had been developed. Based on a high-specific antibody produced by 1- (1-naphthyl) -3-(5-carboxypentyl) ure (8H), a highly sensitive immunoassay for carbaryl was developed by using a new hapten 6-[[(1-naphthyloxy) carbonyl] amino] hexanoic acid (3H) conjugated to HRP. 3H had highly similarity to carbaryl, and was synthesized with a safer and more practical approach. A series of parameters of this method were optimized in this study, including the concentration of coated antibody, concentration of enzyme conjugate and diluent of enzyme conjugate. And the performance of the developed method had also been evaluated systemically.The developed heterlogous ELISA could obtain an IC50 value of 2μg/kg, 12-fold more sensitive than the previous result of homologous ELISA with 8H-HRP. After using ECL-ELISA, the sensitivity was further improved 10-fold to 0.2μg/kg.Various food samples were chosen for the study, including solid and liquid samples: apple juice, orange juice, almond drink, cherry, peach, cucumber and cabbage. Liquid samples could be analyzed directly after diluting with PBS; extraction of solid matrix was very simple and effective. With a lower limit and more convenient pre-treatment, the immunoassay was 75-fold more sensitive in the determination of carbaryl in broad food samples compared to the homologous ELISA, and samples containing 6.4μg kg-1 of carbaryl could be detected. Average recoveries for apple juice, orange juice, almond drink, cherry, peach, cucumber and cabbage were 97.5%, 93.0%, 92.2%, 97.5%, 93.0%, 101.7% and 88.3% respectively, most of the coefficients of variation were under 10%.Validation was conducted by high-performance liquid chromatography (HPLC). The correlation between data obtained by CD-ELISA and HPLC, ECL-ELISA and HPLC was good (R2>0.95), showing that the developed immunoassay and the simple, rapid and efficient method was reliable for the detection of carbaryl and suitable for the rapid quantitative or qualitative determination in food samples.