Study On Enzyme-Linked Immunosorbent Assay (ELISA) For Insecticide Imidacloprid And Fipronil

For study of immunochemistry characteristic of heterocyclic insecticide, we choose imidacloprid and fipronil as study objects, establishing indirect ELISA for imidacloprid and direct ELISA for fipronil. The hapten IM for imidacloprid was obtained by reacting of imidacloprid and 3 -Mercaplopropionic acid in the alkaline solution. And then the hapten was conjugated to bovine serum albumin (BSA) to form an artificial antigen IM-BSA with modified active ester method. The fipronil was hydrolyzed into hapten FH1 under alkaline or acidity condition. The FH1 was covalently conjugated to BSA and ovalbumin (OVA) to prepare immunogens and coating antigens. With the glutaraldehyde (GA) methods, we synthesized the immunogen FH-GD-BSA. The conjugates were identified and the molecular ratios of hapten to carrier protein were determined, by UV spectrophotomertry, to be within the range from 8:1 to 35:1.New Zealand white rabbits were immunized with the synthetic antigens, and the two kinds of antisera that are specific to imidacloprid and fipronil were obtained by using IM-BSA and FH1-BSA as immunogens. Titers of the antisera were from 1: 25,600 to 1:51200 and from 1:25,000 to 1:64,000, respectively. However, the liters of anti-FH-GD-BSA antisera were low, only 1:400. The polyclonal antibodies in serum were salted out by caprylic acid-saturated ammonium sulfate. The enzyme tagged antibodies were prepared by coupling horseradish peroxidase (HRP) to the purified antibody with the modified method of sodium periodate. The titers of enzyme tagged antibodies of anti-FHl-BSA were 1:5000.By using the two antibodies with high liter, two kinds of Enzyme-Linked Immunosorbent Assay (ELISA) have been established, including an indirect ELISA for imidacloprid and a direct ELISA for fipronil. For imidacloprid and fipronil, linear regression curve in terms of concentration and rale of inhibition are as follows: y = 10.129Ln(x) + 18.104 (R2=0.993) and y = 9.3554Ln(x) + 24.36 (R2=0.995), respectively. I50 and I20, which were used to estimate the assay sensilivily, can becalculated from the linear regression equation, and I20 was usually adopted as the limit of detection (LOD). The LOD for imidacloprid and fipronil were 1.2g/L and 0.627g/L respectively. The I50 were 21.8g/L and 15.5g/L, for the two insecticides, respectively. The linear relationship were kept well from 1 to 103g/ml and from 0.1 to 100g/l respectively for imidacloprid and fipronil.The cross-reaction of anti-IM-BSA and anti-FHl-BSA to their corresponding analogs were tested. The maximal concentration for half inhibition of antibody-antigen reaction were 21.8g/L for imidacloprid, 1125g/L for acetaniprid. This meant that the cross-reactivity ratios were 1.9% for acetaniprid. For other analogs the ratios were less than 1.0%. When the concentration of fipronil were 21.8g/L, the inhibition ratio to antibody-antigen reaction were 50%. The cross-reactivity ratios between fipronil and its four metabolities were over 80%.At spiked levels of 0.05, 0.1, and l.0g/ml recoveries of imidacloprid in soil were from 86.5 to 110.2% with C.V.% of 4.67~9.64. In tap water the recoveries were 86.4-104.2%, with C.V.% of 3.51-6.5 . For fipronil the recoveries in soil were 79.5-83.2%, with C.V.% of 2.54~6.25 while in pond water the recoveries were 76.2-79.8% with C.V.% of 5.94-9.85.By the established and optimized ELISA, the residue dynamics of imidacloprid and fipronil in soil and in water could be monitored. The method was satisfied with the requirement of the pesticide residues analysis, so the present study laid the foundation for production of the ELISA kits.