Immunoassay Research For Fenvalerate Residue In Agro-product

Rational design is the basis and premise to improve antibody quality in the course of immunoassayfor pesticide. The position of spacer-arm between the carrier protein and hapten is critical effect on thesensitivity and specificity of antibody. According to the fenvalerate structure, three haptens withdifferent spacer-arm were designed and synthesized. The effects of spacer-arm position and structure tothe character of antibodies were analyzed. Novol fluorescence immunoassay technology was achievedusing antibodies conjugated with quantum dots.Two kind of the antibody and related data were also collected in our group. The last antibody Ab3andthe hapten were obtained in this work. The effects of different spacer-arm positions to the antibodycharacter were analyzed.Three positions were designed to adde spacer-arm between the position of diphenyl ether, α-cyanoand fenvaleric acid, respectively. And the positions of diphenyl ether and α-cyano have been reported.The fenvaleric acid was the first time been researched with the spacer-arm on for fenvalerate. The spaceeffects of three parts were studied on differnent spacer-arm positions of haptens.According to the structure of fenvaleric acid, cyanohydrin was obtained by3-phenoxy-benzaldehyde reaction with hydrocyanic acid. Then the para of α-isopropyl-p-chlorobenzyl cyanide was nitration. The cyano was hydrolyzed. Chloride compound wasobtained via the former reaction products acting with SOCl₂. Esters compound with nitro wasobtained by cyanohydrin reacting with acyl chloride. The nitro of the ester was deoxidized to getone hapten with amino. The esters with nitro reacted with succinic anhydride to get another hapten.The haptens were identified by MS and NMR-H spectroscopy.In the synthesis route of antigen, the amino and carboxyl group of hapten directly were coupled to thecarrier protein via diazotization and active ester method, respectively. The haptens coupled with BSAwas used as immunogen and the haptens conjugated with OVA was used as coating antigen. Theconjugated products were identified by UV spectrum and electrophoresis.Balb/c mice were immunized different haptens. One group of mice were immunized the antigen withlong chain spacer-arm, the other group were immunized the antigen with short chain spacer-arm. TheIC₅₀value was2.4μg/mL and more than20μg/mL, respectively.According to the result of the antiserum competition ELISA, the group of long chain spacer-armhapten was selected to fuse with myeloma cells and hybridoma cell3E7. IC₅₀value was1-2μg/mL andno cells were selected in the result of another mouse with a serum titer （>20μg/mL）.It was found that the hapten with hydrolyzed cyano showed the best immune effect, the limit ofdetection was1.02μg/L and the linear range was from3.81μg/L to714.94μg/L. The cross-reativityof the antibody4E3to fenvalerate analogues were all less than1%. The antibody4E3showed highquality of sensitivity and specificity.Comparing the antibodies and haptens with different positions and structures, it was found that thepositions and structures showed potent effects on the antibody and the cyano group was the bestposition to get the antibody with high quality, which increased the immunization effect of the character of the haptens completely. It was also found that long chain structure of the spacer-arm was better thanaromatic group.CdTe quantum dots were synthesized by a modified method based on the literature. The UVabsorbance and fluorescence spectrum of the quantum dots were measured to determine the excitationand emission wavelength, also the concentration and particle size of the quantum dots. Antibody forfenvalerate （4E3） and deltamethrin （2B12） were conjugated with the quantum dots gsxyrs usingEDC/NHS active ester method. A new fluorescence-linked immunosorbent assay was achieved with theadvantages of time-saving and less consumption of reagents.