Study On Enzyme-linked Immunassay For The Detection Of Avermectins Residues In Food Products

In this paper, the enzyme-linked immunosorbent assay method for the determination of avermectins had been developed.Avermectin(AVM) was modified to make the hapten. First, protection of the5-hydroxy group as the tert-butyldimeth-ylsily ether. The second, succinoylation of the4’-hydroxy group using succinic anhydride and the last, deprotection of the5-hydroxy group. The hapten of avermectin which oppsited the special structure was prepared. The carrier protein BSA was conjugated with hapten to obtain the immunogen by the active ester method and the hapten was coupled to OVA as coated antigen by the N-hydroxysuccinimide. The anti-AVM antibody with high affinity was obtained after immunized to New Zealand white rabbits. The anti-AVM polyclonal antibody had the high cross-reaction with avermectin(100%); ivermectin(21.9%); eprinomectin(123%).Based on the antibody,an ELISA method was developed to detect avermectin, ivermectin, eprinomectin residues in food. The ELISA used in this study exhibited high sensitivity. The sensitivity of ELISA for avermectin, ivermectin, eprinomectin were1.92±0.3ng/mL,8.75±0.5ng/mL,1.56±0.3ng/mL, respectively. The limit of detection of the ELISA for avermectin, ivermectin, eprinomectin were0.13±0.03ng/mL,0.55±0.05ng/mL,0.10±0.02ng/mL, respectively. The average recoveries for AVM from fortified samples including beef, pork, liver, pear at three concentration of10,40,100μg/kg were in the ranges75.20-89.78%.In order to validated of the ELISA, the fortified samples were analyzed by high-performance liquid chromatography (HPLC). Sample extracts were analyzed by ELISA and HPLC, and the results obtained by two methods correlated well, the correlation coefficient was0.9862.The ELISA method described in this paper can determine AVMs in food rapidly. This research will be useful for the development of anti-AVM ELISA kit.