Antibody Capture Immunoassay for the Detection of Permethrin Carboxylesterase in Colorado Potato Beetle, Leptinotarsa decemlineata Say

Polyclonal antibodies were generated against the three denatured forms (30-, 48-, and 59kDa proteins) of permethrin carboxylesterase from Colorado potato beetle. Each antiserum showed cross-reactivities to all the denatured forms of permethrin carboxylesterase as judged by antibody capture immunoassay. An antibody capture immunoassay was developed using the immunoglobulin purified from the 30-kDa antiserum in conjunction with denatured Colorado potato beetle hemolymph as the antigen source. In this antibody capture immunoassay, the hemolymph from the permethrin-resistant strain produced 1.1-5.3 times higher signal levels than that from the susceptible strain depending on assay conditions, suggesting that the hemolymph from the resistant strain contains more permethrin carboxylesterase. These results corroborate our original findings that permethrin carboxylesterase is overexpressed in the hemolymph of the permethrin-resistant strain of Colorado potato beetle and contributes to overall permethrin resistance. The relative quantities of permethrin carboxylesterase in four different strains of Colorado potato beetle with different levels of permethrin resistance were assessed with this immunoassay and with a carboxylesterase activity assay using alpha-naphthyl caproate as substrate. The average levels of permethrin carboxylesterase determined by the antibody capture immunoassay were better correlated with the levels of permethrin resistance than those determined by the carboxylesterase activity. Thus, the antibody capture immunoassay can be utilized as a sensitive means to detect the levels of permethrin carboxylesterase as a resistance marker in field populations of Colorado potato beetle.