High-sensitivity assay for pesticide using a peroxidase as chemiluminescent label

The application of a newly isolated transgenic tobacco peroxidase (TOP) as a chemiluminescent label for immunoassay purposes is described for the first time. The enzyme has been oxidized with m-periodate and subsequently coupled to the model compound 2,4-dichlorophenoxyacetic acid (2,4-D) using a carbodiimide method. As compared to the native horseradish peroxidase used in control experiments, the TOP enzyme showed significantly higher efficiency of coupling to the antigen and no loss of the specific activity was observed. The obtained 2,4-D-TOP conjugate demonstrated unique properties in chemiluminescent detection. The latter allowed the minimization of the conjugate concentration due to the superior chemiluminescent activity of the enzyme. A highly sensitive capillary chemiluminescent immunoassay using the 2,4-D-TOP conjugate as labeled competitor is reported. Direct competitive ELISA has been performed using a specific monoclonal antibody immobilized onto the sol-gel treated glass capillary surface. A modified photomultiplier tube with a special holder for a capillary was used for the resulting chemiluminescent signal detection. The typical standard calibration curve for the 2,4-D pesticide detection is linear between 30 pg and 500 ng/mL.