High-throughput automated luminescent magnetic particle-based immunoassay to monitor human exposure to pyrethroid insecticides

We have developed a sensitive, automated, competitive chemiluminescent immunoassay for the detection of 3-phenoxybenzoic acid (3-PBA), a metabolite common to many pyrethroid insecticides. The system uses a competitive hapten-protein conjugate that has been labeled with an acridinium ester as the chemiluminescent probe and secondary antibody-coated paramagnetic particles for the separation. After the immunoassay reagents and samples are combined for the competitive incubation step, a fully automated system is used to load the postincubation mixture into a delivery cuvette, facilitating the subsequent magnetic separation of the immunocomplex and the measurement of chemiluminescent signal for quantification. The immunoassay format described here supports the requirement for high throughput necessary for monitoring large numbers of samples in population-based studies. The optimized immunoassay was more sensitive than the conventional enzyme immunoassay in buffer (IC(50) = 0.1 and 2 microg/L, respectively). The mixed-mode solid-phase extraction used for sample preparation to reduce possible urinary matrix effects allowed the accurate measurement of 3-PBA levels as low as 1 microg/L. The automated chemiluminescent immunoassay described here is sensitive, simple to use, and more rapid than the previously reported standard microplate assay.