title: Magnetic bead-based phage anti-immunocomplex assay (PHAIA) for the detection of the urinary biomarker 3-phenoxybenzoic acid to assess human exposure to pyrethroid insecticides.
abstract: Noncompetitive immunoassays are advantageous over competitive assays for the detection of small molecular weight compounds. We recently demonstrated that phage peptide libraries can be an excellent source of immunoreagents that facilitate the development of sandwich-type noncompetitive immunoassays for the detection of small analytes, avoiding the technical challenges of producing anti-immunocomplex antibody. In this work we explore a new format that may help to optimize the performance of the phage anti-immunocomplex assay (PHAIA) technology. As a model system we used a polyclonal antibody to 3-phenoxybenzoic acid (3-PBA) and an anti-immunocomplex phage clone bearing the cyclic peptide CFNGKDWLYC. The assay setup with the biotinylated antibody immobilized onto streptavidin-coated magnetic beads significantly reduced the amount of coating antibody giving identical sensitivity (50% saturation of the signal (SC(50))=0.2-0.4ng/ml) to the best result obtained with direct coating of the antibody on ELISA plates. The bead-based assay tolerated up to 10 and 5% of methanol and urine matrix, respectively. This assay system accurately determined the level of spiked 3-PBA in different urine samples prepared by direct dilution or clean-up with solid-phase extraction after acidic hydrolysis with overall recovery of 80-120%.
authors.affiliation: Department of Entomology and UCD Cancer Research Center, University of California, Davis, 95616, USA.
authors.forename: Hee-Joo
authors.initials: HJ
authors.lastname: Kim
authors.forename: Ki Chang
authors.initials: KC
authors.lastname: Ahn
authors.forename: Andrés
authors.initials: A
authors.lastname: González-Techera
authors.forename: Gualberto G
authors.initials: GG
authors.lastname: González-Sapienza
authors.forename: Shirley J
authors.initials: SJ
authors.lastname: Gee
authors.forename: Bruce D
authors.initials: BD
authors.lastname: Hammock
mesh_terms: D000906:Antibodies; D001565:Benzoates / Q000276:immunology; D015415:Biomarkers / Q000652:urine; D006801:Humans; D007118:Immunoassay / Q000379:methods* / Q000592:standards; D007306:Insecticides / Q000652:urine*; D008280:Magnetics; D008722:Methods; D008863:Microspheres; D019151:Peptide Library; D011722:Pyrethrins / Q000652:urine*; D012107:Research Design
chemical_list: D000906:Antibodies; D001565:Benzoates; D015415:Biomarkers; D007306:Insecticides; D019151:Peptide Library; D011722:Pyrethrins; C017618:3-phenoxybenzoic acid