Development of a noncompetitive phage anti-immunocomplex assay for brominated diphenyl ether 47.
Kim-HJ; Rossotti-MA; Ahn-KC; González-Sapienza-GG; Gee-SJ; Musker-R; Hammock-BD
Anal Biochem 2010 Jun; 401(1):38-46
We present a new application of the noncompetitive phage anti-immunocomplex assay (PHAIA) by converting an existing competitive assay to a versatile noncompetitive sandwich-type format using immunocomplex binding phage-borne peptides to detect the brominated flame retardant, brominated diphenyl ether 47 (BDE 47). Three phage-displayed 9-mer disulfide-constrained peptides that recognize the BDE 47-polyclonal antibody immunocomplex were isolated. The resulting PHAIAs showed variable sensitivities, and the most sensitive peptide had a dose-response curve with an SC(50) (concentration of analyte producing 50% saturation of the signal) of 0.7ng/ml BDE 47 and a linear range of 0.3-2ng/ml, which was nearly identical to the best heterologous competitive format (IC(50) of 1.8ng/ml, linear range of 0.4-8.5/ml). However, the PHAIA was 1400-fold better than homologous competitive assay. The validation of the PHAIA with extracts of house furniture foam as well as human and calf sera spiked with BDE 47 showed overall recovery of 80-113%. The PHAIA was adapted to a dipstick format (limit of detection of 3.0ng/ml), and a blind test with six random extracts of local house furniture foams showed that the results of the PHAIA and dipstick assay were consistent, giving the same positive and negative detection.
Antibody-response; Biological-agents; Chemical-analysis; Chemical-binding; Chemical-composition; Dose-response; Environmental-exposure; Humans; Immunochemistry; Immunological-tests; Immunology; Peptides; Protein-biochemistry; Protein-chemistry; Quantitative-analysis; Statistical-analysis;
Author Keywords: Phage anti-immunocomplex assay; Phage-displayed peptide; Phage ELISA; Noncompetitive immunoassay; BDE 47; Brominated flame retardant
Bruce D. Hammock, Department of Entomology and UCD Cancer Research Center, University of California, Davis, CA 95616
Agriculture; Cooperative Agreement
University of California - Davis