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 (SC50) = 0.2-0.4 ng/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%.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 45-52 |
| Number of pages | 8 |
| Journal | Analytical Biochemistry |
| Volume | 386 |
| Issue number | 1 |
| DOIs | |
| State | Published - Mar 1 2009 |
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Keywords
- 3-Phenoxybenzoic acid
- Noncompetitive immunoassay
- Phage anti-immunocomplex assay
- Phage ELISA
- Phage peptide display
- Pyrethroid insecticides
ASJC Scopus subject areas
- Biochemistry
- Biophysics
- Molecular Biology
- Cell Biology
Cite this
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. / Kim, Hee Joo; Ahn, Ki Chang; González-Techera, Andrés; González-Sapienza, Gualberto G.; Gee, Shirley J.; Hammock, Bruce D.
In: Analytical Biochemistry, Vol. 386, No. 1, 01.03.2009, p. 45-52.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - 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
AU - Kim, Hee Joo
AU - Ahn, Ki Chang
AU - González-Techera, Andrés
AU - González-Sapienza, Gualberto G.
AU - Gee, Shirley J.
AU - Hammock, Bruce D.
PY - 2009/3/1
Y1 - 2009/3/1
N2 - 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 (SC50) = 0.2-0.4 ng/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%.
AB - 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 (SC50) = 0.2-0.4 ng/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%.
KW - 3-Phenoxybenzoic acid
KW - Noncompetitive immunoassay
KW - Phage anti-immunocomplex assay
KW - Phage ELISA
KW - Phage peptide display
KW - Pyrethroid insecticides
UR - http://www.scopus.com/inward/record.url?scp=58349117028&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=58349117028&partnerID=8YFLogxK
U2 - 10.1016/j.ab.2008.12.003
DO - 10.1016/j.ab.2008.12.003
M3 - Article
C2 - 19101498
AN - SCOPUS:58349117028
VL - 386
SP - 45
EP - 52
JO - Analytical Biochemistry
JF - Analytical Biochemistry
SN - 0003-2697
IS - 1
ER -