Noncompetitive fluorescent immunoassay for the detection of the human urinary biomarker 3-phenoxybenzoic acid with bench top immunosensor KinExA™ 3000

Hee Joo Kim, Shirley J. Gee, Qing X. Li, Bruce D. Hammock

Research output: Chapter in Book/Report/Conference proceedingConference contribution

1 Citation (Scopus)

Abstract

A sensitive, automated, non-competitive fluorescent immunoassay was developed for quantitative analysis of 3-phenoxybenzoic acid (PBA) in human urine sampes as a putative biomarker of exposure to pyrethroid insecticides using the bench top immunoanalyzer, KinExA™ 3000 system. The key difference between the KinExA system and the enzymelinked immunosorbent assay (ELISA) is to eliminate the PBA-antibody interaction with the coating antigen. This can be achieved by separately capturing the free PBA-antibody onto the hapten-immobilized beads when a constant amount of reaction solution in equilibrium between PBA-antibody and analyte passes through the bead-packed glass capillary column. Optimal dilution of the PBA antibody was determined when fluorescent signals of 0.5-2 were obtained and a sufficient amount of coating antigen was immobilized to ensure the capture of all free antibodies. IC 50S of the two KinExA methods (0.3 and 0.6 ng/mL for one- and two-step KinExA, respectively) were 3- and 6-fold better than the heterologous ELISA and were approximately 650- and 300-fold lower compared to that of the homologous ELISA (IC 50 of 200 ng/mL). The KinExA assay was negligibly affected within tested range of pHs (5-10) and ionic strengths (1, 5, and 10X PBS). Similar urine matrix effects were observed in the two KinExA assays with a 5- to 10-fold increase in IC 50s when 5 and 10% of urine was contained in the reaction buffer. A high correlation (r 2 = 0.99) was observed between detected and spiked concentrations of PBA standard with average recoveries of 88-160%.

Original languageEnglish (US)
Title of host publicationACS Symposium Series
Pages171-185
Number of pages15
Volume966
StatePublished - 2007

Publication series

NameACS Symposium Series
Volume966
ISSN (Print)00976156

Fingerprint

Immunosensors
Biomarkers
Assays
Immunosorbents
Antibodies
Antigens
Coatings
Pyrethrins
Haptens
Insecticides
Ionic strength
Dilution
3-phenoxybenzoic acid
Buffers
Recovery
Glass
Chemical analysis

ASJC Scopus subject areas

  • Chemistry(all)

Cite this

Kim, H. J., Gee, S. J., Li, Q. X., & Hammock, B. D. (2007). Noncompetitive fluorescent immunoassay for the detection of the human urinary biomarker 3-phenoxybenzoic acid with bench top immunosensor KinExA™ 3000. In ACS Symposium Series (Vol. 966, pp. 171-185). (ACS Symposium Series; Vol. 966).

Noncompetitive fluorescent immunoassay for the detection of the human urinary biomarker 3-phenoxybenzoic acid with bench top immunosensor KinExA™ 3000. / Kim, Hee Joo; Gee, Shirley J.; Li, Qing X.; Hammock, Bruce D.

ACS Symposium Series. Vol. 966 2007. p. 171-185 (ACS Symposium Series; Vol. 966).

Research output: Chapter in Book/Report/Conference proceedingConference contribution

Kim, HJ, Gee, SJ, Li, QX & Hammock, BD 2007, Noncompetitive fluorescent immunoassay for the detection of the human urinary biomarker 3-phenoxybenzoic acid with bench top immunosensor KinExA™ 3000. in ACS Symposium Series. vol. 966, ACS Symposium Series, vol. 966, pp. 171-185.
Kim HJ, Gee SJ, Li QX, Hammock BD. Noncompetitive fluorescent immunoassay for the detection of the human urinary biomarker 3-phenoxybenzoic acid with bench top immunosensor KinExA™ 3000. In ACS Symposium Series. Vol. 966. 2007. p. 171-185. (ACS Symposium Series).
Kim, Hee Joo ; Gee, Shirley J. ; Li, Qing X. ; Hammock, Bruce D. / Noncompetitive fluorescent immunoassay for the detection of the human urinary biomarker 3-phenoxybenzoic acid with bench top immunosensor KinExA™ 3000. ACS Symposium Series. Vol. 966 2007. pp. 171-185 (ACS Symposium Series).
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abstract = "A sensitive, automated, non-competitive fluorescent immunoassay was developed for quantitative analysis of 3-phenoxybenzoic acid (PBA) in human urine sampes as a putative biomarker of exposure to pyrethroid insecticides using the bench top immunoanalyzer, KinExA™ 3000 system. The key difference between the KinExA system and the enzymelinked immunosorbent assay (ELISA) is to eliminate the PBA-antibody interaction with the coating antigen. This can be achieved by separately capturing the free PBA-antibody onto the hapten-immobilized beads when a constant amount of reaction solution in equilibrium between PBA-antibody and analyte passes through the bead-packed glass capillary column. Optimal dilution of the PBA antibody was determined when fluorescent signals of 0.5-2 were obtained and a sufficient amount of coating antigen was immobilized to ensure the capture of all free antibodies. IC 50S of the two KinExA methods (0.3 and 0.6 ng/mL for one- and two-step KinExA, respectively) were 3- and 6-fold better than the heterologous ELISA and were approximately 650- and 300-fold lower compared to that of the homologous ELISA (IC 50 of 200 ng/mL). The KinExA assay was negligibly affected within tested range of pHs (5-10) and ionic strengths (1, 5, and 10X PBS). Similar urine matrix effects were observed in the two KinExA assays with a 5- to 10-fold increase in IC 50s when 5 and 10{\%} of urine was contained in the reaction buffer. A high correlation (r 2 = 0.99) was observed between detected and spiked concentrations of PBA standard with average recoveries of 88-160{\%}.",
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