One-step immunoassay for tetrabromobisphenol a using a camelid single domain antibody-alkaline phosphatase fusion protein

Jia Wang, Zuzana Majkova, Candace R S Bever, Jun Yang, Shirley J. Gee, Ji Li, Ting Xu, Bruce D. Hammock

Research output: Contribution to journalArticle

26 Citations (Scopus)

Abstract

Tetrabromobisphenol A (TBBPA) is a ubiquitous brominated flame retardant, showing widespread environmental and human exposures. A variable domain of the heavy chain antibody (VHH), naturally occurring in camelids, approaches the lower size limit of functional antigen-binding entities. The ease of genetic manipulation makes such VHHs a superior choice to use as an immunoreagent. In this study, a highly selective anti-TBBPA VHH T3-15 fused with alkaline phosphatase (AP) from E. coli was expressed, showing both an integrated TBBPA-binding capacity and enzymatic activity. A one-step immunoassay based on the fusion protein T3-15-AP was developed for TBBPA in 5% dimethyl sulfoxide (DMSO)/phosphate buffered saline (PBS, pH 7.4), with a half-maximum signal inhibition concentration (IC<inf>50</inf>) of 0.20 ng mL<sup>-1</sup>. Compared to the parental VHH T3-15, T3-15-AP was able to bind to a wider variety of coating antigens and the assay sensitivity was slightly improved. Cross-reactivity of T3-15-AP with a set of important brominated analogues was negligible (<0.1%). Although T3-15-AP was susceptible to extreme heat (90 °C), much higher binding stability at ambient temperature was observed in the T3-15-AP-based assay for at least 70 days. A simple pretreatment method of diluting urine samples with DMSO was developed for a one-step assay. The recoveries of TBBPA from urine samples via this one-step assay ranged from 96.7% to 109.9% and correlated well with a high-performance liquid chromatography-tandem mass spectroscopy (HPLC-MS/MS) method. It is expected that the dimerized fusion protein, VHH-AP, will show promising applications in human exposure and environmental monitoring.

Original languageEnglish (US)
Pages (from-to)4741-4748
Number of pages8
JournalAnalytical Chemistry
Volume87
Issue number9
DOIs
StatePublished - May 5 2015

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Single-Domain Antibodies
Alkaline Phosphatase
Fusion reactions
Assays
Proteins
Dimethyl Sulfoxide
Flame Retardants
Antigens
High performance liquid chromatography
Escherichia coli
Phosphates
tetrabromobisphenol A
Spectroscopy
Recovery
Coatings
Antibodies
Monitoring

ASJC Scopus subject areas

  • Analytical Chemistry

Cite this

Wang, J., Majkova, Z., Bever, C. R. S., Yang, J., Gee, S. J., Li, J., ... Hammock, B. D. (2015). One-step immunoassay for tetrabromobisphenol a using a camelid single domain antibody-alkaline phosphatase fusion protein. Analytical Chemistry, 87(9), 4741-4748. https://doi.org/10.1021/ac504735p

One-step immunoassay for tetrabromobisphenol a using a camelid single domain antibody-alkaline phosphatase fusion protein. / Wang, Jia; Majkova, Zuzana; Bever, Candace R S; Yang, Jun; Gee, Shirley J.; Li, Ji; Xu, Ting; Hammock, Bruce D.

In: Analytical Chemistry, Vol. 87, No. 9, 05.05.2015, p. 4741-4748.

Research output: Contribution to journalArticle

Wang, J, Majkova, Z, Bever, CRS, Yang, J, Gee, SJ, Li, J, Xu, T & Hammock, BD 2015, 'One-step immunoassay for tetrabromobisphenol a using a camelid single domain antibody-alkaline phosphatase fusion protein', Analytical Chemistry, vol. 87, no. 9, pp. 4741-4748. https://doi.org/10.1021/ac504735p
Wang, Jia ; Majkova, Zuzana ; Bever, Candace R S ; Yang, Jun ; Gee, Shirley J. ; Li, Ji ; Xu, Ting ; Hammock, Bruce D. / One-step immunoassay for tetrabromobisphenol a using a camelid single domain antibody-alkaline phosphatase fusion protein. In: Analytical Chemistry. 2015 ; Vol. 87, No. 9. pp. 4741-4748.
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abstract = "Tetrabromobisphenol A (TBBPA) is a ubiquitous brominated flame retardant, showing widespread environmental and human exposures. A variable domain of the heavy chain antibody (VHH), naturally occurring in camelids, approaches the lower size limit of functional antigen-binding entities. The ease of genetic manipulation makes such VHHs a superior choice to use as an immunoreagent. In this study, a highly selective anti-TBBPA VHH T3-15 fused with alkaline phosphatase (AP) from E. coli was expressed, showing both an integrated TBBPA-binding capacity and enzymatic activity. A one-step immunoassay based on the fusion protein T3-15-AP was developed for TBBPA in 5{\%} dimethyl sulfoxide (DMSO)/phosphate buffered saline (PBS, pH 7.4), with a half-maximum signal inhibition concentration (IC50) of 0.20 ng mL-1. Compared to the parental VHH T3-15, T3-15-AP was able to bind to a wider variety of coating antigens and the assay sensitivity was slightly improved. Cross-reactivity of T3-15-AP with a set of important brominated analogues was negligible (<0.1{\%}). Although T3-15-AP was susceptible to extreme heat (90 °C), much higher binding stability at ambient temperature was observed in the T3-15-AP-based assay for at least 70 days. A simple pretreatment method of diluting urine samples with DMSO was developed for a one-step assay. The recoveries of TBBPA from urine samples via this one-step assay ranged from 96.7{\%} to 109.9{\%} and correlated well with a high-performance liquid chromatography-tandem mass spectroscopy (HPLC-MS/MS) method. It is expected that the dimerized fusion protein, VHH-AP, will show promising applications in human exposure and environmental monitoring.",
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AU - Gee, Shirley J.

AU - Li, Ji

AU - Xu, Ting

AU - Hammock, Bruce D.

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