Details:

Alternative Title:
Anal Chem
Publisher's site:
http://dx.doi.org/10.1021/ac504735p
Personal Author:
Wang, Jia ; Majkova, Zuzana ; Bever, Candace R. S. ; Yang, Jun ; Gee, Shirley J. ; ... More +
Wang, Jia ; Majkova, Zuzana ; Bever, Candace R. S. ; Yang, Jun ; Gee, Shirley J. ; Li, Ji ; Xu, Ting ; Hammock, Bruce D. ; Less -
Description:
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.
Subject:
[+]
Alkaline Phosphatase
Antibodies
Article
Chromatography, High Pressure Liquid
Fusion Protein
Humans
Immunoassay
Polybrominated Biphenyls
Recombinant Fusion Proteins
Tandem Mass Spectrometry
Tetrabromobisphenol A
Urine
Variable Domain Of Heavy Chain Antibody
Alkaline Phosphatase

Source:
Anal Chem. 87(9):4741-4748.
Pubmed ID:
25849972
Pubmed Central ID:
PMC4476793
Document Type:
Journal Article ;
Funding:
2U50OH007550/OH/NIOSH CDC HHS/United States ; P42 ES004699/ES/NIEHS NIH HHS/United States ; P42ES04699/ES/NIEHS NIH HHS/United States ;
2U50OH007550/OH/NIOSH CDC HHS/United States ; P42 ES004699/ES/NIEHS NIH HHS/United States ; P42ES04699/ES/NIEHS NIH HHS/United States ; Less -
Collection(s):
CDC Public Access
Main Document Checksum:
[+]
urn:sha256:efd80d7902ebcab980309c0ca7ec5c5f27239abc790b23500581e3cb20836629
File Type:

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