i
Superseded
This Document Has Been Replaced By:
i
Retired
This Document Has Been Retired
i
Up-to-date Information
This is the latest update:
One-step Immunoassay for Tetrabromobisphenol A Using a Camelid Single Domain Antibody-Alkaline Phosphatase Fusion Protein
-
Published Date:
Apr 22 2015
-
Publisher's site:
-
Source:Anal Chem. 87(9):4741-4748.
-
Details:
-
Alternative Title:Anal Chem
-
Personal Author:
-
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:
-
Pubmed ID:25849972
-
Pubmed Central ID:PMC4476793
-
Document Type:
-
Funding:
-
Collection(s):
-
Main Document Checksum:
- File Type:
-
Supporting Files:
image/gif image/jpeg image/gif image/jpeg image/gif image/jpeg image/gif image/jpeg image/gif image/jpeg application/pdf image/gif image/jpeg image/gif image/jpeg image/gif image/jpeg image/gif image/jpeg image/gif image/jpeg application/octet-stream image/gif image/jpeg image/gif image/jpeg image/gif image/jpeg image/gif image/jpeg
No Related Documents.