One-Step Immunoassay for Tetrabromobisphenol A Using a Camelid Single Domain Antibody–Alkaline Phosphatase Fusion Protein

Abstract

Tetrabromobisphenol A (TBBPA) is a ubiquitous brominated flame retardant, showing widespread environment 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. 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 analogs 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 by this one-step assay ranged from 96.7–109.9% and correlated well with an HPLC-MS/MS method. It is expected that the dimerized fusion protein, VHH-AP, will show promising applications in human exposure and environmental monitoring.

Synergetic signal amplification of multi-walled carbon nanotubes-Fe3O4 hybrid and trimethyloctadecylammonium bromide as a highly sensitive detection platform for tetrabromobisphenol A

“…Up to now, various analytical techniques have been developed for the determination of TBBPA, which includes high-performance liquid chromatography21, liquid chromatography tandem mass spectrometry2223, gas chromatography-mass spectrometry2425, surface-enhanced Raman scattering spectroscopy26 and immunosensor2728. However, the above methods are laborious and time-consuming, which usually suffer from complicated and expensive instruments, multistep and complex sample preparation and skilled operators.…”

Single-Domain Antibodies As Versatile Affinity Reagents for Analytical and Diagnostic Applications

“…Nbs have been produce as recombinant fusion proteins with hyperactive mutated versions of the alkaline phosphatase (AP) of E. coli ( 94 ). The combination of target binding with signal transduction domains reduces the number of steps in immunoassay applications ( 95 ), and due to the spontaneous dimerization of AP the functional affinity of the Nb partner increases significantly, improving the assay sensitivity. Surface plasmon resonance measurement of the interaction of five VHHs to different bacterial toxins showed in all cases that the K D of the VHH–AP fusion was roughly an order of magnitude lower than that of the parent Nb ( 96 ).…”

VHH antibodies: emerging reagents for the analysis of environmental chemicals

“…The VHH was subjected to heating to 90 °C for 10 and 90 min, and it retained 80% and 20% of its activity, respectively. To take advantage of being able to genetically modify a protein, this VHH was genetically fused to alkaline phosphatase to serve as the reporting label [38]. Because the binding protein was directly linked to the reporting protein, the number of incubating and washing steps in the VHH-AP assay were reduced, thus speeding up the analysis time.…”

“…These are common problems in expression technology that are easily solved. Already there are two examples where alkaline phosphatase (AP) has been genetically linked to a VHH [28, 38]. The resulting assays exhibited comparable to slightly improved sensitivities, while also reducing the number of steps in the analytical procedure.…”

“…In addition, the use of AP allows for either a colorimetric or fluorometric product for detection, and Liu and colleagues demonstrated a 10-fold improvement in IC 50 using the fluorometric analysis [28]. Wang and colleagues also report that the fusion construct of VHH-AP is more susceptible to extreme heat (90 °C), but the reagent retained excellent stability at ambient temperature for at least 70 days [38]. Chemical fluorophores that require chemical conjugation techniques are very different than those can be genetically fused, such as green fluorescent protein.…”