title: 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 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.
authors: †Beijing Key Laboratory of Biodiversity and Organic Farming, College of Resources and Environmental Sciences, China Agricultural University, Beijing 100193, China.|§College of Food Science and Technology, Huazhong Agricultural Universiy, Wuhan 430070, China.
authors: Jia
authors: J
authors: Wang
authors: ‡Department of Entomology and UCD Comprehensive Cancer Center, University of California, Davis, California 95616, United States.
authors: Zuzana
authors: Z
authors: Majkova
authors: ‡Department of Entomology and UCD Comprehensive Cancer Center, University of California, Davis, California 95616, United States.
authors: Candace R S
authors: CR
authors: Bever
authors: ‡Department of Entomology and UCD Comprehensive Cancer Center, University of California, Davis, California 95616, United States.
authors: Jun
authors: J
authors: Yang
authors: ‡Department of Entomology and UCD Comprehensive Cancer Center, University of California, Davis, California 95616, United States.
authors: Shirley J
authors: SJ
authors: Gee
authors: †Beijing Key Laboratory of Biodiversity and Organic Farming, College of Resources and Environmental Sciences, China Agricultural University, Beijing 100193, China.
authors: Ji
authors: J
authors: Li
authors: †Beijing Key Laboratory of Biodiversity and Organic Farming, College of Resources and Environmental Sciences, China Agricultural University, Beijing 100193, China.
authors: Ting
authors: T
authors: Xu
authors: ‡Department of Entomology and UCD Comprehensive Cancer Center, University of California, Davis, California 95616, United States.
authors: Bruce D
authors: BD
authors: Hammock
mesh_terms: D000469:Alkaline Phosphatase / Q000737:chemistry* / Q000378:metabolism*; D000906:Antibodies / Q000737:chemistry* / Q000276:immunology*; D002851:Chromatography, High Pressure Liquid; D006801:Humans; D007118:Immunoassay / Q000379:methods*; D011075:Polybrominated Biphenyls / Q000276:immunology / Q000652:urine*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D053719:Tandem Mass Spectrometry
chemical_list: D000906:Antibodies; D011075:Polybrominated Biphenyls; D011993:Recombinant Fusion Proteins; D000469:Alkaline Phosphatase; C020806:tetrabromobisphenol A