Heterologous Antigen Selection of Camelid Heavy Chain Single Domain Antibodies against Tetrabromobisphenol A

Abstract

Tetrabromobisphenol A (TBBPA) is a ubiquitous flame retardant. A high-throughput immunoassay would allow for monitoring of human and environmental exposures as a part of risk assessment. Naturally occurring antibodies in camelids that are devoid of light chain, show great promise as an efficient tool in monitoring environmental contaminants, but they have been rarely used for small molecules. An alpaca was immunized with a TBBPA hapten coupled to thyroglobulin and a variable domain of heavy chain antibody (VHH) T3–15 highly selective for TBBPA was isolated from a phage displayed VHH library using heterologous coating antigens. Compared to the VHHs isolated using homologous antigens, VHH T3–15 had about a 10-fold improvement in sensitivity in an immunoassay. This assay, under the optimized conditions of 10% methanol in the assay buffer (pH 7.4), had an IC50 for TBBPA of 0.40 ng mL–1 and negligible cross reactivity (<0.1%) with other tested analogues. After heating the VHH at 90 °C for 90 min about 20% of the affinity for coating antigen T3-BSA remained. The recoveries of TBBPA from spiked soil and fetal bovine serum samples ranged from 90.3% to 110.7% by ELISA and agreed well with a liquid chromatography–tandem mass spectrometry method. We conclude the many advantages of VHH make them attractive for the development of immunoassays to small molecules.

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

“…The synthesis of haptens T1–T6 (Figure S-1 in Supporting Information) and the selection of anti-TBBPA VHHs were described in previous studies 7, 32 . The TBBPA standard was purchased from TCI Co. Ltd. (Tokyo, Japan).…”

“…The IC 50 value was obtained from a four-parameter logistic equation generated by SigmaPlot 10.0. The indirect competitive VHH-based ELISA was developed in our previous work 7 . In this study, T3-15-AP was selected to optimize and develop a one-step immunoassay.…”

“…As an alternative, immunoassays have been developed for TBBPA for environmental detection with high sensitivities 31–34 . In an earlier work, we reported the selection of VHHs specific for TBBPA from an immunized alpaca VHH-derived library and the development of a VHH-based ELISA with a half-maximum signal inhibition concentration (IC 50 ) of 0.4 ng mL −1 TBBPA 7 .…”

Recent advances in the analysis of TBBPA/TBBPS, TBBPA/TBBPS derivatives and their transformation products

“…Crossreactivity values of the ELISA with TBBPA derivatives were <0.05% [23]. Alpacas were also immunized with a TBBPA hapten coupled to thyroglobulin, which was applied to quantitatively determine the concentration of TBBPA at trace levels (~10 ng/g) in the environment [49]. A selective biotin-streptavidin-amplified ELISA for the measurement of TBBPA within an LOD (IC10) has been developed [50].…”

Deoxynivalenol-mimic nanobody isolated from a naïve phage display nanobody library and its application in immunoassay

“…Furthermore, for development of sensitive and reliable immunoassay for small molecules, the quality and selectivity of hapten-protein conjugates are critical. For detection of hapten-protein conjugates, the heterologous immunoassay system (i.e., the immunizing hapten and the coating hapten differ in their molecular structures) presents a good alternative to homologous assays in achieving higher sensitivity in competitive immunoassay format [15][16][17]. Although heterology format is M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT 4 very useful, the procedures of chemical synthesis of hapten-protein conjugates are over elaborative and complicated.…”