Development and Utilization of Camelid VHH Antibodies from Alpaca for 2,2′,4,4′-Tetrabrominated Diphenyl Ether Detection

Abstract

An antibody-based analytical method for the detection of a chemical flame retardant using antibody fragments isolated from an alpaca has been developed. One specific chemical flame retardant congener, 2,2′,4,4′-tetrabrominated diphenyl ether (BDE-47), is often the major poly-BDE (PBDE) congener present in human and environmental samples and that which is the most frequently detected. An alpaca was immunized with a surrogate of BDE-47 covalently attached to a carrier protein. The resulting mRNA coding for the variable domain of heavy-chain antibodies (VHH) were isolated, transcribed to cDNA, and cloned into a phagemid vector for phage display library construction. Selection of VHHs recognizing BDE-47 was achieved by panning under carefully modified conditions. The assay sensitivity for detecting BDE-47 was down to the part-per-billion (microgram per liter) level. Cross-reactivity analyses confirmed that this method was highly selective for BDE-47 and selected hydroxylated metabolites. When exposed to elevated temperatures, the camelid VHH antibodies retained more reactivity than a polyclonal antibody developed to the same target analyte. The use of this VHH antibody reagent immobilized onto a Au electrode for impedance biosensing demonstrates the increased versatility of VHH antibodies.

Nanobody-based enzyme immunoassay for ochratoxin A in cereal with high resistance to matrix interference

“…To characterize how the anti-OTA Nbs perform compared to a mouse monoclonal antibody (mAb 6H8), temperature stability of these antibodies was evaluated as described by Bever and coworkers [14] with some modifications. Briefly, antibodies were diluted to working conditions (data not shown) and were heated to different temperatures (25, 50, 65, 80, 95 °C) for 5 min and to 90 °C for increasing times (0, 5, 15, 30, 45, 60, 75 min).…”

“…Compared to the conventional antibody fragments, the evolution of hydrophilic amino acids within framwork 2 of the heavy chain variable domain contributes to the high water solubility of nanobodies [9]. Due to their unique properties, more and more nanobodies for toxic and harmful substances were isolated and used as the detection reagent of immunoassays in food analysis [13, 14]. In addition, Nbs are readily genetically manipulated to construct fusions with green fluorescence protein and alkaline phosphatase [15–17], thus expanding their applications in immunoassay.…”

Smartphone-interfaced lab-on-a-chip devices for field-deployable enzyme-linked immunosorbent assay

“…Importantly, we have implemented the electrolytic control and pumping mechanisms to completely replicate an immunoassay (i.e., ELISA) procedure into a microscale, lab-on-achip format with comparable performance to the laboratory-based method. 45 In summary, our integrated mobile-interfaced POC system, as shown in Fig. 1, can be cost-effective, power-efficient, field-deployable, widely accessible, user-friendly, simple-to-use (plug-and-play), which could be offered as a generic biochemical detection platform in the future.…”

“…46 The reagent used for detection was a VHH isolated from an alpaca, described here. 45,47 The VHH was directly labeled with horseradish peroxidase (HRP; EZ-Link Plus Activated Peroxidase, Pierce Biotechnology, Rockford, IL) in phosphate buffered saline (PBS) with slight modifications to the manufacturer's protocol. The VHH was mixed with activated HRP at a molar ratio of 4:1 due to the smaller size of the VHH protein.…”

“…57,58 Heat stability is another feature of VHH especially applicable in mobile POC diagnostics as temperature may fluctuate during travel and geographic locations. 45 Additionally, the storage condition of ELISA devices is less of a constraint. Here, HRP is directly conjugated to our VHH, eliminating the downstream step of using secondary antibodies for visualization.…”