Isolation of Alpaca Anti-Hapten Heavy Chain Single Domain Antibodies for Development of Sensitive Immunoassay

Abstract

Some unique subclasses of Camelidae antibodies are devoid of light chain and the antigen binding site is comprised exclusively of the variable domain of the heavy chain (VHH). Although conventional antibodies dominate current assay development, recombinant VHHs have a high potential as alternative reagents for the next generation of immunoassay. We expressed VHHs from an immunized alpaca and developed a VHH-based immunoassay using 3-phenoxybenzoic acid (3-PBA), a major metabolite of pyrethroid insecticides as a model system. A phage VHH library was constructed and seven VHH clones were selected by competitive binding with 3-PBA. The best immunoassay developed with one of these VHHs showed an IC50 of 1.4 ng/mL (limit of detection (LOD) = 0.1 ng/mL). These parameters were further improved by using the phage borne VHH, IC50 = 0.1 ng/mL and LOD = 0.01 ng/mL. Both assays showed a similar tolerance to methanol and dimethylsulfoxide up to 50% in assay buffer. The assay was highly specific to 3-PBA and its 4-hydroxylated derivative, 4-hydroxy 3-PBA (150% cross reactivity) with negligible cross reactivity with other tested structural analogs and the recovery from spiked urine sample ranged from 80 to 112%. In conclusion, a highly specific and sensitive VHH for 3-PBA was developed using sequences from immunized alpaca and phage display technology for antibody selection.

VHH antibodies: emerging reagents for the analysis of environmental chemicals

“…3-Phenoxybenzoic acid (3-PBA) is a common urinary metabolite of most pyrethroid insecticides that could be used as a biomarker of human or environmental exposure to these pesticides. In this work [31], 3-PBA was used as a model system to study the practical aspects of producing VHH against small molecules. The VHHs were selected using gradually decreasing concentrations of 3-PBA-BSA conjugate and free 3-PBA and more stringent washing.…”

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

“…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.…”

Towards nano-diagnostics for rapid diagnosis of infectious diseases – current technological state

“…In particular, the thermal stability renders them attractive as an alternative reagent in immunoassays in tropical, resource-poor countries. Kim et al (82) developed a VHH-based immunoassay using 3-phenoxybenzoic acid, which is the major metabolite of pyrethroid insecticides, as a model system. They developed several immunoassays, including bacteriophage-born VHH, and compared the results.…”