Abstract
Our objective is to develop a rapid and sensitive assay based on magnetic beads to detect the concentration of influenza H3N2. The possibility of using variable domain heavy-chain antibodies (nanobody) as diagnostic tools for influenza H3N2 was investigated. A healthy camel was immunized with inactivated influenza H3N2. A nanobody library of 8 × 108 clones was constructed and phage displayed. After three successive biopanning steps, H3N2-specific nanobodies were successfully isolated, expressed in Escherichia coli, and purified. Sequence analysis of the nanobodies revealed that we possessed four classes of nanobodies against H3N2. Two nanobodies were further used to prepare our rapid diagnostic kit. Biotinylated nanobody was effectively immobilized onto the surface of streptavidin magnetic beads. The modified magnetic beads with nanobody capture specifically influenza H3N2 and can still be recognized by nanobodies conjugated to horseradish peroxidase (HRP) conjugates. Under optimized conditions, the present immunoassay exhibited a relatively high sensitive detection with a limit of 50 ng/mL. In conclusion, by combining magnetic beads with specific nanobodies, this assay provides a promising influenza detection assay to develop a potential rapid, sensitive, and low-cost diagnostic tool to screen for influenza infections.
Highlights
Influenza A and B viruses cause a pandemic threat to human health throughout the world [1]
We developed a rapid and sensitive immunoassay for influenza H3N2 detection based on single-domain antigenbinding fragments of camelid-like heavy-chain antibodies
Nb3 coupled to magnetic beads and horseradish peroxidase (HRP)-conjugated Nb1 have been used for rapid and sensitive detection of influenza H3N2
Summary
Influenza A and B viruses cause a pandemic threat to human health throughout the world [1]. Sporadic transmission of influenza viruses from birds to humans could lead to unpredictable pandemic outbreaks. Influenza is an infectious disease of the respiratory tract that can infect millions of people and kills hundreds of thousands of them [2]. Humans, infected with influenza A, manifest typically an acute upper respiratory tract illness characterized by fever, cough, and sore throat. Disease severity depends mainly on the virulence of the influenza virus strain and immune competence of the patients [3]. Influenza viruses are members of the Orthomyxoviridae family, and they are further classed as A, B, and C viruses [4]. 17 influenza A hemagglutinin (HA) subtypes have been described.
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