title: Combining magnetic nanoparticle with biotinylated nanobodies for rapid and sensitive detection of influenza H3N2.
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 × 10(8) 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.
authors.affiliation: The Key Laboratory of Developmental Genes and Human Disease, Ministry of Education, Institute of Life Sciences, Southeast University, Nanjing 210096, People's Republic of China ; Jiangsu Nanobody Engineering and Research Center, Nantong 226010, People's Republic of China.
authors.forename: Min
authors.initials: M
authors.lastname: Zhu
authors.affiliation: State Key Laboratory of Materials-Oriented Chemical Engineering, College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, Nanjing 210009, People's Republic of China.
authors.forename: Yonghong
authors.initials: Y
authors.lastname: Hu
authors.affiliation: Jiangsu Nanobody Engineering and Research Center, Nantong 226010, People's Republic of China.
authors.forename: Guirong
authors.initials: G
authors.lastname: Li
authors.affiliation: Jiangsu Nanobody Engineering and Research Center, Nantong 226010, People's Republic of China.
authors.forename: Weijun
authors.initials: W
authors.lastname: Ou
authors.affiliation: 302 Military Hospital of China, Beijing 100039, People's Republic of China.
authors.forename: Panyong
authors.initials: P
authors.lastname: Mao
authors.affiliation: 302 Military Hospital of China, Beijing 100039, People's Republic of China.
authors.forename: Shaojie
authors.initials: S
authors.lastname: Xin
authors.affiliation: The Key Laboratory of Developmental Genes and Human Disease, Ministry of Education, Institute of Life Sciences, Southeast University, Nanjing 210096, People's Republic of China ; Jiangsu Nanobody Engineering and Research Center, Nantong 226010, People's Republic of China ; Sipailou No. 2, Southeast University, Nanjing 210096, People's Republic of China.
authors.forename: Yakun
authors.initials: Y
authors.lastname: Wan
keywords: Biotinylation; Influenza A grade 2 (H3N2); Magnetic nanoparticles; Nanobody