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Combining magnetic nanoparticle with biotinylated nanobodies for rapid and sensitive detection of influenza H3N2.

Zhu M, Hu Y, Li G, Ou W, Mao P, Xin S, Wan Y - Nanoscale Res Lett (2014)

Bottom Line: After three successive biopanning steps, H3N2-specific nanobodies were successfully isolated, expressed in Escherichia coli, and purified.Biotinylated nanobody was effectively immobilized onto the surface of streptavidin magnetic beads.Under optimized conditions, the present immunoassay exhibited a relatively high sensitive detection with a limit of 50 ng/mL.

View Article: PubMed Central - HTML - PubMed

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.

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.

No MeSH data available.


Related in: MedlinePlus

The use of double nanobody sandwich method for influenza A grade 2 detection. (A) We used the magnetic beads linked with streptavidin to capture Nb3-biotin and used the Nb1 coupled to HRP as detector for H3N2 antigen detection. (B) Serial concentrations of H3N2 antigen were used for their detection. The lowest antigen concentration that could be detected was 50 ng/mL (P < 0.05). (C) The relative activity of Nb1 and Nb3 which were incubated at 37°C for different amounts of time. The activity of Nb1 and Nb3 reduced 26.03% and 17.22%, respectively, after heat incubation for 48 h.
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Figure 6: The use of double nanobody sandwich method for influenza A grade 2 detection. (A) We used the magnetic beads linked with streptavidin to capture Nb3-biotin and used the Nb1 coupled to HRP as detector for H3N2 antigen detection. (B) Serial concentrations of H3N2 antigen were used for their detection. The lowest antigen concentration that could be detected was 50 ng/mL (P < 0.05). (C) The relative activity of Nb1 and Nb3 which were incubated at 37°C for different amounts of time. The activity of Nb1 and Nb3 reduced 26.03% and 17.22%, respectively, after heat incubation for 48 h.

Mentions: Biotinylated nanobodies were attached to the magnetic beads via a streptavidin linker. They will be used to capture influenza viral particles. After separation using a magnetic field, HRP-labeled Nb1 was added to detect H3N2. HRP/H2O2 catalyzed the oxidation of TMB to form a blue-colored complex product changing to yellow after the addition of sulfuric acid to the reaction media [24]. The optical density (OD) value of the product was measured to analyze the concentrations of H3N2 in the reaction. The whole sandwich ELISA immunoassay is summarized in Figure 6A. With the increased concentrations of influenza H3N2, the binding ratio relative to BSA (negative control) also increased. As shown in Figure 6B, the OD value of the sample with 50 ng/mL H3N2 was significantly higher than that for the BSA alone (negative control) (P < 0.05). However, 10 ng/mL of H3N2 did not show significant difference from the control (P > 0.05). These results demonstrated that the detection limit in our indirect ELISA assay was about 50 ng/mL, which is more sensitive than conventional double-antibody sandwich enzyme-linked immune sorbent assay (DAS-ELISA). Finally, we checked the stability of two H3N2 nanobodies during different incubation times at 37°C. As shown in Figure 6C,D, after 48 h of heat incubation, Nb1 and Nb3 keep 73.49% and 82.78% of their original antigen-binding activity, respectively. We then investigated whether the decreased signal upon incubation at 37°C is due to inactivation of the antigen-binding capacity of the nanobody or due to degradation of the detection tag. To this end, we analyzed the samples incubated for different amounts of time at an elevated temperature in SDS-PAGE and Western blot. The result of the Western blot (Additional file 1: Figure S2) showed that the detection tag was still present on these nanobodies. Thus, we considered that the decreased signal upon incubation at 37°C is due to the decreased antigen-binding activity of nanobodies. Overall, these data demonstrated that Nbs to H3N2 showed good stability and they will provide promising diagnostic materials for clinical application.


Combining magnetic nanoparticle with biotinylated nanobodies for rapid and sensitive detection of influenza H3N2.

Zhu M, Hu Y, Li G, Ou W, Mao P, Xin S, Wan Y - Nanoscale Res Lett (2014)

The use of double nanobody sandwich method for influenza A grade 2 detection. (A) We used the magnetic beads linked with streptavidin to capture Nb3-biotin and used the Nb1 coupled to HRP as detector for H3N2 antigen detection. (B) Serial concentrations of H3N2 antigen were used for their detection. The lowest antigen concentration that could be detected was 50 ng/mL (P < 0.05). (C) The relative activity of Nb1 and Nb3 which were incubated at 37°C for different amounts of time. The activity of Nb1 and Nb3 reduced 26.03% and 17.22%, respectively, after heat incubation for 48 h.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4199786&req=5

Figure 6: The use of double nanobody sandwich method for influenza A grade 2 detection. (A) We used the magnetic beads linked with streptavidin to capture Nb3-biotin and used the Nb1 coupled to HRP as detector for H3N2 antigen detection. (B) Serial concentrations of H3N2 antigen were used for their detection. The lowest antigen concentration that could be detected was 50 ng/mL (P < 0.05). (C) The relative activity of Nb1 and Nb3 which were incubated at 37°C for different amounts of time. The activity of Nb1 and Nb3 reduced 26.03% and 17.22%, respectively, after heat incubation for 48 h.
Mentions: Biotinylated nanobodies were attached to the magnetic beads via a streptavidin linker. They will be used to capture influenza viral particles. After separation using a magnetic field, HRP-labeled Nb1 was added to detect H3N2. HRP/H2O2 catalyzed the oxidation of TMB to form a blue-colored complex product changing to yellow after the addition of sulfuric acid to the reaction media [24]. The optical density (OD) value of the product was measured to analyze the concentrations of H3N2 in the reaction. The whole sandwich ELISA immunoassay is summarized in Figure 6A. With the increased concentrations of influenza H3N2, the binding ratio relative to BSA (negative control) also increased. As shown in Figure 6B, the OD value of the sample with 50 ng/mL H3N2 was significantly higher than that for the BSA alone (negative control) (P < 0.05). However, 10 ng/mL of H3N2 did not show significant difference from the control (P > 0.05). These results demonstrated that the detection limit in our indirect ELISA assay was about 50 ng/mL, which is more sensitive than conventional double-antibody sandwich enzyme-linked immune sorbent assay (DAS-ELISA). Finally, we checked the stability of two H3N2 nanobodies during different incubation times at 37°C. As shown in Figure 6C,D, after 48 h of heat incubation, Nb1 and Nb3 keep 73.49% and 82.78% of their original antigen-binding activity, respectively. We then investigated whether the decreased signal upon incubation at 37°C is due to inactivation of the antigen-binding capacity of the nanobody or due to degradation of the detection tag. To this end, we analyzed the samples incubated for different amounts of time at an elevated temperature in SDS-PAGE and Western blot. The result of the Western blot (Additional file 1: Figure S2) showed that the detection tag was still present on these nanobodies. Thus, we considered that the decreased signal upon incubation at 37°C is due to the decreased antigen-binding activity of nanobodies. Overall, these data demonstrated that Nbs to H3N2 showed good stability and they will provide promising diagnostic materials for clinical application.

Bottom Line: After three successive biopanning steps, H3N2-specific nanobodies were successfully isolated, expressed in Escherichia coli, and purified.Biotinylated nanobody was effectively immobilized onto the surface of streptavidin magnetic beads.Under optimized conditions, the present immunoassay exhibited a relatively high sensitive detection with a limit of 50 ng/mL.

View Article: PubMed Central - HTML - PubMed

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.

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.

No MeSH data available.


Related in: MedlinePlus