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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

Purification of H3N2-specific nanobodies and specificity of two paired nanobodies. (A) Four nanobodies having different sequences were purified by immobilized metal affinity chromatography (IMAC) using a His-Select column. (B) Isoelectric point, molecular weight, and yield of four nanobodies. (C, D) The Nb1 and Nb3, recognizing two different epitopes as shown via a pairing experiment, were chosen to detect the specificity to five types of different viral proteins by ELISA. One hundred microliters of each inactivated influenza virus (5 μg/mL) was coated onto microtiter plates, and 100 μL nanobodies (10 μg/mL) were added. After reaction with mouse anti-HA tag antibody and then anti-mouse IgG-alkaline phosphatase, the chromogenic solution containing bisphosphate was added, and the absorbance at 405 nm was measured by an ELISA reader. BSA (5 μg/mL) was used as control. Values were the means of three replicates. The values of Nb1 and Nb3 were significantly different from the control (***P < 0.001). (E) Specificity of the nanobodies to the proteins on the surface of the H3N2 virus by ELISA. The H3N2 virus and HA protein were coated, and BSA was used as control. After incubation with Nb1 and Nb3, respectively, anti-His tag mouse monoclonal antibody was added, and then anti-mouse IgG-alkaline phosphatase was added for detection. Finally, absorbance at 405 nm was read.
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Figure 4: Purification of H3N2-specific nanobodies and specificity of two paired nanobodies. (A) Four nanobodies having different sequences were purified by immobilized metal affinity chromatography (IMAC) using a His-Select column. (B) Isoelectric point, molecular weight, and yield of four nanobodies. (C, D) The Nb1 and Nb3, recognizing two different epitopes as shown via a pairing experiment, were chosen to detect the specificity to five types of different viral proteins by ELISA. One hundred microliters of each inactivated influenza virus (5 μg/mL) was coated onto microtiter plates, and 100 μL nanobodies (10 μg/mL) were added. After reaction with mouse anti-HA tag antibody and then anti-mouse IgG-alkaline phosphatase, the chromogenic solution containing bisphosphate was added, and the absorbance at 405 nm was measured by an ELISA reader. BSA (5 μg/mL) was used as control. Values were the means of three replicates. The values of Nb1 and Nb3 were significantly different from the control (***P < 0.001). (E) Specificity of the nanobodies to the proteins on the surface of the H3N2 virus by ELISA. The H3N2 virus and HA protein were coated, and BSA was used as control. After incubation with Nb1 and Nb3, respectively, anti-His tag mouse monoclonal antibody was added, and then anti-mouse IgG-alkaline phosphatase was added for detection. Finally, absorbance at 405 nm was read.

Mentions: In order to express nanobodies in E. coli, the common methods consist in subcloning VHH fragments from the phage display phagemid vector into an expression plasmid. However, in our study, we will use E. coli WK6 strains to express the nanobodies. These cells have unique properties which cannot suppress the amber stop codon between gene III and VHH from plasmid pMECS. In this case, we can directly transform the VHH cloned in phage display phagemid pMECS vector from TG1 to WK6 cells to express nanobodies without the need for a subcloning step. Upon IPTG induction, soluble nanobodies are expressed in the periplasmic region of WK6 cells. The induced nanobodies were further purified by Ni2+-IDA column. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of four purified nanobodies showed a good quality with more than 90% purity after one-step purification (Figure 4A). Yields of our four purified nanobodies ranged from 5 to 10 mg/L TB medium (Figure 4B).These four individual nanobodies showed great diversity based on CDR3 sequences, and they may recognize different epitopes of influenza H3N2. Next, we have performed a nanobody-pairing assay with these four nanobodies. It turned out that Nb1 and Nb3 showed to be a great combination for further diagnostic applications based on sandwich immunoassay. As influenza A viruses have been classified into various subtypes, we want to know whether the isolated Nbs from our library are specific to H3N2. We then coated five different subtypes of influenza A viruses H1N1, H3N2, H5N1, H7N2, and H9N2 onto ELISA plates. Interestingly, Nb1 and Nb3 can only recognize H3N2 (Figure 4C,D). In order to assure which kind of protein on the surface of the H3N2 virus is recognized by these nanobodies, we detected its specificity by ELISA. The result in Figure 4E showed that Nb1 and Nb3 were specific to the hemagglutinin protein of H3N2 virus particles. To our knowledge, this is the first report for the identification of nanobodies against influenza H3N2. These nanobodies might be a promising tool to be used for sensitive detection of influenza H3N2.


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)

Purification of H3N2-specific nanobodies and specificity of two paired nanobodies. (A) Four nanobodies having different sequences were purified by immobilized metal affinity chromatography (IMAC) using a His-Select column. (B) Isoelectric point, molecular weight, and yield of four nanobodies. (C, D) The Nb1 and Nb3, recognizing two different epitopes as shown via a pairing experiment, were chosen to detect the specificity to five types of different viral proteins by ELISA. One hundred microliters of each inactivated influenza virus (5 μg/mL) was coated onto microtiter plates, and 100 μL nanobodies (10 μg/mL) were added. After reaction with mouse anti-HA tag antibody and then anti-mouse IgG-alkaline phosphatase, the chromogenic solution containing bisphosphate was added, and the absorbance at 405 nm was measured by an ELISA reader. BSA (5 μg/mL) was used as control. Values were the means of three replicates. The values of Nb1 and Nb3 were significantly different from the control (***P < 0.001). (E) Specificity of the nanobodies to the proteins on the surface of the H3N2 virus by ELISA. The H3N2 virus and HA protein were coated, and BSA was used as control. After incubation with Nb1 and Nb3, respectively, anti-His tag mouse monoclonal antibody was added, and then anti-mouse IgG-alkaline phosphatase was added for detection. Finally, absorbance at 405 nm was read.
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Related In: Results  -  Collection

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Figure 4: Purification of H3N2-specific nanobodies and specificity of two paired nanobodies. (A) Four nanobodies having different sequences were purified by immobilized metal affinity chromatography (IMAC) using a His-Select column. (B) Isoelectric point, molecular weight, and yield of four nanobodies. (C, D) The Nb1 and Nb3, recognizing two different epitopes as shown via a pairing experiment, were chosen to detect the specificity to five types of different viral proteins by ELISA. One hundred microliters of each inactivated influenza virus (5 μg/mL) was coated onto microtiter plates, and 100 μL nanobodies (10 μg/mL) were added. After reaction with mouse anti-HA tag antibody and then anti-mouse IgG-alkaline phosphatase, the chromogenic solution containing bisphosphate was added, and the absorbance at 405 nm was measured by an ELISA reader. BSA (5 μg/mL) was used as control. Values were the means of three replicates. The values of Nb1 and Nb3 were significantly different from the control (***P < 0.001). (E) Specificity of the nanobodies to the proteins on the surface of the H3N2 virus by ELISA. The H3N2 virus and HA protein were coated, and BSA was used as control. After incubation with Nb1 and Nb3, respectively, anti-His tag mouse monoclonal antibody was added, and then anti-mouse IgG-alkaline phosphatase was added for detection. Finally, absorbance at 405 nm was read.
Mentions: In order to express nanobodies in E. coli, the common methods consist in subcloning VHH fragments from the phage display phagemid vector into an expression plasmid. However, in our study, we will use E. coli WK6 strains to express the nanobodies. These cells have unique properties which cannot suppress the amber stop codon between gene III and VHH from plasmid pMECS. In this case, we can directly transform the VHH cloned in phage display phagemid pMECS vector from TG1 to WK6 cells to express nanobodies without the need for a subcloning step. Upon IPTG induction, soluble nanobodies are expressed in the periplasmic region of WK6 cells. The induced nanobodies were further purified by Ni2+-IDA column. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of four purified nanobodies showed a good quality with more than 90% purity after one-step purification (Figure 4A). Yields of our four purified nanobodies ranged from 5 to 10 mg/L TB medium (Figure 4B).These four individual nanobodies showed great diversity based on CDR3 sequences, and they may recognize different epitopes of influenza H3N2. Next, we have performed a nanobody-pairing assay with these four nanobodies. It turned out that Nb1 and Nb3 showed to be a great combination for further diagnostic applications based on sandwich immunoassay. As influenza A viruses have been classified into various subtypes, we want to know whether the isolated Nbs from our library are specific to H3N2. We then coated five different subtypes of influenza A viruses H1N1, H3N2, H5N1, H7N2, and H9N2 onto ELISA plates. Interestingly, Nb1 and Nb3 can only recognize H3N2 (Figure 4C,D). In order to assure which kind of protein on the surface of the H3N2 virus is recognized by these nanobodies, we detected its specificity by ELISA. The result in Figure 4E showed that Nb1 and Nb3 were specific to the hemagglutinin protein of H3N2 virus particles. To our knowledge, this is the first report for the identification of nanobodies against influenza H3N2. These nanobodies might be a promising tool to be used for sensitive detection of influenza H3N2.

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