Limits...
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

Library construction. (A) The segments containing VHH gene fragments were amplified by a first PCR. (B) The fragments were amplified by a second, nested PCR. (C) Size of the library was determined by counting the number of clones after serial dilutions and plating on plates containing selective antibiotics. (D) Clones were randomly selected to detect the percentage of clones with a phagemid containing an insert of a proper size for a VHH.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4199786&req=5

Figure 2: Library construction. (A) The segments containing VHH gene fragments were amplified by a first PCR. (B) The fragments were amplified by a second, nested PCR. (C) Size of the library was determined by counting the number of clones after serial dilutions and plating on plates containing selective antibiotics. (D) Clones were randomly selected to detect the percentage of clones with a phagemid containing an insert of a proper size for a VHH.

Mentions: In order to isolate nanobodies against H3N2 with high affinity and specificity, a 2-year-old healthy Bactrian camel was immunized with the inactivated H3N2 virus over a period of 7 weeks. After the final immunization, we tested the antigen-specific total IgG titer within the serum from the immunized animal and showed that 1:1,000 serum dilution still gave a good signal. This indicated that immunization with inactivated H3N2 has raised a good immune response. According to the procedure in Figure 1, the heavy-chain antibody variable region (also known as VHH) sequences were amplified from lymphocyte cDNA of the camel. Firstly, two PCR products including a 900 bp fragment for VH-CH1-CH2 exons and 600 bp for VHH-CH2 exons (Figure 2A) were amplified with primers CALL001 and CALL002 [20]. The gene for the VHH domain of about 400 bp (Figure 2B) was amplified with nested primers PMCF, BACK-1, and BACK-2 using as template the 600 bp PCR fragment.


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)

Library construction. (A) The segments containing VHH gene fragments were amplified by a first PCR. (B) The fragments were amplified by a second, nested PCR. (C) Size of the library was determined by counting the number of clones after serial dilutions and plating on plates containing selective antibiotics. (D) Clones were randomly selected to detect the percentage of clones with a phagemid containing an insert of a proper size for a VHH.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Library construction. (A) The segments containing VHH gene fragments were amplified by a first PCR. (B) The fragments were amplified by a second, nested PCR. (C) Size of the library was determined by counting the number of clones after serial dilutions and plating on plates containing selective antibiotics. (D) Clones were randomly selected to detect the percentage of clones with a phagemid containing an insert of a proper size for a VHH.
Mentions: In order to isolate nanobodies against H3N2 with high affinity and specificity, a 2-year-old healthy Bactrian camel was immunized with the inactivated H3N2 virus over a period of 7 weeks. After the final immunization, we tested the antigen-specific total IgG titer within the serum from the immunized animal and showed that 1:1,000 serum dilution still gave a good signal. This indicated that immunization with inactivated H3N2 has raised a good immune response. According to the procedure in Figure 1, the heavy-chain antibody variable region (also known as VHH) sequences were amplified from lymphocyte cDNA of the camel. Firstly, two PCR products including a 900 bp fragment for VH-CH1-CH2 exons and 600 bp for VHH-CH2 exons (Figure 2A) were amplified with primers CALL001 and CALL002 [20]. The gene for the VHH domain of about 400 bp (Figure 2B) was amplified with nested primers PMCF, BACK-1, and BACK-2 using as template the 600 bp PCR fragment.

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