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

H3N2-specific VHH genes were selected from the sdAb library. (A) The enrichment for phage particles in wells coated with antigen versus wells without antigen was detected after each round of panning. Phages collected from each round were incubated with H3N2 virus and NaHCO3 (100 mM, pH 8.0), respectively. Then, the eluted phages were transformed into TG1 cells. +: Phages transformed into cells after panning with H3N2 virus. −: Phages panning with NaHCO3 were used as control. (B) After three rounds of panning, H3N2-specific VHHs were enriched 67-fold compared with control (+/−). (C) Periplasmic extract ELISA for 95 colonies. The colonies, whose absorbance signal (nanobodies incubated with H3N2) is more than twofold higher than that for the negative control (nanobodies incubated with NaHCO3), were considered as positive. (D) Four kinds of different amino acid sequences of anti-H3N2 VHHs were identified.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: H3N2-specific VHH genes were selected from the sdAb library. (A) The enrichment for phage particles in wells coated with antigen versus wells without antigen was detected after each round of panning. Phages collected from each round were incubated with H3N2 virus and NaHCO3 (100 mM, pH 8.0), respectively. Then, the eluted phages were transformed into TG1 cells. +: Phages transformed into cells after panning with H3N2 virus. −: Phages panning with NaHCO3 were used as control. (B) After three rounds of panning, H3N2-specific VHHs were enriched 67-fold compared with control (+/−). (C) Periplasmic extract ELISA for 95 colonies. The colonies, whose absorbance signal (nanobodies incubated with H3N2) is more than twofold higher than that for the negative control (nanobodies incubated with NaHCO3), were considered as positive. (D) Four kinds of different amino acid sequences of anti-H3N2 VHHs were identified.

Mentions: After getting a good quality of nanobody library for phage display, we performed bio-panning to isolate nanobodies against inactivated H3N2. A total of around 2 × 1011 phage particles from the library were used in pannings. In order to evaluate the enrichment during the process of panning, we compared the ratio of colony numbers between pannings on H3N2 influenza viruses and PBS as negative control. As demonstrated in Figure 3A,B, after two and three rounds of biopanning, the ratios have been gradually increased from 5-fold to 67-fold. Based on our experience, high enrichment in only three rounds of biopanning from a large and diverse phage library is important for a successful retrieval of antigen-specific clones [16,22].Next, we have randomly picked 95 colonies after panning and tested whether the PE-ELISA would reveal the presence of nanobodies binding to inactivated H3N2. Of the 95 colonies, 19 showed a good target recognition with binding ratios relative to a non-coated well of more than 2 (Figure 3C). VHH fragments from these 19 colonies were further amplified by PCR and sequenced to confirm the presence of nanobodies. Based on sequencing data, all 19 colonies contained the correct (in frame) VHH fragments. Sequence analysis further classified these 19 nanobodies into 4 classes, which are shown by diversified complementarity determining region 3 (CDR3) sequences (Figure 3D). We define these four classes of nanobodies as Nb1, Nb2, Nb3, and Nb4, respectively.


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)

H3N2-specific VHH genes were selected from the sdAb library. (A) The enrichment for phage particles in wells coated with antigen versus wells without antigen was detected after each round of panning. Phages collected from each round were incubated with H3N2 virus and NaHCO3 (100 mM, pH 8.0), respectively. Then, the eluted phages were transformed into TG1 cells. +: Phages transformed into cells after panning with H3N2 virus. −: Phages panning with NaHCO3 were used as control. (B) After three rounds of panning, H3N2-specific VHHs were enriched 67-fold compared with control (+/−). (C) Periplasmic extract ELISA for 95 colonies. The colonies, whose absorbance signal (nanobodies incubated with H3N2) is more than twofold higher than that for the negative control (nanobodies incubated with NaHCO3), were considered as positive. (D) Four kinds of different amino acid sequences of anti-H3N2 VHHs were identified.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: H3N2-specific VHH genes were selected from the sdAb library. (A) The enrichment for phage particles in wells coated with antigen versus wells without antigen was detected after each round of panning. Phages collected from each round were incubated with H3N2 virus and NaHCO3 (100 mM, pH 8.0), respectively. Then, the eluted phages were transformed into TG1 cells. +: Phages transformed into cells after panning with H3N2 virus. −: Phages panning with NaHCO3 were used as control. (B) After three rounds of panning, H3N2-specific VHHs were enriched 67-fold compared with control (+/−). (C) Periplasmic extract ELISA for 95 colonies. The colonies, whose absorbance signal (nanobodies incubated with H3N2) is more than twofold higher than that for the negative control (nanobodies incubated with NaHCO3), were considered as positive. (D) Four kinds of different amino acid sequences of anti-H3N2 VHHs were identified.
Mentions: After getting a good quality of nanobody library for phage display, we performed bio-panning to isolate nanobodies against inactivated H3N2. A total of around 2 × 1011 phage particles from the library were used in pannings. In order to evaluate the enrichment during the process of panning, we compared the ratio of colony numbers between pannings on H3N2 influenza viruses and PBS as negative control. As demonstrated in Figure 3A,B, after two and three rounds of biopanning, the ratios have been gradually increased from 5-fold to 67-fold. Based on our experience, high enrichment in only three rounds of biopanning from a large and diverse phage library is important for a successful retrieval of antigen-specific clones [16,22].Next, we have randomly picked 95 colonies after panning and tested whether the PE-ELISA would reveal the presence of nanobodies binding to inactivated H3N2. Of the 95 colonies, 19 showed a good target recognition with binding ratios relative to a non-coated well of more than 2 (Figure 3C). VHH fragments from these 19 colonies were further amplified by PCR and sequenced to confirm the presence of nanobodies. Based on sequencing data, all 19 colonies contained the correct (in frame) VHH fragments. Sequence analysis further classified these 19 nanobodies into 4 classes, which are shown by diversified complementarity determining region 3 (CDR3) sequences (Figure 3D). We define these four classes of nanobodies as Nb1, Nb2, Nb3, and Nb4, respectively.

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