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Characterization of single-domain antibodies against Foot and Mouth Disease Virus (FMDV) serotype O from a camelid and imaging of FMDV in baby hamster kidney-21 cells with single-domain antibody-quantum dots probes.

Wang D, Yang S, Yin S, Shang Y, Du P, Guo J, He J, Cai J, Liu X - BMC Vet. Res. (2015)

Bottom Line: Results demonstrated that the two sdAbs were specific for serotype O and shared no cross-reactivity with serotypes A and Asia 1.The equilibrium dissociation constant (KD) values of the two sdAbs ranged from 6.23 nM to 8.24 nM, which indicated high affinity to FMDV antigens.Co-localization with the sdAb-AF488 and sdAb-QD probes indicated the same location of FMDV virions in baby hamster kidney-21 (BHK-21) cells. sdAb-QD probes are powerful tools to detect and image FMDV in BHK-21 cells.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Veterinary Etiological Biology, National Foot and Mouth Diseases Reference Laboratory, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou, Gansu, China. sdau_dw@163.com.

ABSTRACT

Background: Foot-and-mouth disease (FMD) is a highly contagious disease that affects cloven-hoofed animals and causes significant economic losses to husbandry worldwide. The variable domain of heavy-chain antibodies (VHHs or single domain antibodies, sdAbs) are single-domain antigen-binding fragments derived from camelid heavy-chain antibodies.

Results: In this work, two sdAbs against FMD virus (FMDV) serotype O were selected from a camelid phage display immune library and expressed in Escherichia coli. The serotype specificity and affinity of the sdAbs were identified through enzyme-linked immunosorbent assay and surface plasmon resonance assay. Moreover, the sdAbs were conjugated with quantum dots to constitute probes for imaging FMD virions. Results demonstrated that the two sdAbs were specific for serotype O and shared no cross-reactivity with serotypes A and Asia 1. The equilibrium dissociation constant (KD) values of the two sdAbs ranged from 6.23 nM to 8.24 nM, which indicated high affinity to FMDV antigens. Co-localization with the sdAb-AF488 and sdAb-QD probes indicated the same location of FMDV virions in baby hamster kidney-21 (BHK-21) cells.

Conclusions: sdAb-QD probes are powerful tools to detect and image FMDV in BHK-21 cells.

No MeSH data available.


Related in: MedlinePlus

Subcellular localization of FMDV with the sdAb-QD probes. The BHK-21 cells were harvested at 4 h.p.i and processed for immunofluorescence with sdAb-AF488 (green A1 and B1) and sdAb-QDs (red A2 and B2). All nuclei (A3, B3) were stained with DAPI. The merged images show the co-localization of FMDV with different probes. Magnification is 100×
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Fig7: Subcellular localization of FMDV with the sdAb-QD probes. The BHK-21 cells were harvested at 4 h.p.i and processed for immunofluorescence with sdAb-AF488 (green A1 and B1) and sdAb-QDs (red A2 and B2). All nuclei (A3, B3) were stained with DAPI. The merged images show the co-localization of FMDV with different probes. Magnification is 100×

Mentions: The conjugated products run relatively slow when tested with 1 % agarose gel because of their larger molecular weight than QDs alone (data not shown), indicating that the sdAbs were successfully labeled with QDs. The BHK-21 cells were fixed at 4 h.p.i. and monitored for the presence of FMDV virions using an immunofluorescence assay (IFA). As shown in Fig. 7, the FMDV virions can be observed with sdAb-QD probe staining. To identify the specificity of the sdAb-QD probes in vitro, probes sdAb-QDs and sdAb-AF488 were used to stain the fixed cells in a single trial. The merged images in Fig. 7 indicate that FMDV virions were located at the same area as the sdAb-QDs and sdAb-AF488 probes in BHK-21 cells. Most of the virions were located at one side of the nuclei, which indicated a novel invasion mechanism of FMDV when infecting BHK-21 cells. These results suggest that the sdAb-QDs probes are excellent candidates for further research.Fig. 7


Characterization of single-domain antibodies against Foot and Mouth Disease Virus (FMDV) serotype O from a camelid and imaging of FMDV in baby hamster kidney-21 cells with single-domain antibody-quantum dots probes.

Wang D, Yang S, Yin S, Shang Y, Du P, Guo J, He J, Cai J, Liu X - BMC Vet. Res. (2015)

Subcellular localization of FMDV with the sdAb-QD probes. The BHK-21 cells were harvested at 4 h.p.i and processed for immunofluorescence with sdAb-AF488 (green A1 and B1) and sdAb-QDs (red A2 and B2). All nuclei (A3, B3) were stained with DAPI. The merged images show the co-localization of FMDV with different probes. Magnification is 100×
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4489003&req=5

Fig7: Subcellular localization of FMDV with the sdAb-QD probes. The BHK-21 cells were harvested at 4 h.p.i and processed for immunofluorescence with sdAb-AF488 (green A1 and B1) and sdAb-QDs (red A2 and B2). All nuclei (A3, B3) were stained with DAPI. The merged images show the co-localization of FMDV with different probes. Magnification is 100×
Mentions: The conjugated products run relatively slow when tested with 1 % agarose gel because of their larger molecular weight than QDs alone (data not shown), indicating that the sdAbs were successfully labeled with QDs. The BHK-21 cells were fixed at 4 h.p.i. and monitored for the presence of FMDV virions using an immunofluorescence assay (IFA). As shown in Fig. 7, the FMDV virions can be observed with sdAb-QD probe staining. To identify the specificity of the sdAb-QD probes in vitro, probes sdAb-QDs and sdAb-AF488 were used to stain the fixed cells in a single trial. The merged images in Fig. 7 indicate that FMDV virions were located at the same area as the sdAb-QDs and sdAb-AF488 probes in BHK-21 cells. Most of the virions were located at one side of the nuclei, which indicated a novel invasion mechanism of FMDV when infecting BHK-21 cells. These results suggest that the sdAb-QDs probes are excellent candidates for further research.Fig. 7

Bottom Line: Results demonstrated that the two sdAbs were specific for serotype O and shared no cross-reactivity with serotypes A and Asia 1.The equilibrium dissociation constant (KD) values of the two sdAbs ranged from 6.23 nM to 8.24 nM, which indicated high affinity to FMDV antigens.Co-localization with the sdAb-AF488 and sdAb-QD probes indicated the same location of FMDV virions in baby hamster kidney-21 (BHK-21) cells. sdAb-QD probes are powerful tools to detect and image FMDV in BHK-21 cells.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Veterinary Etiological Biology, National Foot and Mouth Diseases Reference Laboratory, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou, Gansu, China. sdau_dw@163.com.

ABSTRACT

Background: Foot-and-mouth disease (FMD) is a highly contagious disease that affects cloven-hoofed animals and causes significant economic losses to husbandry worldwide. The variable domain of heavy-chain antibodies (VHHs or single domain antibodies, sdAbs) are single-domain antigen-binding fragments derived from camelid heavy-chain antibodies.

Results: In this work, two sdAbs against FMD virus (FMDV) serotype O were selected from a camelid phage display immune library and expressed in Escherichia coli. The serotype specificity and affinity of the sdAbs were identified through enzyme-linked immunosorbent assay and surface plasmon resonance assay. Moreover, the sdAbs were conjugated with quantum dots to constitute probes for imaging FMD virions. Results demonstrated that the two sdAbs were specific for serotype O and shared no cross-reactivity with serotypes A and Asia 1. The equilibrium dissociation constant (KD) values of the two sdAbs ranged from 6.23 nM to 8.24 nM, which indicated high affinity to FMDV antigens. Co-localization with the sdAb-AF488 and sdAb-QD probes indicated the same location of FMDV virions in baby hamster kidney-21 (BHK-21) cells.

Conclusions: sdAb-QD probes are powerful tools to detect and image FMDV in BHK-21 cells.

No MeSH data available.


Related in: MedlinePlus