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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

Monoclonal phage ELISA. A total of 131 random clones from the sdAb library were analyzed with monoclonal phage ELISA. FMDV type O antigens at 10 μg/mL were coated in each well. PBS was used as the negative control. A total of 12 clones were selected on the basis of absorbance. The x-axis presents the clone number, and the y-axis shows the absorbance values at 450 nm
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Fig2: Monoclonal phage ELISA. A total of 131 random clones from the sdAb library were analyzed with monoclonal phage ELISA. FMDV type O antigens at 10 μg/mL were coated in each well. PBS was used as the negative control. A total of 12 clones were selected on the basis of absorbance. The x-axis presents the clone number, and the y-axis shows the absorbance values at 450 nm

Mentions: NAL-O was subjected to four rounds of panning for the phage particles against FMDV type O. Phage recovery increased by more than 100-fold after the fourth round of panning than after the first one (Table 2), which demonstrated an efficient enriching factor of antigen-specific sdAbs. A total of 131 individual E. coli clones were randomly chosen from NAL-O and analyzed with monoclonal phage ELISA. Twelve clones yielded high absorbance at 450 nm (Fig. 2), and local sequencing of the 12 clones helped identify two sdAb gene sequences, which were named sdAb-c1 and sdAb-c2.Table 2


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)

Monoclonal phage ELISA. A total of 131 random clones from the sdAb library were analyzed with monoclonal phage ELISA. FMDV type O antigens at 10 μg/mL were coated in each well. PBS was used as the negative control. A total of 12 clones were selected on the basis of absorbance. The x-axis presents the clone number, and the y-axis shows the absorbance values at 450 nm
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig2: Monoclonal phage ELISA. A total of 131 random clones from the sdAb library were analyzed with monoclonal phage ELISA. FMDV type O antigens at 10 μg/mL were coated in each well. PBS was used as the negative control. A total of 12 clones were selected on the basis of absorbance. The x-axis presents the clone number, and the y-axis shows the absorbance values at 450 nm
Mentions: NAL-O was subjected to four rounds of panning for the phage particles against FMDV type O. Phage recovery increased by more than 100-fold after the fourth round of panning than after the first one (Table 2), which demonstrated an efficient enriching factor of antigen-specific sdAbs. A total of 131 individual E. coli clones were randomly chosen from NAL-O and analyzed with monoclonal phage ELISA. Twelve clones yielded high absorbance at 450 nm (Fig. 2), and local sequencing of the 12 clones helped identify two sdAb gene sequences, which were named sdAb-c1 and sdAb-c2.Table 2

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