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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

Western blot analysis of the purified sdAbs. SdAbs were expressed through the fusion of 6 × His tag at the N-terminus. Lanes 1 and 2, purified sdAb-c1 and sdAb-c2. M = pre-stained molecular weight markers
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Fig5: Western blot analysis of the purified sdAbs. SdAbs were expressed through the fusion of 6 × His tag at the N-terminus. Lanes 1 and 2, purified sdAb-c1 and sdAb-c2. M = pre-stained molecular weight markers

Mentions: The sdAb genes were induced and analyzed using SDS–PAGE (Fig. 4a). The sdAbs had an expected molecular weight of approximately 35 kDa. The purified sdAb recombinant protein was analyzed with a SDS–PAGE gel (Fig. 4b). Only minor nonspecific contaminations were detected in the eluted fraction. The sdAbs were confirmed with Western blot, and the target protein bands (approximately 35 kDa) were observed on the PVDF membrane (Fig. 5).Fig. 4


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)

Western blot analysis of the purified sdAbs. SdAbs were expressed through the fusion of 6 × His tag at the N-terminus. Lanes 1 and 2, purified sdAb-c1 and sdAb-c2. M = pre-stained molecular weight markers
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig5: Western blot analysis of the purified sdAbs. SdAbs were expressed through the fusion of 6 × His tag at the N-terminus. Lanes 1 and 2, purified sdAb-c1 and sdAb-c2. M = pre-stained molecular weight markers
Mentions: The sdAb genes were induced and analyzed using SDS–PAGE (Fig. 4a). The sdAbs had an expected molecular weight of approximately 35 kDa. The purified sdAb recombinant protein was analyzed with a SDS–PAGE gel (Fig. 4b). Only minor nonspecific contaminations were detected in the eluted fraction. The sdAbs were confirmed with Western blot, and the target protein bands (approximately 35 kDa) were observed on the PVDF membrane (Fig. 5).Fig. 4

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