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

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.

Fig. 1

Schematic of strategies for constructing a phage display library and panning of sdAbs against FMDV type O

Fig. 2

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

Fig. 3

Multiple amino acid sequence alignment of FMDV type O-specific sdAb antibody clones. The framework and CDR regions and amino acid numbering as defined by Kabat are indicated. The CDR regions are boxed in thin lines, and the four conservative hallmark residues of sdAbs in FR2 (Phe37, Glu44Ala, Arg45, and Gly47Ala) are boxed in thick lines. The missing sequences are marked as the dash

Fig. 4

SDS–PAGE analysis of the expression and purification of sdAbs. a Expression of sdAbs in E. coli. Lanes 1 and 2, bacterial lysates from the sdAb-c1 and sdAb-c2 groups under non-inducing conditions. Lanes 3 and 4, bacterial lysates from the sdAb-c1 and sdAb-c2 groups under inducing conditions. (b) Purification of recombinant sdAbs protein. Lanes 1 and 2, purified sdAb-c1 and sdAb-c2. M = molecular weight markers, size indicated in kilodalton (kDa)

Fig. 5

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

Fig. 6

Serotype specificity identification of sdAbs against FMDV serotype O, Asia 1, and A antigens. All samples were tested for absorbance at 450 nm. The negative camel serum was coated as the negative control, and the polyclonal antibodies against FMDV type O were coated as the positive control. Error bars represent the ± standard deviation of three independent measurements

Fig. 7

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×