Selection and Characterization of CSFV-Specific Single-Domain Antibodies and Their Application along with Immunomagnetic Nanobeads and Quantum Dots

Abstract

Outbreak of classical swine fever (CSF) results in high mortality and thus causes severe economic losses in the swine industry. Single-domain antibody (sdAb) is the smallest antigen-binding molecule derived from camelid heavy-chain antibodies and has the potential to be used as a molecular probe for detection of CSF virus (CSFV). In this study, two sdAb fragments against the E2 antigen of CSFV were obtained, expressed in vitro. e functional characteristics analysis indicated that the recombinant sdAbE2-1 and sdAbE2-2 have excellent binding activity, specificity, and high affinity with equilibrium constant value of 3.34 × 10 − 7 and 1.35 × 10 − 8 M to E2 protein. en, sdAbE2s were conjugated with quantum dots (QD)/AF488 to synthesize two molecular probes for imaging CSFV distribution in cells. e sdAbE2-1 was also labeled with carboxyl-magnetic beads to construct immunomagnetic nanobeads (IMNBs) able to capture CSFV virions and recombinant E2 protein. QD/AF455-sdAbE2s probes colocalised with CSFV virions in swine testis cells, and IMNBs were used as a detection template and proved to bind specifically with CSFV virions and E2 protein. e selected sdAb fragments and sdAb-based molecular probes may be used for the rapid identification of CSFV during field outbreaks and for research on CSFV and host interactions.

Nanobodies in the limelight: Multifunctional tools in the fight against viruses

“…It further eliminates the species restriction for multiple co-staining. Nanobodies conjugated to different fluorophores have been used to detect viral proteins in infected cells [12,35,36]. In comparison to conventional antibodies, the nanobody length of 3-4 nm leads to a reduced linkage error (distance of the fluorophore to the target protein), conferring a higher image resolution [37].…”

“…Doerflinger et al made use of such nanobody binding to an occluded epitope on the capsid protein of human norovirus (hNoV) that was able to recognize four different genotypes [72]. Many other nanobodies found in the literature could be exploited for the development of diagnostics tests, reducing costs, and enhancing the availability of these tests compared with currently available alternatives [35,[73][74][75][76].…”