Identification and Characterization of Nanobodies from a Phage Display Library and Their Application in an Immunoassay for the Sensitive Detection of African Swine Fever Virus

African swine fever (ASF) is one of the most lethal and devastating diseases of domestic and wild swine. The continual spread and frequent outbreaks of ASF have seriously threatened the pig and pig-related industries, causing great socioeconomic losses at unprecedented proportions. Although ASF has been documented for a century, no effective vaccine or antiviral treatment is currently available. Nanobodies (Nbs) derived from heavy-chain-only antibodies in camelids have been discovered to be effective as therapeutics and robust biosensors in imaging and diagnostic applications. In the present study, a high-quality phage display library containing specific Nbs raised against ASFV proteins was successfully constructed, and 19 nanobodies specific to ASFV p30 were preliminarily identified by phage display technology. After extensive evaluation, nanobodies Nb17 and Nb30 were employed as immunosensors and applied to develop a sandwich enzyme-linked immunosorbent assay (ELISA) for the detection of ASFV in clinical specimens. This immunoassay showed a detection limit of approximately 1.1 ng/mL target protein and 10 hemadsorption (HAD /mL) of ASFV and exhibited high specificity with no cross-reaction with the other porcine viruses tested. The performances of the newly developed assay and a commercial kit in testing 282 clinical swine samples were very similar (93.62% agreement). However, the novel sandwich Nb-ELISA showed higher sensitivity than the commercial kit when serial dilutions of ASFV-positive samples were tested. The present study describes a valuable alternative technique for the detection and surveillance of ASF in endemic regions. Furthermore, additional nanobodies specific to ASFV may be developed using the generated VHH library and employed in different biotechnology fields.