Multivalent nanobody-based sandwich enzyme-linked immunosorbent assay for sensitive detection of porcine reproductive and respiratory syndrome virus

Background: The pandemic of the porcine reproductive and respiratory syndrome virus (PRRSV) has caused huge economic losses and continues to threaten the swine industry worldwide. Antibodies are critical for determining the sensitivity and specificity of diagnostic immunoassays. Recently, nanobodies have been increasingly used in diagnostic immunoassays because of their numerous advantages over traditional antibodies, including simple genetic engineering to improve the affinity and fusion with reported agents. This study is the first to develop a sandwich enzyme-linked immunosorbent assay (ELISA) with high sensitivity and specificity to detect the PRRSV. Results: Thirteen nanobodies against PRRSV were prepared by using phage display technology and prokaryotically expressed. Two of those nanobodies with high affinity, Nb12 and Nb35, were selected and employed to develop the sandwich ELISA. To obtain greater sensitivity, a trivalent nanobody (3×Nb12) and a bivalent nanobody-HRP fusion protein (2×Nb35-HRP) were used as the capture antibody and the detecting antibody, respectively, in a subsequently modified sandwich ELISA. This modified ELISA was found to have high sensitivity for detecting PRRSV, with a detection limit of 10 TCID 50 /µL, which was approximately 200-fold greater than the single-copy nanobody-based sandwich ELISA. The developed assay shows high specificity and can detect almost all circulating lineages of PRRSV-2 in China. Conclusions: The trivalent nanobodies and bivalent nanobody-HRP were applied to develop an improved sandwich ELISA for the first time, and the ELISA exhibits high sensitivity and specificity for detecting the target virus. This study provides suggestions for reforming nanobodies and for the further development of multivalent nanobody-based ELISAs for other various viruses.