Characterization and Double-Antibody Sandwich ELISA Application of a Monoclonal Antibody Against Akabane Virus Nucleocapsid Protein

Abstract Background Akabane virus (AKAV) is a Culicoides-borne Orthobunyavirus that is teratogenic to the fetus of cattle and small ruminant species. Objective This study aimed to develop an effective diagnostic assay for the detection of AKAV using produced monoclonal antibody (mAb). Method First, the mAb against N protein of AKAV was produced and characterized by Western blot (WB) and indirect immunofluorescence (IFA) assays. Then, the linear epitope of AKAV N protein against the mAb was identified and the mAb was applied to establish a double-antibody sandwich ELISA (DAS-ELISA). Results One AKAV N-reactive monoclonal mAb was generated and designated as 2D3. WB and IFA assays indicated that 2D3 could react with both recombinant N protein and AKAV isolate TJ2016. The linear epitope recognized by mAb 2D3 was located at amino acids 168–182 of AKAV N protein. The DAS-ELISA established on based mAb 2D3 was able to detect both the purified AKAV N protein (with a detection limit of 6.25 ng/mL) and AKAV-infected cell culture supernatant (with a detection limit of 250 TCID50/mL). Conclusions Taken together, we successfully prepared a mAb 2D3 against AKAV N protein and identified its corresponding linear epitope, and then established a DAS-ELISA for the detection of AKAV antigen. Highlights A produced mAb against AKAV N protein was used to define a linear epitope of AKAV and establish a DAS-ELISA for AKAV antigen detection.