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. Objectives This study aimed to develop an effective diagnostic assay for the detection of AKAV using produced monoclonal antibody (mAb). Methods Firstly, 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 double antibody sandwich ELISA (DAS-ELISA) established based mAb 2D3 was able to detect both the purified AKAV N protein (with detection limit of 6.25 ng/ml) and AKAV-infected cell culture supernatant (with detection limit of 250 TCID50/ml). Conclusion 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.

Generation of Stable Cell Lines Expressing Akabane Virus N Protein and Insight into Its Function in Viral Replication

“…As a member of the orthobunyaviruses, AKAV shares most of its characteristics with the other viruses (such as Schmallenberg virus, SBV) of this genus. In terms of the reason that the S-segment is the most conserved genomic region within individual orthobunyavirus species and the N protein it encodes is the most abundant protein in virions and infected cells [ 10 ], it is currently widely used for virus or antibody detection [ 18 , 19 , 22 , 25 , 26 , 27 ]. In the present study, we successfully established and characterized two stable BHK-21 cell lines (C8H2 and F7E5) in which the AKAV N protein is constitutively expressed ( Figure 4 A).…”

“…The monoclonal antibody (mAb) 2D3 that was raised against the AKAV N protein [ 22 ] was prepared in our laboratory. Mouse anti-β-actin mAb, FITC-labeled goat anti-mouse IgG, and RBITC-labeled goat anti-mouse IgG were purchased from SolarbioLife Sciences (Beijing, China).…”