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.