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

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Abstract
Akabane virus (AKAV) is a Culicoides-borne Orthobunyavirus that is teratogenic to the fetus of cattle and small ruminant species. This study aimed to develop an effective diagnostic assay for the detection of AKAV using produced monoclonal antibody (mAb). 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). 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). 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. 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.
Funding Information
  • Fundamental Research Funds of Chinese Academy of Inspection and Quarantine (2020JK021, 2022JK11)
  • National key research and development program (2022YFD1802000)

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