Research Article Open Access
Volume 4 | Issue 5 | DOI: https://doi.org/10.33696/immunology.4.148

Establishment of an Indirect Enzyme-linked Immunosorbent Assay for Detection of the NS4 Protein of Bluetongue Virus

  • 1College of Animal Science and Technology, Guangxi University, Nanning, Guangxi, 530005, PR China
  • 2College of Veterinary Medicine, Southwest University, Rongchang, Chongqing, 402460, PR China
  • 3Chongqing Dazu Center for Animal Disease Prevention and Control, Dazu, Chongqing, 402360, PR China
+ Affiliations - Affiliations

Corresponding Author

Huashan Yi, dyxyihuashan@swu.edu.cn

Received Date: October 08, 2022

Accepted Date: November 11, 2022


An indirect enzyme-linked immunosorbent assay (iELISA) was established to detect the serological prevalence of bluetongue virus (BTV) infection in ruminant populations. A recombinant NS4 (rNS4) protein was used as the encapsulated antigen. Optimization of the iELISA included the encapsulated antigen, serum dilution, blocking solution, and working concentration of a horseradish peroxidase (HRP)-labeled secondary antibody (Ab) by the square-matrix titration test. The results showed that the optimal reaction conditions for the iELISA were: volume of coating buffer, 2.5 μg/mL; serum dilution, 1:200; blocking solution, 0.25% polyvinyl alcohol + 5% skim milk powder; working concentration of the HRP-labeled secondary Ab, 1:4000; and critical values, sample-to-positive ratios 0.26 and 0.38 for determining positivity and negativity, respectively. The detection sensitivity of the iELISA was up to 1:4000, the intra- and inter-batch reproducibility coefficients of variation were less than 10%, and the positive and negative concordance rates of 56 bovine serum samples were both 100%, confirming excellent sensitivity and reproducibility. The proposed iELISA should prove useful for the diagnosis of BTV infection and future epidemiological investigations.


Clinical immunology, Immunochemistry, Immunochemistry, Immunomodulation, Immunotherapy

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