Development and Application of Indirect ELISA for IBDV VP2 Antibodies Detection in Poultry.

Infectious bursal disease virus (IBDV) is one of the most important immunosuppressive viruses in poultry, causing the global spread of infectious bursal disease (IBD). It poses a significant threat to the healthy development of the poultry industry. Vaccination is an effective approach for controlling IBDV infection. Therefore, reliable immune monitoring for IBDV is critical for maintaining poultry health. The enzyme-linked immunosorbent assay (ELISA) is a common technique used to detect specific antibodies in clinical serum testing and for the serological evaluation of IBDV vaccines. Among the currently available and under development IBDV vaccines, IBD VP2 subunit-based vaccines account for a considerable proportion. These vaccines stimulate the production of antibodies that are specific only to VP2. However, most IBDV antibody ELISA kits approved for use have applied the whole virus as the coating antigen, which does not adequately meet the diverse requirements for IBDV detection across different conditions. This study utilized a prokaryotic expression system to express the VP2 protein of the IBDV epidemic strain, assembling it into virus-like particles to be used as coating antigens. This approach enabled the establishment of an indirect ELISA method for detecting IBDV VP2 antibody (VP2-ELISA). The optimal coated antigen concentration was determined to be 2.5 μg/mL, with overnight coating at 4 °C; sealing with 5% skim milk at 37 °C for 4 h; serum dilution at 1:500 with incubation at 37 °C for 30 min; secondary antibody dilution at 1:4000 with incubation at 37 °C for 40 min; and then incubation with the substrate solution 3,3',5,5'-tetramethylbenzidine at room temperature for 20 min. The criterion for interpreting the detection results was OD ≥ 0.111 indicates IBDV antibody positivity, while OD < 0.111 indicates negativity. The established VP2-ELISA can specifically detect IBDV-positive sera at the lowest serum dilution of 1:6400, with intra- and inter-batch coefficients of variation of <2%. This indicates that the VP2-ELISA exhibits good specificity, sensitivity, and stability. Detection experiments using 20 laboratory-immunized chicken serum samples and 273 clinical serum samples demonstrated that the results of VP2-ELISA were consistent with those of commercial ELISA kits coated with whole virus. In summary, the VP2-ELISA developed in this study offers advantages in immune response detection for IBD VP2 subunit-based vaccines and is appropriate for evaluating the efficacy of IBD vaccines and detecting clinical serum samples.