Development of a sandwich ELISA for the detection of Chinese sacbrood virus infection

Chinese sacbrood disease (CSBD) is a highly pathogenic infectious disease in bees that is caused by Chinese sacbrood virus (CSBV). Although several molecular detection methods have been developed for CSBV, there are no commercially available enzyme-linked immunosorbent assay (ELISA) kits. We therefore developed a sandwich ELISA to detect CSBV antigens. To this end, monoclonal antibodies were produced using VP2 as an immunogen and subsequently characterized. Hybridomas were screened for the secretion of immunoglobulin G (IgG). Using an unlabeled monoclonal antibody (mAb) for coating and a horseradish peroxidase (HRP)-labeled mAb for detection, a CSBV sandwich ELISA method was established. This method showed specificity for CSBV and did not show cross-reactivity with other bee viruses. The detection limit of the sandwich ELISA was 3.675 × 10⁴ copies/µL. Sixty bee larvae were tested using our sandwich ELISA method, and the presence of CSBV was verified by reverse transcription polymerase chain reaction (RT-PCR). The total coincidence rate was 90%. Thus, a sandwich ELISA method with high specificity and accuracy and a detection limit of 3.675 × 10⁴ copies/µL has been successfully developed and can be used for the clinical detection of CSBV. This method will support rapid diagnosis, real-time monitoring, and early warning of CSBD.