China Animal Husbandry & Veterinary Medicine ›› 2025, Vol. 52 ›› Issue (9): 4368-4378.doi: 10.16431/j.cnki.1671-7236.2025.09.033

• Preventive Veterinary Medicine • Previous Articles    

Establishment of Direct Immunofluorescence Assay for IBDV and Its Preliminary Application

JIANG Yanping1, SUN Jian2, LIU Wei3, LI Bolong3, BAI Huitao3, YANG Jing3, LI Jiaxuan3, CUI Wen3, ZHOU Han3, HAN Jianchun1, TANG Lijie3   

  1. 1. Heilongjiang Green Food Science Research Institute, Harbin 150028, China;
    2. The Rural Public Affairs Service Center of Songyuan in Jilin, Songyuan 138000, China;
    3. College of Veterinary Medicine, Northeast Agricultural University, Harbin 150030, China
  • Received:2025-01-12 Published:2025-08-29

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Abstract: 【Objective】 The monoclonal antibody of Infectious bursal disease virus (IBDV) in chickens was labeled with R-phycoglobin fluorescein (R-PE) to establish a direct immunofluorescence assay for the rapid detection of IBDV in tissues and cells. 【Method】 After resuscitation of monoclonal antibody cells 4C12,monoclonal antibody ascites was prepared by immunizing mice.It was purified by chromatography column,identified by indirect immunofluorescence assay and ELISA,and the antibody titer was determined.The monoclonal antibodies were labeled by coupling kit,a direct immunofluorescence assay was established and the reaction conditions were optimized，and its specificity,sensitivity and stability were detected.The established method was used to detect the bursa of Fabricius and spleen of chickens infected with IBDV. 【Result】 The titer of purified 4C12 mab ascites was 1∶108.A direct immunofluorescence assay for 4C12 monoclonal antibody labeled with R-PE was established.The optimal reaction conditions were determined as follows:DT40 cells were infected with IBDV for 48 h,double fixation with 4% paraformaldehyde and anhydrous ethanol as fixants,the working concentration of the monoclonal antibody of R-PE-4C12 was 24 μg/mL,and the antibody was incubated for 90 min.Under these conditions,the fluorescence effect was the best.The established direct immunofluorescence assay had no cross-reaction with ALV,REV,AEV and EDSV,and had good specificity.This method could still detect positive signals when the virus dilution was 103 egg infection dose for 50% (ELD50),and had good sensitivity.The stability test showed that R-PE-4C12 could still produce a stable fluorescence signal after being stored at 4 ℃ for 21 d.In the established direct immunofluorescence assay,R-PE-4C12 could bind to IBDV in the infected tissue to produce specific red fluorescence.Compared with the indirect immunofluorescence assay,there was no significant difference in the fluorescence effect between the two methods. 【Conclusion】 The direct immunofluorescence assay established in this study had good specificity,sensitivity and stability，and could be used for the detection of IBDV in tissues and cells,providing an rapid and effective detection scheme for IBDV in the laboratory.

Key words: Infectious bursal disease virus (IBDV); direct immunofluorescence assay; monoclonal antibody; R-phycoglobin fluorescein

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