鸡传染性法氏囊病病毒直接免疫荧光检测方法的建立及初步应用

doi:10.16431/j.cnki.1671-7236.2025.09.033

摘要/Abstract

摘要： 【目的】利用R-藻红蛋白荧光素(R-PE)标记鸡传染性法氏囊病病毒(Infectious bursal disease virus，IBDV)单克隆抗体，建立快速检测组织和细胞中IBDV的直接免疫荧光方法。【方法】试验取单克隆抗体细胞4C12复苏后，免疫小鼠制备单克隆抗体腹水，用层析柱进行纯化，通过间接免疫荧光法和ELISA方法对其进行鉴定，并测定抗体效价；通过偶联试剂盒对单克隆抗体进行标记，建立直接免疫荧光法并优化反应条件，检测其特异性、敏感性及稳定性。利用建立的方法对感染IBDV的鸡法氏囊和脾脏进行检测。【结果】纯化的4C12单克隆抗体腹水效价为1∶10⁸；用R-PE标记4C12单克隆抗体，成功建立了直接免疫荧光检测方法，确定最佳反应条件为：IBDV感染DT40细胞48 h、4%多聚甲醛和无水乙醇为固定剂进行双重固定、R-PE-4C12株单克隆抗体工作浓度为24 μg/mL、抗体孵育90 min，此条件下荧光效果最佳。建立的直接免疫荧光方法与禽白血病病毒(ALV)、禽网状内皮组织增生症病毒(REV)、禽脑脊髓炎病毒(AEV)和减蛋综合症病毒(EDSV)不发生交叉反应，特异性较好；该方法在病毒含量为10³鸡胚半数感染量(ELD₅₀)时仍能检测到阳性信号，灵敏性较好；稳定性试验显示，R-PE-4C12在4 ℃保存21 d后，仍能产生稳定的荧光信号。建立的直接免疫荧光方法中R-PE-4C12能与感染组织中IBDV结合产生特异性红色荧光；与间接免疫荧光法相比，两者荧光效果没有明显差异。【结论】本研究建立的直接免疫荧光方法具有较好的特异性、敏感性和稳定性，可以用于组织和细胞中IBDV的检测，为实验室IBDV检测提供一种快速有效的检测方案。

关键词: 鸡传染性法氏囊病病毒(IBDV); 直接免疫荧光法; 单克隆抗体; R-藻红蛋白荧光素

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

中图分类号:

S852.65^＋7

姜艳平, 孙健, 刘薇, 李博龙, 白慧涛, 杨景, 李佳璇, 崔文, 周晗, 韩建春, 唐丽杰. 鸡传染性法氏囊病病毒直接免疫荧光检测方法的建立及初步应用[J]. 中国畜牧兽医, 2025, 52(9): 4368-4378.

JIANG Yanping, SUN Jian, LIU Wei, LI Bolong, BAI Huitao, YANG Jing, LI Jiaxuan, CUI Wen, ZHOU Han, HAN Jianchun, TANG Lijie. Establishment of Direct Immunofluorescence Assay for IBDV and Its Preliminary Application[J]. China Animal Husbandry & Veterinary Medicine, 2025, 52(9): 4368-4378.

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