鹿慢性消耗性疾病免疫组织化学检测方法的建立与优化

Establishment and Optimization of Immunohistochemistry Assay for Detection of Chronic Wasting Disease

【目的】在自主研制的抗鹿朊蛋白单克隆抗体基础上,建立和优化了鹿慢性消耗性疾病(chronic wasting disease,CWD)的免疫组织化学(immunohistochemistry,IHC)诊断方法。【方法】按照常规石蜡病理切片制作方法制备阴性和阳性切片,应用ABC法对获得的5株单抗进行免疫组织化学检测。【结果】试验结果表明,5株单抗(编号分别为5A5、3B2、6D12、5E3、1F5)中只有5E3和3B2可以与CWD的阳性鹿延髓切片发生特异性免疫反应,而且5E3的反应较强烈,3B2的反应较弱,其它3株单抗则在试验的浓度范围内无特异性免疫反应。此外,5E3的稀释比例为1:10 000时,免疫反应效果达到最佳状态。【结论】实验结果表明,单抗5E3可以用于我国CWD的监测。

[Objective] The purpose of this study is to establish and optimize immunohistochemical assay for detection of chronic wasting disease （CWD） based on the monoclonal antibodies （McAbs） against Elk cellular prion protein which were independently developed by China Animal Health and Epidemiology Center. [Method] Negative and positive slices were made by conventional paraffin method,and ABC method of IHC was used to detect the 5 McAbs. [ Result ] The results indicated that only two （No. 5E3 and 3B2） from 5 McAbs （No. 5A5, 3B2, 6D12, 5E3 and 1F5, respectively ） could be used for the immunohistochemical detection of positive spinal cord section of Elk. It was showed strong specific immunoreactivity with cervid abnormal prion protein by 5E3 and weak specific immunoreactivity by 3B2. There was no any immunoreactivity by other McAbs with various experimental concentrations. Additionally, the best immunoreactivity was present when McAb 5E3 was used at 1/10000 dilution. [ Conclusion ] The study demonstrated that 5E3 can be used in national surveillance of CWD.