鸭肝组织中DHBcAg表达水平定量免疫组织化学方法的建立

Establishment of the Quantitative Immunohistochemical Examination of DHBcAg in Duck Liver

目的：建立鸭肝组织中鸭乙肝病毒核心抗原（DHBcAg）表达水平定量的免疫组织化学方法。方法比较热修复、微波修复、胃蛋白酶修复和不修复四种不同抗原修复方法的免疫组化检测技术，建立 DHBcAg抗原修复的最佳方案。采用Image-Pro Plus 6.0软件，设置优化图像采集参数，通过扣除空白区域的面积计算二氨基联苯胺（DAB）黄染的平均光密度（MD），客观评价不同切片中阳性表达的程度。采用该方法对 DHBV感染的鸭肝组织中 DHBcAg表达水平进行定量，评价DHBcMAb-TAT PTD穿膜抗体的抗病毒作用。结果抗原不修复的方式进行 DHBcAg免疫组化染色优于其他三种抗原修复的方法。建立优化的图像采集参数，进行DHBcAg抗原表达的 MD值计算。比较受试麻鸭治疗前后肝组织中DHBcAg的 MD差值，结果显示随着DHBcMAb-TAT PTD穿膜抗体给药剂量的增加，鸭肝细胞内 DHBcAg的表达逐渐降低，存在明显剂量依赖关系。表明该方法可有效评价药物对DHBcAg的影响。结论建立DHBcAg抗原不修复的定量免疫组织化学方法可以更加客观、准确的评价DHBcAg表达水平。

Objective To develop a quantitative immunohistochemistry assay for duck hepatitis B virus core antigen （DHB-cAg）in duck liver tissue.Methods By comparison with no repair antigen and repair antigen with high pressure,microwave and trypsin,the best solution of antigen retrieval was determined.By optimizing the parameter of image acquisition and de-ducting blank area,mean density of yellow areas was calculated using Image-Pro Plus 6.0 software.Using the assay devel-oped to determine the level of DHBcAg in liver tissue from duck infected by DHBV,anti-DHBV activity of DHBcMAb-TAT PTD conj ugate was examined.Results SABC method with no repair antigen was selected,which was better than other methods.DHBcAg expression in duck liver tissue could be objectively and accurately quantified by setting Image-Pro Plus 6.0 software parameters and calculating mean density of yellow areas.By comparison with the differences between mean densityat baseline of treatment and end of treatment,it was showed that DHBcMAb-TATPTD conjugate treatment dose-de-pendently reduced the levels of DHBcAg in liver tissue,which show that the assay developed could effectively evaluate the anti-DHBV activity of agent.Conclusion The immunohistochemistry assay developed in this study can objectively and accu-rately evaluate the level of DHBcAg in duck liver tissue.