多克隆及单克隆P24抗体在石蜡组织切片上检测BDV-P24蛋白质的应用

Application of the antigen-antibody(P24 polyclonal and monoclonal antibody)binding in detecting BDV-P24 protein in paraffin tissue sections

目的:验证本课题组前期制备的多克隆及单克隆P24抗体能否在石蜡组织切片上检测博尔纳病病毒(Borna disease virus,BDV)磷蛋白(P24蛋白质)以及探索其运用的条件。方法:选择出生24 h内的SD大鼠20只为实验组颅内注射BDV病毒液,对照组大鼠20只在相同部位注射磷酸盐缓冲液(phosphate buffered saline,PBS),未经注射的SD大鼠和C57小鼠各40只作为阴性实验对照,饲养到60 d取脑石蜡包埋制片;免疫组化染色用Envision两步法,一抗为多克隆或单克隆P24抗体,以PBS代替一抗作为空白对照,P24抗体的稀释浓度分别从1∶50、1∶100、1∶200、1∶400直到1∶5 000;以高倍镜下神经细胞细胞质呈棕黄色或棕褐色颗粒为阳性,将每张切片阳性细胞比例评分乘以阳性强度评分计算总分。结果:多克隆P24和单克隆P24抗体分别在BDV感染大鼠实验组与阴性对照组的染色评分差异有统计学意义(多克隆抗体Z=-3.108,P=0.000;单克隆抗体Z=-4.605,P=0.000);在多克隆P24抗体稀释度中,1∶200、1∶400的敏感性好而背景着色较轻,在单克隆P24抗体稀释度中,1∶100、1∶200、1∶400的敏感性好而无背景着色。结论:多克隆及单克隆P24抗体在石蜡组织切片上能检测出BDV P24蛋白质;并且多克隆P24抗体推荐的浓度为1∶200和1∶400,单克隆P24抗体的推荐浓度为1∶100、1∶200和1∶400。

Objective：To verify the antigen-antibody（P24 polyclonal and monoclonal antibody） binding in detecting Borna disease virus （BDV）-P24 protein in paraffin tissue sections and to explore the work conditions. Methods： Forty Sprague-Dawley rats born within 24 h were intracranially（i.e.） inoculated in the right cerebral hemisphere with either BDV solution or phosphate-buffered saline （con- trol：sham inoculated）. Forty Sprague-Dawley rats and 40 C57 rats without injection were chosen as negative controls. The brains of rats on 60 d after birth were perfused and were subsequently embedded into paraffin. Then the immunohistoehemical staining was per- formed following Envision two-step protocol. The primary antibody was chosen as P24 polyclonal and monoclonal antibody and the di- lution ranged from 1：50 to 1：5 000. The primary antibody was replaced by PBS as blank control. The brownish yellow or brown parti- cles distributing in the cells were considered as positive and total score was got by multiplying positive cell proportion score and posi-tive intensity score. Results：There were significant differences in staining scores between experimental group and control group （polyclonal P24 group Z=-3.108,P=0.000,monoclonal P24 group Z=-4.605,P=0.000. It showed a high sensitivity with slightly stained background in dilution 1：200,1：400 using polyclonal P24 staining while a high sensitivity without stained background in dilution 1：100, 1：200,1：400 using monoclonal 1＇24 staining. Conclusion：BDV P24 polyclonal and monoclonal antibody can bind the antigen distributed in paraffin sections of BDV infectious rat. The recommended dilution of polyclonal P24 antibody is 1：200 and 1：400. The recommended dilution of monoclonal P24 antibody is 1：100,1：200 and 1：400.