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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 whether the polyclonal and monoclonal P24 antibodies prepared in our previous study can detect the P24 protein of Borna disease virus (BDV) on paraffin sections and to explore the conditions for its application. Methods: Twenty SD rats were selected intradermal injection of BDV virus solution within 24 hours after birth. Twenty rats in the control group were injected phosphate buffered saline (PBS) at the same site, without injection of SD Forty rats in C57 and C57 mice were used as negative experimental control and fed on to paraffin embedded in paraffin for 60 days. Immunohistochemistry was performed by Envision two-step method. The first antibody was polyclonal or monoclonal P24 antibody, Primary antibody as a blank control, P24 antibody dilution concentrations were 1:50,1: 100,1: 200,1: 400 until 1: 5000; under high magnification, the cytoplasm of neuronal cells was brown or brown particles were positive , The score of each section of positive cells multiplied by the score of positive intensity score to calculate the total score. RESULTS: The staining scores of polyclonal P24 and monoclonal P24 were significantly different between experimental group and negative control group (polyclonal antibody Z = -3.108, P = 0.000; monoclonal antibody Z = -4.605 , P = 0.000). In the dilution of polyclonal P24 antibody, the sensitivity of 1: 200 and 1: 400 was good and the background was lighter. In the dilution of monoclonal P24 antibody, 1: 100, 1: 200 and 1 : 400 Sensitivity is good without background coloring. CONCLUSION: Polyclonal and monoclonal P24 antibodies can detect BDV P24 protein on paraffin sections; the recommended concentrations of polyclonal P24 antibody are 1: 200 and 1: 400, the recommended concentrations of monoclonal P24 antibody are 1: 100, 1: 200 and 1: 400.