应用鼠单抗显示鼠组织中特异性抗原的免疫细胞化学方法

AN IMPROVED IMMUNOCYTOCHEMICAL METHOD FOR DETECTION OF THE SPECIFIC ANTIGENS IN RAT TISSUES BY THE USE OF MOUSE MONOCLONAL ANTIBODIES

作者在应用小鼠抗肾综合征出血热病毒(HFRSV)单克隆抗体(MAbs)免疫细胞化学法定位HFRSV自然感染大鼠组织中的病毒抗原时发现,部分大鼠组织的特异性HFRSV MAbs的染色切片及无关MAb对照和空白对照染色的切片中均出现血液。血细胞,血管壁,单核吞噬细胞系统,肾小球毛细血管及散在的心肌和肝细胞阳性,说明组织中有交叉反应。免疫双扩试验,羊抗鼠IgG桥抗可与大鼠Ig产生免疫沉淀反应,提示,组织中的交叉反应是由桥抗与大鼠细胞中本身存在的Ig(EIg)反应所致。为了避免EIg对特异性病毒抗原染色的干扰,我们以HFRSV MAbs预先分别与二抗羊抗鼠IgG结合,再用正常鼠Ig结合可能存在的二抗中尚未结合MAbs的Fab结合位点,制成一抗分子复合物,并以此作为一个新的相当于羊源性单克隆抗体孵育组织,以抗羊抗体连接羊PAP的多重PAP法显示一抗分子复合物的特异性抗原结合位点,结果显示该方法具有一抗的特异性,以有多重PAP法的敏感性,可有效的避免因EIg产生的交叉反应。用小鼠抗大鼠Kappa轻链MAb按同样方法制备一抗分子复合物用于染色证实,产生交叉阳性的部位确为大鼠组织中的Ig。病毒抗原及Ig的定位结果说明,HFRSV自然感染大鼠组织中的病毒抗原有广泛分布,也存在免疫复合物,但后者有别于HFRS人体组织的分布,另外,心肌及肝细胞Ig阳性说明该组织存在因HFRSV感染所造成的组织损伤,该损伤可能与机体的免疫反应有关。

When the indirect immunocytochemical methods were employedto demonstrate the specific antigens in tissues by the use of the primary antibodies prepared from the same species of animals as the target tissues come from, the specific results were frequently difficult to be interpreted due to the binding of the second antibody to the endogenous immunoglobulin(EIg) as well as any primary antibodies present in the tissues. This problem also occured in the tissues of the rats naturally infected with hemorrhagic fever with renal syndrome virus (HFRSV) stained by mouse monoclonal antibodies (MAbs) against HFRSV with the conventional repeated PAP immunocytochemical method to detect the viral antigens. This binding of second anti—mouse IgG antibody to the EIg, which was identified by the immunodiffusion analysis, occured both in the HFRSV MAb stained and negative control sections and was localized not only in the blood serum and blood cells but also in the vasculatures especially in the renal glomerular tufts in large quantities, reticuloendothelial Cells, myocardial cells and hepatocytes. In order to eliminate the interference of EIg with the viral antigen staining, an improved immunocytochemical method was developed. A molecular complex was first prepared by mixing the mouse MAbs against HFRSV(primary antibodies), goat anti—mouse IgG second antibody and normal mouse Ig, and then was incubated with the tissue sections, the specific binding of the complex to the viral antigen was demonstrated by the more sensitive repeated PAP method using antigoat IgG antibody and goat PAP. The EIg, which caused the nonspecific binding in the viral antigen staining sections, was further confirmed from the same method using the molecular complex formed from the MAb against kappa light chain of rat Ig(MAb Mark—L). This method was found to be more specific and sensi tive, and suitable for detection of the specific antigens in the tissues from the same species of animals as the primary antibodies were prepared from. The specific antigen detection results in this paper also showed that the viral antigens distributed in the tissues of the HFRSV infected rats were similar to that in the tissues of the HFRS patients, and the immunocomplexes were also present in the HFRSV infected rats but different from that in the HFRS human tissues in distribution, In addction, the abnormal Ig positive in the myocardial cells and hepatocytes indicated that the cellular damage was in existance, and was suggested to be related to the HFRSV infection, and might be caused by the immune response of the bodies against HFRSV infection.