Gost J I, Insausti R, Gonzalo L M
Departamento de Anatomía, Universidad de Navarra.
Rev Med Univ Navarra. 1993 Apr-Jun;38(2):9-20.
The development of a monoclonal antibody that selectively labels the astrocitic population of the central nervous system in the three species tested (human, monkey and rat) is described. The tissue used as immunogen consisted in a sector of the human hippocampus (CA1) from a brain removed shortly after death. The neuropathological examination in this case revealed an congophilic angiopathy. The tissue was frozen until homogenezation and intraperitoneal injection in 8 weeks old BALB/C mice, after which lymphocytes from the spleen were fused with NS-1 myeloma cells devoid of the enzyme Hypoxantine Guanine Phosphoribosyl Transferase (HPGT (-)) following standard procedures. The resulting hybridomas were tested by ELISA assays, immunofluorescence and immunohistochemical (PAP and ABC) techniques. The monoclonal antibody obtained was identified as an IgM and the Western blot analysis indicated that it recognizes a protein of 43,000 dalton. Immunohistochemistry was performed in 30 or 50 microns sections of human temporal lobe perfused through the carotid arteries with a mixture of 4% paraformaldehyde and 0.02% picric acid. Monkey tissue (sectioned at 50 microns) was obtained from a brain with the same fixation. Rat tissue followed the same protocol, although better results were seen after shifting to 1% paraformaldehyde and 1.25% glutaraldehyde (sections were 30 microns thick). Paraffin embedded human tissue (fixed in either 10% buffered formalin or in 4% paraformaldehyde) was also tested. All immunoreactions were intensified in a solution of osmium tetroxide and thiocarbohydrazide. The monoclonal antibody gave best results at a dilution of 1:50-100, where it gave a sharp labeling of both fibrous and protoplasmic astrocytes up to the most delicate processes, both in white and the grey matter. Rat tissue fixed in only 4% paraformaldehyde resulted in less optimal labeling, mostly restricted to limiting glia and Bergmann glia. The other fixation protocol (1% paraformaldehyde and 1.25% glutaraldehyde) gave a labeling comparable to monkey and human tissue. Paraffin embedded tissue was positive and similar to that obtained in frozen tissue, only when used the 4% paraformaldehyde fixation, but not when it was fixed in 10% formalin. Therefore, this monoclonal antibody has high specificity for the astrocitic population in the nervous system of mammals and it can be useful in a variety of studies of the normal and pathological situations of the nervous system.
本文描述了一种单克隆抗体的研发过程，该抗体能够选择性标记受试的三种物种（人类、猴子和大鼠）中枢神经系统中的星形胶质细胞群体。用作免疫原的组织取自一名死后不久取出的大脑中的人类海马体（CA1）区域。该病例的神经病理学检查显示存在嗜刚果红血管病。将组织冷冻至匀浆，然后注射到8周龄的BALB/C小鼠腹腔内，之后按照标准程序将脾脏中的淋巴细胞与缺乏次黄嘌呤鸟嘌呤磷酸核糖转移酶（HPGT (-)）的NS-1骨髓瘤细胞进行融合。通过酶联免疫吸附测定（ELISA）、免疫荧光和免疫组织化学（PAP和ABC）技术对所得杂交瘤进行检测。获得的单克隆抗体被鉴定为IgM，蛋白质印迹分析表明它识别一种43,000道尔顿的蛋白质。免疫组织化学在通过颈动脉灌注4%多聚甲醛和0.02%苦味酸混合物的人类颞叶30或50微米切片上进行。猴子组织（切成50微米厚的切片）取自经过相同固定处理的大脑。大鼠组织遵循相同方案，不过改用1%多聚甲醛和1.25%戊二醛后效果更佳（切片为30微米厚）。还对石蜡包埋的人类组织（用10%缓冲福尔马林或4%多聚甲醛固定）进行了检测。所有免疫反应在四氧化锇和硫代碳酰肼溶液中得到增强。该单克隆抗体在1:50 - 100的稀释度下效果最佳，在此稀释度下，它能清晰地标记纤维性和原浆性星形胶质细胞，直至最细微的突起，在白质和灰质中均如此。仅用4%多聚甲醛固定的大鼠组织标记效果欠佳，大多局限于界膜胶质细胞和伯格曼胶质细胞。另一种固定方案（1%多聚甲醛和1.25%戊二醛）的标记效果与猴子和人类组织相当。石蜡包埋组织仅在使用4%多聚甲醛固定时呈阳性且与冷冻组织的结果相似，而用10%福尔马林固定时则不然。因此，这种单克隆抗体对哺乳动物神经系统中的星形胶质细胞群体具有高度特异性，可用于多种神经系统正常和病理情况的研究。
