Shojaeian Sorour, Zarnani Amir Hassan, Jeddi-Tehrani Mahmood, Fereidooni Forouzandeh, Torkabadi Ebrahim, Akhondi Mahammad Mehdi, Tabatabaei-Panah Akram Sadat, Allameh Abdolamir
Department of Biochemistry, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran.
Iran J Immunol. 2009 Dec;6(4):174-85.
Monoclonal antibodies (mAbs) are essential tools for many molecular immunology investigations, epitope mapping and molecular modelling, clinical laboratory diagnostic tests and immunotherapy. Humoral immune response of immunized animals largely depends on the nature of antigen and the immunization technique. Polysaccharides and heavily-glycosylated proteins are very elusive targets incapable of mounting long-lasting, high affinity antibody responses. Carcinoma antigen 125 (CA 125), a well known tumor marker of ovarian cancer, is a mucin type antigen consisting of repetitive units of heavily glycosylated moieties which render production of mAbs very difficult.
To evaluate the efficacy of heterologous antigen preparations as a way of mouse immunization in the production of anti-CA 125 mAb.
Two different protocols of immunization were used for priming of NMRI mice. In the first method, mice conventionally immunized by three intraperitoneal injections of purified CA 125 and boosted by the antigen three days before fusion. In the second approach, mice were primed by three intraperitoneal injections of living CA 125 positive cells of OVCAR-3 cell line, and boosted by intravenous injection of the purified extracellular domain of CA 125. Production of mAb was performed by standard hybridoma technology and mAbs were characterized by different immunoassays.
The first method failed to produce stable clones despite six time fusion. A total of ten stable clones, however, were produced in the second approach. Some of the clones were characterized and found to have excellent immunoreactivity when tested by ELISA assay, western blotting, intracellular and surface immunofluorescent staining of OVCAR-3 cell line and immunohistochemical staining of ovarian cancer tissues.
Altogether the results of the present study clearly showed that heterologous antigen preparation is the method of choice for immunization when production of monoclonal antibody against highly glycosylated poorly immunogenic antigens is concerned.
单克隆抗体（mAb）是许多分子免疫学研究、表位作图和分子建模、临床实验室诊断测试及免疫治疗的重要工具。免疫动物的体液免疫反应很大程度上取决于抗原的性质和免疫技术。多糖和高度糖基化的蛋白质是非常难以捉摸的靶点，无法引发持久、高亲和力的抗体反应。癌抗原125（CA 125）是一种著名的卵巢癌肿瘤标志物，是一种粘蛋白型抗原，由高度糖基化部分的重复单元组成，这使得单克隆抗体的产生非常困难。
评估异源抗原制剂作为小鼠免疫方法在抗CA 125单克隆抗体生产中的效果。
采用两种不同的免疫方案对NMRI小鼠进行初次免疫。第一种方法，小鼠通过腹腔内注射三次纯化的CA 125进行常规免疫，并在融合前三天用该抗原进行加强免疫。第二种方法，小鼠通过腹腔内注射三次OVCAR-3细胞系的活CA 125阳性细胞进行初次免疫，并通过静脉注射纯化的CA 125细胞外结构域进行加强免疫。通过标准杂交瘤技术生产单克隆抗体，并用不同的免疫测定法对单克隆抗体进行表征。
尽管进行了六次融合，第一种方法仍未能产生稳定的克隆。然而，第二种方法共产生了10个稳定的克隆。对其中一些克隆进行了表征，通过ELISA测定、蛋白质印迹、OVCAR-3细胞系的细胞内和表面免疫荧光染色以及卵巢癌组织的免疫组织化学染色测试，发现它们具有优异的免疫反应性。
总之，本研究结果清楚地表明，当涉及生产针对高度糖基化、免疫原性差的抗原的单克隆抗体时，异源抗原制剂是免疫的首选方法。
Zotero 插件