Hajighasemi Fatemeh, Shokri Fazel
Department of Immunology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran ; Department of Immunology, School of Medicine, Shahed University, Tehran, Iran.
Avicenna J Med Biotechnol. 2010 Jan;2(1):37-45.
Different IgG subclass profiles are produced in response to different antigenic stimuli in a variety of diseases. IgG subclass levels may reflect disease severity. Quantification of IgG subclasses depends on the availability of specific Monoclonal antibodies (MAbs). In the present study seven hybridoma clones producing MAbs reactive with multiple subclasses of human IgG were established. Splenocytes from Balb/c mice immunized with Fc fractions of human IgG1 or IgG2 myeloma proteins were fused with mouse myeloma cells. Fused cells were selected and cloned by limiting dilution assay. Antibody secreting cells were screened by Enzyme-linked immunosorbent assay (ELISA) and the specificity of secreted MAbs was further analyzed, using a panel of purified human myeloma paraproteins of different IgG subclasses by ELISA and immunoblotting. Cross-reactivity to immunoglobulins (Igs) of other species was studied by indirect ELISA using serum samples collected from 9 animals. The MAbs were found to react with triple IgG subclasses, including IgG1,2,4 (n=4) and IgG1,2,3 (n=3). Immunoblotting studies revealed recognition of linear (n=4) or conformational (n=3) epitopes by these MAbs. The most abundant cross-reactivity (71.4%) was observed with monkey Ig while no cross-reactivity was detected with hen and cat sera. The MAbs mostly displayed a restricted pattern of cross-reactivity and one of them did not bind to any of the animal sera tested. The affinity constant of 3 MAbs was measured by ELISA. Based on the data obtained from this study, mouse MAbs reactive with multiple human IgG subclasses are directed to a variety of immunogenic epitopes, mostly shared with IgG of other species. These MAbs are valuable tools for purification of non-reactive IgG subclasses through negative affinity chromatography. These MAbs could also provide an opportunity for epitope mapping of the Fc region of IgG, as well as serological phylogenetic studies.
在多种疾病中，针对不同的抗原刺激会产生不同的IgG亚类谱。IgG亚类水平可能反映疾病的严重程度。IgG亚类的定量取决于特异性单克隆抗体（MAb）的可用性。在本研究中，建立了7个产生与人类IgG多个亚类反应的MAb的杂交瘤克隆。用人类IgG1或IgG2骨髓瘤蛋白的Fc片段免疫的Balb/c小鼠的脾细胞与小鼠骨髓瘤细胞融合。通过有限稀释法选择并克隆融合细胞。通过酶联免疫吸附测定（ELISA）筛选抗体分泌细胞，并使用不同IgG亚类的纯化人类骨髓瘤副蛋白通过ELISA和免疫印迹进一步分析分泌的MAb的特异性。通过使用从9只动物收集的血清样本进行间接ELISA研究与其他物种免疫球蛋白（Ig）的交叉反应性。发现这些MAb与三种IgG亚类反应，包括IgG1、2、4（n = 4）和IgG1、2、3（n = 3）。免疫印迹研究揭示了这些MAb对线性（n = 4）或构象（n = 3）表位的识别。观察到与猴Ig的交叉反应性最为丰富（71.4%），而在鸡和猫血清中未检测到交叉反应性。这些MAb大多表现出有限的交叉反应模式，其中一种不与任何测试的动物血清结合。通过ELISA测量了3种MAb的亲和常数。基于本研究获得的数据，与多种人类IgG亚类反应的小鼠MAb针对多种免疫原性表位，这些表位大多与其他物种的IgG共享。这些MAb是通过负亲和色谱法纯化非反应性IgG亚类的有价值工具。这些MAb还可为IgG Fc区域的表位作图以及血清学系统发育研究提供机会。
