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. 2009 Apr;1(1):19-26.
Mammalians express several subclasses of the IgG molecule. In human being there are four homologous IgG subclasses, each of which is structurally unique and has different functions. Quantification of IgG subclasses is fundamental to clinical assessment and diagnosis of many diseases as such assessments depends on the availability of subclassspecific antibodies (Abs), particularly monoclonal antibodies (MAbs). In the present study, we produced and characterized two murine MAbs specific for human IgG3 molecule. These MAbs were obtained by the fusion of myeloma cells with splenocytes from Balb/c mice immunized with heavy chain of a human IgG3 myeloma protein. Fused cells were selected in hypoxanthine, aminopterine and thymidine (HAT) medium and cloned by limiting dilution assay. Ab-secreting cells were screened by enzyme-linked immunosorbent assay (ELISA) and the specificity of secreted MAbs was further analyzed, using a panel of purified myeloma proteins by ELISA and immunoblotting. Two stable hybridomas designated 1F18G7 and 1F18A11 were obtained secreting MAbs specific for Fc fragment of human IgG3. None of these MAbs showed cross-reactivity with other immunoglobulin isotypes derived from human and nine other animals, except 1F18A11 which displayed a weak cross-reactivity with only dog serum. Immunoblotting results indicate that these MAbs react with linear epitope(s) located in the heavy chain of human IgG3 molecules. The affinity constant of 1F18G7 and 1F18A11 MAbs was found to be 0.81×10(9) Mol (-1) and 0.71×10(9) Mol (-1), respectively, as measured by ELISA. These two MAbs with relatively high affinity can be useful tools for quantification of IgG3 subclass levels in human serum.
哺乳动物表达几种IgG分子亚类。在人类中，有四种同源IgG亚类，每一种在结构上都是独特的，并且具有不同的功能。IgG亚类的定量对于许多疾病的临床评估和诊断至关重要，因为此类评估取决于亚类特异性抗体（Abs），特别是单克隆抗体（MAbs）的可用性。在本研究中，我们制备并鉴定了两种针对人IgG3分子的鼠单克隆抗体。这些单克隆抗体是通过骨髓瘤细胞与用人类IgG3骨髓瘤蛋白重链免疫的Balb/c小鼠的脾细胞融合而获得的。融合细胞在次黄嘌呤、氨基蝶呤和胸腺嘧啶核苷（HAT）培养基中进行筛选，并通过有限稀释法进行克隆。通过酶联免疫吸附测定（ELISA）筛选分泌抗体的细胞，并使用一组纯化的骨髓瘤蛋白通过ELISA和免疫印迹进一步分析分泌的单克隆抗体的特异性。获得了两个稳定的杂交瘤，命名为1F18G7和1F18A11，它们分泌针对人IgG3 Fc片段的单克隆抗体。除了1F18A11与狗血清有弱交叉反应外，这些单克隆抗体均未与源自人类和其他九种动物的其他免疫球蛋白同种型表现出交叉反应。免疫印迹结果表明，这些单克隆抗体与位于人IgG3分子重链中的线性表位发生反应。通过ELISA测定，发现1F18G7和1F18A11单克隆抗体的亲和常数分别为0.81×10⁹ Mol⁻¹和0.71×10⁹ Mol⁻¹。这两种具有相对高亲和力的单克隆抗体可作为定量人血清中IgG3亚类水平的有用工具。
