Hajighasemi Fatemeh, Khoshnoodi Jalal, Shokri Fazel
Department of Immunology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran ; Department of Immunology, Faculty of Medicine, Shahed University, Tehran, Iran.
Avicenna J Med Biotechnol. 2012 Oct;4(4):170-7.
Pan-IgG specific monoclonal antibodies (MAbs) are essential tools for assessment of humoral immunity, immune deficiency and gammopathy. In this study, four hybridoma clones producing MAbs with different specificities for human IgG isotypes were established.
Splenocytes from Balb/c mice immunized with Fc fractions of human IgG were fused with SP2/0 myeloma cells. Hybridoma cells were selected in HAT selective medium 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 myeloma IgG proteins by ELISA and immunoblotting. Cross-reactivity to immunoglobulins (Igs) of other species was studied by indirect ELISA using serum samples collected from 9 animals.
Immunoblotting studies revealed recognition of either linear or conformational epitopes by these MAbs. The most abundant cross-reactivity (100%) was observed with monkey Igs, while no cross-reactivity was detected with rabbit, guinea pig, sheep, goat, cat and hen sera. Two of the MAbs cross-reacted with either dog or horse sera. The affinity constant of two MAbs was measured by ELISA and found to be 0.74×10(8)M(-1) and 0.96×10(7)M(-1).
Our results indicate that these pan-IgG specific MAbs recognize restricted linear or conformational epitopes located on all human IgG subclasses with no cross-reactivity to IgG from most species studied. These MAbs are potentially useful tools for quantification of total or antigen-specific IgG levels.
全IgG特异性单克隆抗体（MAbs）是评估体液免疫、免疫缺陷和丙种球蛋白病的重要工具。在本研究中，建立了四个产生对人IgG同种型具有不同特异性的单克隆抗体的杂交瘤克隆。
用人类IgG的Fc片段免疫的Balb/c小鼠的脾细胞与SP2/0骨髓瘤细胞融合。杂交瘤细胞在HAT选择培养基中进行选择，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IgG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。通过使用从9只动物收集的血清样本进行间接ELISA研究对其他物种免疫球蛋白（Igs）的交叉反应性。
免疫印迹研究显示这些单克隆抗体识别线性或构象表位。观察到与猴Igs的交叉反应性最高（100%），而与兔、豚鼠、绵羊、山羊、猫和鸡血清未检测到交叉反应性。其中两种单克隆抗体与狗或马血清发生交叉反应。通过ELISA测量了两种单克隆抗体的亲和常数，发现分别为0.74×10(8)M(-1)和0.96×10(7)M(-1)。
我们的结果表明，这些全IgG特异性单克隆抗体识别位于所有人IgG亚类上的受限线性或构象表位，对大多数研究物种的IgG无交叉反应性。这些单克隆抗体是定量总IgG或抗原特异性IgG水平的潜在有用工具。
