Shahmohammadi-Farid Sima, Ghods Roya, Jeddi-Tehrani Mahmood, Bayat Ali-Ahmad, Mojtabavi Nazanin, Razavi Alireza, Zarnani Amir-Hassan
Department of Pathobiology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran AND Hematology and Oncology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran.
Oncopathology Research Center, Iran University of Medical Sciences, Tehran, Iran AND Department of Molecular Medicine, Faculty of Advanced Technologies in Medicine, Iran University of Medical Sciences, Tehran, Iran.
Iran J Allergy Asthma Immunol. 2017 Dec;16(6):460-470.
Siglec-F (SF) is a surface glycoprotein expressed by mouse eosinophils and induces caspase- and mitochondria-dependent apoptosis after engagement with its cognate ligand or specific antibodies. This targeting eosinophils by monoclonal antibodies may help diverse diseases associated with increased frequency of eosinophils including allergy and asthma. In this paper, production of murine and rat monoclonal antibodies (mAbs) against Siglec-F has been addressed. Balb/c mice were immunized with siglec-F1 (SF1) and siglec-F2 (SF2) synthetic peptides conjugated to a carrier protein. Rats were immunized with Chinese hamster ovary CHO cells overexpressing Siglec-F (CHO-SF) or with Siglec-F-human immunoglobulin FC fusion protein (CHO-SF-Ig). Hybridomas were produced by standard protocol and screened for their reactivity by enzyme-linked immunosorbent assay (ELISA), western blotting (WB), and flow cytometry. In parallel, polyclonal antibodies were generated in New Zealand White rabbits immunized with SF1 and SF2 peptides. Three mouse and three rat mAbs were generated against synthetic peptides and SF-Ig, respectively. All mouse monoclonal and rabbit polyclonal antibodies reacted well with immunizing molecules in ELISA and detected specific band of Siglec-F in WB. However, they failed to detect native molecule in flow cytometry analysis. Quite the contrary, rat mAbs did not reacted with the denatured protein in WB, instead exhibited significant reactivity with CHO-SF cells in flow cytometry. Based on the heavily glycosylated nature of Siglec-F, it seems that generation of anti-SF antibodies able to detect native protein needs a properly folded molecule for immunization. Monoclonal antibodies reported here are invaluable tools for studying linear and conformation epitopes of SF and tracing mouse eosinophils.
唾液酸结合免疫球蛋白样凝集素F（Siglec-F，SF）是一种由小鼠嗜酸性粒细胞表达的表面糖蛋白，在与同源配体或特异性抗体结合后可诱导半胱天冬酶和线粒体依赖性凋亡。通过单克隆抗体靶向嗜酸性粒细胞可能有助于治疗与嗜酸性粒细胞频率增加相关的多种疾病，包括过敏和哮喘。本文阐述了针对Siglec-F的鼠源和大鼠单克隆抗体（mAb）的制备。用与载体蛋白偶联的唾液酸结合免疫球蛋白样凝集素F1（SF1）和唾液酸结合免疫球蛋白样凝集素F2（SF2）合成肽免疫Balb/c小鼠。用过量表达Siglec-F的中国仓鼠卵巢（CHO）细胞（CHO-SF）或唾液酸结合免疫球蛋白样凝集素F-人免疫球蛋白FC融合蛋白（CHO-SF-Ig）免疫大鼠。按照标准方案制备杂交瘤，并通过酶联免疫吸附测定（ELISA）、蛋白质印迹法（WB）和流式细胞术筛选其反应性。同时，用SF1和SF2肽免疫新西兰白兔制备多克隆抗体。分别产生了三种针对合成肽和SF-Ig的小鼠和大鼠单克隆抗体。所有小鼠单克隆抗体和兔多克隆抗体在ELISA中均能与免疫分子良好反应，并在WB中检测到Siglec-F的特异性条带。然而，它们在流式细胞术分析中未能检测到天然分子。恰恰相反，大鼠单克隆抗体在WB中不与变性蛋白反应，而是在流式细胞术中与CHO-SF细胞表现出显著反应性。基于Siglec-F高度糖基化的性质，似乎能够检测天然蛋白的抗SF抗体的产生需要一个正确折叠的分子用于免疫。本文报道的单克隆抗体是研究SF的线性和构象表位以及追踪小鼠嗜酸性粒细胞的宝贵工具。
