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Iran J Allergy Asthma Immunol. 2017 Dec;16(6):460-470.

Generation and Characterization of Siglec-F-Specific Monoclonal Antibodies.

Author information

1
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.
2
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.
3
Monoclonal Antibody Research Center, Avicenna Research Institute, Academic Center for Education, Culture and Research (ACECR), Tehran, Iran.
4
Monoclonal Antibody Research Center, Avicenna Research Institute, Academic Center for Education, Culture and Research (ACECR), Tehran, Iran.
5
Department of Immunology, Faculty of Medicine, Iran University of Medical Sciences, Tehran, Iran.
6
Department of Pathobiology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran.
7
Reproductive Immunology Research Center, Avicenna Research Institute, Academic Center for Education, Culture and Research (ACECR), Tehran, Iran AND Immunology Research Center, Iran University of Medical Sciences, Tehran, Iran.

Abstract

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.

KEYWORDS:

Epitopes; Flow cytometry; Monoclonal antibody; Native protein; Siglec-F; Western blotting

PMID:
29338152
[Indexed for MEDLINE]
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