Generation and Characterization of Siglec-F-Specific Monoclonal Antibodies

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Shahmohammadi Farid Sima; GHODS ROYA; JEDDI TEHRANI MAHMOOD; BAYAT ALI AHMAD; Mojtabavi Nazanin; RAZAVI ALIREZA; ZARNANI AMIR HASSAN

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Journal Paper

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Journal: Iranian Journal of Allergy, Asthma and Immunology Year:2017 | Volume:16 | Issue:6 Page(s): 460-470

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Title

Generation and Characterization of Siglec-F-Specific Monoclonal Antibodies

Author(s)

Keywords

Epitopes

Flow cytometry

Monoclonal antibody

Native protein

Siglec-F

Western blotting

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.

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APA: Copy

Shahmohammadi Farid, Sima, GHODS, ROYA, JEDDI TEHRANI, MAHMOOD, BAYAT, ALI AHMAD, Mojtabavi, Nazanin, RAZAVI, ALIREZA, & ZARNANI, AMIR HASSAN. (2017). Generation and Characterization of Siglec-F-Specific Monoclonal Antibodies. IRANIAN JOURNAL OF ALLERGY, ASTHMA AND IMMUNOLOGY (IJAAI), 16(6), 460-470. SID. https://sid.ir/paper/291586/en

Vancouver: Copy

Shahmohammadi Farid Sima, GHODS ROYA, JEDDI TEHRANI MAHMOOD, BAYAT ALI AHMAD, Mojtabavi Nazanin, RAZAVI ALIREZA, ZARNANI AMIR HASSAN. Generation and Characterization of Siglec-F-Specific Monoclonal Antibodies. IRANIAN JOURNAL OF ALLERGY, ASTHMA AND IMMUNOLOGY (IJAAI)[Internet]. 2017;16(6):460-470. Available from: https://sid.ir/paper/291586/en

IEEE: Copy

Sima Shahmohammadi Farid, ROYA GHODS, MAHMOOD JEDDI TEHRANI, ALI AHMAD BAYAT, Nazanin Mojtabavi, ALIREZA RAZAVI, and AMIR HASSAN ZARNANI, “Generation and Characterization of Siglec-F-Specific Monoclonal Antibodies,” IRANIAN JOURNAL OF ALLERGY, ASTHMA AND IMMUNOLOGY (IJAAI), vol. 16, no. 6, pp. 460–470, 2017, [Online]. Available: https://sid.ir/paper/291586/en

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