The AJ517 antibody detects the mouse CD8β protein by flow cytometry

Aubry-Lachainaye, Jean-Pierre

Bulla, Monica

D’Esposito, Alessia

Giusti, Teresa

Guilhen, Cyril

Manoukian, Arec

Pirek, Cyril

Sassi, Ali

Verdon, Margaux

English

Open Access Publications (Univ. de Genève)

doi:10.24450/journals/abrep.2020.e114  Antibody Reports, 2020, vol. 3, e114 This work is licensed under a Creative Commons Attribution 4.0 International License. Geneva University Library Open Access Publications https://oap.unige.ch/journals/abrep  |  ISSN 2624-8557 The AJ517 antibody detects the mouse CD8β protein by flow cytometry Alessia G. D’Esposito1, Arec H. Manoukian1, Cyril Pirek1, Margaux Verdon1, Teresa M. Giusti1, Monica Bulla1, Ali Sassi1, Jean-Pierre Aubry-Lachainaye2, Cyril Guilhen1 1 Bachelor in Biomedical Sciences, Faculty of Medicine, University of Geneva, 1 rue Michel Servet, CH-1211, Geneva, Switzerland 2 Flow Cytometry Core Facility, University of Geneva, 1211 Geneva 4, Switzerland  Abstract The AJ517 antibody detects the mouse CD8β protein by flow cytometry.  Introduction CD8 is a membrane-bound glycoprotein complex expressed primarily in cytotoxic T lymphocytes. It is composed of two transmembrane subunits, a and b, that associate to form a disulfide-linked heterodimer (Parnes, 1989) present at the cell surface. Here, we describe the ability of the AJ517 antibody, a single chain fragment (scFv) derived from the 35.17.2 hybridoma, to successfully detect the CD8β protein (Uniprot #P10300) by flow cytometry in HEK293 cells expressing CD8a and CD8b.  Materials & Methods Antibodies: ABCD_AJ517 antibody (ABCD nomenclature, web.expasy.org/abcd/; Lima et al., 2019) was produced by the Geneva Antibody Facility (www.unige.ch/antibodies/) as mini-antibody with the antigen-binding scFv fused to a rabbit IgG Fc. The synthesized scFv sequence (GeneArt, Invitrogen) correspond to the sequence of the variable regions of the 35.17.2 hybridoma (Pierres et al., 1982) joined by a peptide linker (GGGGS)3. The sequencing of the 35.17.2 hybridoma was performed by the Geneva Antibody Facility. HEK293 suspension cells (growing in serum-free FreeStyleTM 293 Expression Medium, Gibco #12338) were transiently transfected with the vector coding for the scFv-Fc. AJ517 supernatant (30 mg/L) was collected after 4 days.  Antigen: The 35.17.2 hybridoma was originally raised against murine leukocytes in Lou/WSl rats (Pierres et al., 1982). HEK293 suspension cells (growing in FreeStyleTM 293 Expression Medium, Gibco #12338) were transiently transfected 3 days before the experiment with the vectors coding for the full-length mouse CD8a (Uniprot #P01731) and CD8β protein. Co-transfection with the CD8a encoding plasmid was performed to guarantee proper protein dimerization and trafficking.  Protocol: The whole procedure was carried out at 4°C. 500’000 transfected cells were pelleted and washed once with washing buffer (PBS + 0.2% BSA (w/v). Cells were then incubated for 20 minutes with the primary antibody AJ517 (3 µg/ml). After two washes in washing buffer, cells were incubated for 20 minutes with secondary goat anti-rabbit IgG conjugated to Alexa Fluor 488 (dilution 1:400, Molecular Probes #A11034). After two washes in washing buffer, cells were resuspended in 500 µL of washing buffer and analyzed with a flow cytometer (BD AccuriTM C6).  Results The antibody AJ517 specifically detects the CD8β protein at the cell surface of transfected HEK293 cells expressing CD8a and CD8b. No signal was detected in mock transfected cells (Fig. 1).   Fig. 1. The AJ517 antibody specifically binds at the cell surface of cells expressing CD8a and CD8b (red). No signal was detected in mock transfected cells incubated (blue).  References Lima WC, Gasteiger E, Marcatili P, Duek P, Bairoch A, Cosson P. The ABCD database: a repository for chemically defined antibodies. Nucleic Acids Res. 2019; pii:gkz714. PMID:31410491 Parnes JR. Molecular biology and function of CD4 and CD8. Adv Immunol. 1989; 44:265‑311. PMID:2493728 Pierres M, Goridis C, Golstein P. Inhibition of murine T cell-mediated cytolysis and T cell proliferation by a rat monoclonal antibody immunoprecipitating two lymphoid cell surface polypeptides of 94 000 and 180 000 molecular weight. Eur J Immunol. 1982; 12(1):60-9. PMID:6977452  Conflict of interest The authors declare no conflict of interest. 

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The AJ517 antibody detects the mouse CD8β protein by flow cytometry

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