The AJ519 antibody detects the human TAC/ILR2A protein by flow cytometry

Authors

  • Boris Gueorguiev
  • Elisa Domingos
  • Vincent Gaspoz
  • David Arsimoles
  • Mustafa Haraj
  • Monica Bulla
  • Ali Sassi
  • Jean-Pierre Aubry-Lachainaye
  • Cyril Guilhen

DOI:

https://doi.org/10.24450/journals/abrep.2020.e117

Abstract

The AJ519 antibody detects the human TAC protein by flow cytometry.

Introduction

The alpha subunit of the interleukin 2 receptor, also known as the TAC antigen (Uniprot #P01589), is a protein displayed notably at the surface of T lymphocytes (Uchiyama et al., 1981; Malek and Castro, 2010). Here, we describe the ability of the AJ519 antibody, a single chain fragment (scFv) derived from the 7G7 hybridoma, to successfully detect the TAC protein by flow cytometry in TAC-transfected HEK293 cells.

Materials & Methods

Antibodies: ABCD_AJ519 antibody (ABCD nomenclature, http://web.expasy.org/abcd/; Lima et al., 2019) and IgG produced by 7G7 hybridoma were produced by the Geneva Antibody Facility (http://unige.ch/medecine/antibodies/). AJ519 antibody was produced as mini-antibody with the antigen-binding scFv fused to a rabbit IgG Fc. The synthesized scFv sequence (GeneArt, Invitrogen) corresponds to the sequence of the variable regions of the 7G7 hybridoma (Rubin et al., 1985) joined by a peptide linker (GGGGS)3. The sequencing of the 7G7 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. AJ519 supernatant (50 mg/L) was collected after 4 days.

Antigen: The 7G7 hybridoma was originally raised against human influenza virus-stimulated PBMC in BALB/cJ mice (Rubin et al., 1985). HEK293 suspension cells (growing in FreeStyleTM 293 Expression Medium, Gibco #12338) were transiently transfected 3 days before the experiment with the vector coding for the full-length human TAC protein.

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 either the original mouse hybridoma 7G7 supernatant (dilution 1:2 in PBS-BSA) or with the reformatted scFv antibody AJ519 (5 mg/L). After two washes in washing buffer, cells were incubated for 20 minutes with either secondary goat anti-mouse or anti-rabbit IgG conjugated to Alexa Fluor 488 (dilution 1:400, Molecular Probes #A11029 and #A11034 respectively). 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

Both the IgG produced by the mouse hybridoma 7G7 and the reformatted scFv AJ519 detect the TAC protein at the cell surface of HEK293 transfected cells. No signal was detected in mock transfected cells (Fig. 1).

Figure 1. Both IgG produced by the mouse hybridoma 7G7 (orange) and the reformatted scFv AJ519 (blue) label HEK293 cells overexpressing the TAC protein. No signal was detected in mock transfected cells incubated with the AJ519 antibody (red).

Conflict of interest

The authors declare no conflict of interest.

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

Malek TR, Castro I. Interleukin-2 receptor signaling: at the interface between tolerance and immunity. Immunity. 2010; 33(2):153-65. PMID: 20732639

Rubin LA, Kurman CC, Biddison WE, Goldman ND, Nelson DL. A monoclonal antibody 7G7/B6, binds to an epitope on the human interleukin-2 (IL-2) receptor that is distinct from that recognized by IL-2 or anti-Tac. Hybridoma. 1985; 4(2):91‑102. PMID:2408992.

Uchiyama T, Broder S, Waldmann TA. A monoclonal antibody (anti-Tac) reactive with activated and functionally mature human T cells. I. Production of anti-Tac monoclonal antibody and distribution of Tac (+) cells. J Immunol. 1981; 126(4):1393‑7. PMID:6970774.

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Published

2020-01-18

Section

Article

How to Cite

1.
Gueorguiev B, Domingos E, Gaspoz V, Arsimoles D, Haraj M, Bulla M, Sassi A, Aubry-Lachainaye J-P, Guilhen C. The AJ519 antibody detects the human TAC/ILR2A protein by flow cytometry. Antib. Rep. [Internet]. 2020 Jan. 18 [cited 2024 May 24];3(1):e117. Available from: https://oap.unige.ch/journals/abrep/article/view/117

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