The AJ072 antibody against the human transferrin receptor labels HeLa cells by surface immunofluorescence

Abstract

The AJ072 antibody against the human transferrin receptor labels the cell membrane of HeLa cells by surface immunofluorescence; AM236 does not.

Introduction

TfR (Transferrin receptor protein 1, CD71; Uniprot P02786) is a type II transmembrane glycoprotein that binds the iron-carrier glycoprotein transferrin (Tf). Cellular uptake of iron occurs via receptor-mediated endocytosis of diferric Tf/TfR complexes (Candelaria et al., 2021). Here, we describe the ability of the AJ072 recombinant antibody against human TfR1 to stain the cell membrane of HeLa cells by surface immunofluorescence; AM236 does not, presumably due to the fact that this antibody is poorly produced.

Materials & Methods

Antibodies: ABCD_AJ072 and ABCD_AM236 antibodies (http://web.expasy.org/abcd/, ABCD nomenclature) were produced by the Geneva Antibody Facility (http://unige.ch/medecine/antibodies/) as mini-antibodies with the antigen-binding scFv fused to a rabbit IgG Fc. The synthesized scFv sequences (GeneArt, Invitrogen) correspond to the sequences of the variable regions of the clones ch128.1 (for AJ072; Friden, 1994) and M16 (for AM236; Shusta and Tillotson, 2016) joined by a peptide linker (GGGGS)₃. HEK293 suspension cells (growing in FreeStyle™ 293 Expression Medium, Gibco 12338) were transiently transfected with the vector coding for the scFv-Fc. Supernatants were collected after 4 days; both antibodies have low production yields (<5 mg/L).

Antigen: HeLa cells were cultured on glass coverslips (Menzel-Gläser, 22x22 mm) and grown in DMEM GlutaMAX^TM (Gibco 31966) supplemented with 8% Fetal Bovine Serum (Gibco 10270).

Protocol: Cells were rinsed once with cold PBS, and kept for 30 min at 4 ^oC (ice+water bath). Cells were then incubated with the tested antibodies (undiluted, i.e. final concentration 5 mg/L in PBS + 0.2% (w/v) BSA (PBS-BSA)) for 15 min at 4 ^oC, fixed with PBS + 4% paraformaldehyde (w/v) (Applichem A3013) for 15 min at room temperature, and blocked with PBS + 40 mM ammonium chloride (NH₄Cl) (Applichem A3661) for 5 min. After 1 wash (5 min) with PBS-BSA, cells were incubated for 30 min with secondary goat anti-rabbit IgG conjugated to AlexaFluor-488 (1:300, Molecular Probes A11034). After 3 washes (10 min) with PBS-BSA, cells were incubated during 5 min with DAPI (1:500, Molecular Probes D1306), washed twice with PBS-BSA and once with PBS, and mounted on slides (Menzel-Gläser, 76x26 mm) with Möwiol (Hoechst) + 2.5% (w/v) DABCO (Fluka 33480). Pictures were taken using a Zeiss LSM700 confocal microscope, with a 63x Neofluar oil immersion objective.

Results

Using a cell surface labeling protocol, AJ072 successfully labeled the plasma membrane of HeLa cells (Fig. 1). No staining was seen with AM236 antibody; this might be due to the fact that this antibody is poorly produced. No staining was observed when the primary antibody was omitted (Fig. 1, No Ab).

Figure 1. AJ072 successfully labeled the cell membrane of HeLa cells (in green); in blue, nuclei were stained with DAPI. No labelling was seen for AM236 antibody, or when the primary antibody was omitted (No Ab panel). Scale bar: 20 µm.

Conflict of interest

The authors declare no conflict of interest.