AC650, AC653, AC656 and AD460 antibodies label the mouse CD8α protein by immunofluorescence

Abstract

The recombinant antibodies AC650, AC653, AC656 and AD460 detect by immunofluorescence the mouse CD8α protein.

Introduction

The CD8 glycoprotein is composed of two transmembrane subunits, alpha and beta. Its expression, on the surface of cytotoxic T lymphocytes, contributes to drive the specific interaction between the T-cell receptor and class-I major histocompatibility complex molecules on target cells (Wong and Pamer, 2003). Here, we describe the ability of four recombinant antibodies (AC650, AC653, AC656 and AD460) to successfully detect the mouse CD8-alpha protein (Uniprot P01731) by immunofluorescence in CD8-alpha-transfected HEK293 cells. Two other tested antibodies (AD461 and AJ518) did not.

Materials & Methods

Antibodies: ABCD_AC650, ABCD_AC653, ABCD_AC656, ABCD_AD460, ABCD_AD461 and ABCD_AJ518 antibodies (ABCD nomenclature, http://web.expasy.org/abcd/) were produced by the Geneva Antibody Facility (http://unige.ch/medecine/antibodies/) as mini-antibodies with the antigen-binding portion fused to a rabbit IgG Fc. The synthesized scFv sequences (GeneArt, Invitrogen) correspond to the sequences of the variable regions joined by a peptide linker (GGGGS)₃ (see Table 1 for clone names and references). HEK293 suspension cells (growing in FreeStyle™ 293 Expression Medium, Gibco 12338) were transiently transfected with the vector coding for the scFv-Fc of each antibody. Supernatants (see Table 1 for individual yields) were collected after 4 days.

Table 1. Clone number, reference and production yields for the antibodies used in this study.

ABCD	Clone	Reference	Yield ( mg /L)
AC650	F03	Schofield et al.,2007	100
AC653	E10	Schofield et al.,2007	60
AC656	B06	Schofield et al.,2007	70
AD460	YTS 105.18	Shore et al., 2006	<5
AD461	OKT8	Kung and Goldstein, 1982	<5
AJ518	19.178	Hennecke and Cosson, 1993	10

Antigen: HEK293cells (growing in DMEM, Gibco 11960044, supplemented with 10% FBS) were transiently transfected 2 days before the experiment with a vector coding for the full-length mouse CD8-alpha. Cells transfected with an irrelevant plasmid (mock-transfected) were used as a negative control.

Protocol: The whole procedure was carried out at room temperature. Transfected HEK293 cells were fixed with PBS + 4% paraformaldehyde (w/v) (Applichem A3013) for 30 min, and blocked with PBS + 40 mM ammonium chloride (NH₄Cl) (Applichem A3661) for 5 min. Cells were then permeabilized in PBS + 0.2% saponin (w/v) (Sigma S7900) for 5 min, washed once (5 min) with PBS + 0.2% (w/v) BSA (PBS-BSA), and incubated for 30 min with the recombinant antibodies (5 mg/L in PBS-BSA). After 3 washes (5 min) with PBS-BSA, cells were incubated for 30 min in PBS-BSA with secondary goat anti-rabbit IgG conjugated to AlexaFluor-488 (1:400, Molecular Probes A11034). After 3 washes (5 min) with PBS-BSA, cells were 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

Antibodies AC650, AC653, AC656 and AD460 recognized the mouse CD8 protein addressed at the cell surface of CD8-alpha transfected cells (Fig. 1). The weak signal observed with the antibody AD460 is probably due to the fact that this antibody is poorly produced. The absence of staining in mock-transfected cells indicates the specificity of the signal observed (Fig. 1). AD461 and AJ518 did not recognize the CD8-alpha protein; for AD461, this might be due to the fact that this antibody is poorly produced.

Figure 1. AC650, AC653, AC656 and AD460 specifically labeled transfected HEK293 cells expressing the mouse CD8-alpha protein. AD461 and AJ518 did not recognize the CD8-alpha protein. No labeling was seen in mock-transfected cells. Scale bar: 20 μm.

Conflict of interest

The authors declare no conflict of interest.