AC650, AC653, AC656 and AD460 antibodies recognize the mouse CD8α protein by flow cytometry

Abstract

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

Introduction

CD8 protein, composed of two subunits (alpha and beta), is a transmembrane glycoprotein complex expressed primarily in cytotoxic T lymphocytes (Parnes, 1989). Here, we describe the ability of four recombinant antibodies (AC650, AC653, AC656 and AD460) to successfully detect the CD8-alpha protein (Uniprot P01731) 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: HEK293suspensioncells (growing in FreeStyle™ 293 Expression Medium, Gibco 12338) were transiently transfected 2 days before the experiment with a vector coding for the full-length human CD8-alpha protein. Cells transfected with an irrelevant plasmid (mock) were used as a negative control.

Protocol: The whole procedure was carried out at 4°C. 1x10⁶ 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 recombinant antibodies (5 mg/L in PBS-BSA). After two washes in washing buffer, cells were incubated for 20 minutes with secondary goat anti-rabbit IgG conjugated to AlexaFluor-488 (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 (Beckman Coulter CytoFLEX).

Results

Antibodies AC650, AC653, AC656 and AD460 detected the CD8-alpha protein in CD8-alpha-transfected HEK293 cells. No signal was detected in mock-transfected cells (Fig. 1). AD461 and AJ518 did not recognize the CD8-alpha protein by flow cytometry. For AD461, this might be due to the fact that this antibody is poorly produced. All these antibodies were also tested at lower concentration (1 mg/L) in PBS-BSA. Only antibodies AC653, AC656 and AD460 exhibited a positive signal at this concentration (data not shown).

Figure 1. Mono-parametric representation of flow cytometry analysis depicting the Alexa Fluor 488 signal. AC650, AC653, AC656 and AD460 antibodies labeled HEK293 transfected cells overexpressing the CD8-alpha protein. No signal was detected in mock-transfected cells. AD461 and AJ518 did not recognize the CD8-alpha protein by flow cytometry. Transfection efficiency was estimated to be around 20%.

Conflict of interest

The authors declare no conflict of interest.