RB174, RB175, RB176, RB177 and RB178 antibodies recognize a fragment of the Dictyostelium discoideum CMF protein by ELISA

Abstract

The recombinant antibodies RB174, RB175, RB176, RB177 and RB178 detect by ELISA a fragment of the Dictyostelium discoideum Conditioned Medium Factor (CMF) fused to a GST protein.

Introduction

The Conditioned Medium Factor (CMF, gene cmfA, Uniprot #P34090, DDB_G0275007) is a secreted protein involved in cell density sensing in Dictyostelium discoideum (Gomer et al., 1991; Mehdy and Firtel, 1985). Here we describe the ability of five recombinant antibodies (RB174, RB175, RB176, RB177 and RB178) to detect by ELISA a GST-fused fragment of the CMF protein.

Materials & Methods

Antibodies: ABCD_RB174, ABCD_RB175, ABCD_RB176, ABCD_RB177 and ABCD_RB178 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 scFv fused to a mouse IgG2A Fc (MRB174, MRB175, MRB176, MRB177 and MRB178). HEK293 adherent cells (growing in DMEM, Gibco #11960044 supplied with 8% FBS) were transiently transfected with the vectors coding for each scFv-Fc. Supernatants (~1-5 mg/l) were collected after 5 days.

Antigen: The antibodies were originally raised against a GST protein fused to the residues 415-514 of the CMF protein (KNVQVSDLDTFIPLSNTTSTSNVKMVGEYKDVRTIVHSPPL HEITKEMRDGKMPKELADRIGKSTGNGKLILTHGYCSEGVWPIEDFENSVEFQDFNQNR). This chimeric GST-CMF was used as antigen for ELISA detection. GST was used as negative control.

Protocol: The whole procedure was carried out at room temperature. Bacterial lysates containing GST proteins were incubated in a glutathione-coated 96-well plate (Pierce #15240) for 30 min. Each well was rinsed three times with 100 μl of washing buffer (PBS + 0.5% (w/v) BSA + 0.05% (w/v) Tween20), then incubated for 1 hour with 50 µl of MRB antibody-containing supernatant diluted in washing buffer (Fig. 1). After rinsing 3 times (100 µl washing buffer), wells were incubated with horseradish peroxidase-coupled goat anti-mouse IgG (Bio-Rad #170-6516, dilution 1:1000, 50 μl per well) for 30 min. After 3 rinses, Tetramethylbenzidine (TMB) substrate (Sigma #T5569) was added (50 μl per well). The reaction was stopped by the addition of 25 μl of 2 M H₂SO₄. The absorbance (OD) was measured at 450 nm, and the absorbance at 570 nm was subtracted.

Results

Antibodies RB174, RB175, RB176, RB177 and RB178 bound in a concentration-dependent manner to the GST-CMF antigen, but not to the GST negative control (Fig. 1). Note that this antigen only encompasses a small portion of the CMF protein, and that it is presumably not properly folded. Being produced in bacteria, it also lacks several post-translational modifications (disulfide bridges, glycosylations) typical of secreted proteins. Further experiments will be necessary to determine if and in what conditions these antibodies recognize the full CMF protein.

Figure 1. Specific binding of RB antibodies to the target GST-CMF protein, but not to GST (shown only for RB174; all the other background curves are superimposed), as detected by ELISA.

Conflict of interest

The authors declare no conflict of interest.