RB740, RB741 and RB742 antibodies recognize the Dicytostelium discoideum Talin B protein by ELISA

Abstract

The recombinant antibodies RB740, RB741 and RB742 detect by ELISA the Dictyostelium discoideum Talin-B fused to a GST protein.

Introduction

Talin-B (TalB, UniProt #Q54K81) is a D. discoideum actin-binding protein which is involved in phagocytosis (Niewöhner et al., 1997) and morphogenesis (Tsujioka et al., 2004). Here we describe the ability of three recombinant antibodies (RB740, RB741 and RB742) to detect by ELISA a GST-fused TalB protein.

Materials & Methods

Antibodies: ABCD_RB740, ABCD_RB741 and ABCD_RB742 antibodies (ABCD nomenclature, http://web.expasy.org/abcd/) were discovered by the Geneva Antibody Facility (http://unige.ch/medecine/antibodies/) and produced as mini-antibodies with the antigen-binding scFv used to a mouse IgG2A Fc (MRB740, MRB741 and MRB742). HEK293 suspension cells (growing in HEK TF medium, Xell#861-0001, supplemented with 0.1% Pluronic F68, Sigma#P1300) were transiently transfected with the vectors coding for each scFv-Fc. Supernatants (~100 mg/L) were collected after 5 days.

Antigen: The antibodies were originally raised against a GST protein fused to the residues 737 to 876 of TalB protein. This chimeric GST-TalB 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 MRB740, MRB741 and MRB742 bound in a concentration-dependent manner to the GST-TalB antigen, but not to the GST negative control (Fig. 1).

Figure 1. Specific binding of MRB antibodies to the target GST-TalB protein, but not to GST (shown only for MRB740; MRB741 and MRB742 background curve is superimposed), as detected by ELISA.

Conflict of interest

The authors declare no conflict of interest.