RB252, RB253, RB254 and RB255 antibodies recognize the human Miner1/CISD2 protein by ELISA

Abstract

The recombinant antibodies RB252, RB253, RB254 and RB255 detect by ELISA the human Miner1/CISD2 fused to a GST protein.

Introduction

Miner1/CISD2 (CDGSH iron-sulfur domain-containing protein 2, MitoNEET-related 1 protein, UniProt #Q8N5K1) is an integral protein of the endoplasmic reticulum. Here we describe the ability of four recombinant antibodies (RB252, RB253, RB254 and RB255) to detect by ELISA a GST-fused Miner1 protein.

Materials & Methods

Antibodies: ABCD_RB252, ABCD_RB253, ABCD_RB254 and ABCD_RB255 antibodies (ABCD nomenclature, http://web.expasy.org/abcd/) were produced by the Geneva Antibody Facility (http://unige.ch/medecine/antibodies/; Blanc et al., 2014) as mini-antibodies with the antigen-binding scFv portion fused to a mouse IgG2A Fc (MRB252, MRB253, MRB254 and MRB255). HEK293T cells (growing in DMEM GlutaMAX^TM (Gibco, #31966) supplemented with 8% Fetal Bovine Serum (Gibco, #10270)) were transiently transfected with the vector coding for the scFv-Fc of each antibody. Supernatants (~5 mg/L) were collected after 3 days.

Antigen: The antibodies were originally raised against a GST protein fused to the 75 C-terminal residues of Miner1 protein (61-135), corresponding to the entire presumptive cytosolic domain of the protein. This chimeric GST-Miner1 protein 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 MRB252, MRB253, MRB254 and MRB255 bound in a concentration-dependent manner to the GST-Miner1 antigen, but not to the GST negative control (Fig. 1).

Figure 1. Specific binding of MRB antibodies to the target GST-Miner1 protein, as detected by ELISA. ‘Control’ indicates the binding of MRB252 to GST (all other control curves were superimposed).

Conflict of interest

The authors declare no conflict of interest.