RB250 and RB251 antibodies recognize the human MitoNEET/CISD1 protein by ELISA

Authors

  • Philippe Hammel
  • Wanessa Cristina Lima
  • Oliver Hartley
  • Pierre Cosson

DOI:

https://doi.org/10.24450/journals/abrep.2018.e1

Abstract

The recombinant antibodies RB250 and RB251 detect by ELISA the human MitoNEET/CISD1 fused to a GST protein.

Introduction

MitoNEET/CISD1 (CDGSH iron-sulfur domain-containing protein 1, UniProt #Q9NZ45) is a human transmembrane protein localized in the outer mitochondrial membrane (Vernay et al., 2017). Here we describe the ability of two recombinant antibodies (RB250 and RB251) to detect by ELISA a GST-fused MitoNEET protein.

Materials & Methods

Antibodies: ABCD_RB250 and ABCD_RB251 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 fused to a mouse Fc (MRB250 and MRB251). HEK293T cells (growing in DMEM GlutaMAXTM (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 almost full-length MitoNEET protein (missing the initial 31 residues). This chimeric GST-MitoNEET 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 MRB250 and MRB251 bound in a concentration-dependent manner to the GST-MitoNEET antigen, but not to the GST negative control (Figure 1).

Figure 1. Specific binding of MRB antibodies to the target GST-MitoNEET protein, but not to GST (shown only for MRB250; MRB251 background curve is superimposed), as detected by ELISA

Conflict of interest

Pierre Cosson, Wanessa Cristina Lima and Oliver Hartley are editors of the Antibody Reports journal.

References

Blanc C, Zufferey M, Cosson P. Use of in vivo biotinylated GST fusion proteins to select recombinant antibodies. ALTEX. 2014; 31(1):37-42. PMID:24100547

Vernay A, Marchetti A, Sabra A et al. MitoNEET-dependent formation of intermitochondrial junctions. Proc Natl Acad Sci USA. 2017; 114(31):8277-8282. PMID:28716905

Downloads

Published

2018-11-01

Section

Article

How to Cite

1.
Hammel P, Lima WC, Hartley O, Cosson P. RB250 and RB251 antibodies recognize the human MitoNEET/CISD1 protein by ELISA. Antib. Rep. [Internet]. 2018 Nov. 1 [cited 2024 May 23];1(1):e1. Available from: https://oap.unige.ch/journals/abrep/article/view/1

Most read articles by the same author(s)

1 2 3 4 5 6 > >>