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

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

HAMMEL, Philippe, et al.

Abstract

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

HAMMEL, Philippe, et al . RB250 and RB251 antibodies recognize the human MitoNEET/CISD1 protein by ELISA. Antibody Reports , 2018, vol. 1, no. 1, p. e1

DOI : 10.24450/journals/abrep.2018.e1

Available at:

http://archive-ouverte.unige.ch/unige:110464

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doi:10.24450/journals/abrep.2018.e1 Antibody Reports, 2018, vol. 1, e1

This work is licensed under a Creative Commons Attribution 4.0 International License.

Geneva University Library Open Access Publications https://oap.unige.ch/journals/abrep | ISSN 2624-8557

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

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

Geneva Antibody Facility, Faculty of Medicine, University of Geneva, 1 rue Michel Servet, CH-1211, Geneva, Switzerland

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: RB250 and RB251 antibodies (ABCD nomenclature, https://web.expasy.org/abcd/) were produced by the Geneva Antibody Facility (www.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 (Fig. 1).

Fig. 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.

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

Conflict of interest

The authors declare no conflict of interest.

MRB250/GST

MRB250/GST-MitoNEET MRB251/GST-MitoNEET

ELISA signal (OD)

0.0 0.5 1.0 1.5

1:10 1:100 1:1000

Antibody Dilution

Références

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