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RB519, RB520, RB521 and RB522 antibodies recognize the human ISOC1 protein by ELISA

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RB519, RB520, RB521 and RB522 antibodies recognize the human ISOC1 protein by ELISA

CLÉMENT LEBOUBE, Sophie, GOOSSENS, Nicolas

Abstract

The recombinant antibodies RB519, RB520, RB521 and RB522 detect by ELISA the human ISOC1 fused to a GST protein.

CLÉMENT LEBOUBE, Sophie, GOOSSENS, Nicolas. RB519, RB520, RB521 and RB522 antibodies recognize the human ISOC1 protein by ELISA. Antibody Reports , 2019, vol. 2, no.

4, p. e64

DOI : 10.24450/journals/abrep.2019.e64

Available at:

http://archive-ouverte.unige.ch/unige:131067

Disclaimer: layout of this document may differ from the published version.

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doi:10.24450/journals/abrep.2019.e64 Antibody Reports, 2019, vol. 2, e64

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

RB519, RB520, RB521 and RB522 antibodies recognize the human ISOC1 protein by ELISA

Sophie Clément

1

, Nicolas Goossens

2

1Clinical Pathology and 2Gastroenterology and Hepatology, Geneva University Hospitals, Rue Gabrielle-Perret-Gentil 4, 1205 Geneva, Switzerland

Abstract

The recombinant antibodies RB519, RB520, RB521 and RB522 detect by ELISA the human ISOC1 fused to a GST protein.

Introduction

ISOC1 (Isochorismatase domain-containing protein 1, UniProtKB #Q96CN7) is a protein expressed in the peroxisome of different subsets of human cells (Gronemeyet et al., 2013; Islinger et al., 2007). Here we describe the ability of four recombinant antibodies (RB519, RB520, RB521 and RB522) to detect by ELISA a GST-fused ISOC1 protein.

Materials & Methods

Antibodies: ABCD_RB519, ABCD_RB520, ABCD_RB5521 and ABCD_RB522 antibodies (ABCD nomenclature, 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 rabbit IgG Fc (RRB519, RRB520, RRB521 AND RRB522). HEK293 suspension cells (growing in FreeStyle™ 293 Expression Medium, Gibco #12338) were transiently transfected with the vectors coding for each scFv-Fc. Supernatants (~20-100 mg/l) were collected after 4 days.

Antigen: The antibodies were originally raised against a GST protein fused to the full-length ISOC1 protein (residues 2-298). This chimeric GST-ISOC1 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 RRB 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-rabbit 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 RRB519, RRB520, RRB521 and RRB522 bound in a concentration-dependent manner to the GST- ISOC1 antigen, but not to the GST negative control (Fig.

1).

Fig. 1. Specific binding of RRB antibodies to the target GST-ISOC1 protein, as detected by ELISA. ‘Control’ indicates the binding of RRB519 to GST (all other control curves were superimposed).

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 Gronemeyer T, Wiese S, Ofman R, et al. The proteome of human liver peroxisomes: identification of five new peroxisomal constituents by a label-free quantitative proteomics survey. PLoS One. 2013; 8(2):e57395.

PMID:23460848

Islinger M, Lüers GH, Li KW, Loos M, Völkl A. Rat liver peroxisomes after fibrate treatment. A survey using quantitative mass spectrometry. J Biol Chem. 2007;

282(32):23055-69. PMID:17522052

Conflict of interest

The authors declare no conflict of interest.

0.0 0.5 1.0 1.5

1:100 1:1000 1:10000

ELISA Signal (OD)

Antibody Dilution

Control RB519 RB520 RB521 RB522

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