• Aucun résultat trouvé

RB172 and RB173 antibodies recognize a fragment of the Dictyostelium discoideum CMF protein by ELISA

N/A
N/A
Protected

Academic year: 2022

Partager "RB172 and RB173 antibodies recognize a fragment of the Dictyostelium discoideum CMF protein by ELISA"

Copied!
2
0
0

Texte intégral

(1)

Article

Reference

RB172 and RB173 antibodies recognize a fragment of the Dictyostelium discoideum CMF protein by ELISA

PIETERS, Jean

Abstract

The recombinant antibodies RB172 and RB173 detect by ELISA a fragment of the Dictyostelium discoideum Conditioned Medium Factor (CMF) fused to a GST protein.

PIETERS, Jean. RB172 and RB173 antibodies recognize a fragment of the Dictyostelium discoideum CMF protein by ELISA. Antibody Reports , 2020, vol. 3, no. 3, p. e183

DOI : 10.24450/journals/abrep.2020.e183

Available at:

http://archive-ouverte.unige.ch/unige:138771

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

1 / 1

(2)

doi:10.24450/journals/abrep.2020.e183 Antibody Reports, 2020, vol. 3, e183

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

RB172 and RB173 antibodies recognize a fragment of the Dictyostelium discoideum CMF protein by ELISA

Philippe Hammel1,

Jean Pieters

2

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

2 Biozentrum, University of Basel, Klingelbergstrasse 50, 4056 Basel, Switzerland

Abstract

The recombinant antibodies RB172 and RB173 detect by ELISA a fragment of the Dictyostelium discoideum Conditioned Medium Factor (CMF) fused to a GST protein.

Introduction

The Conditioned Medium Factor (CMF, gene cmfA, Uniprot #P34090, DDB_G0275007) is a secreted protein involved in cell density sensing in Dictyostelium discoideum (Gomer et al., 1991; Mehdy and Firtel, 1985).

Here we describe the ability of two recombinant antibodies (RB172 and RB173) to detect by ELISA a GST-fused fragment of the CMF protein.

Materials & Methods

Antibodies: ABCD_RB172 and ABCD_RB173 antibodies (ABCD nomenclature, https://web.expasy.org/

abcd/) were produced by the Geneva Antibody Facility (https://www.unige.ch/medecine/antibodies/) as mini- antibodies with the antigen-binding scFv fused to a mouse IgG2A Fc (MRB172 and MRB173). HEK293 adherent cells (growing in DMEM, Gibco #11960044 supplied with 8% FBS) were transiently transfected with the vectors coding for each scFv-Fc. Supernatants (~1-5 mg/l) were collected after 5 days.

Antigen: The antibodies were originally raised against a GST protein fused to the residues 99-154 of the CMF protein (KTSLSSSKSESEHKSKFYKVIKSIYQTPTVTNGSFGIDGA TTPSFSLTWDKPVVG). This chimeric GST-CMF 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 RB172 and, to a lesser extent, RB173 bound in a concentration-dependent manner to the GST-CMF antigen, but not to the GST negative control (Fig. 1). Note that this antigen only encompasses a small portion of the CMF protein, and that it is presumably not properly folded. Being produced in bacteria, it also lacks several post-translational modifications (disulfide bridges, glycosylations) typical of secreted proteins. Further experiments will be necessary to determine if and in what conditions these antibodies recognize the full CMF protein.

Fig. 1. Specific binding of RB antibodies to the target GST-CMF protein, but not to GST (shown only for RB172; RB173 background curve is superimposed), as detected by ELISA.

References

Gomer RH, Yuen IS, Firtel RA. A secreted 80 x 10(3) Mr protein mediates sensing of cell density and the onset of development in Dictyostelium. Development. 1991;

112:269-78. PMID:1663029

Mehdy MC, Firtel RA. A secreted factor and cyclic AMP jointly regulate cell-type-specific gene expression in Dictyostelium discoideum. Mol Cell Biol. 1985; 5:705-13.

PMID:2985966

Conflict of interest

The authors declare no conflict of interest.

0.0 0.3 0.6 0.9

1:10 1:100 1:1000

ELISA Signal (OD)

Antibody Dilution Control

RB172

RB173

Références

Documents relatifs

The recombinant antibodies RA11 and RA12 detect by western blot a peptide of the Dictyostelium discoideum Mucolipin protein fused to a GST protein.. ZUFFEREY, Madeleine,

(A) AI239 and MRB95 labeled the plasma membrane of HeLa cells expressing the GST-tagged TAC protein (in white); the signal co-localized (arrows) with the signal generated by

Antibodies MRB155, MRB156 and MRB189 bound in a concentration-dependent manner to the GST-Tsg101 antigen, but not to the GST negative control (Fig. Note that this antigen

The recombinant antibodies RB513, RB514, RB515, RB516, RB517 and RB518 detect by ELISA antigens in a purified mixture of Dictyostelium discoideum putative lysosomal proteins..

The recombinant antibodies RB376 and RB377 detect by Western blot the full-length AlyA protein from Dictyostelium discoideum.. RB376 and RB377 antibodies recognize the

The recombinant antibodies RB431, RB432, RB433, RB434, and RB435 detect by ELISA a synthetic peptide from the Dictyostelium RapA protein.. HAMMEL, Philippe, LIMA,

The recombinant antibodies RB436, RB437 and RB439 detect by ELISA a synthetic peptide from the Dictyostelium Nup133 protein.. HAMMEL, Philippe, LIMA,

The recombinant antibodies RB376, RB377 and RB378 detect by ELISA a synthetic peptide from the Dictyostelium AlyA protein.. HAMMEL, Philippe,