RB815, RB816 and RB817antibodies recognize the Ripply2 protein from Danio rerio by ELISA

Abstract

The recombinant antibodies RB815, RB816 and RB817 detect by ELISA the Danio rerio Ripply2 protein fused to a Twin-Strep-Tag.

Introduction

Ripply2 (UniProt #Q2WG79) is a Danio rerio protein involved in somitogenesis (Kawamura et al., 2005). Here we describe the ability of three recombinant antibodies (RB815, RB816 and RB817) to detect by ELISA the Ripply2 protein.

Materials & Methods

Antibodies: ABCD_RB815, ABCD_RB816 and ABCD_RB817 antibodies (ABCD nomenclature, http://web.expasy.org/abcd/) were discovered by the Geneva Antibody Facility (http://unige.ch/medecine/antibodies/) and produced as mini-antibodies with the antigen-binding scFv portion fused to a rabbit IgG Fc. The synthesized scFv sequences (GeneArt, Invitrogen) comprise the sequence of the variable regions joined by the linker (GGGS)3. HEK293 suspension cells growing in HEK TF medium (Xell#861-0001, Sartorius) supplemented with 0.1% Pluronic F68 (Sigma #P1300) were transiently transfected with the vector coding for the scFv-Fc of each antibody. Supernatants (~70 mg/L, 80 mg/L and 40 mg/L for RB815, RB816 and RB817, respectively) were collected after 5 days.

Antigen: Antibodies were raised against the full-length Ripply2 protein, fused at its C-terminus with a Twin-Strep-Tag (RIPP2-TST) and produced in transiently transfected HEK293 cells. The same construction was used for the ELISA experiment. A Twin-Strep-Tagged peptide from the human tenascin protein served as a negative control (UniProt #P24821, residues 1619-1710, TNC-TST).

Protocol: The whole procedure was carried out at room temperature. TST tagged proteins at saturating concentration (10 pmol/well) were incubated on MaxiSorp 96-well plates (Nunc # 44-2404-21) 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 RB 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 (Sigma #A8275, 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 RB815, RB816 and RB917 bound in a concentration-dependent manner to the RIPP2-TST protein against which they were raised, but not to the negative control protein (Fig. 1).

Figure 1. Specific binding of RB antibodies to the target RIPP2-TST protein, as detected by ELISA. ‘Control’ indicates the binding of RB815 to the negative control peptide TNC-TST (all other control curves were superimposed).

Conflict of interest

Philippe Hammel is a cofounder and a shareholder of ABCD Antibodies SA.