The RB395 antibody recognizes a Dictyostelium AplA peptide by ELISA

Abstract

The recombinant antibody RB395 detects by ELISA a synthetic peptide from the Dictyostelium AplA protein (amino acids 370-388).

Introduction

AplA (Amoeba Saposin A, DDB_G0284043, UniProt #Q54Q68) is a member of the saposin family of proteins (IPR008373). Saposins aid in the activation of hydrolase enzymes involved in the breakdown of sphingolipids (Rorman et al., 1989). Here we describe the ability of a recombinant antibody (RB395) to detect by ELISA a synthetic biotinylated peptide from the AplA protein.

Materials & Methods

Antibodies: The ABCD_RB395 antibody (ABCD nomenclature, http://web.expasy.org/abcd/) was 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 mouse IgG Fc (MRB395). HEK293 suspension cells (growing in FreeStyle™ 293 Expression Medium, Gibco #12338) were transiently transfected with the vector coding for the scFv of each antibody. Supernatant (~60g/mL) was collected after 5 days.

Antigen: The antibody was raised against a N-biotinylated synthetic peptide of 19 amino acids corresponding to residues 370-388 of the AplA protein (EYLEFAVTQLEAKFNPTEI, AplA.b). As a negative control, a N-biotinylated peptide corresponding to residues 302-318 from the same protein (PAPTPTSTPSTIKIDVN, AplA.a) was used.

Protocol: The whole procedure was carried out at room temperature. Biotinylated peptides at saturating concentration (10 pmol/well) were immobilized on streptavidin-coated ELISA plates (Pierce #15124) 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

The MRB395 antibody bound the AplA.b peptide against which it was raised, but not to the negative control AplA.a peptide (Fig. 1). Although this antibody recognizes specifically the AplA.b peptide in ELISA, its ability to bind the full-length protein should be determined in future experiments.

Figure 1. Specific binding of the MRB antibody to the target AplA.b peptide, as detected by ELISA. ‘Control’ indicates the binding of MRB395 to the negative control AplA.a.

Conflict of interest

The authors declare no conflict of interest.