RB198, RB199 and RB200 antibodies recognize a fragment of the hepatitis E virus ORF3 protein by ELISA

Abstract

The recombinant antibodies RB198, RB199 and RB200 detect by ELISA a fragment of the hepatitis E virus ORF3 protein fused to a GST protein.

Introduction

Hepatitis E virus (HEV) ORF3 protein (UniProt #E9N3C1) is a small palmitoylated protein required for the secretion of infectious viral particle (Gouttenoire et al., 2018). Here we describe the ability of three recombinant antibodies (RB198, RB199 and RB200) to detect by ELISA a GST-fused fragment of the HEV (Kernow-C1 isolate, genotype 3) ORF3 protein.

Materials & Methods

Antibodies: ABCD_RB198, ABCD_RB199 and ABCD_RB200 antibodies (ABCD nomenclature, http://web.expasy.org/abcd/; Lima et al., 2020) were produced by the Geneva Antibody Facility (http://unige.ch/medecine/antibodies/; Blanc et al., 2014) as mini-antibodies with the antigen-binding scFv fused to a mouse IgG2A Fc (MRB198, MRB199 and MRB200). 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 62-113 (QPTPSPPISFHNPGLELALGSRPAPLAPLGVTSPSAPPLPPAVDLPQLGLRR) of the HEV ORF3 protein. This chimeric GST-ORF3 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 RB198, RB199 and RB200 bound in a concentration-dependent manner to the GST-ORF3 antigen, but not to the GST negative control (Fig. 1). RB198 and RB200 recognize the native antigen in HEV-infected cells by immunofluorescence and immunoblotting (Gouttenoire et al., 2018).

Figure 1. Specific binding of RB antibodies to the target GST-ORF3 protein, as detected by ELISA. ‘Control’ indicates the binding of RB198 to GST (all the other background curves are superimposed).

Conflict of interest

The authors declare no conflict of interest.