RB196 and RB197 antibodies recognize a fragment of the RdRp region of the hepatitis E virus ORF1 protein by ELISA

Abstract

The recombinant antibodies RB196 and RB197 detect by ELISA a fragment of the RdRp region of the hepatitis E virus ORF1 protein fused to a GST protein.

Introduction

Hepatitis E virus (HEV) ORF1 protein (UniProt #H9E9C7) is the viral replicase responsible for the neosynthesis of viral RNA genomes during infection (Debing et al., 2016). Here we describe the ability of two recombinant antibodies (RB196 and RB196) to detect by ELISA a GST-fused fragment of the RdRp region of the HEV (Kernow-C1 isolate, genotype 3) ORF1 protein.

Materials & Methods

Antibodies: ABCD_RB196 and ABCD_RB197 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 (MRB196 and MRB197). 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 1414-1474 (QATTCELYELVEAMVEKGQDGSAVLELDLCNRDVSRITFFQKDCNKFTTGETIAHGKVGQG) of the HEV ORF1 polyprotein. This chimeric GST-HEVRdRp 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 RB196 and RB197 bound in a concentration-dependent manner to the GST-HEVRdRp antigen, but not to the GST negative control (Fig. 1).

Figure 1. Specific binding of RB antibodies to the target GST-HEVRdRp protein, as detected by ELISA. ‘Control’ indicates the binding of RB196 to GST (RB197 curve is superimposed).

Conflict of interest

The authors declare no conflict of interest.