Antibodies ABCD_RB989-RB998 recognize the Dictyostelium discoideum GAA protein by ELISA

Abstract

The 10 recombinant antibodies ABCD_RB989 to ABCD_RB998 detect by ELISA the purified Dictyostelium discoideum GAA protein.

Introduction

The Dictyostelium discoideum GAA protein (UniProt #Q55D50) is a putative alpha-glucosidase, orthologous to the human lysosomal alpha-glucosidase (UniProt #P10253, Persson et al., 2023), which is essential for glycogen degradation in lysosomes (Hermans et al., 2004). Here we describe the ability of ten recombinant antibodies (ABCD_RB989-RB998) to detect by ELISA a purified Twin Strep-Tagged GAA protein from D. discoideum.

Materials & Methods

Antibodies: ABCD_RB989 to ABCD_RB998 antibodies (ABCD nomenclature, http://web.expasy.org/abcd/, referred to collectively as RB989-998) were discovered by the Geneva Antibody Facility (http://unige.ch/medecine/antibodies/). Briefly, a synthetic VHH phage display library (in-house) was panned against a Twin Strep-tagged GAA protein (see antigen section). After three rounds of panning, selected phage vectors were isolated using a plasmid preparation kit (Qiagen), and the VHH inserts were subcloned into custom-made expression vectors and sequenced. The selected antibodies were produced as minibodies with the antigen-binding VHH fused to a rabbit IgG Fc. HEK293 suspension cells growing in HEK TF medium (Sartorius #861-0001), supplemented with 0.1% Pluronic F68 (Sigma #P1300) were transiently transfected with the vectors coding for each VHH-Fc. Supernatants were collected after 3 days. Estimated production yields were approximately 100-110 mg/L for antibodies RB989, RB995, RB997 and RB998, and between 70-80 mg/L for RB990, RB991, RB992, RB993, RB994 and RB996.

Antigen:We used a fusion protein composed of a human IL2 signal sequence for insertion in the ER followed by the coding sequence of the D. discoideum GAA protein without sequence signal (amino acids 24 to 867) and fused at its C-terminus to a Twin-Strep-Tag^® (IBA Lifesciences, GAA-TST). The fusion protein was produced in transiently transfected HEK293 cells and purified using MagStrep Streptactin XT beads according to the manufacturer instruction (IBA Lifesciences #2-5090-002). A TSTtagged PldX protein from D. discoideum(PldX-TST, UniProt #Q55CF8, residues 20-427) was produced the same way and used as a negative control.

Protocol: The whole ELISA procedure was carried out at room temperature. Purified TST tagged proteins were coated directly on Maxisorp Elisa plates (Nunc #44-2404-21) at 3 µg/ml in PBS for 45 min. As a saturation agent, 50 µL of PBS-BSA 3% (w/v) was added, followed by 20 minutes of incubation. Each well was then 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 with 100 µL of 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 for background correction.

Results & discussion

Antibodies RB989–RB998 bound to the GAA-TST antigen in a concentration-dependent manner but did not bind to the PldX-TST negative control (Fig. 1).

Figure 1. Specific binding of RB antibodies to the target GAA-TST protein, but not to the negative control (shown only for RB989; other background curves are superimposed), as detected by ELISA.

Conflict of interest

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

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.