RB712 and RB713 antibodies recognize the Spargel protein by ELISA

Authors

  • Philippe Hammel
  • Daniela Ungureanu

DOI:

https://doi.org/10.24450/journals/abrep.2022.e998

Keywords:

Recombinant antibody, ELISA, RB712, RB713

Abstract

The recombinant antibodies RB712 and RB713 detect by ELISA the Drosophila melanogaster Spargel protein (or GCH) fused to a GST protein.

Introduction

Drosophila melanogaster Spargel protein (UniProt #Q8IPM1) is involved in mitochondria biogenesis. Spargel is a homolog of PGC-1α, a human protein involved in Parkinson disease (Merzetti and Staveley 2015). Here we describe the ability of two recombinant antibodies (RB712 and RB713) to detect a GST-fused Spargel protein by ELISA.

Materials & Methods

Antibodies: ABCD_RB712 and ABCD_RB713 antibodies (ABCD nomenclature, http://web.expasy.org/abcd/) were produced by the Geneva Antibody Facility (http://unige.ch/medecine/antibodies/) as mini-antibodies with the antigen-binding scFv fused to a rabbit IgG Fc (MRB712 and MRB713). HEK293 suspension cells (growing in HEK TF medium, Xell #861-0001, supplemented with 0.1% Pluronic F68, Sigma #P1300) were transiently transfected with the vectors coding for each scFv-Fc. Supernatants (~20 - 100 mg/l) were collected after 5 days.

Antigen: The antibodies were originally raised against a GST protein fused to the RNA recognition motif (RRM, amino acids 916 to 999) of Drosophila melanogaster Spargel protein. This chimeric GST-Spargel 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 RRB 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 (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 RRB712 and RRB713 bound in a concentration-dependent manner to the GST-Spargel antigen, but not to the GST negative control (Fig. 1).

Figure 1. Specific binding of RRB antibodies to the target GST-Spargel protein, but not to GST (shown only for MRB712; MRB713 background curve is superimposed), as detected by ELISA.

Conflict of interest

The authors declare no conflict of interest.

References

Merzetti, Eric M., and Brian E. Staveley. 2015. “Spargel, the PGC-1α Homologue, in Models of Parkinson Disease in Drosophila Melanogaster.” BMC Neuroscience 16 (1): 70. PMID: 26502946.

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Published

2022-11-15

Section

Article

How to Cite

1.
Hammel P, Ungureanu D. RB712 and RB713 antibodies recognize the Spargel protein by ELISA. Antib. Rep. [Internet]. 2022 Nov. 15 [cited 2024 Jul. 14];5(2):e998. Available from: https://oap.unige.ch/journals/abrep/article/view/998