Introduction
HMtA (UniProt #A5UJP0) together with the HMtB protein form the histone-related protein complex (HMt). This complex has been shown to be able to bind and compact DNA to form nucleosome-like structures that contain positive DNA supercoils (Tabassum et al., 1992). Here we describe the ability of five recombinant antibodies (RB714, RB715, RB716, RB717, RB718) to detect by ELISA a GST-fused HMtA protein.
Materials & Methods
Antibodies: ABCD_RB714, ABCD_RB715, ABCE_RB716, ABCD_RB717 and ABCD_RB718 antibodies (ABCD nomenclature, http://web.expasy.org/abcd/) were discovered by the Geneva Antibody Facility (http://unige.ch/medecine/antibodies/; Blanc et al., 2014) and produced as mini-antibodies with the antigen-binding scFv fused to a rabbit IgG Fc (RRB714, RRB715, RRB716, RRB717 and RRB718). 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 (~50-80 mg/l) were collected after 5 days..
Antigen: The antibodies were originally raised against a GST protein fused to the almost full-length HMtA protein (missing the initial methionine residue). This chimeric GST-HMtA was used as antigen for ELISA detection. As a negative control, an irrelevant GST-fused peptide (amino acids 916 to 999 of Drosophila melanogaster Spargel protein; UniProt #Q8IPM1) was used.
Protocol: The whole procedure was carried out at room temperature. Bacterial lysates containing in vivo N-biotinylated GST proteins 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 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 (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.
Results
Antibodies RRB714, RRB715, RRB716, RRB717, RRB718 bound in a concentration-dependent manner to the GST-HMtA antigen, but not to the GST-fused peptide negative control (Fig. 1).
Figure 1. Specific binding of RRB antibodies to the target GST-HMtA protein, but not to the GST-fused peptide negative control (shown only for RRB714, background curve is superimposed), as detected by ELISA.
Conflict of interest
The authors declare no conflict of interest.