Recombinant rtta protein

Detection of anti-rtTA antibodies was conducted using an enzyme-linked immunosorbent assay. Nunc MaxiSorp P96 plates (Sigma-Aldrich, France) were coated overnight at 4 °C with recombinant rtTA protein (at 5 µg/ml, Proteogenix, France). After 3 phosphate-buffered saline washings and 0.1% Tween 1% gelatin saturation for 1 hour at 37 °C, wells were incubated for 2 hours at 37 °C with 12 serial twofold dilutions of macaque sera (from 1/10 to 20,480) then for 1 hour at 37 °C with goat horseradish peroxidase-conjugated anti-rhesus IgG (Cliniscience, France). Positive controls consisted in a serum from an anti-rtTA immunized macaque and a commercial polyclonal anti-TetR antibody (MoBiTec, Germany). Revelation was performed using 2.2-3,3′,5,5′-Tetramethylbenzidine (TMB, BD OptEIA, BD Biosciences) and absorbance of duplicate samples were read at 450 nm with a correction at 570 nm on a Multiskan Go reader (Thermo Scientific, France). Threshold of positivity was determined using 15 negative sera obtained from unrelated naïve macaques as mean of optic density for each dilution +2*SD. IgG titers for experimental animals was defined as the last sera dilution with optic density remaining above the threshold curve.