Star54

Levels of anti-luciferase activity in sera were measured by Elisa. Microtitre plates were coated overnight at 4°C with 100 μl of recombinant luciferase (2 μg/ml Roche Diagnostics Ltd) prepared in bicarbonate buffer (pH 9.5). Plates were washed with PBS and then blocked with 2% marvel solution in PBS for 1 hour at room temperature. Plates were again washed with PBS/Tween (0.05%) and then day 28 serum (diluted from 1:10 up to 1:1x10⁸ with PBS Tween) was incubated for 3 hours at room temperature. After washing with PBS/Tween, bound antibody was detected using HRP conjugated sheep anti-mouse IgG diluted 1:1000 (OBT 1508P; AbDSerotec, Kidlington, UK). After 1 hour the signal was detected using the TMB microwell substrate system (Kirkegaard and Perry Laboratories Inc., Gaithersburg, MD, USA) and the reaction stopped by addition of 4M sulphuric acid (100 μl). Absorbance measurements were performed at 450 nm using a TecanGENios microplate reader (Tecan Group Ltd; Männedorf, Switzerland) with Magellan 4 software. Absorbance readings were compared against a standard generated with a rabbit anti-luciferase IgG fraction of sera which was detected with a secondary HRP conjugated sheep anti-rabbit IgG diluted 1:1000 (Star 54; AbDSerotec).