Figure 1

Characterisation of monoclonal anti-RAI3 antibodies. A:. Direct ELISA of a serial dilution of BALB/c mouse serum carried out at several time points during immunisation. Increasing antbody titre indicates a good humoral immune response. Measurements are from triplicates, expressed as means ± S.D. B: Direct ELISA of hybridoma supernatants from four selected monoclones showing a concentration dependent RAI3 signal and no cross-reactivity to control proteins. BSA: bovine serum albumin; CP1: control protein 1, non-related human protein (CD30 ligand); CP2: control protein 2, related human protein, GPCR (GPR30); No: Negative control, no protein coated. Measurements are from triplicates, expressed as means ± S.D. C: Competitive ELISA of Mabs pre-incubated with a serial dilution of soluble antigen RAI3. Only unbound antibody can bind to the immobilised antigen on the ELSA plate allowing the comparison and determination of affinities based on the absorption values. Measurements are from triplicates, expressed as means ± S.D. D: Western blot analysis of cell extracts from transfected HEK293T cells. Cells expressed either RAI3 (lane 1), mock protein (lane 2), RAI3-GFP (lane 3) or mock-GFP protein (lane 4). Aliquots (10 μg) of total protein from cell extracts are loaded as indicated, with β-actin as the loading control. At least three experiments for Mab 24 2.3 are represented. Equal binding pattern was observed in all experiments.