Competition ELISA to test for specificity for mutated B-Raf. ELISA was performed using 2 positive sera from melanoma patients (A, B) and 1 positive sera from the control group (C) as well as a human serum with known background reactivity (D; serum from a non-melanoma patient of the dermatology department with reactivity against BSA used for blocking). Plates were coated with B-Raf (■, ▲) or B-Raf V599E (▼, ◆) and competition was performed by titrating in increasing concentrations (0.0, 0.1, 1.0 and 5.0 μg/ml) of B-Raf (■ ▼) or B-Raf V599E (▲, ◆). Data is presented using relative OD405 values that are adjusted relative to the first experimental OD values of the corresponding dataset. For the positive sera with specific Raf antibodies (A-C), competition reveals no differences between B-Raf and B-Raf V599E regardless of the combination suggesting that the antibodies can not discriminate between wt and mutated B-Raf. In contrast, the non-specific serum no. 79 showed, as expected, no sign of competition with whatever antigen combination. n.d. not determined.