Raf specific antibody response in different patients' sera. Analysis of 2 representative positive (42) and negative (12) sera using a secondary antibody detecting human IgG, IgA and IgM is depicted in A. Sera no. 42 and 12 were tested for specificity against B-Raf, B-Raf V599E (V599E), an unrelated antigen (PSA) and without the addition of antigen (-). Serum 42 reacted against B-Raf and B-Raf V599E and not against an unrelated antigen. Sera 12 showed no measurable response. Sera withdrawn at different time points from patient no. 106 were tested at different dilutions in an ELISA and OD405 was determined (B). In the case of this patient, sera were negative at the first two time points and became strongly positive within three months. Specificity of the response was confirmed using different concentrations of antigen for coating (0, 0.1, 1 and 2 μg/ml) and different serum dilutions (C). All 371 patient and 119 control serum samples were analysed in a similar way. In D, serum was analyzed in a similar way then in C using an IgG specific secondary antibody. Serum #42 is weakly positive for B-Raf specific IgG antibodies, serum #1 is representative for most other sera of healthy donors and melanoma patients which are negative for B-Raf specific IgG antibodies.