Sample processing
The last sample obtained from each of the 1006 patients was screened for anti-p53 antibodies at a 1/10 dilution. Those patients who proved to be positive for p53 autoantibodies at this low dilution were then tested for autoantibody status using all samples from each patient; here a 1/500 dilution of plasma was used because this was found to give good specificity and sensitivity for known positive and negative controls. Of those patients who were antibody negative in the initial screen, 60 had had a plasma sample obtained around the time of their primary diagnosis of breast cancer; these primary samples were assayed (1/10 dilution) to look for any positive to negative switches in autoantibody levels during clinical follow up.