A Novel Method For Detecting P53 Autoantibodies From Patients With Non-small Cell Lung Cancer

Background and objectiveSerum autoantibody is significant for the early diagnosis and prognosis of cancer. p53 autoantibodies were found in sera of patients with a variety of different cancers. So our objective is to synthesize peptide array to analyse p53 autoantibodies in the sera of patients with non-small cell lung cancer (NSCLC).MethodTo evaluate its role in lung cancer progression, we analyzed p53 autoantibodies from patients with non-small cell lung cancer by ELISA, Western blot and peptide library of p53. The peptide library consisted of 12 amino acids long peptides with nine amino acids overlapping with the adjacent peptides immobilised on a cellulose membrane. The membrane was incubated with 1/400 dilutions of p53 monoclonal antibody (DO-2) whose epitopes were already known to establish a new approach to detect p53 antibody. Then, we analysed the p53 autoantibodies from the sera of NSCLC and controls by ELISA, Western blot and peptide array.ResultWe synthesized on cellulose membranes twelve-amino-acid overlapping peptides which included all of the sequences of the polypeptide chain of p53. By ELISA, we found that 21 of the 84 patients developed antibodies against p53 and non of the healthy individuals was positive. However, 14 patients'sera had p53 autoantibodies which were detected by peptide array. Furthermore, these p53 autoantibodies recognized common epitopes. The common epitopes are located in regions on the polypeptide chain of p53, which are functionally important for the role of p53 in growth control. All the 14 positive lung cancer cancer sera that were picked out by the peptide array screening were used as first antibody to detect the pure p53 protein transferred to the NC membrane, and 14 of them got a band with a similar mass when compared to the result from the p53 monoclonal antibody DO-2, and then we got the Gray value from both the spots got from the screening membrane and bands from the western blot membrane, and employed the Pearson Correlation Test to analysis the correlation, and we got a significant positive correlation between the two results.ConclusionTaken together, the peptide array could be applied not only to detect the autoantibodies in the sera of patients with lung cancer, but also to map the epitopes of the autoantibodies. Further more, these findings implicate that p53 autoantibodies might be involved in lung cancer progression and the epitopes might offer important information of the prognosis.