Two nitrocellulose membrane disks were placed on and then lifted from the same phage grown plate of biopan 4. and 87 normal serum samples. A logistic regression model and leave-one-out validation were used to evaluate predictive accuracies with a single marker as well as with combined markers. Identities of those selected proteins were revealed through the sequence BLAST program. Results We harvested 100 putative tumor-associated phage clones after biopan enrichment. Sequencing analysis revealed that six phage proteins were inframe and unique. Antibodies to these six phage-expressed proteins were TP-10 measured by ELISAs, and the results showed that three of the phage clones TP-10 had statistical significance in discriminating patients from normal individuals. BLAST results of the three proteins showed great matches to ASB-9, SERAC1, and RELT. Measurements of the three predictive phage proteins were combined in a logistic regression model that achieved 80% sensitivity and 100% specificity in prediction of sample status, whereas leave-one-out validation achieved 77.0% sensitivity and 82.8% specificity among 87 patient samples and 87 control samples. Receiver operating characteristic curve analysis and the leave-one-out method both showed that combined measurements of the three antibodies were more predictive of disease than any of the single antibodies studied, underscoring the importance of identifying multiple potential markers. Conclusion Serum autoantibody profiling is a promising approach for early detection and diagnosis of breast cancer. Rather than one autoantibody, a panel of autoantibodies appears preferable to achieve superior accuracy. Further refinements will need to be made to further improve the accuracy. Once refined, the assay must be applied to a prospective patient population to demonstrate applicability. Introduction In women, breast cancer is the most common malignancy and the second most common cause of cancer-related mortality [1]. Further reduction in the mortality will require successful strategies for early detection and screening of the disease. A sensitive assay to identify biomarkers that can accurately determine the onset of breast cancer C especially if the technique is of low risk to the patient, such as blood drawing C is ideal for TP-10 early cancer detection. Much of the effort in the past has centered on the discovery and characterization of single tumor-associated antigens as cancer markers. Two clinically used breast cancer antigens, CA 15-3 and CA 27.29, are elevated in less than 10% of early-disease patients and in about 75% of advanced-disease patients. Neither antigen is recommended for screening or diagnosis of onset breast cancer [2]. On the basis of the marked heterogeneity of most human cancers, it is doubtful that a single gene, chromosome aberration, or protein will provide sufficient accuracy for early detection. In contrast to the TP-10 detection of serum antigens, the detection of serum antibodies to tumor antigens may provide reliable serum markers for cancer diagnosis and prognosis [3-6]. Changes in the level of gene expression [3, 7-9] and aberrant expression of tissue-restricted gene products [10,11] are factors in the development of a humoral immune response in cancer patients. There are several advantages of using serum antibodies as markers for tumor development. First, tumor-associated autoantibodies circulate in the blood much earlier than serum antigens. Autoantibodies to p53 have been reported in patients with early-stage ovarian or colorectal cancers [12,13], and a panel of serum antibodies can detect nonsmall-cell lung cancer 5 years prior to autoradiograph detection [14]. Second, antibodies may be more abundant than antigens, especially at low tumor burden. Thirty percent of patients with ductal carcinoma em in situ /em in which the proto-oncogene HER-2/ em neu /em was overexpressed Keratin 18 (phospho-Ser33) antibody had serum antibodies specific to this protein [7,15]. In this respect, serologic analysis of recombinant cDNA expression libraries of tumors with autologous serum has identified some relevant tumor antigens: MAGE [16], SSX2 [17], and NY-ESO-1 [18]. We previously reported the use of combined autoantibodies as markers for early detection of nonsmall-cell lung cancer [14,19,20]. We achieved over 90% sensitivity and specificity in diagnosing stage I nonsmall-cell lung cancer using five antibody markers in serum samples. Can the previously described technique be applied to breast cancer? If so, can the technique provide sufficient sensitivity and specificity to be clinically useful? In the present study, we used similar techniques on a breast cancer T7 cDNA phage library to identify tumor-associated proteins. We then measured autoantibody reactivities to identified phage-expressed TP-10 proteins in breast cancer patient sera and normal sera by immunochemistry and ELISA to evaluate the sensitivity and specificity of single versus combined antibody measurements for predicting probability of disease. We further present the identities of corresponding proteins.