Development of natural protein microarrays for diagnosing cancer based on an antibody response to tumor antigens

The detection of autoantibodies to tumor antigens has potential utility for the early diagnosis of cancers. In previous studies, we have identified tumor antigens based on Western blot analysis of tumor cell lysates that were incubated with subject sera to identify proteins that elicit specific reactivity in sera from patients with the corresponding tumor type. More recently, we have explored the use of microarrays spotted with tumor proteins as an alternative to Western blots. Microarrays provide a high throughput, high sensitivity alternative to the use of Western blots for tumor antigen profiling. In this study, we have assessed the reproducibility of natural protein microarrays and their ability to distinguish between lung cancer sera and controls. Protein lysates from the A549 human lung adenocarcinoma cell line were separated into 1840 fractions that were spotted in duplicate, along with various controls, on nitrocellulose coated slides. Sera from 18 newly diagnosed patients with lung cancer and from 15 healthy controls were each hybridized to an individual microarray. The reactivity of arrayed proteins with Ig was determined by incubation with biotinylated goat-anti-human-Ig followed by phycoerythrin-conjugated streptavidin. The intensity measures of duplicate spots (within-slide) and duplicate slides (between-slides) were highly reproducible, exhibiting correlation values >0.9. A total of 63 of the 1840 arrayed fractions demonstrated increased reactivity in cancer patients relative to controls as measured by a rank-based statistic (p<0.008). Microarrays of tumor-derived proteins provide the means for uncovering a repertoire of tumor antigens that have induced an antibody response in patients with specific cancers.