[PubMed] [Google Scholar] 10. are quantified indirectly – by detecting the presence of auto-antibodies against tumor proteins in human serum. The result of the optimization and validation process was in the case of ecPKA a reproducible and stable assay. In case of NNMT the assay was probably not sensitive enough. Keywords: Auto-antibody, tumor marker, ELISA, colorectal cancer INTRODUCTION Serum tumor markers have been the most widely used approach for cancer detection. Currently, the majority of the available tumor markers represent cancer antigens. For example, the PSA represents a marker for prostate cancer, the CEA for colorectal cancer, the cancer antigen CA15-3 for breast cancer, the cancer antigen CA19-9 for gastrointestinal Galactose 1-phosphate Potassium salt cancer, the cancer antigen CA125 for the diagnosis of ovarian cancer, AFP is a liver cancer marker or human hCG, a breast cancer marker (1, 2). Quantification of all markers is based on the antigen determination Galactose 1-phosphate Potassium salt method and generally exhibits limited specificity and sensitivity. Therefore, new cancer biomarkers or better screening methods are necessary to improve the diagnostics of the disease. There is increasing evidence that the immune system of patients with cancer responds to tumor antigens with the production of antibodies. The observed accumulation of such auto-antibodies in cancer patients suggests them to be of high diagnostic/prognostic value (1, 3). Therefore, specifically developed auto-antibody detecting ELISAs could be used to quantify tumor markers, indirectly. As shown in Figure 1, the serum auto-antibodies that will be measured are bound between the coating antigen (tumor marker) and the enzyme labeled antibody, directed against human IgG. An increase in signal indicates the presence of auto-antibodies in the serum samples. It has been shown previously that the auto-antibody ELISAs exhibit enhanced sensitivity and specificity for some of the tumor markers, compared to ELISAs detecting the tumor antigen. In general, the auto-antibody ELISA is superior to antigen-determining kits concerning rapidity and reproducibility, is easy to perform and represents a cost-saving method (4, 5). In this context, two interesting candidates for tumor markers are ecPKA and NNMT. In normal mammalian cells, the cAMP-dependent PKA is located strictly intracellular. In cancer cells of various cell types, however, PKA has been shown to be secreted into the medium. The speculation is that ecPKA excretion might elicit the induction of serum auto-antibodies against Galactose 1-phosphate Potassium salt ecPKA, indicating that ecPKA might be a cancer antigen, and that the presence of such auto-antibodies could serve as a diagnostic marker. This has already led to the development of a novel enzyme immunoassay method that measures the level of IgG auto-antibodies generated against ecPKA (1), which we intended to confirm and optimize with our antibodies (Figure 1). Open in a separate window FIGURE 1 Schematic Rabbit Polyclonal to MGST1 illustration of an ELISA to detect auto-antibodies against serum tumor markers as e.g. PKA and NNMT (graph adapted from (1)). NNMT has recently attracted much interest in the field of different types of cancer. In humans, NNMT is predominantly expressed in the liver and is predicted to be located in the cytoplasm (6, 7). It could represent a new marker for colorectal cancer (CRC) that will further enhance detection of the disease and trigger a follow-up colonoscopy, as CRC patients showed elevated NNMT levels in serum. Furthermore, NNMT showed higher sensitivity than the established CRCtumor marker CEA, indicating that NNMT could be more suitable to discriminate between patients with CRC and apparently healthy individuals (8, 9, 10, 11). But further studies are needed to address the potential correlation of NNMT-serum levels with the presence of CRC. As NNMT was described to be secreted into serum, we tested whether it elicited the development of auto-antibodies as well. All these facts indicate the probable utility of this new auto-antibody assay as a cancer screening tool without the false positives often associated with conventional testing. It could also provide a novel technology for cancer detection (1). MATERIALS AND METHODS The serum samples used for the optimization of the detection method were obtained from subjects with CRC (n = 8, mean age 59 years 21 years). Tested cancer patients were of different age and sex, with a wide range of stages of malignancy and different therapies. Control serum was obtained from healthy volunteers (n = 7, mean age 30 years 27 years). The serum samples were kept at -80C until use and were thawed only once before use. The anti-ecPKA auto-antibodies and NNMT-levels were measured by solid phase ELISA. For this purpose, flat bottom polystyrene 96-well Immunolon-4 HBX microtiter plates were used (binding capacity 100-200 Galactose 1-phosphate Potassium salt ng IgG/cm2). Each single.