A26 Four protocols based on proprietary and/or published detection strategies and reagents have been applied to a cohort of 115 subjects comparing patients with diagnosed lung cancer, smokers not diagnosed with lung cancer and healthy individuals. These protocols were based on measurements of levels of tNOX, a pancancer cancer-specific member of the ECTO-NOX family of growth related cell surface quinol (NADH) oxidases with protein disulfide-thiol interchange activity in plasma or serum. Standard ELISA protocols compared two different polycolonal antibodies and a dot blot ELISA protocol utilized a tNOX-specific single chain variable region fragment (scFv) based on a previously developed tNOX-specific monoclonal antibody (scFv) along with 2-D gel separations with detection using the anti-tNOX scFv. The dot blot ELISA values and the 2-D gels were quantified using computer driven algorithms developed by one of us (MPM) to eliminate operator bias. Use of the dot-blot ELISA and standard ELISAs were restricted to sera. The 2-D gel assay was utilized with plasma and sera. Values of two standard deviations greater than the values for healthy individuals were scored as indicating cancer presence. Circulating tNOX as revealed by 2-D gel analysis was regarded as a definitive indication of lung cancer presence. With 25 healthy individuals, the dot blot ELISA, the standard ELISA and 2-D gel results were uniformly negative. With sera from 65 smokers, 7 gave values both by the dot-blot ELISA and the standard ELISA greater than 2 standard deviations above the average for healthy individuals. Both the dot-blot ELISA and the standard ELISA allow for rapid high-throughput screening of sera of smokers or other groups at risk for lung cancer and yield a low incidence of false positives. For samples yielding positive values, tNOX presence, was confirmed using the specific 2-D gel protocol. Sera or plasma from 25 lung cancer patients contained the tNOX protein whereas tNOX was not detected by this method in any healthy individuals comparing the 25 specimens thus far analyzed in parallel by the 2-D gel protocol and more than 200 specimens analyzed for tNOX presence by other methods.