Abstract: Objective To develop and verify a diagnostic enzyme-linked immunosozbent assay（ELISA） kit in vitro for lung cancer by using lung cancer-related markers of neuron-specific enolase（NSE） and cytokeratin 19 fragment（CYFRA21-1） with high specificity and sensitivity.
Methods In vitro diagnostic ELISA kits were prepared by coating anti-NSE and anti-CYFRA21-1 antibodies at the bottom of the microtiter plates at a proper proportion. NSE and CYFRA21-1 antigens were used as research subjects. Coating buffer， packet time and packet temperature were investigated by detecting the index of stability， sensitivity and accuracy of ELISA kits. Results Period of validity of in vitro NSE and CYFRA21-1-based dual-color diagnostic ELISA kit for lung cancer was 6 months at 4 ℃. The sensitivity，specificity and accuracy of in vitro NSE and CYFRA21-1-based, dual-color diagnostic ELISA kit were 83.8%（129/154），53.8%（57/106） and 71.5%（186/260），which were higher than that of anti-NSE or anti-CYFRA21-1 antibodies diagnostic ELISA kits. And the areas under the receiver operating characteristics curves was 0.90. Conclusion NSE and CYFRA21-1-based lung cancer dual-color diagnostic ELISA kit was successfully prepared， which could improve the sensitivity，specificity and accuracy of detecting lung cancer. And it was confirmed that it has the potential of developing into a new diagnostic molecular imaging reagent for lung cancer.