Monoclonal antibody serum-based immunoassay for lung cancer early detection

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Lung cancer is a leading cause of cancer death in both men and women, yet there is no recommended screening method. ACS estimates that during 2006 and in the US there will be about 174,470 new diagnosed cases and 162,460 deaths, accounting for 29% of all cancer deaths. An easy-to-use immunodiagnostic assay would have clinical applications in lung cancer diagnosis as well as in lung cancer screening.

At present, symptomatic patients are referred to chest X ray or CT to detect pulmonary nodules that need further confirmation via CT, PET scan or biopsies. CT, in addition to its cost, has a high rate of incidental nodule detection. This is a particular drawback because incidental nodules are often seen in smokers and former smokers which represent the lung cancer high risk population, amounting to 120 million individuals in the US alone.

We have previously developed lung cancer serum biomarkers using polyclonal antibodies with high sensitivity for early stage adenocarcinoma and squamous cell carcinoma of the lung (resectable stages I/II), and high specificity when comparing lung cancer versus non-lung cancer controls, i.e. serum from high risk smokers, normal and benign controls with small lung nodules. The serum biomarkers were validated on over 700 serum samples of lung and other cancers, benign, high risk and normal controls, as well as through multiple third party validations including immunohistochemistry, tissue array, and a blind study.

We have now generated monoclonal antibodies to the lung cancer serum biomarkers. The monoclonal antibodies specifically stained lung cancer tissues by immunohistochemistry, with membrane, nuclear or cytoplasmic localization.

Six monoclonal antibodies were selected with optimal sensitivity and specificity using 194 serum samples from 68 lung cancer patients and 126 non-lung cancer patients, i.e. prostate and colon cancers and normal controls. Specifically, monoclonal antibodies were reacted with serum samples using our proprietary multiplex protein array technology (MPAT), an immunoassay linked to a data acquisition and imaging system. Briefly, the same matrix of protein samples was simultaneously interrogated by a given antibody, then a secondary antibody linked to a fluorescent dye was used to visualize antigen-antibody reaction for each clinical sample. A scanned image of all spots was produced with a Li-Cor Odissey infrared imaging system and then analyzed. Using these clinical samples, mAb1 yielded 65% sensitivity at 90% specificity in the comparison lung cancer versus non-lung cancer, or 95% sensitivity at 70% specificity.

Western blot analysis using lung cancer adenocarcinoma cell line total protein extract, showed that the six selected monoclonal antibodies recognize single protein bands. We have developed an ELISA-based prototype immunodiagnostic assay for lung cancer screening or diagnosis. Data on the validation of this immunodiagnostic assay will be presented.