Sandwich ELISA serum-based immunoassay for lung cancer detection

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Lung cancer is a leading cause of cancer death, yet there is no recommended screening method. ACS estimates that during 2007 and in the US there will be about 213,380 new diagnosed cases and 160,390 deaths, accounting for 29% of all cancer deaths. 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. An easy-to-use immunodiagnostic assay would have clinical applications in lung cancer diagnosis as well as in lung cancer screening.
 In recent years, we have discovered biomarkers for early detection of lung cancer (AACR 2007 # 2685). We have generated a battery of monoclonal antibodies (mAbs) and tested them with 194 serum samples including 68 lung cancer and 126 non-lung cancer patients using our proprietary multiplex protein array technology (MPAT). Several mAbs with high sensitivity for lung cancer were selected.
 To further confirm mAb reactivity on lung cancer, we have tested the selected mAbs with in-house and commercial lung cancer tissue microarrays (75 cases). mAbs specifically stain lung cancer tissues, and mAb staining includes membrane, nuclear and cytoplasmic localization.
 We have then tested the selected lung cancer specific mAbs by antibody capture immunoassay. First, mAbs are reacted with 32 clinical samples comprising 16 lung Adca and 16 high risk smokers. mAb-biomarker reaction is detected by cheluminescence using a CCD camera, and quantification of mAb binding is performed from the scanned image. The 95th percentile of intensities in the control group is used as a cut-off value for positive signal. The best mAbs performers with binding intensities above the cut-off value, in at least 50% of lung cancer samples are selected for further studies.
 The selected mAbs are then tested with 250 clinical samples comprising 64 early stage non small cell lung carcinoma (24 Adca, 24 squamous cell, and 16 large cell carcinoma) and 186 non-lung cancer samples (41 normal, 25 colon Adca, 120 prostate Adca).
 Sensitivity and specificity are determined from receiver operating curves (ROC) using the statistical package GB-STAT (Dynamic microsystem). When comparing the lung cancer to the non-lung cancer group, sensitivity for most mAbs ranges from 60-80% at 80% specificity. The best lung serum biomarkers are now being further validated by the antibody capture assay on over 700 serum samples, including early lung cancer (n=150), late lung cancer (n=100), benign lung nodules (n=50), high risk smokers (n-50), normal controls (n=50), as well as other cancer (n=300). We are also developing two-antibody sandwich assays using optimal capture/detecting antibody pairs.
 These assays could be valuable to complement CT diagnosis in symptomatic individuals or to complement CT screening of high risk individuals.