Monoclonal antibody serum-based immunoassay for colon cancer detection

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Colorectal cancer (CRC) is a major cause of cancer death in both men and women. It is estimated that there will be about 148,610 new diagnosed cases and 55,170 deaths in 2006 in the US. There are several recommended screening methods for CRC, including colonoscopy and FOBT. Colonoscopy is the current gold standard, yet it is invasive and expensive. It is recognized that CRC is curable by resection when localized, however, only 44% of US adults over the age of 50 undergo screening. An easy-to-use immunodiagnostic assay would have clinical applications in CRC diagnosis as well as in screening: it would be safer, more cost-effective, and would gain patient preference over any other available screening method.

We have previously developed CRC serum biomarkers using polyclonal antibodies with high sensitivity and specificity for early and late stage of CRC versus normal and benign controls. The CRC serum biomarkers were validated on over 500 serum samples of colon and other cancers, benign, and normal controls, as well as through multiple third party validations including immunohistochemistry, and a blind study.

We have now generated monoclonal antibodies to the CRC serum biomarkers. Monoclonal antibodies to CRC serum biomarkers specifically stained colon 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 CRC patients, and 126 non-CRC patients, including prostate and lung cancer, 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 and mAb 2 yielded 80% sensitivity at 90% specificity, and 75% sensitivity at 80% specificity respectively, in the comparison CRC versus non-CRC (normal, benign and other cancers).

Western blot analysis using the DLD-1 colon adenocarcinoma cell line total protein extract, showed that the six selected monoclonal antibodies recognize single protein band, strengthening the CRC specificity of these biomarkers. We have developed an ELISA-based prototype immunodiagnostic assay for CRC screening or diagnosis. Data on the validation of this immunodiagnostic assay will be presented.