Development of a novel multiplex beads-based assay for autoantibody detection for colorectal cancer diagnosis

Humoral response in cancer patients can be used for early cancer detection. By screening high-density protein microarrays with sera from colorectal cancer (CRC) patients and controls, we identified 16 tumor-associated antigens (TAAs) exhibiting high diagnostic value. This high number of TAAs requires the development of multiplex assays combining different antigens for a faster and more accurate prediction of CRC. Here, we have developed and optimized a bead-based assay using nine selected TAAs and two controls to provide a multiplex test for early CRC diagnosis. We screened a collection of 307 CRC patients' and control sera with the beads assay to identify and validate the best TAA combination for CRC detection. The multiplex bead-based assay exhibited a similar diagnostic performance to detect the humoral response in comparison to multiple ELISA analyses. After multivariate analysis, a panel composed of GTF2B, EDIL3, HCK, PIM1, STK4, and p53, together with gender and age, was identified as the best combination of TAAs for CRC diagnosis, achieving an AUC of 89.7%, with 66% sensitivity at 90.0% fixed specificity. The model was validated using bootstrapping analysis. In summary, we have developed a novel multiplex bead assay that after validation with a larger independent cohort of sera could be utilized in a high-throughput manner for population screening to facilitate the detection of early CRC patients.