3). Again, the scoring cutoff was determined by the non-averaged, replicate MFI values, resulting in a more stringent analysis (i.e. MFI values of the three different assay runs for the normal patient samples were not averaged before determining cutoff). Results are shown in Supplementary Fig. 3. Despite this stringent cutoff, all five previously known p53-positive samples (based on ELISA in Fig. 2) remained positive on the VeraCode™ beads in this rigorous inter-assay setting. Furthermore, the two additional p53-positives

picked up only by the VeraCode™ assay (and not ELISA) shown in Fig. 2 (single assay), also remained positive in this rigorous inter-assay setting. The average inter-assay CV was 20% across all sample-protein pairs (see error bars in Supplementary Fig. 3 for more detail). Finally, as an additional metric of inter-assay reproducibility, linear regression selleck products analysis of two separate assay runs of Obeticholic Acid in vitro the 94 samples for two different

TAAs (assayed in multiplex) showed R2 values ≥ 0.96 in both cases (Supplementary Fig. 4; p53, as well as insulin-like growth factor 2 mRNA binding protein 2 [IGF2BP2] (Reuschenbach et al., 2009)). Next, to show compatibility of the VeraCode™ based assay with multiple biomarker classes and to increase the overall diagnostic sensitivity for CRC beyond TAAs alone, we combined three distinct biomarkers, i) autoantibodies against the aforementioned p53 TAA, as well as detection of serum Evodiamine levels of ii) carcinoembryonic antigen (CEA) and iii) the cytokine

GDF15. While CEA is very well known as a CRC biomarker for disease monitoring and prognostics, its diagnostic use alone or as part of a biomarker panel is currently under investigation ( Creeden et al., 2011 and Su et al., 2012). GDF15 (MIC-1) is perhaps not as well as established for CRC, however, several recent studies suggest it may be useful as a prognostic and diagnostic marker ( Xue et al., 2010, Wallin et al., 2011 and Brown et al., 2012). First, we sought to demonstrate that sandwich immunoassay detection of non-antibody serum protein biomarkers could also be achieved on the VeraCode™ system. As with the p53 TAA, this was done by comparison to conventional ELISA. In this case CEA was used as a model system (with 52 CRC and 25 normal serum/plasma samples). For the VeraCode™ assay, an anti-CEA capture antibody was attached to the bead surface. Following incubation with the serum/plasma samples to capture the CEA, detection was with a biotin labeled anti-CEA antibody followed by a fluorescently labeled streptavidin. ELISA (sandwich immunoassay format) was simply performed using a commercially available kit (see Materials and methods). Results are shown in Fig. 3. Clear agreement is apparent between the two assays as seen by the overlaid bar graphs in Fig. 3A.