While the inter-assay CVs were acceptable, future improvements in reproducibility may be achieved with the development of rigorous assay-to-assay normalization controls and with better mixing approaches for the large and relatively dense 240 micron glass beads (cylinders), which tend to settle quickly and may result in poor and inconsistent mixing and binding kinetics. Likewise, the VeraCode™ system was also technically validated against ELISA selleck kinase inhibitor for detection of non-antibody circulating protein biomarkers using a sandwich immunoassay format. In this case, the CRC biomarker CEA was used as a model system. Here, 94% hit concordance was seen between the two assay types in 52 CRC samples (and quantitative correlation of

R2 = 0.9 when a linear regression is performed between the assays). Not surprisingly, the only discordant hits were borderline positive or negative CRC samples that fell extremely close to the cutoffs (see red asterisks in Fig. 3A), as the consistently low background click here in the normal patients resulted in a very low scoring

cutoff (both assays show 100% specificity against normal samples). Next, by combining the most robust TAA observed in our studies, p53, with sandwich immunoassay based quantification of the well-known CRC biomarker CEA, and the cytokine GDF15 in a hybrid multiplexed assay, we achieved a composite diagnostic sensitivity and specificity of 54% and 98%, respectively (186 samples CRC and normal). Thus, we demonstrate the ability to measure, in multiplex, two distinctly different biomarker types using different assay formats, simultaneously, on the VeraCode™ beads. As with the TAAs alone, the additive benefit of combing multiple biomarkers stems from the lack of complete redundancy, with each biomarker detecting several patients (9 to 29) which the others ADP ribosylation factor did not, and with no single biomarker exceeding 38% sensitivity (GDF15). It is important to emphasize that while the particular biomarkers used here were chosen to exemplify the immunoassay method, the clinical studies

performed here were only preliminary, retrospective validation studies on a particular cohort of CRC and normal patient samples, and that the results of these studies would need further validation using larger patient cohorts, as well as non-target disease controls (e.g. inflammatory bowel disease and cancers other than CRC) and ultimately, blinded studies and prospective clinical studies. In the future, it is expected that the CRC biomarker panel not only would expand, but also would be refined through elimination of biomarkers as further studies are performed using the VeraCode™ immunoassay methods presented here. For example, GDF15 is a stress-induced cytokine and in addition to CRC has been shown to be a biomarker for a variety of conditions such as heart disease (reviewed in Wollert and Kempf, 2012) and worsening albuminuria in patients with type 2 diabetes (Hellemons et al., 2012).