Investigating a novel multiplex proteomics technology for detection of changes in serum protein concentrations that may correlate to tumor burden

Abstract

Background: To account for cancer heterogeneity, we previously introduced the concept of “personalized” tumor markers, which are biomarkers that are informative in subsets of patients or even a single patient. Recent developments in various multiplex protein technologies create excitement for the discovery of markers of tumor burden in individual patients, but the reliability of the technologies remains to be tested for this purpose. Here, we sought to explore the potential of a novel proteomics platform, which utilizes a multiplexed antibody microarray, to detect changes in serum protein concentration that may correlate to tumor burden in pancreatic cancer. Methods: We applied the Quantibody® Human Kiloplex Array to simultaneously measure 1,000 proteins in sera obtained pre- and post-surgically from five pancreatic cancer patients. We expected that proteins which decreased post-surgery may correlate to tumor burden. Sera from two healthy individuals, split into two aliquots each, were used as controls. To validate the multiplexed results, we used single-target ELISA assays to measure the proteins with the largest serum concentration changes after surgery in sera collected pre- and post-surgically from the previous five patients and 10 additional patients. Results: The multiplexed array revealed nine proteins with more than two-fold post-surgical decrease in at least two of five patients. However, validation using single ELISAs showed that only two proteins tested displayed more than two-fold post-surgical decrease in one of the five original patients. In the independent cohort, six of the proteins tested showed at least a two-fold decrease post-surgery in at least one patient. Conclusions: Our study found that the Quantibody® Human Kiloplex Array results could not be reliably replicated with individual ELISA assays and most hits would likely represent false positives if applied to biomarker discovery. These findings suggest that data from novel, high-throughput proteomic platforms need stringent validation to avoid false discoveries.

Uncovering the Depths of the Human Proteome: Antibody-based Technologies for Ultrasensitive Multiplexed Protein Detection and Quantification

“…The micro-ELISA array technology from RayBiotech combines 25 non-overlapping glass-slide arrays to perform up to 1,000 miniature sandwich ELISAs simultaneously, targeting mostly inflammation-related proteins, including cytokines, chemokines, growth factors, tumor markers, and transcription factors (37)(38)(39)(40)(41)(42)(43). Each 75mm x 25mm glass slide contains 16 identical antibody Performance data by the manufacturer reports high precision with an average intra-assay CV of 7-10% and inter-assay CV of 10-15%, and a dynamic range of around 3-to 4-log concentration.…”

“…A latest study employed the micro-ELISA array from Raybiotech to identify new diagnostic biomarkers in ovarian cancer and validated nine candidates using ELISA (143). However, Ren et al reported incongruities between the micro-ELISA array and ELISA data for nine protein candidates in pancreatic cancer (41). The overall findings illustrated that inconsistencies in protein quantification between different ultrasensitive multiplex immunoassays are highly analyte dependent.…”

“…For the latter it is imperative to evaluate new multiplex proteomics technologies by including technical replicates and positive and J o u r n a l P r e -p r o o f negative controls for checking analytical sensitivity, specificity, and precision to verify the claims of the manufacturers. Otherwise, seemingly impressive technologies, if used under a black-box approach, can generate presumably promising but fallacious data (41,(153)(154)(155).…”