Comparison of Commercial ELISA Kits, a Prototype Multiplex Electrochemoluminescent Assay, and a Multiplex Bead-Based Immunoassay for Detecting a Urine-Based Bladder-Cancer-Associated Diagnostic Signature.

Hideki Furuya,Regan S Wong,Charles J Rosser,Ian Pagano,Riko Lee,Keanu Chee,Takashi Kobayashi

doi:10.3390/diagnostics9040166

Abstract

The ability to accurately measure multiple proteins simultaneously in a single assay has the potential to markedly improve the efficiency of clinical tests composed of multiple biomarkers. We investigated the diagnostic accuracy of the two multiplex protein array platforms for detecting a bladder-cancer-associated diagnostic signature in samples from a cohort of 80 subjects (40 with bladder cancer). Banked urine samples collected from Kyoto and Nara Universities were compared to histologically determined bladder cancer. The concentrations of the 10 proteins (A1AT; apolipoprotein E—APOE; angiogenin—ANG; carbonic anhydrase 9—CA9; interleukin 8—IL-8; matrix metalloproteinase 9—MMP-9; matrix metalloproteinase 10—MMP10; plasminogen activator inhibitor 1—PAI-1; syndecan—SDC1; and vascular endothelial growth factor—VEGF) were monitored using two prototype multiplex array platforms and an enzyme-linked immunosorbent assay (ELISA) according to the manufacturer’s technical specifications. The range for detecting each biomarker was improved in the multiplex assays, even though the lower limit of quantification (LLOQ) was typically lower in the commercial ELISA kits. The area under the receiver operating characteristics (AUROC) of the prototype multiplex assays was reported to be 0.97 for the multiplex bead-based immunoassay (MBA) and 0.86 for the multiplex electrochemoluminescent assay (MEA). The sensitivities and specificities for MBA were 0.93 and 0.95, respectively, and for MEA were 0.85 and 0.80, respectively. Accuracy, positive predictive values (PPV), and negative predictive values (NPV) for MBA were 0.94, 0.95, and 0.93, respectively, and for MEA were 0.83, 0.81, and 0.84, respectively. Based on these encouraging preliminary data, we believe that a multiplex protein array is a viable platform that can be utilized as an efficient and highly accurate tool to quantitate multiple proteins within biologic specimens.

Highlights

While there continues to be increasing reports of biomarker discovery and early validation studies being published, few new biomarkers have entered clinical practice over the past 30 years.There are many reasons for this, but one of the overwhelming reasons has been the reliance on single biomarkers for the evaluation of cancers that we know can have a broad range of molecular changes.When coupling the tumor’s heterogeneity with the observed variation, even between individuals with similar tumors, it is no wonder very few accurate biomarkers have progressed to clinical relevance.a shift has occurred to molecular signatures comprised of multiple biomarkers being favored over single biomarkers
The range for detecting analytes was improved in the multiplex assays, even though the level of quantification (LLOQ) was typically lower in the commercial enzyme-linked immunosorbent assay (ELISA) kits
SDC1 in multiplex electrochemoluminescent assay (MEA) and A1AT and SDC1 in multiplex bead-based immunoassay (MBA), coefficient of variation (CV) were lower in commercial ELISA assays compared to the multiplex array platforms

Summary

Introduction

While there continues to be increasing reports of biomarker discovery and early validation studies being published, few new biomarkers have entered clinical practice over the past 30 years.There are many reasons for this, but one of the overwhelming reasons has been the reliance on single biomarkers for the evaluation of cancers that we know can have a broad range of molecular changes.When coupling the tumor’s heterogeneity with the observed variation, even between individuals with similar tumors, it is no wonder very few accurate biomarkers have progressed to clinical relevance.a shift has occurred to molecular signatures comprised of multiple biomarkers being favored over single biomarkers. While there continues to be increasing reports of biomarker discovery and early validation studies being published, few new biomarkers have entered clinical practice over the past 30 years. There are many reasons for this, but one of the overwhelming reasons has been the reliance on single biomarkers for the evaluation of cancers that we know can have a broad range of molecular changes. When coupling the tumor’s heterogeneity with the observed variation, even between individuals with similar tumors, it is no wonder very few accurate biomarkers have progressed to clinical relevance. A shift has occurred to molecular signatures comprised of multiple biomarkers being favored over single biomarkers. The advent of advanced molecular profiling techniques has enabled the derivation of molecular signatures that can more accurately diagnose cancer and make individualized patient evaluation and care feasible. Several molecular signature assays (e.g., Oncotype DX breast, Oncotype DX prostate, and MammaPrint) are being incorporated into clinical practice [1,2,3]

Objectives

Methods

Results

Discussion

Conclusion