There is currently a gap in the translational research between identification of new biomarkers and development and commercialization of FDA-approved diagnostic tests. A large number of potential cancer serum/plasma biomarkers have emerged from academic research projects. Although none of them has sufficient sensitivity and specificity to be used alone as a screening assay, a combination of markers might allow identification of individuals who are at the earliest and most treatable stage of cancer.

Discovery and validation of biomarker panels for early detection requires samples, ideally collected before cancer diagnosis, with cases and controls collected using the same protocol. Stand-alone ELISAs require a significant amount of sample, e.g. 100 μL or more per replicate and per assay. To make maximum use of these precious samples, assays must be developed to minimize sample volume requirements.

We developed electrochemiluminescence (ECL)-based, multiplexed, serum/plasma immunoassay panels to measure more than 40 cancer-related biomarkers using a 96-well, 7-spot format. Due to the high sensitivity of ECL technology, these assays can be used with diluted serum or plasma bringing the total sample volume required to run all 40 assays down to approximately 40 μL per replicate.

These MSD® MULTI-ARRAY assay panels contain most of the classical cancer markers (AFP, Ca125, Ca15.3, Ca19.9, Ca125, CEA, Cyfra 21.1, Her-2, NSE) as well as a number of cancer-associated markers such as growth factors and their receptors (e.g., VEGF, sFlt-1, cMet, SCF, cKit, EGFR); cytokines and chemokines (e.g., IL6, IL-2R); MMPs; and inflammation markers.

The assay format is simple: diluted sample or calibrator is added to blocked and washed plates. After a 2-hour incubation with agitation, plates are washed, and detection antibody reagent is added. After a 1-hour incubation, plates are washed and read on an MSD SECTOR® Imager (read time, 1-3 minutes per plate).

The assays are sensitive enough to measure these biomarkers in normal samples, and the dynamic range extends beyond the elevated levels expected in disease states. Multiplexing does not affect accuracy; each analyte is measured accurately even when other analytes are present in high abundance. Spike recovery and dilution linearity for most assays were in the range of 80% to 120% of expected values. Detection limits for a majority of the assays were between 1 and 10 pg/mL, and dynamic ranges were between 3 and 4 logs.

In conclusion, MSD MULTI-ARRAY® assay panels have been developed and validated for measurement of serum biomarkers of relevance to early detection, diagnosis, prognosis, and/or monitoring of various cancers. The versatility, ease of use, and high-throughput features of the technology make it ideally suited for large scale clinical studies.

Citation Format: Martin K. Stengelin, Anahit Aghvanyan, Gelu Dobrescu, Mingyue Wang, Anu Mathew, Eli N. Glezer, Jacob N. Wohlstadter. Multiplex panels for cancer-associated biomarkers: quantify 40 biomarkers from 40 μL of serum or plasma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3496. doi:10.1158/1538-7445.AM2013-3496