Multiplex assays for biomarker research and clinical application: translational science coming of age

Abstract

Over the last decade, translational science has come into the focus of academic medicine, and significant intellectual and financial efforts have been made to initiate a multitude of bench-to-bedside projects. The quest for suitable biomarkers that will significantly change clinical practice has become one of the biggest challenges in translational medicine. Quantitative measurement of proteins is a critical step in biomarker discovery. Assessing a large number of potential protein biomarkers in a statistically significant number of samples and controls still constitutes a major technical hurdle. Multiplexed analysis offers significant advantages regarding time, reagent cost, sample requirements and the amount of data that can be generated. The two contemporary approaches in multiplexed and quantitative biomarker validation, antibody-based immunoassays and MS-based multiple (or selected) reaction monitoring, are based on different assay principles and instrument requirements. Both approaches have their own advantages and disadvantages and therefore have complementary roles in the multi-staged biomarker verification and validation process. In this review, we discuss quantitative immunoassay and multiple reaction monitoring/selected reaction monitoring assay principles and development. We also discuss choosing an appropriate platform, judging the performance of assays, obtaining reliable, quantitative results for translational research and clinical applications in the biomarker field.

Figure 1.

A schematic of comparison of two quantitative multiplex analysis approaches: antibody-based immunoassay and MS-based MRM. Advantages and disadvantages for both approaches are summarized and compared.

Figure 2.

Blood from a healthy individual was collected with BD Vacutainer* plastic red top tubes and sat at room temperature for either 1 h or 3 h (clotting time). After centrifugation, serum was collected, aliquoted and stored at −80°C. Equal amounts of delipidated HSA/IgG-deleted serum proteins (150 μg) were loaded onto 18 cm IEF first-dimension gels, pH 4–7 IPG strip. The second-dimension SDS-PAGE was carried out with 10% polyacrymide Bis-Tris neutral gels. Following the second-dimension separation, gels were visualized by silver staining. Boxes (which appear to the right) show an expanded view of the indicated region on each gel.

Figure 3.

A standard curve of IL-6 was generated with a luminex bead array plateform (Bio-Plex human 9-Plex kit, GM-CSF, IFN-g, IL-1β, IL-2, IL-6, IL-8, IL-10, IL-12p70, TNF-α from Bio-Rad). The standard 9 cytokine mixture was obtained from Bio-Rad and a 9-point standard curve was established through a 1:4 serial dilution with the diluent supplied. The actual and expected concentrations of IL-6 were calculated, and then % recovery (observed concentration/expected concentration × 100) was calculated. The Quantifiable range is defined by a concentration range where CV was < 15% and % recovery was < 100 ± 20% from standard calibrators. It should be noted that LLOQ and LOD are not overlapping. LLOQ was defined by % recovery.

Figure 4.

Sample matrix affects assay performance. Standard curves of fourfold serial dilutions of purified human glial fibrillary acidic protein in 1% BSA in PBS (▲) or serum cytokine assay diluent (●) from MesoScale Discovery were analyzed using a MesoScale Discovery platform. Plates coated with monoclonal capture antibody (SMI-26, Covance, Denver, PA, USA) at 100 ng per well in PBS. Polyclonal anti-GFAP (Dako, Carpinteria, CA, USA) was used for GFAP detection.

Figure 5.

The schematic illustrates the major steps in MRM assay development with a Sciex Q 4000 TRAP^™ linear ion trap mass spectrometer. The first step is to design signature peptides (precursor ion for Q1) for proteins of interest. The peptide sequence can be selected through a LC-MS/MS experiment or through in silico design. The second step is to test the predefined transitions (Q1/Q3 pairs) and retention times, and peptide parent ion’s identity is confirmed by a full-scan precursor ion MS/MS. If an absolute quantitation is needed, the isotopically labeled signature peptide should be synthesized and used as internal standard. The third step is to optimize and multiplex the desired MRM assay. The fourth step is to quantitate the peak area of selected fragment ion and establish LOQ and LOD.