Abstract
Due to insufficient biomarker validation and poor performances in diagnostic assays, the candidate biomarker verification process has to be improved. Multi-analyte immunoassays are the tool of choice for the identification and detailed validation of protein biomarkers in serum. The process of identification and validation of serum biomarkers, as well as their implementation in diagnostic routine requires an application of independent immunoassay platforms with the possibility of high-throughput. This review will focus on three main multi-analyte immunoassay platforms: planar microarrays, multiplex bead systems and, array-based surface plasmon resonance (SPR) chips. Recent developments of each platform will be discussed for application in clinical proteomics, principles, detection methods, and performance strength. The requirements for specific surface functionalization of assay platforms are continuously increasing. The reasons for this increase is the demand for highly sensitive assays, as well as the reduction of non-specific adsorption from complex samples, and with it high signal-to-noise-ratios. To achieve this, different support materials were adapted to the immobilized biomarker/ligand, allowing a high binding capacity and immobilization efficiency. In the case of immunoassays, the immobilized ligands are proteins, antibodies or peptides, which exhibit a diversity of chemical properties (acidic/alkaline; hydrophobic/hydrophilic; secondary or tertiary structure/linear). Consequently it is more challenging to develop immobilization strategies necessary to ensure a homogenous covered surface and reliable assay in comparison to DNA immobilization. New developments concerning material support for each platform are discussed especially with regard to increase the immobilization efficiency and reducing the non-specific adsorption from complex samples like serum and cell lysates.
Highlights
Due to insufficient biomarker validation and poor performances in diagnostic assays, the candidate biomarker verification process has to be improved
This paper focuses on microarrays, bead assays and array-based surface plasmon resonance (SPR) assays and discusses their application in serum biomarker discovery
Multi-analyte immunoassays have the potential to be employed as the state-of-the-art technology for the in-depth serum biomarker discovery including validation studies and assay development
Summary
Validation of candidate biomarkers has a single goal: to determine, if there is sufficient evidence for a potential clinical utility of a given biomarker candidate to ensure further investment in that candidate for clinical trials. Insufficient diagnostic test accuracy is just the tip of the iceberg It is the consequence of poorly designed biomarker candidate discovery and validation phases without clear understanding of the nuances of interpreting high dimensional data sets which often leads to biases and high false discovery rates [3]. The ELISA is limited by the fact that the classic approach allows only a single antigen detection and often requires relatively large volumes of sample material compared with state-of-the-art methods This concern becomes especially acute when clinical studies require access to limited amounts of biological material. ELISA, multiplex arrays have several advantages, including their high-throughput nature, requirement for smaller sample volume, efficiency in terms of time and cost, the ability to evaluate one antigen in the context of multiple others, and the ability to reliably detect different proteins across a broad dynamic range [20]. Multi-analyte immunoassays might be the tool of choice for the identification and detailed validation of protein biomarkers in serum
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