Validation Processes of Protein Biomarkers in Serum—A Cross Platform Comparison

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.

Methodical and pre-analytical characteristics of a multiplex cancer biomarker immunoassay

“…As well-known for many research-use-only (RUO)-assays, there often is only limited data available concerning the methodological performance despite being distributed and used for study and research purposes [20][21][22] . This is quite relevant for the scientific community as the same names of the parameters are used in these compound tests as in regular in-vitro diagnostic (IVD) labeled assays which are commonly used in clinical laboratory routine diagnostics for which the methodical quality and clinical validity has been investigated thoroughly in most cases.…”

Immunoproteomics technologies in the discovery of autoantigens in autoimmune diseases

“…Unfortunately, discovery efforts employing MS-based proteomics technologies have yet to yield any FDA-approved biomarkers (83). A number of issues may contribute to this shortcoming that is increasingly recognized in the field of proteomics (84,85), including: [1] incomplete validation of biomarker candidates, [2] use of suboptimal statistical methods, and [3] technical or strategical limitations.…”

“…The discovery of autoantigens/autoantibodies happens roughly in three phases: [1] screening for specific autoantibody/autoantigen combinations in patients, [2] molecular identification and characterization of the autoantigen, and [3] characterization of the candidate autoantigen's immunogenicity and the corresponding autoantibody signatures. A common theme in the screening phase is testing the autoreactivity of circulating antibodies within bodily fluids [such as serum, cerebrospinal fluid (CSF) or synovial fluid] from patient cohorts against proteomes sourced from primary cell culture, tissue/ cell culture, tissue micro-dissection, or artificially generated peptide libraries/arrays.…”

The Use of Antibodies in Small-Molecule Drug Discovery

“…11 Antibody arrays have recently been reported in multiple different formats, ranging from commercially available planar arrays, through qualitative multiplexed bead-based arrays and array-based surface plasmon resonance systems (which enable rapid, label-free, high-throughput analysis in low sample volume), to the greatly enhanced sensitivity of nanostructured immunoassays. [12][13][14][15] These technologies have been successfully applied in identifying global protein changes associated with a range of diseases including cancer [16][17][18][19] and neurodegenerative disorders. 20 The detailed technical aspects of these methodologies including some of the different technological characteristics have been reviewed previously by Borrebaeck and Wingren.…”