Biomarker discovery via monoclonal antibody microarrays

Biomarkers have a broad range of applications, from the diagnosis, prognosis and monitoring of disease progression through to the monitoring of clinical responses to a therapeutic intervention and the development and delivery of personalised treatments.

In drug trials, biomarkers provide qualitative measures of biological effects that provide links between the mechanism of action and clinical effectiveness. This link can assist companies in evaluating the biological response to a novel biopharmaceutical at each stage of development. This ability is crucial as each phase of drug development requires analysis and critical ‘go/no go decisions’, regrettably still based today on a relatively limited suite of biomarkers.

While gene array analysis has transformed research, diagnostics and the wider healthcare industry, proteomics is considered the next step in the study of biological systems
This is because distinct genes are expressed in distinct cell types and this varies from time to time. While mRNA analysis was utilised to monitor these changes in the past, it is known that mRNA is not always translated into protein, and the amount of protein produced can depend on the physiological state of the cell. Also, post translational modification means that proteins are subjected to a wide variety of chemical modifications including phosphorylation and glycosylation. Not only can these alter a proteins function, but also its structure, so the use of specific monoclonal antibodies, developed without bias and applied in arrays can enable rapid, multiplexed analysis and enable biomarker discovery.

This approach for serum based protein biomarkers has many advantages, not least due to their relative ease of detection and minimally invasive sample collection.

However, the widespread use of multiplex arrays for biomarker discovery has been limited to date by the technology. With the range of abundance of proteins in blood covering about 12 orders of magnitude, with albumin by far the most abundant, devising a platform for protein biomarker discovery has been challenging.

One such development has been the proteomic profiling of human plasma by shotgun mass spectrometry. While undoubtedly a popular approach for the discovery of novel biomarkers, it suffers from limitations in reproducibility and sensitivity due to the wide dynamic range of protein concentration in plasma, often necessitating extensive upfront processing to remove abundant proteins.

Furthermore, mass spectrometry based approaches require a costly and extensive infrastructure and results are often further hampered by the lack of available immuno-affinity reagents.

A viable and economical alternative is the use of monoclonal antibody microarrays directed against natural human plasma proteins.

One such system is Randox’s Biochip Protein Profiling Array, utilising Randox’s award-winning Evidence Investigator analyser system and Biosystems International (BSI) QuantiPlasma™ antibody library.

The monoclonal antibodies utilised are raised against a mixture of native human serum proteins, offering the benefit of detecting these polypeptides in their native configuration, harbouring their numerous potential post-translational modifications and carrying the ‘imprints’ of their numerous interactions with other (macro) molecules including proteins.
This combination allows for precise proteome profiling for Biomarker discovery without the need for sample labelling.

The groundwork for this innovative process combines several key technologies including the chromatographic processing of human plasma, the high throughput generation of monoclonal antibodies (mAbs), and high throughput screening (HTS) to identify those antibodies which are suitable for further development for the production of microarrays and/or novel diagnostic products. A key advantage is that once successful in detection, the antibody(s) are available to identify the cognate antigen.

Monoclonal antibody library generation overview

Plasma Collection & normalisation → HT Hybridoma Cloning → mAb production & validation → Biochip microarray spotted


Biochip Technology Overview

Following protein and epitope redundancy analysis, the mAB’s are spotted onto a proprietary ceramic Biochip, a key component of the Protein Profiling Array. The Biochip is an activated ceramic support, onto which the monoclonal antibodies are precisely spotted into discrete test regions, which enables biomarker multiplexing from as little as 5µls of sample, providing a throughput of 1500 test results in under 3hrs.

Assay Protocol Overview

Plasma samples from clinical cohorts ready for profiling are added to the Biochip in the presence of labelled tracers (provided with the kit).
Following addition of the sample(s) under investigation, the mAB’s specifically bind to any target serum proteins, thereby reducing the binding of tracer in a standard inhibition assay, leading to a reduction in signal output. The sample’s signal is detected using HRP based chemiluminescence via a Charge Couple Device (CCD).

The samples are automatically quantified by the in-built software, enabling a comparison of plasma proteome profiles between the control and study cohorts resulting in the identification of mAB(s) that detect quantifiable differences between biologically different sample sets.

Data analysis

In-house testing utilising the Biochip Protein Profiler clearly shows that different plasma protein levels are distinguishable between normal and abnormal samples.

The illustration above shows percentage inhibition for two plasma samples (indicated by the green and red bar graphs) for a selection of just 5 of the mAB’s available on the array.
This graph clearly illustrates a number of antibodies where the inhibition is markedly different, indicating significant variation in plasma protein concentrations between the two samples under study as recognised by the specific mAB’s.

Once validated, these protein biomarkers could then be utilised in additional research and ultimately for diagnostic and theranostic applications; either in isolation or as part of a multi-biomarker (multiplex) array to further enhance specificity and sensitivity.

The first Randox Biochip Protein Profiler Array contains 300 mABs, each reacting to a different epitope of the human plasma proteome, enabling extensive proteomic analysis.

Given the urgent requirement for the identification of biomarkers and their adoption into the clinic, protein biomarkers are highly sought after. For pharma, their application as surrogate endpoints in clinical studies and as companion diagnostics has only raised their importance further, especially against the backdrop of a well publicised drug pipeline problem. Platforms such as the Biochip Protein Profiler Array are a major step forward in this regard and have the potential to revolutionise healthcare.