Extending sensitivity of biomarker quantitation immunoassays

Patricia Ahrweiler, Field Marketing Manager, Gyros Protein Technologies explains how its Gyrolab Bioaffy 4000 CD extends Gyrolab assay sensitivity to the low picogram level for improved analysis of biomarkers

Biomarker research has accelerated in recent years, driving the development of novel biomarker candidates and panels for the study of a range of diseases. A case in point is the cytokine storm that can contribute to the development of acute respiratory distress syndrome (ARDS) in patients suffering from COVID-19.

A nanoliter-scale approach to biomarker analysis

Current manual immunoassay methods have drawbacks of lengthy assay times, and limited dynamic range and sensitivity. Miniaturising and automating biomarker immunoassays on Gyrolab nanoliter-scale microfluidic platforms significantly reduces sample volume requirements and assay run time; utilising automation increases reproducibility through limiting manual interventions. Gyros Protein Technologies’ Gyrolab platform has been designed for biopharmaceutical applications to provide high-performance, robust and rapid immunoassays, with the compact disc (CD) at the heart of the ingenuity of the platform.

Gyrolab Bioaffy 4000 CD extends sensitivity of Gyrolab immunoassays down to the low picogram per milliliter (pg/mL) range, or a 2-6 fold increased sensitivity compared with the Bioaffy 1000 CD.

How to achieve multiple biomarker analysis: multiplex or singleplex?

A common approach to analysis of multiple biomarkers in one sample is by multiplexing. This approach can simplify analysis, but there are disadvantages to multiplexing immunoassays that makes singleplex an attractive alternative. One of the biggest obstacles, says Gyros Protein Technologies,  is the widespread level of cross-reactivity between antibody reagents that can lead to long assay development times. There are also challenges in measuring different analytes over widely different concentrations in the same reaction vessel, or failure of a single analyte assay requiring re-analysing the multiplex reaction or individual assays. These drawbacks suggest that it can be more productive to run singleplex assays that are time- and volume-efficient.

Gyroplex panels multiply efficient singleplex immunoassays

Gyrolab assays run in series on sequential CDs is an efficient strategy to generate more reliable biomarker data (Figure 1). This approach, called the Gyroplex panel, has a number of advantages:

  • Data reproducibility: No cross-talk or reagent interference since each data point is generated from a dedicated microstructure
  • Flexible assay setup: Well-optimised and individually tailored assays using in-house or proprietary reagents enable customisation
  • Productivity: If one assay fails, there is no need to rerun all assays as would be the case with multiplexing

Gyroplex panel allows each assay to be individually validated. In addition, the need for small sample volumes supports preclinical studies where serum sample volumes are limited.

Multiple CDs can be run overnight on Gyrolab xPand 5-CD system, which allows 5 biomarker assays to be measured in duplicate for 37 samples in one continuous run. For example, 2 x 5 CD runs, with one Gyrolab Bioaffy 4000 CD taking approximately 80 minutes to run enables 370 biomarker levels to be determined in 24 hours.

Application: Five cytokine Gyroplex panel performance

To illustrate the power of running Gyroplex panels using Gyrolab Bioaffy 4000 CD, five different cytokine biomarkers were analysed by running each assay on a dedicated CD in an overnight run on Gyrolab xPand system (Figure 2). The panel included five human cytokines involved in the TH1 response: IL-2, IL-6, IL-10, IFN-gamma and TNF-alpha.

The broad dynamic ranges of the individual assays in the 5-cytokine Gyroplex panel based on Gyrolab Bioaffy 4000 enabled the efficient determination of biomarker levels over 3 logs, from low pg/mL to over 1,000 pg/mL. The biomarker panel was run unattended overnight, using 12 µL per duplicate.

The introduction of Gyrolab Bioaffy 4000 CD extends Gyrolab assay sensitivity to the low picogram level for improved analysis of biomarkers and for use in pharmacokinetic assays where detection of low levels of antibody-based therapeutics is needed.

Gyroplex panels enable multiple biomarkers to be analysed using very low volumes of samples. Individual, optimised assays with no cross-talk between assays or samples enables the confident analysis of different analytes with widely different concentrations in the same run, optimised to the requirements of the biomarker assay. This approach provides flexibility during the development of biomarker panels and simplifies assay design and analysis, where multiple assay conditions can be used for different analytes with unique assay requirements.



Figure 1




Figure 2


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