Olink proteomics FAQ: Platform, applications & comparisons

Olink® Reveal data is now verified for readout on MGI T1+ and G400 sequencers, demonstrating consistent performance with Illumina systems and expanding access to high-quality, NGS-based proteomics.

The identification and analysis of protein biomarker signatures play a critical role in precision medicine. Recent years have marked a significant leap forward in this domain, thanks to significant technological advancements in multiplex protein analysis. This blog post explores these developments, highlighting key technologies that have transformed our approach to protein measurements including bead-based immunoassay and electrochemiluminescence (ECL), their critical role in cytokine profiling, biomarker panels, and the challenges that persist in optimizing specificity and sensitivity. Multiplex Immunoassay vs. ELISA: What’s the Difference? Before we explore the advancements, let’s clarify a common question: How does a multiplex immunoassay differ from the traditional ELISA (Enzyme-Linked Immunosorbent Assay)? ELISA is a widely used immunoassay method for detecting and quantifying substances such as proteins, using a single target per assay. Its specificity and sensitivity have made it a benchmark in immunoassay platforms for clinical diagnostics. However, ELISA is limited by its ability to analyze only one biomarker per sample, which can be time-consuming and sample-intensive for comprehensive biomarker profiling. Multiplex Immunoassay, on the other hand, enables measurement of multiple targets simultaneously within a single sample. Techniques like bead-based immunoassay and electrochemiluminescence (ECL) were useful for the efficient study of complex biomarker signatures and cytokine profiling. This method conserves sample volume and significantly reduces the time and cost associated with biomarker analysis. Key Technologies in Multiplex Immunoassays Bead-Based Immunoassay: This technique utilizes color-coded microspheres (beads) linked to distinct fluorophores, enabling the quantification of hundreds of distinct proteins. It is pivotal for protocols for multiplex immunoassay, especially in cytokine profiling and biomarker panel analysis. Electrochemiluminescence (ECL): ECL-based multiplex immunoassays employ an array of analyte-specific capture antibodies on a solid matrix. These assays are chosen for high specificity and sensitivity. Microfluidics Cartridge-Based Assays: Leveraging microfluidic devices, these assays separate and detect multiple proteins in a single sample. Innovations in immunoassay multiplexing 1980 – 1990’s Flourescent-based Light-absorbing reactions to quantify protein concentrations. Small degree of multiplexing can be achieved by using distinct colors of flourescence for different proteins. 1990’s Chemiluminescent-based Light-producing reactions to quantify protein concentrations. Some degree of multiplexing can be achieved by using distinct chemiluminescent tags or physical separation of reaction. 2000’s Bead-based Color-coded microspheres to allow for quantification of hundreds of distinct proteins in a single sample. Electrochemiluminescence-based Electric current to stimulate a light-producing reaction that is proportional to the protein concentration. Multiplexing of up to ten proteins can be achieved by using patterned arrays where each spot on the array is coated with a different capture antibody. 2010’s Microfluidics cartridge-based Microfluidic devices to separate and detect multiple proteins in a single sample. Degree of multiplexing is lower than bead-based assays. Proximity Extension Assay (PEA) Pairs of antibodies labeled with DNA oligonucleotides, which upon target binding and hybridization form a new DNA molecule by proximity extension. The resulting DNA barcode is then quantified using qPCR or NGS, allowing for highly multiplexed and sensitive protein measurements. The Imperative for a Higher Degree of Multiplexing The highlighted multiplex technologies have been instrumental in allowing simultaneous measurement of multiple target analytes. However, it needs to be acknowledged that there is a growing demand for technologies capable of achieving even greater levels of multiplexing. Such advancements are essential for comprehensive biomarker signatures, understanding disease mechanisms and propelling the field of precision medicine. However, as we push the boundaries of multiplexing, the performance of these assays — in terms of specificity, sensitivity, and accuracy — becomes increasingly critical. Inaccuracies or lack of assay specificity and sensitivity can lead to misinterpretations of data, impacting research findings. What is the solution to multiplexing with optimal performance? Among the various technologies available, the Proximity Extension Assay (PEA) combines antibody- and DNA-based methodologies to provide a unique immunoassay that delivers unwavering performance regardless of the number of analytes that are being multiplexed. A critical differentiator of PEA as a tool for biomarker discovery and validation is the ability to quality control all steps of the reaction in each individual sample, and the ability to extract data from only 1 μL of sample. For researchers interested in clinical diagnostics and therapeutic development facilitated by multiplexing technologies or interested in benchmarking immunoassay platforms, downloading our detailed ebook will provide you with a wealth of information. This includes challenges and opportunities in multiplexing, study planning and experimental design, data analysis and interpretation and case studies to uncover biomarker signatures with potential clinical utility.

Multiplex immunoassay technologies enable the simultaneous detection of multiple target analytes in the same sample and assay workflow. These powerful methods support cytokine profiling or the development of protein biomarker panels for the advancement of precision medicine. Furthermore, one can expect multiplexing increases analytical efficiency, cost-effectiveness, and reduces sample volume requirements. The transition from the traditional analysis of single biomarkers to more comprehensive biomarker signatures brings a more holistic approach to disease understanding and improves the discrimination power of statistical models. How to ensure data quality when multiplexing? Leveraging the full potential of multiplex immunoassays in biomarker research is not completely straightforward. Researchers may encounter challenges with cross-reactivity, signal interference, low sensitivity, high variability, limited dynamic range and other issues that affect the assay’s performance and reliability. Addressing these challenges is crucial for harnessing the capabilities of multiplex immunoassays in biomarker panel development and cytokine profiling. Key strategies include the careful selection of highly specific capture and detection antibodies to reduce cross-reactivity, and the optimization of assay conditions to minimize signal interference and variability. Leverage the full potential of Multiplex Immunoassays in Biomarker Research This Ebook offers a detailed exploration of the main challenges in multiplexing immunoassays and how to bypass them by using the Proximity Extension Assay (PEA) – a technology that sets a new quality standard by effectively addressing the challenges commonly encountered. PEA technology has the revolutionary capability to measure multiple target analytes in just 1 μL of sample. The Ebook also addresses practical tips on study design, data analysis, and interpretation. Table of contents: Chapter 1: Multiplexing: Challenges and Opportunities Chapter 2: Study Planning and Experimental Design Chapter 3: Data Analysis and Interpretation Chapter 4: Case Studies: Robust Biomarker Signatures with Potential Clinical Utility Main challenges in multiplex immunoassays Cross-Reactivity Cross-reactivity represents a significant hurdle, where capture and detection antibodies may bind to non-target antigens, leading to false-positive results. Minimizing cross-reactivity is essential for ensuring the assay’s specificity and accuracy. Signal Interference Signal interference, arising from the assay’s components, readout measures or the sample itself, can obscure the detection of target analytes, compromising the assay’s sensitivity and overall performance. Low Sensitivity The challenge of low sensitivity is particularly acute when detecting low-abundance biomarkers, making it difficult to identify crucial, yet scarce, analytes in complex samples. High Variability High variability in assay results undermines the reproducibility and reliability of multiplex immunoassays. Limited Dynamic Range The analytes combined in a single assay can span a wide range of concentrations. This variability in concentration of each analyte, coupled with differing dilution requirements, narrows the overall dynamic range available for the assay. Consequently, this limitation complicates the simultaneous analysis of samples with highly variable biomarker levels. Lack of Appropriate Quality Controls The inherent complexity of multiplex assays calls for robust quality controls to validate and ensure the reliability of results. Complex Data Analysis The vast amount of information generated from a single multiplex immunoassay requires sophisticated statistical methods to accurately interpret the findings. Laborious Validation Extensive validation is necessary to confirm the specificity, sensitivity, and reproducibility of multiplex immunoassays, demanding significant time and resources.

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