The Evolution of Multiplex Immunoassay Technologies

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.

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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.

Setting New Quality Standards for Multiplex Immunoassays

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