Overcome the Challenges of Multiplex Immunoassays

Immunoassays in Clinical Research Biomarkers are fundamental to accelerate clinical research and drug development. They broadly fall into two categories: those linked to disease (such as susceptibility, diagnostic, prognostic or disease activity biomarkers) and those associated with drug action (such as predictive, pharmacodynamic, safety or efficacy biomarkers). Among these, proteins are especially valuable due to their central role in all biological functions and direct involvement in disease mechanisms. The importance of proteins as biomarkers highlights the need for methods that can accurately and precisely quantify them. Immunoassays are commonly used for their accessibility and versatility, ranging from traditional methods like enzyme-linked immunosorbent assay (ELISA) to more advanced technologies. They have known drawbacks, however, including shortcomings on dynamic range, analytical sensitivity, and target specificity. Despite these challenges, immunoassay methods are constantly evolving, with ongoing efforts to standardize and improve quality, so that a complete realization of the great potential of protein biomarkers in clinical research can be achieved. Advantages of a multi-analyte approach There are limitations associated with traditional immunoassays that hinder progression from single biomarkers to biomarker signatures consisting of multiple analytes. Several innovations in multiplex immunoassay formats are fortunately changing this landscape. Measuring multiple analytes from a single sample simplifies the identification, validation and implementation of protein signatures. A biomarker signature not only offers a more holistic approach to understanding diseases, but also potentially increases discrimination power of statistical models. Additionally, multiplex immunoassays offer practical benefits, such as a reduction in assay costs, time to results, and sample volume requirements. Diseases are often the result of a complex network of biological changes, not just a single pathological process. Consequently, protein signatures provide a more nuanced and comprehensive understanding of the disease state and treatment response. Chapter 1 Multiplexing: Challenges and Opportunities 4 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. Chapter 1 – Multiplexing: Challenges and Opportunities Technological advances that enable multiplexing In recent years, we have witnessed a series of technological advancements that progressively unlocked the potential of multiplex protein analysis. Refer to the timeline below for key events. Among critical developments are the creation of planar (electro)chemiluminescence and bead-based immunocapture platforms. These technologies have been extensively applied and have successfully demonstrated the value of multiplexing to more efficiently identify protein signatures with biological and clinical relevance. However, with time it became evident that there are still technical challenges to be addressed to make multiplexing a tool that researchers can confidently rely on. 5 Chapter 1 – Multiplexing: Challenges and Opportunities Common Challenges in Current Methods Limited dynamic range Immunoassay panels need to measure target analytes with compatible abundance. If not, dilution of the samples for high-concentration analytes lead to undetectability of low-level analytes. Poor sensitivity of multiplex assays with higher plex grade also limits the dynamic range. Lack of quality controls Quality control materials are traditionally not well developed for multiplex immunoassays. Ensuring the accuracy, reliability, and reproducibility of multiplex immunoassays requires stringent quality control measures at various stages of the assay process. Complex data analysis Multiplex immunoassays produce complex datasets that require advanced software capabilities for accurate interpretation of data and for close monitoring of data quality. Laborious validation More steps in the analytical validation are required to ensure that quantification of each analyte in the presence of all other analytes is accurate. Cross-reactivity Non-specific binding of antibodies to the wrong antigen is more likely to happen with increasing antibody pairs present in the assay. Cross-reactivity impairs assay specificity and can give false-positive results. Signal interference Fluorescence-based readouts may have overlapping emission spectra that interfere with results. Light-based readouts may have light bleeding to adjacent spots and wells. Low sensitivity It is very difficult to maintain high sensitivity with increasing plex grade in an assay. Higher cross-reactivity and signal interference cause background noise, deteriorating ability to distinguish low abundance proteins from background. High variability When scaling up, issues like poor specificity, low sensitivity and signal interference increase. These may cause results to vary from run to run and deteriorate reproducibility of results. Multiplex Immunoassays Challenges associated with multiplexing immunoassays Although each multiplex immunoassay platform has its unique strengths and limitations, there are some common challenges that cause users to be unable or reluctant to implement panels with higher plex grade. Refer to the image above for details. While the illustrated challenges in multiplex protein biomarker detection seem daunting, they can be overcome. In fact, the next generation of multiplex immunoassays address these very issues. The new solutions aim to bring high confidence to multiplex analysis by increasing specificity and sensitivity, expanding the dynamic range, and simplifying the processes of quality control, data normalization and analysis. In the following section, we will explore the last innovation mentioned in our timeline of multiplex advances: the proximity extension assay (PEA) technology. 6 Pre-Readout steps in PEA Binding of antibody pairs, which are labeled with complementary DNA oligonucleotides, to the target antigen in the solution. A Oligonucleotides that come into proximity are hybridized and extended with a DNA polymerase. B The newly created DNA barcode is then amplified through PCR, making it ready for readout through NGS or qPCR methods. C Chapter 1 – Multiplexing: Challenges and Opportunities White Paper View article here Proximity extension assay (PEA) technology PEA is an innovative method based on a dual-recognition immunoassay, where two matched antibodies labelled with unique DNA oligonucleotides simultaneously bind to the target analyte in solution. When the two antibodies are brought into proximity their complementary DNA oligonucleotide hybridize, serving as template for a DNA polymerase-dependent extension step. This creates a double-stranded DNA barcode which is unique for the specific antigen and quantitatively proportional to the initial concentration of target protein. PEA technology combines antibody- and DNA-based methodologies to provide a unique immunoassay that delivers unwavering performance regardless of the number of targets that are being multiplexed.