Profiling Inflammation Biomarkers Using Proteomics Approaches

Inflammation is like a double-edged sword for the body. On the one hand, inflammation “serves as the natural defense mechanism that fights disease,” explains Vanitha Margan, Global Product Manager at Bio-Rad Laboratories. Conversely, an overreactive or attenuated immune response has been linked to a host of chronic diseases and acute infections such as cardiovascular disease (CVD) and COVID-19.^1,2 Chronic low-grade inflammation is also “getting more and more attention as an underlying factor to many diseases, such as dementia/Alzheimer’s, cancer, and other diseases that contribute to more than half of all deaths worldwide,” adds Katarina Hornaeus, Product Manager for Olink’s Flex and Focus product lines.

Since inflammation plays such a critical role in disease onset and outcomes, researchers need a way to elucidate this relationship further. Profiling the proteins participating in inflammation will help clinicians diagnose disease and manage the negative impacts that inflammation incurs. While previous technological developments have enabled researchers to measure protein biomarkers, emerging technologies have increased the throughput of these efforts. Developments in those technologies have, in turn, increased the discovery and quantification of novel and existing inflammation biomarkers.

Multiplex immunoassays

Initial efforts to isolate and quantify proteins began with enzyme-linked immunosorbent assays (ELISAs) that measure the activity of a reporter enzyme bound to a detection antibody.³ ELISAs require optimized test conditions, including target antibody, capture antibody selected, the instruments used to detect activity, and the buffer being used. Furthermore, ELISAs can only measure one analyte per reaction. To circumvent these issues, multiplex immunoassays such as the xMAP bead-based immunoassay use the principle of ELISA on polystyrene beads that are free to float in suspension, interacting throughout the sample. Margan notes that “the xMAP multiplex immunoassay technology uses distinctly color-coded beads as a substrate capturing analytes in solution and employs the fluorescent dye phycoerythrin for detection. The unique bead color code allows simultaneous detection of multiple immune system mediators from one sample in a single reaction well. Multiplex immunoassays provide many advantages over single-plex ELISAs, including higher throughput, shorter assay time, and reduced sample volume consumption.”

Multiplex immunoassays have helped identify pro-inflammatory cytokines implicated in the progression of multiple diseases such as depression, cancer, and infectious diseases.⁴ Even so, multiplex immunoassays have their own set of technical challenges that must be overcome to maximize their capabilities. Margan expands on this further: “End users should be aware of cross-reactivity in their multiplex immunoassays especially during their own multiplex assay development. This originates from two sources: the reagents and the samples. In reagent-driven cross-reactivity, a combination of antibody-antibody, antibody-antigen, or antigen-antigen binding among proteins results in a false positive signal or background noise. In sample-driven cross-reactivity, the clinical specimens may contain antibodies (endogenous) or sample collection additives (exogenous) that interfere with the multiplex immunoassay.” Although cross-reactivity can interfere with an immunoassay’s results, there are ways to mitigate these problems. Margen explains that “for reagent-driven cross-contamination, single-detection and single-antigen cross-reactivity studies can be used to evaluate and minimize cross-reactivity.” She also states that “proper sample clean-up procedures or blocking agents can help mitigate sample-driven cross-reactivity.”

Proximity extension assay

The technical limitations of existing multiplex immunoassays necessitate new technologies that improve sensitivity and specificity further for inflammation-based diagnostics. Among these technologies is the proximity extension assay (PEA). The immunoassay employs two matched antibodies, each labeled with unique oligonucleotides that can hybridize with each other. The double-stranded DNA barcode that forms from these oligonucleotides can then be quantified with qPCR or NGS since its abundance is proportional to the target reagent’s concentration.

The power of PEAs as a multiplex immunoassay lies in its increased sensitivity, specificity, and scalability. Hornaeus expands on the benefits of Olink’s technology further: “The increased sensitivity of the assay lies in generating a workflow that does not require sample pre-processing, pre-amplifies signals using PCR, and quantifying protein abundance using NGS- and qPCR-based techniques. We provide a range of options including an Explore platform for monitoring the abundance of thousands of proteins to the Target 48 platform that quantifies 48 proteins with qPCR and our lower plex customizable offerings with Flex and Focus. The Olink PEA technology also provides exceptional read-out specificity. The use of dual antibodies with coupled DNA-tags that have to be in close proximity (i.e., bind to the correct target protein) reduces the risk of false positive data. Finally, Olink’s PEA kits allow end users to measure everything from tens of proteins to over 3000 on almost all kinds of sample matrices while still maintaining high data quality. Our newest addition to the portfolio, Olink Flex, allows users to build their own protein biomarker panel by selecting up to 21 biomarkers from an inflammation focused library of over 200 pre-validated assays with a combinability of 99%. This makes our Olink library the largest and most flexible inflammation library available on the market today.”

The efficacy of PEAs has been demonstrated in R&D efforts to study inflammation in atopic dermatitis (AD), the most common inflammatory skin disease. In a 2020 study, a population of 20 individuals with moderate-to-severe AD and 28 healthy controls had their blood proteomes and skin transcriptomes compared.⁵ Using Olink’s multiplex assay, the researchers surveyed the levels of 354 protein inflammation markers to characterize inflammatory responses associated with AD. The study identified increased expression of multiple inflammatory markers such as matrix metalloproteinase 12 and other cardiovascular-associated proteins. Additionally, the transcriptome data corroborated the findings from the blood proteomes. Put together, PEAs act as an upgrade for the multiplex quantification of protein-based biomarkers.

Proteomics

Multiplex immunoassays and PEAs enable the high-throughput quantification of inflammatory biomarkers. Nevertheless, there may be novel proteins contributing to inflammation that remain uncharacterized. Proteomics aim to rectify this issue by characterizing the complete set of proteins present within a sample. Proteomics efforts have aided single-day workflows to identify inflammatory responses associated with severe COVID-19 disease.⁶ Proteomics has also been used to assess potential inflammatory biomarkers for neurodegenerative diseases such as multiple sclerosis. One such effort identified a coagulation factor—“Tissue factor”—and protein C inhibitor as proteins that drive pro-inflammatory signaling pathways and are observed in chronic plaque lesions.⁷ Future efforts to hone hypotheses for identifying novel inflammatory biomarkers will hence require a robust proteomics pipeline.

Conclusions

Inflammation plays a critical role in protecting the human body from various insults. However, aberrations in the inflammatory response place the host at risk of sickness and death. According to Hornaeus, “A deeper understanding of the underlying inflammatory pathways involved in specific diseases could lead to more efficient treatment options. If we could combine this information with biomarkers for early diagnosis, we would be able to give the patient the correct treatment early in their disease and thereby largely improve the outcome.” Although multiplex immunoassays provide a quantitative approach for measuring inflammatory proteins, other techniques such as PEAs heighten sensitivity and specificity for these analytes. Proteomics efforts have also generated additional leads in the study of novel pro- and anti-inflammatory pathways. With the wide array of technologies available for identifying and characterizing biomolecules, the future is bright for efforts seeking to study and harness inflammatory responses for patient well-being.

References

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7. Han MH, Hwang SI, Roy DB, et al. Proteomic analysis of active multiple sclerosis lesions reveals therapeutic targets. Nature. 2008;451(7182):1076-1081. doi:10.1038/nature06559