Bioassay “Bake-off” to Assess Ability to Quantify Inflammatory Signatures in Blood for Brain Disorders

Bioassay “Bake-off” to Assess Ability to Quantify Inflammatory Signatures in Blood for Brain Disorders

Cross-platform comparison of highly sensitive immunoassay technologies for cytokine markers

The Challenge:

The role of inflammation and the immune system across neuropsychiatric and neurodegenerative diseases is important to understand, and depends on an ability to measure, with precision, cytokines in the various fluids including blood, cerebro-spinal fluid (CSF), and saliva.

What are cytokines?
Cytokines are a large group of proteins, peptides or glycoproteins that are secreted by specific cells of immune system and serve as signaling molecules to mediate and regulate the system.


Characterizing distinct cytokine profiles in brain disease may provide an objective measure, or biomarker, for disease diagnosis or understanding likely disease progression. To enable the discovery and validation of cytokine-based biomarkers, research must utilize reproducible, accurate, quantitative and highly sensitive assays.

Quote from CSO Dr. Andreas Jeromin

This is challenging given that cytokine production is tightly regulated by the immune system such that cytokines are typically at very low levels in human blood and require highly sensitive technologies to be detected. For researchers engaged in biomarker research, this is a critical consideration. Meta-analyses comparing findings from many research studies found that the type of assay used, amongst other variables, accounts for discrepant findings across studies. Because a reference “gold standard” does not exist for accurately measuring inflammatory markers, cross-platform comparisons are necessary to understand platform performance characteristics. In addition to assay sensitivity and precision, other performance parameters include assay performance between repeat runs and over time, differences in real-world application across multiple research sites, and practical concerns, such as cost and ease of running the assay.

Given these complexities, results from the immunoassay “bake-off” will be important for enabling researchers to identify the optimal assay and platform based on the cytokine of interest, with the understanding that these technologies will continue to evolve over time.


Evaluate the performance of five leading immunoassay platforms to inform the selection of immunoassays in the design of research studies and the interpretation of results and improve the consistency of findings across research studies.

CVB Approach:

CVB’s Research Alliance for PTSD/TBI Innovation and Development Diagnostics (RAPID-Dx) Program performed a comprehensive evaluation of five leading platforms to understand their relative and absolute performance to accurately detect cytokines of interest. Our study included blood-based samples from healthy subjects and individuals with a traumatic brain injury, comorbid Post-traumatic Stress Disorder (PTSD)/Traumatic Brain Injury (TBI) or a neurodegenerative condition, Parkinson’s Disease (PD) to understand variation in clinical populations.

Results from this study, conducted in collaboration with the Michael J. Fox Foundation, were recently published in Cytokine X

Blood sample collectionHighlights:
  • Cross-platform comparisons were conducted across five leading immunoassay platforms.
  • Plasma and serum were obtained from healthy controls and clinical populations.
  • Analytic parameters included sensitivity, precision, and performance correlation.
  • Platform performance was highly variable, particularly for low-abundant cytokines.
  • Findings highlight what metrics should be taken into account in selecting immunoassays in future research.
Next Steps:

The RAPID-Dx program will continue to conduct additional cross-platform comparisons that inform biomarker discovery efforts. In our next phase we intend to evaluate metabolomic and transcriptomic platforms.


This work was supported primarily by Cohen Veterans Bioscience and generous donations from Steven A. Cohen for the RAPID-DX program. Additional funding was provided by the Michael J. Fox Foundation (MJFF).

Cohen Veterans Bioscience would also like to acknowledge the MJFF for its contribution of Parkinson’s disease samples. Parkinson’s disease data used in the preparation of this article were obtained from the MJFF-sponsored LRRK2 Cohort Consortium (LCC). For up-to-date information on the study, visit The LRRK2 Cohort Consortium is coordinated and funded by The Michael J. Fox Foundation for Parkinson’s Research. In addition, PTSD biospecimens were donated from the longitudinal cohort study of the Translational Research Center for TBI and Stress Disorders (TRACTS), a VA Rehabilitation Research and Development Traumatic Brain Injury Center of Excellence (B3001-C).

We are immensely grateful to the Indiana University Genetics Biobank for their expertise in the processing and plating of all samples as well as the technology vendors for participating in this cross-platform comparison and providing their technical expertise in processing immunoassays. 

What is a Bake-Off?
Scientifically, a bake-off refers to a rigorous approach for objectively evaluating and comparing tools, platforms or methods so as to objectively establish best practices in research and inform the selection of optimal technology for future use. In some cases, a single tool, platform or method may emerge as the gold standard. In other cases, aspects of tools, platforms or methods may be combined for optimal use in research or clinical medicine.

How do we conduct our Blinded Bake-off of Assays?
We applied the same principles to compare the quality of different technologies for detecting molecules of interest (bioassays).

What is an immunoassay?
An immunoassay is a biochemical test that measures the presence or concentration of a molecule in a solution (e.g., blood) by using an antibody or an antigen. The molecule detected by the immunoassay is often referred to as an “analyte” and is a protein in many cases. Analytes in biological liquids such as serum or urine are frequently measured using immunoassays for medical and research purposes.

What is metabolomics?
Metabolomics is the systematic study of small molecules – or metabolites – that are present in a cell, tissue, or biofluid and are produced due to cellular processes or in response to an environmental stress. The metabolome is the total complement of metabolites present in a biological sample under given genetic, nutritional or environmental conditions.

What is transcriptomics?
Transcriptomics refers to the study of the transcriptome, or the set of all RNA transcripts, in an individual or a population of cells. Because RNA sequences mirror the sequence of the DNA from which it was transcribed, analyzing the transcriptome enables researchers to determine when and where each gene is turned on or off in the cells and tissues of an organism. The term can refer to all RNAs, including coding and non-coding RNA, although some studies focus on messenger (m)RNA, which only reflects genes that are being actively expressed at a given time or situation.

What is systems biology?
As defined by the National Institutes of Health, “systems biology” is an approach in biomedical research to understanding the larger picture—be it at the level of the organism, tissue, or cell—by putting its pieces together. This field of study focuses on complex interactions within biological systems, using a holistic approach to biological research.