The Role of Immunogenicity in Disease & Therapy Response: A Comparative Analysis of Assessment Methods

Christine Bain, Ph.D. September 18, 2025

What is immunogenicity?

Immunogenicity refers to the ability of a substance, such as a New Biological Entity (NBE) or a vaccine, to provoke an immune response when administered in the body. The recognition of the substance as foreign triggers an immune response, which involves the production of antibodies and the activation of immune cells to fight against or neutralize the perceived threat. Immunogenicity can be either desired, in the case of vaccination for instance, or unwanted when an immune reaction develops against a therapeutic antibody or a replacement therapy that can be harmful to the patient.

Why is Immunogenicity Data Important?

In both settings, immunogenicity data will help compare a compound to a placebo or standard-of-care, different populations of patients, different doses, and dose regimens, and eventually help select the safest candidate, dose, and regimen.

Technological Platforms for Measuring Immunogenicity Biomarkers

To assess the immunogenicity of a drug, one can rely on various technologies that include multiplex immunoassays/Ligand Binding Assays (LBA), multiparametric flow cytometry, ELISpot, and hybrid LC-MS/MS for analysis at the protein level, and qPCR or RNAseq for analysis at the transcript level. These assays can each provide specific and/or complementary information on the immunogenicity of a compound, based on different read-outs such as the production of cytokines or cytotoxic mediators, cell proliferation, and activation/exhaustion status.

Herein, we will focus on the comparison of platforms that can measure protein biomarkers in the cell-based immunogenicity setting, with serology testing not being addressed.

How Can Protein Biomarkers Be Measured?

Measuring Soluble Mediators

Cytokines, or more globally, immune mediators, can be measured in different ways. Profiling of soluble mediators can be done in supernatants from PBMC that have been stimulated with the appropriate antigen. Supernatants can then be analyzed on multiple immunoassay/LBA platforms such as Meso Scale Discovery® (MSD) ECL (ElectroChemiLuminescence), Luminex™, Ella™ Automated Immunoassay System, HD-X ™ analyzer, ARGO™ HT/NULISA^TM, or classical ELISA. The choice of platform typically depends on assay sensitivity, multiplexing capacity, range of detectable analytes, and the volume of sample available. These immunoassays provide information on the levels of soluble biomarkers (cytokines, chemokines, growth factors…) secreted by stimulated cells, but not on which type of cells are producing which biomarker.

Quantifying Antigen-Reactive Cells

Technologies such as flow cytometry or ELISpot allow quantification of antigen-specific cells, with only multiparametric flow cytometry being able to provide information on the antigen-reactive cell type [1,2]. Cell-surface markers used to characterize the responding cell subsets (e.g., CD4, CD8 T cells), their differentiation (e.g. Effector, Naïve, effector memory, central memory) and activation status (e.g. expression of CD137, CD154, CD25, CD69….) and even their specificity (e.g. Multimer/Tetramer staining), can be combined with intracellular markers. These will more deeply depict the functional profile of antigen-reactive cells, i.e. their capacity to proliferate (expression of Ki-67, dilution of fluorescent labeled cells), produce cytokines (e.g. IFN-γ, TNF-α, IL-2, IL-4, IL-17A) or lyse target cells (e.g. Granzyme B, perforin, CD107a/b).

Flow Cytometry vs. ELISpot: Which to Choose?

While polychromatic flow cytometry is a powerful tool for measuring multiple markers at the single-cell level, its sensitivity is limited by the number of events that can be acquired and the staining background. Conversely, ELISpot offers high sensitivity, making it the assay of choice for antigens known to be poorly immunogenic, such as in chronic diseases.

Choosing the Right Technology

Ultimately, choosing the right technology involves a good knowledge of the disease, the immunopathological processes, the mechanism of action of the therapeutic compound, that is the context-of-use of the biomarkers to be analyzed. Being able to implement most of these methodologies in a high-quality environment, we can comprehensively explore the immunogenicity of your substance and help you understand its efficacy, safety, and potential for triggering immune responses in individuals.

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