Validating ELISpot for clinical trials
Published: August 18, 2025
Updated: August 22, 2025
17 minute read
Authored by: Tyler Sandberg
ELISpot and FluoroSpot have been widely used in clinical trials for over two decades to support the development of novel vaccines, immunotherapies, and cell and gene therapies. At the time of writing, over 150 active clinical trials list ELISpot or FluoroSpot as an outcome measure to generate data supporting therapy success.
At Mabtech, we are proud to have contributed tools and expertise to over 400 clinical trial publications worldwide. In this post, we will cover essential considerations for successfully integrating ELISpot and FluoroSpot into clinical trials. While we are not regulatory validation experts and cannot account for specific regional requirements, we hope this guide serves as a strong starting point to help you navigate assay validation and implementation.
What are ELISpot and FluoroSpot?
Both ELISpot and FluoroSpot have been instrumental in characterizing immune responses across various clinical applications, from evaluating vaccine immunogenicity to assessing T cell persistence in CAR-T cell therapies. The high sensitivity of ELISpot and FluoroSpot makes them particularly valuable for detecting low-frequency antigen-specific cells, even in patient samples where immune responses might be weak or undetectable in other methods.
ELISpot has been trusted in clinical trials for over 20 years giving quality insights into immune responses elicited by novel therapies. (Paramithiotis et al., Frontiers in Immunology, 2023)
Why validate ELISpot and FluoroSpot for your clinical trial?
Just like any other bioanalytical method used in clinical research, ELISpot and FluoroSpot require a thorough validation to ensure they produce accurate and meaningful data. If these assays are included as primary or secondary endpoints in a clinical trial, assay validation is critical, as regulatory agencies often require evidence that the generated data is robust, reliable, and fit for its intended purpose.
A properly validated ELISpot or FluoroSpot assay can:
• Differentiate true responses from background noise (specificity)
• Provide consistent results within and across assay runs (precision)
• Detect immune responses across a defined dynamic range (sensitivity and limits of quantification)
• Remain consistent despite minor variations in protocols (robustness)
The goal of this white paper is to guide you through the key considerations for validating and implementing ELISpot and FluoroSpot in your next clinical trial. We will cover their applications across different study types, critical validation steps, and best practices for standardization and data acquisition. Our hope is to help you understand and navigate all aspects of the assay to help incorporate them into your research to get your vaccine, immunotherapy, or gene therapy approved.
Applications of ELISpot and FluoroSpot in trials
By now, we hope you have a clear understanding of how ELISpot and FluoroSpot can be powerful tools in clinical trials across various therapeutic areas. Before diving into validation procedures, let’s explore how these assays have been successfully used in different clinical trial settings, along with real-world case studies from published research.
Vaccine clinical trials
ELISpot and FluoroSpot are probably best known for their use in evaluating vaccine-elicited immune responses in basically all stages of vaccine research and development. These are the most common areas where the assays are used:
• Immunogenicity assessment: measure antigen-specific T cells and B cell responses to determine vaccine efficacy.
• Epitope mapping: identify specific peptide sequences that elicit the best T cell responses aiding in vaccine design.
• Correlates of protection: establish immune markers associated with long-term protection.
The COVID-19 pandemic brought a new spotlight on the efficacy of vaccines to combat global outbreaks. ELISpot and FluoroSpot were used by major pharmaceutical companies to evaluate effective vaccine formulations in development as well as immune monitoring during clinical trials. In the Phase I/II Pfizer-BioNTech COVID-19 mRNA vaccine trial, ELISpot Plus: Human IFN-γ was used with RBD peptide stimulation to measure and evaluate T cell responses post-vaccination. Together with intracellular cytokine staining results, it was found that the novel mRNA vaccines elicited robust CD4+ and CD8+ T cell responses in 95% and 76% of participants, respectively.
Wells from an ELISpot Plus: Human IFN-γ kit showing pre- and post-vaccination samples of trial participants receiving the COVID-19 mRNA vaccine. CEF and CEFT peptide pools were used as important positive controls to ensure cells were responsive to peptide stimulation. Sahin et al., Nature 2020.
Immunotherapy clinical trials
When evaluating novel immunotherapies, ELISpot and FluoroSpot can be excellent solutions for immune monitoring. The assays can be used in a number of ways:
• Monitoring immune activation: evaluate T cell responses post-immunotherapy administration.
• Predicting therapeutic efficacy: correlate immune responses with patient outcomes
• Detecting immune-related adverse events: identify unintended immune activation.
A recent publication from 2024 highlighted results from a Phase I clinical trial evaluating MVX-ONCO-1, a personalized immunotherapy. ELISpot Plus: Human IFN-γ was used to monitor T cell responses against irradiated autologous tumor cells. The trial results showed that 50% of patients surviving beyond six months had increased IFN-γ spot-forming units (SFUs) compared to baseline. These results demonstrate how ELISpot results can correlate with patient outcomes from novel immunotherapies.
Vernet et al., Cancer Res. Commun. 2024
Cell therapy clinical trials
Cell therapies have taken the oncology field by storm in recent years. These therapies harness the power of the patient’s own immune cells to combat and kill cancer and tumor cells. ELISpot and FluoroSpot are well suited to evaluate these novel therapies in multiple stages of development and in clinical trials:
• Evaluating CAR-T and TIL functionality and potency: measure cytokine release upon antigen encounter.
• Assessing persistence: monitor the longevity and activity of infused cell products.
• Detecting off-target effects: identify unintended immune responses that could affect safety.
A clinical trial published in 2018 used ELISpot results as an outcome in their results of evaluating TIL therapy for HPV-associated cancers. ELISpot Plus: Human IFN-γ was used to assess HPV-reactive T cell expansion pre- and post-TIL therapy by coculturing patient T cells with autologous HPV-oncoprotein loaded DCs. Pre-screening revealed minimal HPV-reactive T cells before the administration of the TIL therapy. One-month post-treatment, 8 out of 24 patients showed significant increases in HPV-specific T cells. The proof-of-concept clinical trial demonstrated the successful functionality of novel TIL therapies against epithelial cancers.
TIL functionality can be assessed by incubating the cell product with tumor-specific antigen peptides (TSA) and measuring relative analyte secretion in either ELISpot or FluoroSpot. We recommend our FluoroSpot Path: Human immunotherapy potency (3‑color).
Stevanovic et al., Clin Canc Res 2018
Gene therapy clinical trials
The introduction of gene therapy has changed the lives of thousands of patients worldwide and show great promise. The use of ELISpot and FluoroSpot in clinical trials have been useful screening tools to ensure the viral vector and transgene don’t elicit unwanted immune responses. The methods can be helpful for:
• Immune monitoring: detect T cell responses to viral vectors and transgene products.
• Assessing immunogenicity: evaluate immune-mediated clearance of gene therapy vectors.
• Guiding immunosuppressive strategies: determine if immunosuppressive interventions are needed.
An excellent Phase I-IIa clinical trial published their results in the New England Journal of Medicine in 2018, reporting their findings of an AAV vector-based hemophilia B gene therapy treatment. The study used IFN-γ ELISpot over time to screen patients receiving the AAV vector and coagulant factor gene for unwanted immunogenicity. T cells from patients were stimulated with both AAV- and transgene-derived stimuli to assess immunogenicity. To their delight, no patients developed significant cellular responses against the transgene product IX-R338L over the course of the trial.
ELISpot and FluoroSpot assays play a crucial role in immune monitoring across clinical trials, offering insights into vaccine responses, immunotherapy effectiveness, and the safety of cell and gene therapies. The ability to detect antigen-specific responses at a single-cell level makes these assays indispensable for trial success.
Immune cells can be isolated before and after AAV gene therapy to assess their immunogenicity using AAV or transgene-derive peptide pools. Additionally, innate responses to the AAV and transgene themselves are tested, specifically plasmacytoid dendritic cells (pDCs) and other innate drivers of IFN type I responses.
George et al., NEJM 2018
Validation considerations for ELISpot and FluoroSpot in clinical trials
To ensure that ELISpot and FluoroSpot assays generate reliable, reproducible, and regulatory-compliant data, they must undergo thorough validation before being used in clinical trials. Proper validation ensures the assay is fit for purpose and produces consistent, accurate results across different laboratories, operators, and conditions.
In this section, we will outline the key validation parameters, explain why each is important, and describe how to test them, ensuring your assay meets the necessary quality standards for clinical applications.
Regulatory guidelines for validation
There are numerous publications and guidelines out there for the validation of bioanalytical assays, and we’ve done our best to outline and summarize them here for you that are relevant to ELISpot and FluoroSpot.
• FDA and ICH Q2(R1) and Q14: provide general validation principles for bioanalytical methods.
• FDA Bioanalytical Method Validation (2018): defines precision, accuracy, specificity, and sensitivity for assays used in clinical trials.
• Global CRO Council (GCC) White Paper: offers specific validation recommendations for ELISpot and FluoroSpot assays.
We highly recommend you read these resources in their entirety when planning your validation studies. These are listed at the end of the white paper for your convenience.
ELISpot and FluoroSpot procedures
Before validation can begin, optimization of standard operating procedures (SOPs) for the assay and sample handling, as well as assessment of critical reagents and quality controls, are needed.
Sample preparation
Let’s start with sample preparation. Most variability reported in ELISpot and FluoroSpot assays are tied to improper sample preparation and handling. We’ve optimized these SOPs and share them on our website for download to make sure you get the best samples possible. But here are additional considerations to make when planning your clinical trial and validation study:
Sample collection:
• Time frame: <8 hours from blood draw
• Unprocessed blood in optimal shipping containers with temp >22°C, preferably near 30°C
PBMC isolation:
• Ficoll gradient centrifugation (SepMate tubes) is still considered standard for collecting high quality PBMCs.
• CPT tubes are an excellent alternative if collecting blood from multiple sites.
Counting and viability assessment:
• Exclude apoptotic cells from your counting with optimized cell counters.
• Make sure you have the appropriate cell numbers needed for the ELISpot or FluoroSpot included replicates.
Cryopreservation:
• Use an optimal freezing media and slow freezing procedures.
Thawing & resting:
• Overnight resting thawed PBMCs improve ELISpot and FluoroSpot performance.
• Use pre-tested serum to ensure low background spots in negative controls.
Assay procedure
Now, we will move on to establishing the assay protocol itself. Mabtech offers a large variety of ELISpot and FluoroSpot kits validated with real samples before making them available to customers. Our Plus, Pro, and Path kit formats include pre-coated plates, reducing plate-to-plate variability. These kits generally include everything you need with an already optimized protocol and are ready to use.
An important consideration in planning your experiment is your choice of stimuli. You, of course, need high-quality peptides from your antigen of investigation. These can be purchased from a wide range of providers, but be sure they use capping techniques to reduce the risk of unspecific peptides that could skew results. The choice of stimuli for positive controls is also important. Mitogens like PHA are quite popular, ensuring the assay is working as designed, but including positive control peptide pools also ensures that your cells are responsive to peptide formulations as well. We have a multitude of PepPools that are excellent as positive controls, like our recently launched PepPool: CEFRAS Global pools that include a large number of pathogen epitopes covering a wide range of HLA types from diverse ethnic backgrounds.
Attention CROs!
It’s critical to be involved with the trial’s sponsor as early as possible to establish quality cell handling SOPs to ensure ELISpot/FluoroSpot success
Plate reading
The final consideration to make before validation is plate reading. You want an instrument that is user-friendly, has low user-to-user variability, and, most importantly, is accurate. Mabtech’s IRIS 2 and ASTOR 2 fit the bill perfectly. Count settings can and should be optimized and saved for use throughout the entire study, ensuring quality results. An SOP for artifact removal should be established – or let our new automated artifact removal function do this for you. The instrument software should also generate user logs for improved traceability and limit user interference. What makes IRIS 2 and ASTOR 2 work so well is our patented RAWSpot algorithm that detects the spot center every single time. The algorithm is also what gives IRIS 2 and ASTOR 2 unrivaled upper limits of quantification.
With the optimized assay protocol and data acquisition in place, you’re ready for validation.
ELISpot and FluoroSpot validation
There are eight main validation parameters to consider for ELISpot and FluoroSpot. In this section, we’ll define each parameter, how to test them, and recommended CV cutoffs or acceptance criteria.
The eight main validation parameters to consider for ELISpot and FluoroSpot.
Precision
Precision measures the consistency of results within and between assay runs. Validating precision ensures the reproducibility and reliability of the ELISpot or FluoroSpot data. The Global CRO Council recommends a minimum of 10 donors used when testing precision with a %CV value <30% desirable. There are three parts of measuring precision:
Intra-assay precision
Goal: measure variability between replicate wells on the same plate
How to test: Plate samples in at least triplicates (bonus if low, medium, and high responders are included) in a single plate and run with established protocol and calculate the %CV between replicates for each condition.
Inter-assay precision
Goal: Measure variability across different assay runs performed on separate days and plates
How to test: Run the same samples on at least three different days and calculate %CV across runs.
Operator precision
Goal: Measure variability between different operators.
How to test: Have multiple operators run the same samples to assess variability and %CV between results.
Sensitivity (LOD and LLOQ)
There are two main parts to validating sensitivity in ELISpot and FluoroSpot. The limit of detection and the lower limit of quantification:
Limit of detection (LOD): the lowest number of spots that can be reliably distinguished from background noise. Responses below LOD cannot be detected.
How to determine LOD: run the assay with cells + assay medium-only wells in at least triplicates
i. The resulting SFU median x 2 = LOD
ii. SFU median x 3 can also be acceptable if very few spots are present
Well-optimized IFN-γ ELISpot SOPs usually have an LOD of 5-12 spots. Another method to establish and define a positive response is to use the DFR method available at rundfr.fredhutch.org
Lower limit of quantification (LLOQ): the smallest SFU per well that can be reliably quantified with acceptable accuracy and precision.
It’s distinct from the LOD as it emphasizes quantifiable results rather than mere detection
LLOQ is typically low in ELISpot as it describes the SFU below which CV increases above 25–30%.
How to determine LLOQ: utilize PBMC in medium only replicates for donors.
i. SFU replicates with CV > 25-30% = LLOQ (SFU below this are of low precision)
ii. A widely accepted 20-30 SFU for IFN-γ ELISpot
Limit of blank: an additional measure to include to test unspecific spots caused by reagents
How to test: add all reagents and no cells – LOB should ALWAYS be 0.
If you’re experiencing spots in blank wells, revising your protocol is advised. You can also check out our useful guides:
• 7 common ELISpot errors and how to avoid them
• 7 common errors in FluoroSpot and how to avoid them
Upper limit of quantification (ULOQ)
The ULOQ is ultimately determined by the ELISpot or FluoroSpot reader. It determines the upper limit where spot counting is reliable or how many SFUs an ELISpot reader can reliably count.
How to determine ULOQ: Use PBMCs from a high-responding donor with an optimal positive control stimuli – mitogen or PepPool: CEFRAS Global. Read these plates in an automated ELISpot reader and check reader limitations.
Advanced algorithms like RAWSpot in IRIS 2 and ASTOR 2 can handle higher spot densities than traditional image analysis systems.
Specificity
If the last 30 years have taught us anything about immunoassays, it’s that ELISpot (and now FluoroSpot) are extremely sensitive and specific assays. Specificity validates the assay’s response to the intended antigen of interest. So, it’s imperative to have high-quality antigens from a reputable supplier. What you’re really asking when testing specificity is if your experimental antigen elicits responses in naïve or healthy donors. Generally, you want to pre-test your antigen peptide pool in 20-30 naïve donors.
How to test specificity: Use sample PBMCs and test the following conditions (in replicates, of course):
• No antigen
• Antigens of interest
• Positive control peptide pool
• Negative control peptide pool
Robustness and ruggedness
Robustness measures how well the assay performs under small variations in conditions. In other words, it tests your SOPs and protocol’s allowed flexibility without affecting SFUs. A generally accepted CV is <30% for each parameter below.
How to test robustness: a better question is optimization considerations for your SOPs.
Incubation times
• Make sure to choose times for optimal kinetics of analyte secretion.
• In FluoroSpot, when assessing multiple analytes, choose the slower analyte’s recommended incubation time.
Development time in ELISpot
• Critical to determine the optimal time for substrate addition and spot formation. Find an optimal range that doesn’t affect SFUs.
• Added benefit of FluoroSpot since development step is excluded.
Cell resting periods
• If implemented, what ranges are acceptable to give proper results?
Make sure to understand the kinetics of the secreted analyte or analytes of interest when planning your ELISpot or FluoroSpot experiments. Some analytes don’t reach peak secretion until 48 or 72 hours after stimulation!
Linearity
Linearity can be a tricky concept to understand in ELISpot and FluoroSpot. You can stimulate increasing numbers of PBMCs with PHA to see that the response is truly linear, but this doesn’t tell you much about the linearity of the cells’ ability to respond to your experimental peptide.
Therefore, to test the linearity of your experimental setup, you want to assess if the responses are linear within the assay’s range – this depends on proper cell-to-cell contact. This is illustrated well in the book, ELISpot for Rookies by Sylvia Janetzki. Without proper cell-to-cell contact, T cells will not be able to generate a linear response. A widely accepted linear range for peptide stimulation in ELISpot and FluoroSpot is 200k-300k cells per well.
Körber et al., The Journal of Translational Medicine 2016
Adapted from Janetzki, ELISpot for Rookies 2016
Reagent stability
Reagent and sample stability has been covered earlier in establishing proper SOPs and protocols for cell handling as well as using quality reagents. These can be tested for validation and aim for a CV <30% between reagent batches and reference samples.
Reagents
• Quality mAb pairs – Mabtech’s 1-D1K + 7-B6-1 highly cited IFN-γ pair for ELISpot
• Antigen synthesis – look for high-quality peptide manufacturers
• Lot-to-lot variation testing for reagents and serum
Samples
• Cell quality: viability >90% preferred
• Triplicates are a must for quality ELISpot and FluoroSpot data
• Optimized SOPs for cell isolation and handling
Accuracy
Although not listed in the GCC white paper on validation parameters, accuracy is another important part to consider with your ELISpot and FluoroSpot. Accuracy measures the closeness of the observed results to the true value.
Results from previous proficiency panels organized by Immudex.
How to test accuracy: Participate in a proficiency panel – EQAPOL currently offers these types of testing.
1. Enroll.
2. Receive PBMCs, serum, antigens, and reagents from the organizer.
3. Run your ELISpot SOP compared to EQAPOL’s reagents.
4. Report results to the organizer.
5. Your results are compared with other participants, providing a relative accuracy measure.
Conclusions
This post has covered quite a lot of information, but we hope it helps guide you on your way to successful ELISpots and FluoroSpots! There are a number of additional resources that you can find to learn more about the assays, proper cell handling, and more from the following sources:
• Mabtech.com – Knowledge hub: Step-by-step guides, webinars, tutorials, White papers, and more.
• ELISpot Resource Group workshops – A consortium of Millipore, JPT, Zellnet Consulting, and Mabtech for half-day workshops covering everything you need to get your ELISpot up and running.
• Zellnet Consulting – Sylvia Janetzki offers expert consultation services for ELISpot/FluoroSpot implementation and validation support for CROs and pharmaceutical companies.
Also, sorry for coming with this so late, maybe you've already read everything, but if you want to hear our Product Manager Tyler Sandberg take you through the whole thing instead, please check out his webinar:
References
- Paramithiotis et al., Integrated antibody and cellular immunity monitoring are required for assessment of the long term protection that will be essential for effective next generation vaccine development, Frontiers in Immunology 2024
- Vernet et al., A First-in-Human Phase I Clinical Study with MVX-ONCO-1, a Personalized Active Immunotherapy, in Patients with Advanced Solid Tumors, Cancer Research Communications 2024
- Stevanovic et al., A phase II study of tumor-infiltrating lymphocyte therapy for human papillomavirus-associated epithelial cancers, Clin Cancer Res. 2019
- Gorovits et al., Evaluation of Cellular Immune Response to Adeno‐Associated Virus‐Based Gene Therapy, The AAPS Journal 2023
- George et al., Hemophilia B Gene Therapy with a High-Specific-Activity Factor IX Variant, N Engl J Med 2018
- Janetzki, Important Considerations for ELISpot Validation, The Handbook of ELISpot 4th Edition 2024
- Islam et al., Recommendations on ELISpot assay validation by the GCC, Bioanalysis 2022
- Maecker et al., Precision and linearity targets for validation of an IFNγ ELISPOT, cytokine flow cytometry, and tetramer assay using CMV peptides, BMC Immunology 2008
- Körber et al., Validation of an IFNγ/IL2 FluoroSpot assay for clinical trial monitoring, J Transl Med 2016