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Molecular & Genomic Testing
HIV Reservoir & Virology
Immunology
Sample Processing & Biobanking
Therapeutic Modalities
When an antigen-specific T-cell population is rare among PBMCs, bulk methods that average signal across the whole sample may return a null result for a response that is present. Low frequency immune responses of this kind are common in early vaccine studies, cell therapy monitoring, and chronic infection research. An ELISpot (Enzyme-Linked ImmunoSpot) assay addresses this by counting responding cells directly at the single cell level. Each cell that secretes the target cytokine produces a single spot on a capture membrane, and the result is reported as spot-forming units (SFU) per million input cells. If your program needs to know how many cells respond to an antigen, rather than how much cytokine a sample contains, ELISpot answers that question.
The ELISpot method measures the secretion of cytokines, chemokines, and other soluble proteins by individual immune cells. An enzymatic detection step turns each secreting cell into a visible spot on the ELISpot plate, so responses are counted cell by cell rather than averaged across the sample.
Capture Antibody and Membrane Surface
Detection Antibody and Enzymatic Reaction
Why direct counting is sensitive
How FluoroSpot and ELISpot testing work together
Each well of the ELISpot plate contains a membrane coated with a specific capture antibody against the target cytokine. Cells are placed in short-term cell culture on that membrane surface and stimulated ex vivo with peptide pools, viral antigens, tumor antigens, or novel immunogens. Antigen-presenting cells in the PBMC fraction, including monocytes and dendritic cells, present the antigen to T cells, and each responding cell releases cytokine that the capture antibody binds immediately around it.
After the cells are removed, a biotinylated detection antibody binds the captured cytokine, followed by streptavidin conjugated to an enzyme, commonly horseradish peroxidase or alkaline phosphatase. The enzymatic reaction with a precipitating substrate leaves a spot at the site of each secreting cell. An automated reader counts the spots, and the result is reported as SFU per million input cells.
Because the readout is direct enumeration rather than an amplified bulk signal, a small responding population is not diluted by the non-responding cells in the well. This makes ELISpot a sensitive readout for responses concentrated in a small subset, as in early vaccine trials, HIV reservoir studies, and CAR-T monitoring. ELISpot is also used in organ transplantation research to measure donor-reactive T-cell responses.
FluoroSpot applies the same capture principle but replaces enzymatic detection with fluorescent detection, allowing simultaneous detection of two or more secreted proteins using labels of different wavelengths. A cell that secretes both IFN-γ and IL-2 produces overlapping spots that the reader resolves, so polyfunctional cells are counted directly. ELISpot establishes how many cells respond, and FluoroSpot characterizes the functional breadth of those responders. Running FluoroSpot and ELISpot testing on matched samples gives both measures from one study. Our FluoroSpot services offer simultaneous measurement of several cytokines from the same cell and panels built around your study questions. Both platforms run in our CAP-accredited, CLIA-certified laboratory as analytically qualified assays.
Moving a method from a preclinical vendor to a clinical laboratory can mean requalifying it mid-program. We partner with the vivarium of your choice for mouse and non-human primate studies, then carry the work into analytically qualified testing for clinical trials in the same laboratory.
Splitting limited PBMCs across vendors consumes sample and separates datasets that need to be read together. We pair ELISpot with intracellular cytokine staining, immune phenotyping, T-cell proliferation, and dPCR, qPCR, or NGS from the same blood draw.
Accelevir is a 100% scientist-run laboratory. One team works alongside yours from protocol design to final report, so study designs can adapt as the biology evolves.
Antigens, cytokines, timepoints, and controls are chosen to answer what your program needs to know.
Our ELISpot services run as analytically qualified assays in our CAP-accredited, CLIA-certified laboratory in Baltimore, with qualification documentation available to support regulatory filings.
Accelevir began as a Johns Hopkins spinout focused on detecting the rare, intact HIV that persists in people on therapy, and nearly a decade of HIV reservoir work gave our immunologists extensive experience designing assays for rare signals.
We now apply that approach across clinical development in vaccine development, gene therapy, cell therapy, immunotherapy, and infectious disease programs.
ELISpot assay testing is a direct way to assess immune responses that are cell mediated. It answers a question bulk methods cannot: not whether cells produce cytokine in aggregate, but how many individual immune cells respond. Counting cells one at a time gives the assay high sensitivity for small responding populations, which is why it is widely used for immune monitoring.
For vaccine programs, ELISpot quantifies antigen-specific immune responses as the number of T cells a candidate generates, which may add information that total antibody titer alone does not capture. For cell therapy programs, it assesses T cell function, specifically whether engineered T cells retain the ability to secrete effector cytokines after expansion and cryopreservation. For infectious disease research, it distinguishes a broad but weak response from a focused, high-magnitude response to specific epitopes.
Intracellular cytokine staining (ICS) by multiparameter flow cytometry shows which cell subset is producing which cytokine, but it generally requires more cells per condition, which limits its use when responders are rare and sample is limited. When the question centers on frequency rather than phenotype, ELISpot is the appropriate readout.
Our ELISpot services, together with FluoroSpot, cover single-cell cytokine detection and immune monitoring across preclinical and clinical programs.
to target the cytokines or immune responses your study requires
for sensitive detection of individual responding cells
to support larger studies and clinical trials
for peptide pools, viral antigens, tumor antigens, or novel immunogens. Peptide pools can be commercially available or client-provided, and we can qualify custom panels.
for rare or precious PBMC samples, a common constraint in early-phase trials and rare disease programs
to measure functional expansion in response to antigen, as a companion readout to ELISpot
including PBMC isolation, cryopreservation, and secure storage from collection to analysis
across human, non-human primate (NHP), and murine models
with flow cytometry, cytokine multiplex panels, and molecular assays, so immune data can be read alongside vector genome persistence, transgene expression, or viral load
through qualification documentation available to support regulatory filings
ELISpot clinical testing supports vaccine development and immunotherapy programs across several trial designs. In Phase I vaccine trials, ELISpot quantifies the frequency of antigen-specific T cells responding to the immunogen, giving an early read on cellular immunogenicity before neutralizing antibody titers plateau. In vaccine efficacy studies, it can track cellular immunogenicity as a secondary or exploratory endpoint across timepoints. In therapeutic vaccine programs for cancer, HIV, and chronic viral infections, it measures whether the candidate generates detectable T-cell responses. For cell therapy programs, it assesses whether CAR-T or TCR-engineered cells retain cytokine secretion capacity after manufacturing and cryopreservation. In checkpoint inhibitor trials, it tracks tumor antigen-specific T-cell expansion during treatment.
We profile T cell, NK cell, and myeloid responses in preclinical and clinical studies using ICS, ELISpot, and cytokine multiplex assays. ICS shows phenotype and subset, ELISpot establishes absolute frequency, and multiplex cytokine panels capture the soluble environment. Each captures a different property of the same response, which is why programs often run them together.
T-cell readouts can also be run alongside our neutralizing antibody and anti-drug antibody assays. In vaccine programs, that pairs ELISpot and ICS immunogenicity data with neutralizing antibody data. In gene therapy programs, it pairs anti-capsid T-cell responses with anti-capsid antibody testing and pre-existing immunity screening.
Each ELISpot test moves from PBMC isolation to a background-subtracted SFU count:
PBMCs are isolated from fresh or cryopreserved whole blood, then rested, counted, and plated at a defined input onto membrane-bottom plates pre-coated with capture antibody.
Peptide pools spanning the target antigen, whole proteins, or viral lysates are added. Peptide pools can be commercially available or client-provided, and we can qualify custom panels.
A positive control and negative controls run in every assay to confirm cell responsiveness and establish background.
Plates incubate for a period matched to the secretion kinetics of the target cytokine.
Cells are removed. A biotinylated detection antibody binds the captured cytokine, then enzyme-conjugated streptavidin and a substrate convert each secreting cell into a visible spot.
An automated ELISpot reader enumerates spots, reported as SFU per million input cells with background subtracted.
T cell ELISpot assays quantify CD4⁺ and CD8⁺ T cells producing IFN-γ, TNF-α, IL-2, IL-4, IL-17, or other cytokines, with capability across human, NHP, and murine samples.
ImmunoSpot is used interchangeably with ELISpot (Enzyme-Linked ImmunoSpot), and sometimes for the broader family of spot-based assays that includes FluoroSpot. These highly sensitive immunoassays share one principle: single-cell enumeration of secreted proteins. An ImmunoSpot assay can detect cytokines such as IFN-γ, TNF-α, IL-2, IL-4, and IL-17 from CD4⁺ and CD8⁺ T cells, as well as granzyme B, perforin, antibody isotypes from B cells, chemokines, and other soluble proteins for which capture and detection antibodies are available. The choice of marker follows the biological question. Each ImmunoSpot configuration is analytically qualified to the sample type and program stage.
| Configuration | Where it is used |
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IFN-γ ELISpot
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The standard readout for antigen-specific T-cell responses in vaccine trials |
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IL-17 ELISpot
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Autoimmune and Th17-driven disease research
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Antibody-secreting cell (ASC) ELISpot
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Quantifies B-cell responses in humoral immunity studies
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Standard ELISpot counts cells secreting one cytokine. FluoroSpot counts cells co-secreting two or more (for example IFN-γ, TNF-α, and IL-2), identifying the subset with the broadest effector capacity. ICS shows which subset is polyfunctional; ELISpot and FluoroSpot quantify how many cells meet that definition and at what frequency.
Polyfunctional T-cell responses have been associated with immune control in HIV, where nonprogressors appear to maintain more highly functional HIV-specific CD8⁺ T cells than progressors (Betts et al., Blood, 2006), and with vaccine-mediated protection in preclinical models (Darrah et al., Nature Medicine, 2007). The relationship between polyfunctionality and outcome is context-dependent and not fully established, so polyfunctionality data are most informative alongside proliferation, cytotoxicity, and phenotyping readouts.
ELISpot assays are designed starting from the biological question: which cytokine, which antigen, which cell population, and at what stage of the program. The platform is then matched to the question and the sample available. Standard ELISpot fits questions about responder frequency, FluoroSpot fits studies where polyfunctionality is the endpoint, and low-input optimization applies when sample volume is constrained in preclinical studies or clinical trials.
Cell culture and stimulation conditions are qualified to the antigen and species. Peptide pools are titrated to find the concentration that maximizes antigen-specific signal without inducing nonspecific activation. Incubation time is matched to the kinetics of each cytokine, since different cytokines are secreted on different timescales after stimulation. Human, NHP, and murine assays use cross-reactive or species-specific antibodies as appropriate.
Our ELISpot assays run under documented standard operating procedures in our CAP-accredited, CLIA-certified laboratory. Plate layouts and acceptance criteria are defined before the first clinical sample is tested, and qualification documentation is available to support regulatory filings.
ELISpot counts cytokine-secreting cells. We combine it with ICS by multiparameter flow cytometry, which shows polyfunctionality by subset, and with multiplex cytokine and chemokine panels for plasma or culture supernatants. High-parameter immunophenotyping, from focused panels to deep profiling of immune subsets, adds context on which populations are present and responding.
In an AAV gene therapy study, for example, qPCR quantifies vector genomes in PBMCs, ELISpot measures anti-capsid T-cell responses, and a cytokine multiplex panel captures inflammatory signals. Read together, those datasets can help show whether an immune response may be limiting transgene expression.
In checkpoint inhibitor and cell therapy studies, these platforms can run at serial timepoints to show how the immune landscape shifts during treatment. ICS and phenotyping can also be combined with qPCR, dPCR, or NGS from the same sample, relating immune function to vector genome persistence, transgene expression, or viral load.
B cell ELISpot, also called antibody-secreting cell (ASC) ELISpot, quantifies B cells actively secreting immunoglobulin. The capture antibody is specific to an antibody isotype (IgG, IgA, IgM) or to the antigen itself, and results are reported as ASC per million input cells.
B-cell ELISpot is used in vaccine programs to measure the early antibody response before serum titers rise, in mucosal immunity studies to quantify IgA-secreting cells, and in autoimmune disease research to track antibody-producing B cells. Because it measures the response at the cellular level, it can reveal antigen-specific secreting cells that serum measurements may not yet reflect. Dual-color FluoroSpot can distinguish cells secreting IgG from those secreting IgA in the same well.
Protocols are qualified to the sample type and program question and run in our CAP-accredited, CLIA-certified laboratory, with qualification documentation available to support regulatory filings.
ELISpot counts cells that secrete the target cytokine during the in vitro stimulation window. That makes it a direct measure of responder frequency and cell function, but several properties fall outside what it reports:
It does not identify whether a responding cell is CD4⁺ or CD8⁺, memory or effector. ICS and phenotyping address that gap.
Spot size gives only a relative indication of how much cytokine each cell secretes. The primary readout is frequency.
Responses reflect secretion under defined ex vivo stimulation conditions and may not mirror activity in tissue.
Granzyme B or perforin secretion is a surrogate for cytotoxic potential, not a direct measure of target cell killing.
Results depend on cell viability and recovery, particularly after cryopreservation. Proper controls and consistent sample handling are part of every study design because reliable results depend on them.
ELISpot is performed on fresh or cryopreserved PBMCs. Whole blood is not used directly; PBMCs are isolated first. Total cell input depends on the number of antigens, replicates, and controls in the design, and our scientists scope requirements with you before the study begins. Low-input workflows can reduce requirements when sample is limited, although signal may decline at very low cell counts.
ELISpot and FluoroSpot assays supporting clinical trials are analytically qualified under our quality management system, with qualification documentation available to support regulatory filings. Preclinical and discovery-stage work can be run as research-use-only when the sponsor does not require a quality framework.
The three platforms answer different questions and are often run in parallel:
ELISpot: The primary question is how many cells respond, and responders may be rare
FluoroSpot: Polyfunctionality is the endpoint and you need to know how many cells secrete two or more cytokines
Flow cytometry (ICS): Phenotype matters more than frequency, or you need to know which subset (CD4⁺ vs. CD8⁺, memory vs. effector) is producing the cytokine
ELISpot counts individual cytokine-secreting cells and reports a frequency. Multiplex ELISA (enzyme-linked immunosorbent assay) measures total cytokine concentration in plasma or culture supernatant and reports pg/mL. ELISpot resolves rare responses at the cell level; multiplex ELISA is faster and needs less sample handling but cannot distinguish a few highly secreting cells from many low-secreting cells. For antigen-specific cellular responses in vaccine and cell therapy programs, ELISpot is the usual readout, while multiplex ELISA is used for systemic cytokine profiling and safety monitoring.
Bring your antigens, cohort or species, sample availability, endpoints, and the critical question for your next milestone to a scoping conversation with our scientists. We will recommend the readout or combination (ELISpot, FluoroSpot, ICS, or integrated molecular endpoints) and send an itemized proposal matched to your study design.
Accelevir Diagnostics, LLC is a CAP-accredited, CLIA-certified laboratory. Services are provided for research use in support of clinical and preclinical studies and are not intended for diagnostic, therapeutic, prognostic, or patient-management use.
References
Betts MR, et al. HIV nonprogressors preferentially maintain highly functional HIV-specific CD8⁺ T cells. Blood. 2006;107(12):4781–4789.
Darrah PA, et al. Multifunctional TH1 cells define a correlate of vaccine-mediated protection against Leishmania major. Nature Medicine. 2007;13:843–850.
