The Enzyme-Linked Immunosorbent Spot (ELISpot) is a common method for monitoring cellular immune responses to antigen stimulation. The general setup consists of a 96-well microtiter plate (strip or solid format), with a PVDF membrane at the bottom of each well. A capture antibody is immobilized onto the membrane to bind cytokines secreted by the antigen-stimulated cells. After applying an enzyme-conjugated detection antibody and following an enzymatic staining reaction, spots are revealed whereby one spot represents the unique cellular activity of antigen-specific cytokine-secreting cells at a single-cell level.

ELISpot step by step

Membrane coating with capture antibodies

Capture antibodies (yellow mAb) specific for the cytokine of interest are immobilized on the PVDF membrane at the bottom of each well (grey well).

Cell stimulation followed by cytokine secretion

A defined number of isolated cells (e.g. PBMCs, blue dot) is added to each well and incubated with selected stimulants (red structure). This leads to the secretion of cytokines (dark red dots) by antigen-specific reactive cells.

Cytokines bound by capture antibodies

Secreted cytokines are immediately captured by specific antibodies.Cells are eventually removed and  membranes are washed several times to eliminate unbound cytokines.

Binding of detection antibodies

A specific detection antibody (red mAb) is added, either directly conjugated to an enzyme (one-step detection, as shown in the picture, green dot) or biotinylated, requiring the addition of a biotin-specific enzyme-conjugated polypeptide (two-step detection, not shown).

Enzymatic precipitation of substrate

A soluble substrate (A) is added to each well. The substrate is then converted to a colored precipitating product (B) by the Ab-conjugated enzyme (green dot).Thereby, visible spots are generated on the surface of the membrane, each representing the release of cytokines by one single cell.

Read-out of spots

A calibrated ELISpot reader or a microscope is used to quantify the spots (grey dots). According to this read-out step, the number of cytokine-secreting cells is calculated.


ELISpot experimental outline

Day 1

Plate setup

  • Choose an ELISpot plate format (96-well solid or 12 x 8-well strip)
  • Configure your setup (Lophius' plate template) and include appropriate controls.

PBMC isolation


  • Stimulation of isolated PBMC in each well with the antigens of your choice
  • Use Lophius' T-activated® proteins for enhanced stimulatory properties of proteins


Day 2

Washing procedure and detection


Drying procedure

(After drying, plates can be stored at room temperature for several weeks in the dark before analysis) 


Why using an ELISpot assay?

The ELISpot assay measures the functionality of antigen-reactive cells and determines the frequency of cytokine-secreting cells at the single-cell level. This technology is one of the most sensitive, specific and reproducible available today to quantify particularly low frequencies of cellular activity, roughly down to one in a million cells.


Lophius' ELISpot solutions

Diagnostic ELISpot kit

T-Track CMV®

  • Highly-standardized and ready-to-use ELISpot assay
  • Monitoring of CMV-specific immune reconstitution
  • Guidance of antiviral therapy decision-making
  • CE-marked IVD in Europe


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Research-use ELISpot kit

T-Track® IFN-γ HiSpecificityPRO

  • All-in-one and ready-to-use ELISpot kit
  • Highest specificity
  • Low background
  • Flexible setup configuration
  • Standardized read-out  



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    Enhanced stimulatory properties

    T-activated® proteins

    • Enhanced immune-stimulatory capacity and immunogenicity of proteins
    • HLA-antigen-independent stimulation of a broad spectrum of clinically-relevant effector cells (Th, CTL, NK, NKT-like cells)
    • Applicable for multiple immunological assays and read out systems, including ELISpot, FACS and ELISA

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    • Kalyuzhny AE (2005). Handbook of ELISPOT: methods and protocols (Vol. 302). Springer Science & Business Media.
    • Abbas AK, Lichtman AH, Pillai S (2014). Cellular and molecular immunology. Elsevier Health Sciences.
    • Murphy K, Travers P, Walport M, Janeway C (2012). Janeway’s Immunobiology. New York: Garland Science.


    Review Article

    • Calarota SA, Baldanti F (2013). Enumeration and Characterization of Human Memory T Cells by Enzyme-Linked Immunospot Assays. Clin. Dev. Immunol. 2013:637649. (Read more)


    Research Paper / Articles

    • Tassignon J et al. (2005). Monitoring of cellular responses after vaccination against tetanus toxoid: comparison of the measurement of IFN-gamma production by ELISA, ELISPOT, flow cytometry and real-time PCR. J. Immunol. Methods 305:188-198. (Read more)
    • Hagen J et al. (2015). Comparative Multi-Donor Study of IFNγ Secretion and Expression by Human PBMCs Using ELISPOT Side-by-Side with ELISA and Flow Cytometry Assays. Cells 4:84-95. (Read more)
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