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ELISPOT Protocol

The CTL ImmunoSpot® platform permits maximized scientifically-validated single cell ELISPOT analysis. At the unprecedented resolution of up to 1 in 800,000, ImmunoSpot® assays measure effector molecule secretion by individual T cells that have been stimulated by an antigen.

ImmunoSpot® assays therefore provide information on the numbers of the antigen-specific T cells present in a test cell population, typically PBMC. The frequency of the specific T cells within the PBMC reflects the magnitude of T cell immunity. Spot sizes record the amount of cytokine produced by the individual cells. Spot sizes can be increased for polyfunctional T cells and for recently activated T cells and can be decreased in states of anergy or immune suppression. Measuring different molecules that T cells secrete provides information on the effector lineage of T cell immunity.

Several helpful online videos are available on working with ELISPOT assays and PBMC. These videos are linked throughout the text and indicated by the symbol "⦿." You may also visit the ImmunoSpot® YouTube channel to view our complete library. Subscribe to our channel to be notified when new videos are posted.


How ELISPOT Assays Work Principle of the Test Procedure Precautions Technical Tips

How ELISPOT Assays Work

The principle of the enzyme-linked immunosorbent spot assay (ELISPOT) for cytokine detection is shown. 

How ELISPOT Assays Work

(A) An ELISPOT plate with a PVDF membrane is coated with an analyte-specific antibody. 

(B)Freshly isolated, thawed or cultured cells are plated together with the antigens of interest and incubated to allow for the activation of the antigen-specific T cells and the induction of their cytokine secretion.

(C) The cells are washed away leaving the antibody-bound analyte in the well. 

(D) A detection antibody that is specific for a different determinant of the analyte is added – in this example this antibody is directly enzyme-coupled. The labeling of the detection antibody can also be done indirectly, via a streptavidin-biotin reaction.

(E) The addition of a chromogenic substrate leads to an enzyme-catalyzed spot development.


Principle of the Test

The assay can be done with freshly isolated PBMC, or with PBMC that have been frozen and thawed following optimized protocols. Along with the trial ImmunoSpot® Kit, CTL offers PBMC that have pre-defined reactivity to antigen along with the antigen (ePBMC® Reference Samples QC Set™). Such reference samples are indispensable for assay development, qualification, and validation.

Specifically designed 96-well PVDF plates are coated with a cytokine-specific monoclonal capture antibody (e.g., specific for IFN-γ). PBMC (or purified T cell subsets with APC) are pipetted into the pre-coated wells, and are cultured with the test antigen(s). Negative control wells contain either irrelevant antigen, or media alone. As positive control we recommend the use of CEF peptides to elicit CD8 cells, inactivated CMV virus for activation of CD4 cells, or PHA to stimulate both T cell subsets. The ePBMC® Reference Samples QC Set™ is ideally suited as reference standards for the assay’s performance. The activated T cells secrete molecules that are captured by the membrane-bound antibody.  

For the best results in an ELISPOT assay, the cells should be cultured with antigen for a time period that induces maximal cytokine (or other analyte) production: 24 hours are optimal for measuring IFN-γ secretion by T cells. After the activation culture, the cells are discarded (or can be transferred for further characterization/ propagation), and a labeled cytokine-specific detection antibody is added to the plate. Subsequently, the plate-bound detection antibody is visualized via an enzymatic reaction or fluorescence.ImmunoSpot® assays are optimized to detect and quantify in each color precipitation (“spot”) the footprint of a single cell’s secretory activity. Spot numbers therefore denote the accurate frequency of the antigen-specific T cells among the plated cells; spot sizes and morphology provide additional information on the magnitude and kinetics of the cells’ secretory activity. 

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Day 0 (Sterile conditions)

  • Prepare Capture Solution by diluting the Capture Antibody according to your specific protocol.   
  • Many cytokines benefit from pre-wetting the PVDF membrane with 70% ethanol for 30sec and washing with 150μl of PBS three times before adding 80μl of the Capture Solution into each well.
  • ⦿ Incubate plate overnight at 4°C in a humidified chamber.

Day 1 (Sterile conditions)

Day 2

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  • For in vitro and research use only – Not intended for use in diagnostic procedures.
  • Blood samples, PBMC, or cell isolates should be treated as potentially infectious biohazard. Handle everything that comes in contact with specimen as potential biohazard, level II.
  • Wear disposable gloves and personal protection throughout the entire procedure and thoroughly wash hands after handling the test reagents.
  • Wipe any spills immediately with a laboratory-approved disinfectant, such as 10% bleach followed by 70% ethanol.
  • All test specimens and materials used in the procedure must be disposed of as biohazardous waste.


Technical Tips

  • Deviations from specified temperatures, timing requirements, number of washing steps, and specified reagent preparation volumes may alter the performance of the assay.
  • Plates can be washed manually or by a suitable automated plate washer with adjusted pin length and flow rate so membranes and spots are not damaged.  
  • To avoid damage to the PVDF membrane in the wells, avoid touching the membrane with pipette tips or with the plate washer. The PVDF membrane is permeable and protected by an underdrain. Avoid direct contact between the well bottom and wet surfaces, including paper towels or any other materials that can absorb liquid.   
  • While processing plates, the PVDF membrane at the bottom of the wells must remain wet.    
  • When underdrain and gloves are wet, the underdrain may be slippery and difficult to remove. Wipe gloves and underdrain with paper towel before removing.
  • ⦿ After completion of the experiment, do not dry the ELISPOT assay plates at temperatures exceeding 37°C as this may cause the membrane to crack.
  • Spots may not be readily visible while the membrane is still wet. Scan and count plates with a CTL ImmunoSpot® Analyzer only after membranes have completely dried.
  • Higher background appearing in the control wells can be potentially overcome by the following steps:
    • When working with pre-cultured cells, wash the cells thoroughly in CTL-Wash™ prior to the experiment in order to avoid carryover of cytokines and other substances; use  CTL-Test™ for testing PBMC.
    • The SmartCount™ module of the ImmunoSpot® Software automatically recognizes spots over high background or uneven background and will correct background deviations. The Autogating™ module will help discern between T cell-derived and background spots. The CTL technical support team will gladly assist you with using the ImmunoSpot® Software for the analysis of complicated test results. 
  • Data analysis. The CTL ImmunoSpot® Analyzers along with the ImmunoSpot®  Software have advanced features that permit automated, objective recognition of spots, gating and counting. An ELISPOT data management tool, SpotMap™ , is also available for facilitation of high-throughput ELISPOT work.
  • The CTL team will gladly assist you with data analysis and troubleshooting, as well as customizing ELISPOT assays to suit your needs. Please contact us via email at  


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