The Enzyme Linked Immunospot technique was developed by Cecil Czerkinskdy in 1983. ELISPOT is used for the detection of secreted proteins, such as cytokines and growth factors. It is therefore used primarily in immunology research in the following areas:
- Transplantation – prediction of infectious risk
- Vaccine development (IFNγ)
- Th1/Th2, T-cell regulation analysis
- Monocyte and Dendritic cell analysis
- Autoimmune disease
- Cancer – tumor antigens
- Viral infection monitoring and treatment
Description of the ELISPOT
Cells are grown in 96-well plates which have PVDF or nitrocellulose membrane instead of plastic at the bottoms of the wells. Before adding cells, the membrane is coated with primary antibody. Next, the cells are added, they settle on the membrane, they secrete protein, and the secreted protein of interest will bind to the antibody. When this is detected using a detection antibody, the protein will be seen in the locality of that secreting cells as a spot of color (one spot = 1 cell). The membranes are then scanned in and analyzed using one of various software programs that are available for this purpose. These are able to quantify the number and percentage of cells secreting the protein. Therefore, immune responses to various stimuli can be quantified and compared.
Advantages of using ELISPOT
- Sensitive assay
- Functional assay
- Many laboratories now standardize and validate their results by sending the same sample to several reference labs using the same procedure, and comparing results to ensure they are reproducible.
- A large sample of PBMC from a patient or animal that has provided good results can be stored frozen and used as a standard control sample in subsequence experiments to assess inter assay variability.
- Samples should be run in duplicate or preferably triplicate if possible.
Abcam ELISPOT kits
The procedure below is a general guideline procedure for ELISPOT. Abcam ELISPOT kits have been designed for detection of various cytokines and growth factors from PBMC cells (Peripheral Blood Mononuclear cells) or rodent splenocytes. Optimized procedures are supplied with these kits and we recommend that these methods are used.
The researcher will, however, still need to optimize the cell numbers used, the length of cell culture incubation and the cell stimulant that is used.
ELISPOT pair products will require the researcher to use their own reagents and optimize the procedure.
Choosing antibodies: We recommend using antibodies tested in ELISPOT to ensure that they are suitable for this application and that they will be covered by the Abcam 12 month guarantee.
Sample preparation tips
General ELISPOT procedure
- Prepare PVDF membranes in the 96-well plates by incubating in 35% ethanol for 30 seconds.
Ensure the ethanol is washed off well with PBS. Any ethanol left on the membrane can affect cell viability and also antibody binding.
- Coat 96-well plate with capture antibody diluted in PBS. Incubate at 4oC overnight.
A general guideline is that approximately 0.5-1 µg per well of antibody should result in well-defined spots. Kits are optimized with capture concentrations for best performances (100 µl per well).
- Empty the wells, tapping them dry, and wash with PBS
ELISPOT plates should be handled more carefully than ELISA plates. When tapping dry, do this gently. Do NOT use a plate washer at this stage.
- Block the plates by adding 100 µl per well 2% dry skim milk. Incubate 2 hours at room temperature.
- Wash wells in PBS once. If necessary, the plates can be stored at this stage. Wash twice in PBS and leave to dry. Store at 4oC for not more than 2 weeks, in a sealed plastic pouch with a dessicant
- Prepare PBMCs from fresh blood on a FicollTM gradient. Count the cells. They should be over 95% viable. Dilute cells to the required concentration and add cell suspension to wells. If optimizing the assay for cell number, dilute 1:2 down the plate as in an ELISA. DO NOT SHAKE THE PLATES.
The number of cells will require optimization. For example, if a low percentage of cells are expected to secrete the target cytokine, a larger cell number will be required. Refer to specific target kit protocols for recommendations on assay controls and cell number per well. Cell numbers should usually range between 1X105 to 2X105 cells per well.
If possible, try to use serum free media as serum contains mitogens and inhibitors which could affect the results. Alternatively, several batches of serum can be tested to find one with optimal response to noise ratio. This batch can then be stored and used in subsequent experiments.
- Culture overnight 37oC in CO2 incubator. DO NOT SHAKE THE PLATES
*If cells take time to respond to stimulation, please see indirect method below.
Don’t move the plates while the cells are culturing. This will lead to ‘snail trail’ spots that will not be well defined.
Don’t stack the plates if you have more than one. The temperature will not even out across the plate and each well will not be at the same temperature, giving an edging effect as cells will respond less well in the center of the plate where it is cooler.
- During the overnight incubation the cells will secrete cytokine, which will become bound to the primary antibody.
- Wash cells and unbound cytokine away by incubating with PBS 0.1% Tween 20 for 10 minutes. Then wash the plates 3 times with PBS 0.1% Tween 20.
Ensure you include Tween 20 in the wash buffer. Some cells will have started attaching after culture overnight (e.g. some stem cells are known to do this). Tween 20 will help wash these off the membrane. Do NOT use a plate washer at this stage.
- Add the conjugated detection antibody. Dilute the antibody in PBS 1% BSA. Incubate for 1 to 2 hours at room temperature. (Optimization is required).
For our ELISPOT kits, detection antibody concentrations have been determined for best performances and this information is provided on the datasheet. Filtering the detection antibody is recommended when protein aggregates are observed, as this may result in non-specific spot formation.
- Enzyme substrate color development
For enzymatic detection protocols, the base should be taken off the bottom of the plate to enable thorough washing of the membrane before adding substrate/chromogen. For example, after incubation with the streptavidin alkaline phosphatase conjugate that most Abcam ELISPOT kits utilize, remove the base and wash both sides of the membrane under running distilled water. This helps to prevent high background as some reagents can leak through the membrane into the bottom tray of the plate.
- After replacing the base and adding the substrate, monitor spot formation visually. As soon as the membrane looks optimally developed, stop the reaction immediately by gently washing the plate. It is not a good idea to walk away while the spots are developing and the timing for optimal results will be slightly different each time. Take the base off the plates and wash both sides of the membrane with distilled water to stop the spot formation.
- Dry the plates and let membranes dry completely.
Spots may become sharper if membranes are stored overnight at 4oC at this stage before reading, and even better up to 2 weeks after staining (we do not recommend to store longer than 2 weeks). Membranes can be wrapped in foil and stored at 4oC in the dark.
- Punch the membranes out of the wells onto a sticky plastic sheet. This step will depend on your plate reader’s requirements. Consult plate reader manual.
- Scan the sheet and analyze the membrane circles.
Set parameters in the analysis software to allow one to measure:
- Size/spot diameter
- Spot development/slope
These parameters can be saved and used for subsequent experiments for standardized results.
We recommend reading each plate 3 times and averaging the results in order to minimize anomalies in the readings.
If the cells take some time to respond to stimulation, then they will need to be pretreated with the stimulant for an optimized amount of time (e.g. 24 hours) in a separate 96 well culture dish. They can then be transferred to the ELISPOT 96 well assay plate.
Positive control stimulation
Experiments to detect cytokines using ELISPOT will require use of a positive control. In these positive control wells, the cells should be stimulated with an agent known to induce expression of the cytokine being detected. This can then be used to compare to the negative control and to compare as a positive control to other stimulant treatments used.
Ensure you are stimulating the PBMCs with the correct stimulant for detection of your target cytokine.
Stimulation of IL1β, IL-6: LPS, Activates Macrophages via the Toll-Like Receptor 4/complement pathways
Stimulation of IL-2, IL-4: PMA and Ionomycin, Cytokine lymphotoxins
PHA, 10 µg/ml
Concavalin A, 6-10 µg/ml (PBMC) or 0.5 µg/ml (rodent splenocytes)
Anti CD3/CD28 Antibodies (effective only for IFNγ, IL-4, IL-10 and Granzyme B)