This procedure should be used as a guideline only. Please keep in mind that Creative Biolabs cannot guarantee specific results for ELISPOT assays.
ELISPOT is a technique related to ELISA for the detection of secreted proteins, such as cytokines and growth factors. It is also known as an enzyme-linked immunospot. It is a method for the quantitative assessment of protein secretion. Creative Biolabs offers specialized antibody development services for ELISPOT assays.
ELISPOT is performed using PVDF or nitrocellulose membrane 96-well plates. These plates are pre-encapsulated with antibodies specific for the secreted protein. Cells are added to the plates and attached to the encapsulated membranes. The cells are stimulated to secrete the proteins, which bind to the antibody. Next, a detection antibody that binds specifically to the bound protein is added.
The etection of the resulting antibody complexes is performed by the action of enzymes that produce colored substrates or by using fluorescent labels. One advantage of using fluorescence is the ability to recognize multiple secreted proteins simultaneously.
Membranes can be analyzed by manually counting spots or by using an automated reader designed for this purpose. Each secretory cell is shown as a colored or fluorescent spot, so this is a quantitative method for assessing protein secretion.
In the analysis software, set the following measurement parameters:
These parameters can be saved and used for subsequent experiments to obtain standardized results.
We recommend reading each plate 3 times and averaging the results to minimize measurement errors.
If the cells take some time to respond to the stimulus, they may need to be pretreated with the stimulus in a separate 96-well culture dish prior to transfer to the ELISPOT plate.
Experiments using ELISPOT for cytokine detection will require the use of positive controls. In these positive control wells, the cells should be stimulated with an agent known to induce expression of the cytokine being assayed. This can be used to compare with a negative control—no treatment or stimulation of a different secreted protein.
Make sure you stimulate PBMCs with the correct stimuli to detect your target cytokines. Typical stimuli include:
Most ELISPOT experiments are performed with isolated PBMC (peripheral blood mononuclear cells). Both freshly prepared and cryopreserved cells can be used for this experiment. However, it is recommended that frozen cells be allowed to rest for at least one hour after thawing to allow for debridement of cellular debris prior to addition to the plate.
PBMCs should be prepared and plated within 8 hours of blood sample collection to preserve cellular function. If blood samples are left longer than this time, granulocytes (neutrophils) mixed with PBMCs will be activated. When PBMCs are separated from granulocytes, this may alter their buoyancy profile, and therefore granulocytes may contaminate the PBMC layer. Activated granulocytes may also start to activate some PBMCs (they can downregulate the CD3 signaling zeta chain and inhibit T cell function.)
If preparation and plating cannot be performed within 8 hours:
Note: Consistent results are obtained if splenocytes are pre-stimulated with the appropriate cytokine release stimulant for 24–48 hours and further incubated for 8–16 hours to allow for spot formation prior to the addition of splenocytes to ELISPOT plates.
Note: Rapid freezing can damage the cells. Keeping a large number of frozen samples of cells from a good donor as a control in a series of ELISPOT experiments can be a useful tool for checking the standardization of results.
Wash steps not sufficient | Before and after color development, wash both sides of the membrane with distilled water. Some reagents may leak through the membrane to the bottom of the plate, and these reagents can cause high background if not washed off. |
Too many cells secreting cytokine/protein of interest | Reduce the number of cells per well. This will need to be optimized. You may also need to optimize the concentration of the stimulus used. |
Plate not dried properly | Keep the plate out of the light by drying it more often before reading. Overnight drying at 4°C may help increase the contrast between the background and the spots. |
Over-developed plate |
Reduce the developing time. Exceeding 1-hour incubation with the enzyme substrate may result in increased background color. |
Not enough cells secrete the cytokines/proteins of interest | Increase the number of cells. The cell count should typically be between 1–2 X 105 cells per well, which may require some optimization. |
Ensure the cells are stimulated correctly. | Use a positive stimulus control—a stimulus that you know will induce the expression of the cytokine/protein you are interested in. |
Cells are not cultured long enough or may need time to respond to stimuli. | Increase cell incubation time or use indirect methods—pre-treatment of cells with stimulants. |
Inadequate color development | Monitor color development with a high-powered microscope and ensure that the color development reagents are stored correctly and have not lost their activity. |
Not enough primary or secondary antibodies | The concentration of primary and/or secondary antibodies will need to be increased. This will need to be optimized. |
Membrane not pre-treated | Ensure that the membrane is adequately pretreated with 35% ethanol. Afterwards, wash 3 times with PBS. |
Membrane not washed adequately after ethanol treatment | Clean the membrane thoroughly. Sometimes this can happen if ethanol is trapped between the membrane and the bottom of the plate (leaking). |
Membrane has dried out at some stage | Ensure the membrane does not dry. |
Cells unevenly distributed | Gently mix the cells to obtain a homogeneous suspension before transferring them into the wells. |
Pipette tip touched the membrane | Pay attention to the pipetting steps, especially the cleaning, including the automatic cleaning. |
Formation of foam | Foam may form during the cleaning process. A water gun with a narrow bottle opening can produce excessive foam, preventing effective and uniform cleaning. |
Damage from washing | Flow rate on an automated washer may be too high, or manual pipetting may be too harsh. A gentler washing procedure is required. |
Secondary antibody aggregates | Cells still on the membrane, cell debris |
Cells still on the membrane, cell debris | Ensure all the cells are washed from the membrane with PBS Tween 20 before secondary antibody incubation. Cells left on the membrane will form irregular-shaped spots. |
Contaminating platelets (when using PBMC prepared from blood samples) | PBMC preparation needs to be efficient. Wash the plate well after the cell culture stage. |
Cell culture contamination (dust and microbes) | Keep reagents as sterile and clean as possible. Make sure your cell culture technique is sterile. Reagents can be filtered using a low protein binding syringe with a 0.2 µm pore size. |
Mitogens and other factors in the serum are stimulating the cells. |
Heat inactivates the serum. See also "Poorly Defined Spots" regarding plate movement. |
Poor coating, too much antibody | Reduce the primary antibody concentration. |
Prolonged cell culture | The longer the cells are incubated, the more cytokines/proteins they secrete. This will cause larger spots to start merging and become indistinguishable. Shorten the incubation time for the cell culture step. (No more than 24 hours is generally recommended.) |
Cells over-stimulated | Overstimulation will cause the cells to secrete large amounts of cytokines/proteins. This will produce spots that begin to merge and become indistinguishable. Reduce the amount of stimulus in the medium or shorten the incubation time. |
Membrane not pre-treated | The film must be pretreated with ethanol, otherwise, the result may be faint, ill-defined spots. It will be difficult for the reader to distinguish between them. |
Plate movement during cell incubation | Do not allow the plate to move during cell incubation, as multiple spots can be produced. If possible, use a dedicated incubator that is not opened during the incubation. Do not tap the plate after adding the cells. |
Coating antibody not concentrated enough |
Increase the concentration of the encapsulated antibody. The spot quality can help determine if the capture antibody is too diluted or if there is a problem in the coating step. |
White spots in the middle of a normal spot (more usual with enzymatic detection) |
This means that the enzyme conjugate has run out of substrate. Therefore, higher concentrations of secondary antibody and substrate are needed. For fluorescence, increase the concentration of antibody. |
Inconsistency in results between wells |
During cell culture, do not stack plates. This will create an edge effect within the plate due to the non-uniformity of the temperature distribution. When adding cells to the wells, ensure that a well-mixed single cell suspension is used. |
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