The ELISPOT assay - general questions |
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What is an ELISPOT assay? | |
The enzyme-linked immunospot (ELISPOT) assay is a widely used method for monitoring immune responses. The assay is a highly sensitive method for the ex vivo quantification of cytokine or antibody secreting cells after stimulation with an appropriate stimulus in vitro (Cox et al. 2006). In fact, each cell can be detected by the assay as long as a characteristic protein is released and specific high affinity antibodies recognizing the protein are available. The ELISPOT assay is very similar to an enzyme-linked immunosorbent assay (ELISA) and is based on the same immunochemical 'sandwich' principle. The major difference is that an ELISPOT is a combination of both an immunoassay and bioassay because living cells are cultured directly in the wells of the ELISPOT plate. |
In which area of research should an ELISPOT assay be used? | |
The ELISPOT assay can be used in many areas of research such as cancer, infectious disease, autoimmune disease, allergy and organ transplantation. The assay is particularly effective for the measurement of antigen-specific responses post-vaccination in peripheral blood cell preparations. For more information, please see ELISPOT applied in research. |
The ELISPOT assay - specific questionsWhich cell types can be analyzed by ELISPOT? In principle any type of cell that secrete proteins can be investigated in the ELISPOT assay at single cell level. Although so far the technique has mainly been used to identify cytokine secretion by antigen-activated T cells and antibody secretion by B cells from peripheral blood or spleen cell preparations. Also adherent cells can be analyzed in the ELISPOT assay. However, when working with adherent cells then please note that a lysis step is necessary to remove all cells from the ELISPOT plate before proceeding with the detection step. |
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Are there special requirements and storage conditions for blood? | |
Whole blood should be kept at room temperature (RT) until processing. Do not refrigerate. If collected elsewhere, samples should be shipped at ambient temperature to the desired laboratory. Samples should not be at RT for more than 8h after draw. The recommended anti-coagulants are citrate and heparin. For both substances there are no reported adverse effects on cellular function in the ELISPOT assay. EDTA, on the other hand, inhibits coagulation through calcium chelation which may impair cytokine induction during antigen-specific re-stimulation and is therefore dissuaded for use as an anti-coagulant. |
What about ELISPOT assay controls? | |
Appropriate controls are important for the validation of the ELISPOT assay. Choice of controls depends on the cells if interest and type of experiment. Positive control in the T cell ELISPOT assay Both antigen-specific and polyclonal stimuli can be used. However, antigen-specific stimuli are preferred since cells of various species respond differently to polyclonal stimuli. For the human system, vaccine proteins (Tetanus toxoid, Hepatitis B, etc.) or a pool of synthetic peptides of common human viral epitopes are excellent positive controls. When using vaccine proteins, it should of course be known whether individuals have been vaccinated. Negative control in the T cell ELISPOT assay Background control in the T cell ELISPOT assay Positive control in the B cell ELISPOT assay (Memory B cells only) Background control in the B cell ELISPOT assay To: An overview of general cell stimuli commonly used in ELISPOT and FluoroSpot assays |
How critical are the incubation times mentioned in the ELISPOT manual? | |
For optimal results, the coating antibodies should be left in the wells of the ELISPOT plate for minimal 16 h at 4°C. The blocking buffer, detector antibodies and conjugate can be incubated for 1 h at 37°C, 2 h at room temperature or overnight at 4°C without any problem. |
What type of plate should I use? | |
ELISPOT assays can be performed using either 96-well transparent polystyrene-bottomed plates or 96-well PVDF membrane-bottomed plates. Both solid supports bind proteins by hydrophobic interactions (Van der Waal's forces). There are both sterile and non-sterile microtiter plates available for ELISPOT analyses. Since ELISPOT requires short culture times and includes the use of antibiotics in the culture medium, non-sterile plates can be safely used without contamination problems. When using PVDF membrane-bottomed plates a prewetting step with 70% ethanol is required. The treatment with ethanol makes the membrane hydrophilic and ensures optimal binding of the coating antibody resulting in better sensitivity (increased spot number) and more accurate quantitation (more sharply defined spots) (Weiss A.J. 2012). After maximum 2 minutes of prewetting with 25 µl 70% ethanol, the membrane should be rinsed thoroughly by adding coating buffer to the well to efficiently wash out the ethanol before the coating antibody is added to the wells. Once the membrane is ethanol-treated, it must be kept wet for the entire assay procedure. Overtreatment with larger volumes of ethanol, more concentrated ethanol and longer exposure time can lead to trapping of residual liquid between the membrane and underdrain, which may result in poor assay performance. This prewetting step is not necessary for polystyrene-bottomed plates. The U-CyTech ELISPOT kits are either suited for the use with PVDF membrane-bottomed plates (96-well plates from Millipore [cat. no. MSIP S4510] are recommended) or contain 96-well polystyrene-bottomed plates. When needed, extra polystyrene-bottomed plates can be ordered separately (cat. no. CT350). All our 2-plate format T cell ELISPOT kits do contain 96-well plates. |
My question is not mentioned here, how can I contact U-CyTech? | |
The researchers of U-CyTech have over 20 years experience with the development and performance of the ELISPOT assay. Not only for human but also for monkey, mouse and rat species. They are more than happy to provide you with additional advice and to share their experiences with you. Please contract our customer service: U-CyTech biosciences Phone: +31.85 073 1460 |