Help & FAQs: ELISpot and FluoroSpot Kits

  • Can adherent cells be used in ELISpot kits and ELISpot Development Modules?
  • Adherent cells will not block the detection of antigen, so they may be used. The preparation of different cell dilutions are advised. If an excessive number of cells is used, the spots will overlap.
  • Can Dual ELISpot and FluoroSpot Kits be stored after development for later analysis?
  • R&D Systems has not performed a formal study to address this question and the following suggestions are based on anecdotal evidence. Dual-Color ELISpot kits allow for the measurement of two cytokines simultaneously via two different colorimetric substrates: BCIP/NBT and AEC. While BCIP/NBT is very stable, the AEC will fade over time. For this reason, extended storage of Dual-Color ELISpot plates is not recommended, but the plates may be stored at 4 degrees C for at least a week and possibly as much as a month if protected from light. It may be possible to store FluoroSpot plates after development for up to 1 week at 4 degrees C if the plates are protected from light.
  • Can I use a portion of the ELISpot plate and save the rest for later assays?
  • Using partial plates is not recommended. Sterility will be an issue if wells are left empty for one ELISpot assay and then used in a following ELISpot assay.
  • How is an ELISpot assay different from an ELISA assay?
  • Both assays employ the sandwich ELISA format utilizing capture and detection antibodies. The traditional ELISA is designed measure the concentration of analyte in biological samples such as serum, plasma, and conditioned medium. In contrast, the ELISpot assay provides information about the relative number of cells secreting the specific analyte. In an ELISpot assay the cells are added to the plate and the immobilized capture antibody in the immediate vicinity of the cell binds the secreted protein of interest.The sites of cytokine secretion are visualized as spots in the bottom of the well.
  • How many cells do I need to use in the ELISpot kits and ELISpot Development Modules?
  • The number of cells required to achieve optimal results depends on a number of variables. The variables to consider are cell size, type of cell, number of secreting cells, and the method of stimulation. We recommend cell dilutions from 10,000,000 to 10,000 cells per well.
  • How many samples can I run on one plate in the ELISpot kits?
  • There are 96 wells in a plate. Eight wells are required for positive, negative, background, and detection controls. This leaves 88 wells, which is sufficient for 44 samples in duplicate.
  • How should ELISpot data be collected?
  • Chromogenic or fluoroscent spots can be manually quantified using a dissection microscope or automatically quantified by an ELISpot reader. The ELISpot Labs at R&D Systems uses the QuantiHub™ ELISpot Plate Reader from MVS Pacific.
  • Is there an advantage to using the FluoroSpot versus Dual-Color ELISpot kit for the same target cytokines? 
  • There is no advantage relating to sensitivity with using FluoroSpot detection kits over the chromogenic Dual-Color ELISpot for the same target cytokines. However, the FluoroSpot kits allow for a more precise co-localization of two different cytokines secreted by the same cells.
  • What are R&D Systems recommendations to reduce or eliminate high background on ELISpot plates?
  • If the cells are cultured and stimulated in a tube before being plated, the cells must be rinsed with culture media before they are added to the ELISpot plate. If the cells are not rinsed, then cytokine secreted into the culture media could bind to the specific capture antibodies, resulting in high background. Another solution to eliminate background and obtain consistent well-to-well staining is to use an aluminum foil method as reported in Journal of Immunological Methods 257 (2001); "A simple method to reduce the background and improve well-to-well reproducibility of staining in ELISpot assays”. Authored by R&D Systems in-house ELISpot scientists.
  • What are recommended controls for the ELISpot assay?
  • For a positive control, it is recommended to use a recombinant protein of the analyte(s) being tested. For an unstimulated/negative control, it is recommended to use the same number of unstimulated cells as stimulated cells. Use sterile culture media as the background control. For the detection antibody control, it is recommended to substitute PBS for the detection antibody.
  • What is an ELISpot Assay?
  • ELISpot is an abbreviation for Enzyme Linked Immunospot Assay. A capture antibody specific for the protein of interest is coated onto a membrane and cells are added. During an incubation period, the protein secreted from individual cells is captured and detected using a biotinylated antibody specific for a different epitope of the protein. The use of an SA-AP conjugate followed by the chromogen BCIP/NBT results in a colorimetric spot on the membrane at the site of the antigen-secreting cell.The number of antigen secreting cells can be quantified using an automated ELISpot plate reader or manually using a microscope.
  • What is R&D Systems doing to reduce its use of packaging from non-renewable resources?
  • Environmental stewardship is important to R&D Systems and its employees. R&D Systems is ISO 14001:2015 Certified and as part of this we are continuously reviewing our sustainability practices and "green" options. First and foremost, the energy expended to ship back the styrofoam box is more detrimental to the environment than having the facility re-use or recycle it. Our stance is to encourage our customers to implement a recycling program locally. At R&D Systems we have chosen to reduce the use of styrofoam as much as possible by: doing extensive stability testing in order to determine which products can be shipped with minimal packaging at ambient temperatures, converting the use of non-recyclable packaging materials to recyclable plastics, cardboard, or biodegradable materials, and continuing to investigate alternatives to dry ice shipments, the use of re-usable containers, and gel packs that allow for smaller styrofoam containers. Employees were key to initiating a recycling program at our facilities. Internally, we recycle paper, plastic, cardboard, aluminum, and glass.
  • What is the difference between ELISpot kits and ELISpot Development Modules?
  • Complete ELISpot kits are ready-to-run, and require no assay development or refinement. Complete kit components include: Antibody-coated PVDF-backed 96-well Microplate, Detection Antibody, Streptavidin-AP, BCIP/NBT, Positive Control, Dilution Buffers, and Wash Buffer.ELISpot Development Modules and the ELISpot Blue Color Module contain the necessary components for analyte detection and visualization, respectively. These modules can be used together but are sold separately. Each module contains enough reagents for at least five 96-well microplates. Additional supplies are required and assay development expertise is recommended. ELISpot Development Modules include: Capture Antibody Concentrate and Detection Antibody Concentrate. The ELISpot Blue Color Module includes: Streptavidin-AP and BCIP/NBT.
  • What is the difference between the Dual-Color ELISpot and the Dual-Color Fluorospot kit?
  • The Fluorospot kit is a fluorescence-based assay that uses two NorthernLights™ fluorescent probes to detect distinct cell populations, whereas the Elispot kit is a colorimetric based assay that uses Streptavidin with NBT/BCIP and horseradish peroxidase with AEC for detection.
  • Why is the positive control spot dark?
  • The positive control is recombinant protein and it is normal that the wells containing the positive control are dark. This indicates that the kit is working as expected.

ELISpot Development Modules

  • Can an antibody-coated ELISpot plate be stored?
  • Yes, plates can be stored under the proper conditions. Block the plate as described in the protocol and aspirate the blocking solution. Place the plate in a vacuum oven at 30 °C for 1 hour. Store the dried plates in a ziplock bag at 4 °C with a desiccant. The plates can be stored in this manner for 1 week. Before the experiment condition the wells by filling them with sterile culture medium and incubating for approximately 20 minutes. Aspirate this medium before adding the cells. Alternatively, if there is no vacuum oven available, block the plate and aspirate the blocking solution. Add a volume of PBS to each well, cover the plate, and store at 4 °C for a maximum of 2 days. Condition the wells with cell culture medium as described above before adding cells.
  • Do PVDF-bottom immunospot plates need to be pre-wet with ethanol for activation?
  • It is not recommended to pre-wet the plate membrane with ethanol because this can result in an inconsistent number of spots. 
  • The assay insert indicates to incubate the Detection antibody at 2-8°C overnight. Is there a specific time range that is recommended?
  • R&D Systems incubates the Detection antibody overnight, for about 16-18 hours, at 2-8°C.
  • What are R&D Systems recommendations to reduce or eliminate high background on ELISpot plates?
  • If the cells are cultured and stimulated in a tube before being plated, the cells must be rinsed with culture media before they are added to the ELISpot plate. If the cells are not rinsed, then cytokine secreted into the culture media could bind to the specific capture antibodies, resulting in high background. Another solution to eliminate background and obtain consistent well-to-well staining is to use an aluminum foil method as reported in Journal of Immunological Methods 257 (2001); "A simple method to reduce the background and improve well-to-well reproducibility of staining in ELISpot assays”. Authored by R&D Systems in-house ELISpot scientists.
  • What grade BSA is recommend for use in ELISA and ELISpot Development Systems?
  • The grade of the BSA used has been found to be a critical component for running a successful ELISA. BSA with a minimum purity of 98% is recommended. R&D Systems recommends using the same BSAs used in development of the DuoSet; Catalog # DY995 Reagent Diluent Concentrate 2, Catalog # DY997 Reagent Diluent Concentrate 1. Millipore Probumin® Diagnostic Grade BSA (Millipore, Catalog # 820451) is another example.
  • Why do I observe ring shapes at the bottom of ELISPOT plate?
  • The rings develop because this is where the membrane touches the underdrain. To avoid the ring development: I) Use aluminium foil on the bottom of the plate for distribution of heat across the membrane since the cells like to settle in the spots where it's warmer. II) When rinsing the plate after chromagen substrate removal, make sure to take off the underdrain and rinse both sides thoroughly. III) Completely dry membrane before analysis.