Analytical Methods: INF-Y ELISpot for Study Of T cell Response/Activation

The enzyme-linked immunospot (ELISpot) assays are widely used for studying cellular immunology. It provides both quantitative and qualitative information on cellular cytokine responses to defined antigens. It enables the comprehensive screening of patient-derived peripheral blood mononuclear cells to detect the antigenic restriction of T-cell responses for infectious diseases. This technology is has emerged as the gold standard for identifying T cell-mediated response against viral capsid protein and transgenes. 

Principle of ELIspot




Basic Principle

A species-specific monoclonal antibody for IFN-γ has been pre-coated onto a PVDF (polyvinylidene difluoride)-backed microplate. Appropriately stimulated PBMCs/ T cells are pipetted into the wells and the microplate is placed into a humidified 37 °C CO2 incubator for an optimized period of time. During this incubation period, the immobilized antibody in the immediate vicinity of the secreting cells binds secreted IFN-γ. After washing away any cells and unbound substances, a biotinylated monoclonal antibody specific for mouse IFN-γ is added to the wells. Following a wash to remove any unbound biotinylated antibody, alkaline-phosphatase conjugated to streptavidin is added. The unbound enzyme is subsequently removed by washing and a substrate solution (BCIP/NBT) is added. A blue-black colored precipitate forms at the sites of cytokine localization and appear as spots, with each individual spot representing an individual IFN-γ secreting cell. The spots can be counted with an ELISpot reader system. 

General Protocol

Required Reagents 
Pre-coated 96 well PVDF bottomed Microplate
Biotinylated Detection antibody 
Streptavidin-Alkaline Phosphatase Conjugate
Bovine Serum Albumin (BSA) 
BCIP/NBT substrate buffer

Preparation of ELISpot Plate
  • Add 100 μL of 1X PBS to each well. 
  • Incubate plate at room temperature for 10 minutes. 
  • Empty the wells by flicking the plate over a sink and gently tapping on absorbent paper.
Cell Stimulation
  • Add 100 μL of sample, positive or negative controls cell suspension to appropriate wells providing the required concentration of cells and stimulant (cells may have been previously stimulated). 
  • Cover the plate and incubate at 37ºC in a CO2 incubator for an appropriate length of time (10-15 hours). Note: Do not agitate or move the plate during this incubation. 
  • Empty the wells and remove excess solution then add 100 μL of PBS-T to each well. 
  • Incubate the plate at 4ºC for 10 minutes. 
  • Empty the wells as previous and wash the plate 3x with 100 μL of PBS-T.
Color Development & Detection 
  • Add 100 μL of diluted detection antibody to each well. 
  • Cover the plate and incubate at room temperature for 1 hour 30 minutes. 
  • Empty the wells as previous and wash the plate 3x with 100 μL of PBS-T.
  • Add 100 μL of diluted Streptavidin-AP conjugate to each well. 
  • Cover the plate and incubate at room temperature for 1 hour. 
  • Empty the wells and wash the plate 3x with 100 μL of PBS-T.
  • Peel off the plate bottom and wash both sides of the membrane 3 x under running distilled water. 
  • Add 100 μL of ready-to-use BCIP/NBT buffer to each well 
  • Incubate the plate for 5-15 minutes monitoring spot formation visually throughout the incubation period to assess sufficient color development. 
  • Empty the wells and rinse both sides of the membrane 3x under running distilled water. Completely remove any excess solution by gentle repeated tapping on absorbent paper. 
  • Allow the wells to dry and then read results. The frequency of the resulting colored spots corresponding to the cytokine producing cells can be determined using an appropriate ELISPOT reader and analysis software or manually using a microscope.
Kits
Mabtech INF-Y ELISpot Kit 
R& D System INF-Y ELISpot Kit 
Abcam INF-Y ELISpot Kit 

ELISpot Instruments Suitable For Your Laboratory

 Immunspot CTL S6 Analyzer 

ImmunoSpot® Analyzers combine accuracy, flexibility, and scientific methodology, making them invaluable research tools. In addition to ELISPOT assays, the analyzer can also be used for analyzing other microtiter plate-based bioassays. These include bacterial, yeast, stem cell, and tumor colonies, as well as viral plaque assays and more.
In addition to providing high-resolution visible light analysis, multiple fluorescent light sources enable fluorescent applications — FluoroSpot, cell cycle analysis, cell viability tests, apoptosis tests etc. The automated filter changer allows up to 8 wavelengths of fluorescent analysis.

AID vSpot Spectrum (VSR078IFL)

The AID vSpot Spectrum is the new high-end EliSpot/FluoroSpot device from AID. It combines AID iSpot 96-well FluoroSpot analyzing with enzymatic multiple plate evaluation. On the enzymatic side the AID vSpot Spectrum can handle a variety of different assay types including Viral Plaque Assays and Neutralization Assays. It will also read 6, 12, 24, 48, and 384-well plates. The insertion of high-resolution digital cameras provides well images of unprecedented quality. The capabilities of this technology include EliSpot, FluoroSpot, Virus Plaque Assays, Colony Counting etc.

Mabtech IRIS - Spot analysis

A next-generation, 4-color FluoroSpot, and ELISpot reader using RAWspotTM technology that ensures reliable multiplexing. A new dimension of data The proprietary RAWspotTM technology extracts the volume of every spot, which corresponds to the relative amount of secreted analyte.FluoroSpot combines the sensitivity of ELISpot with the capacity to analyze the secretion of several analytes simultaneously. This highly sensitive cellular assay is robust, easy to perform, and suitable for both single tests and large-scale screening. The capabilities include:
  • Capture- analyte secretions at the single-cell level. 
  • Detect immune responses without manipulation of intracellular processes.
  • Discover the true potential of FluoroSpot and see a new dimension in your research. 

Pages You May Like