ELISpot Assay Principle

ELISpot assays employ the sandwich enzyme-linked immunosorbent assay (ELISA) technique.Either a monoclonal or polyclonal antibody specific for the chosen analyte is pre-coated onto a   PVDF (polyvinylidene difluoride)-backed microplate.

Appropriately stimulated cells are pipetted into the wells and the microplate is placed into a humidified 37 °C CO2 incubator for a specified period of time.

During this incubation period, the immobilized antibody, in the immediate vicinity of the secreting cells, binds secreted analyte.

After washing away any cells and unbound substances,a biotinylated polyclonal antibody specific for the chosen analyte is added to the wells.

Following a wash to remove any unbound biotinylated antibody, alkaline-phosphatase conjugated to streptavidin is added.

Unbound   enzyme is subsequently removed by washing and a substrate solution (BCIP/NBT) is added.

A blue-black colored precipitate forms and appears as spots at the sites of cytokine localization, with each individual spot representing an individual analyte- secreting cell.The spots can be counted with an automated ELISpot reader system or manually, using a stereomicroscope.

Technical Notes: ELISpot

The enzyme-linked immunospot (ELISpot) assay was originally developed for the detection of individual B cells secreting antigen-specific   antibodies.This method has since been adapted for the detection of individual cells secreting specific cytokines or other antigens.

ELISpot assays employ the quantitative sandwich enzyme-linked immunosorbant assay (ELISA) technique.

A monoclonal antibody   specific for a   cytokine has been pre-coated onto a PVDF (polyvinylidene difluoride)-backed microplate.

Appropriately stimulated cells are pipetted into the   wells and the   microplate is placed into a humidified 37° C CO2 incubator for a specified period of time. During this incubation period, the immobilized antibody inthe immediate vicinity of the secreting cells binds secreted cytokine. After washing away cells and any unbound substances, a biotinylated polyclonal antibody specific for the cytokine is added to the wells. Following a wash to   remove any unbound biotinylated antibody, alk aline-phosphatase conjugated to streptavidin is added.

Unbound enzyme is subsequently removed by washing and a substrate solution (BCIP/NBT) is added.

Blue-black colored precipitate orms at the sites of cytokine localization and appears as spots,with each individual spot representing an individual   cytokine-secreting cell.The spots can be counted with automated ELISpot reader systems or manually, using a stereomicroscope.

ELISpot Assay Procedure     

Bring all reagents to room temperature, except the diluted Detection Antibody Mixture,which should remain at 2-8 °C. All samples and controls   should be assayed at least in duplicate.

1. Fill all wells in the microplate with 200 μL of sterile culture media and incubate forapproximately 20 minutes at room temperature.
   

2. When cells are ready to be plated, aspirate the culture media from the wells. Immediately add 100 μL of the appropriate cells or   controls   to each well (see Technical Hints for appropriate controls).

3. Incubate cells in a humidified 37 °C CO2 incubator. Optimal incubation time for each stimulus should be determined by the investigator. Do not disturb the cells during the incubation period.

4. Aspirate each well and wash, repeating the process three times for a total of four washes. Wash by filling each well with Wash Buffer (250-300 μL) using a squirt bottle, manifold dispenser, or autowasher. Complete removal of liquid at each step is essential to good   performance.   After the last wash, remove any remaining Wash Buffer by aspirating or decanting. Invert the plate and blot it   against clean paper towels.   Note: Adjust the height of the prongs of the manifold dispenser or autowasher to prevent damage to the membranes.

5. Add 100 μL of diluted Detection Antibody Mixture into each well, and incubate overnight at 2-8 °C. Alternatively, incubation   with detection   antibodies can be done for 2 hours at room temperature on a rocking platform.

6. Repeat the wash procedure described in step 4.

7. Add 100 μL of diluted Streptavidin-AP Concentrate A into each well, and incubate for 2 hours at room temperature.

8. Repeat the wash procedure described in step 4.

9. Add 100 μL of BCIP/NBT Substrate into each well, and incubate for 1 hour at room temperature. Protect from light.

10. Decant the BCIP/NBT Substrate from the microplate and rinse the microplate with deionized water. Invert the microplate and tap to remove   excess water. Remove the flexible plastic underdrain from the bottom of the microplate, wipe the bottom of the plate thoroughly   with paper towels and dry completely either at room temperature (60-90 minutes) or 37 °C (15-30 minutes).




The ELISpot (Enzyme-Linked Immunospot) assay is a highly sensitive method used to detect and quantify individual cells   secreting   specific molecules, such as antibodies, cytokines, or other antigens. Here’s a concise breakdown of the key steps and principles:




Key Steps in ELISpot:

1. Coating:

   • A monoclonal antibody specific to the target cytokine (or antigen) is pre-coated onto a PVDF-backed microplate.

     
     

2. Cell Stimulation & Incubation:

   • Cells (e.g., T cells, B cells) are added to the wells and stimulated (e.g., with antigens or mitogens).

   • The plate is incubated at 37°C in a CO₂ humidified incubator, allowing cells to secrete the cytokine.

   • The immobilized antibody captures secreted cytokine near the secreting cells.

     
     

3. Detection:

   • After washing away cells and unbound molecules, a biotinylated polyclonal antibody (specific to the same cytokine) is added.

   • Streptavidin-alkaline phosphatase is then added, binding to the biotinylated antibody.

   • A substrate (BCIP/NBT) is added, producing an insoluble blue-black precipitate where cytokine was secreted.

     
     

4. Spot Visualization & Quantification:

   • Each spot represents an individual cytokine-secreting cell.

   • Spots are counted using an automated ELISpot reader or manual microscopy.




Advantages of ELISpot:

• High sensitivity – Can detect low-frequency cytokine-secreting cells.

• Single-cell resolution – Each spot corresponds to one active cell.

• Wide applications – Used in immunology (e.g., vaccine studies, autoimmune diseases, infectious diseases like HIV/TB).




Comparison to ELISA:

• ELISA measures total cytokine concentration in solution.

• ELISpot detects frequency of secreting cells (more functional readout).