The ELISPOT assay is a highly sensitive technique to study cell responses to various drugs, stimuli, and inhibitors and allows the detection of individual cells secreting a particular protein. ELISPOT is often used to investigate the Th1/Th2 response, cancer, vaccine development, viral infection monitoring, infectious diseases, autoimmune diseases and transplantation, among others. The relationship between the expression of two cytokines can also be observed with a dual ELISPOT. Figure 1 shows the principle of a dual ELISPOT using fluorescence detection.

The ELISPOT assay is a highly sensitive technique to study cell responses to various drugs, stimuli, and inhibitors and allows the detection of individual cells secreting a particular protein. ELISPOT is often used to investigate the Th1/Th2 response, cancer, vaccine development, viral infection monitoring, infectious diseases, autoimmune diseases and transplantation, among others. The relationship between the expression of two cytokines can also be observed with a dual ELISPOT. Figure 1 shows the principle of a dual ELISPOT using fluorescence detection.

Materials

Human Dual INF-γ + IL4 Fluorospot Kit (Abcam, ab48724)

Kit includes capture antibodies for interferon gamma and IL-4, FITC-conjugated detection antibody for INF-γ and biotinylated detection antibody for IL-4, anti-FITC antibody green fluorescence conjugate, and streptavidin-phycoerythrin conjugate, bovine serum albumin, and dry skimmed milk.

15 × 96-well Polysterene Plate (Nunc Maxisorp, Cat# 468667)

Peripheral Blood Monocytes

ELISPOT Reader

Figure 1. Dual ELISPOT principle. (Click to enlarge)

Method Coat 96-well Plate

1. Incubate PVDF-bottomed well plates with 25 µl of 70% ethanol for 30 s at RT.

2. Empty wells and wash three times with 100 µl/well of PBS pH 7.4.

3. Add 100 µl of INF-γ capture antibody and 100 µl of IL-4 capture antibody in 10 mL of PBS. Mix and dispense 100 µl per well, cover the plate, and incubate overnight at +4°C.

4. Empty wells and wash once with 100 µl of PBS. Blocking

5. Add 100 µl/well of 2% dry skimmed milk in PBS into wells, cover, and incubate for 2 h at RT.

6. Empty wells, tapping them dry, and wash plate once with PBS. Cell Stimulation

7. Add 100 µl/well of peripheral blood monocytes containing between 1 × 105 and 2 × 10⁵ cells per 100 µl and appropriate concentration of stimulator.

8. Cover the plate with a standard 96-well plate plastic lid and incubate cells at 37°C in a CO₂ incubator for 15–20 h. During this period do not disturb the plate. Detection

9. Empty the wells, tapping them dry, and dispense 100 µl of PBS-0.1% Tween 20 into wells. Incubate for 10 min at +4°C.

10. Wash wells three times with PBS-0.1% Tween 20.

11. Mix 100 µl of INF-γ detection antibody and 100 µl of reconstituted IL-4 detection into 10 ml of PBS containing 1% BSA. Distribute 100 µl into wells, cover the plate, and incubate 90 min at 37°C.

12. Empty wells and wash three times with PBS-0.1% Tween 20.

13. Add 100 µl of anti-FITC green fluorescence conjugate/Streptavidin-phycoerythrin solution in each well. Seal the plate and incubate for 1 h at 37°C.

14. Empty wells and wash three times with PBS-0.1% Tween 20. Remove all residual buffer.

15. Read spots on an ELISPOT reader under a UV light source.

16. Store the plate at +4°C away from light. Results

Colored spots generated represent individual secreting cells. In the example below (Figure 2), peripheral blood monocytes have been stimulated by PMA/ionomycin: green spots represent IFN-γ-secreting cells, red spots show IL-4-secreting cells, and orange spots identify IFN-γ/IL-4-secreting cells. An ELISPOT reader is then used to scan, count, and analyze the Spot Forming Cells. Secreting cells can be then quantified.

Figure 2. Results from a dual ELISPOT using fluorescence. (Click to enlarge)

For more information on ELISPOT: www.abcam.com/elispot.

Correspondence

Address correspondence to Abcam, plc, [email protected]; [email protected]; Europe +441223 696000; USA and Canada (866) 739-9884. www.abcam.com/elispot.