Sponsored,vendor-submitted protocol Sponsored by Abcam plc Published in BioTechniques Protocol Guide 2010 (p.9) DOI: 10.2144/00013302
Abstract
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 (ab48724; Abcam). The 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 (Cat. no. 468667; Nunc Maxisorp)
Peripheral blood monocytes
ELISPOT reader
Method
Coat 96-well plate
1. Incubate polyvinylidene fluoride–bottomed well plates with 25 L 70% ethanol for 30 s at RT.
2. Empty wells and wash three times with 100 L/well PBS, pH 7.4.
3. Add 100 L INF-γ capture antibody and 100 L IL-4 capture antibody in 10 mL of PBS. Mix and dispense 100 L per well, cover the plate, and incubate overnight at +4°C.
4. Empty wells and wash once with 100 L PBS.
Blocking
5. Add 100 L/well 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 L/well peripheral blood monocytes containing between 1 × 105 and 2 × 105 cells per 100 L 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 L 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 L INF-γ detection antibody and 100 L reconstituted IL-4 detection into 10 ml PBS containing 1% BSA. Distribute 100 L 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 L 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 above (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 then be quantified.
