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BioTechniques' Protocol Guide 2010
is a collection of detailed, optimized procedures/protocols for use in the lab. With BioTechniques' Protocol Guide, you have access to detailed product information in a downloadable PDF format-which can be easily added to a notebook for ready access. Each protocol is accompanied by a link to the company web site and an e-mail link to contact the company directly.
ELISPOT and Dual ELISPOT: a cell-based assay to assess immune response
Abcam, plc
Sponsored,vendor-submitted protocol    Sponsored by Abcam plc    Published in BioTechniques Protocol Guide 2010 (p.9) DOI: 10.2144/00013302


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.


Figure 1. Dual ELISPOT principle. Cells are incubated in 96-well plates on a membrane previously coated with primary antibodies (mAb capture 1 and 2). Proteins secreted by these cells bind to the closest primary antibody (cytokines 1 and 2). Application of the detection antibodies and subsequent development will result in a colored spot at the site of the secreting cell (spot-forming cell), where one spot = 1 cell.

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


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.


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.


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.


Figure 2. Results from a dual ELISPOT using fluorescence (Fluorospot).Peripheral blood monocytes stimulated with PMA/Ionomycin are detected for secreted human INF-γ and IL-4.

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.

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