to BioTechniques free email alert service to receive content updates.

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.
PROTOCOLS
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

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

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

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

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.

More Information
 
Do you have question about this protocol? Would you like more information about this protocol?
Submit your questions here and Biotechniques will forward your question and contact information from our database to an expert at Abcam plc who will contact you.
Yes, please forward my question, along with my name, title, company, and e-mail address to Abcam plc
BioTechniques will forward your question to the company, who will contact you directly by email.
Request more information
 
Name Question
Job Title
Company
Email
Abcam plc" />
Contact Information
Protocol Categories
rightcontent.jsp called otherwise condition rightcontent.jsp called 222222222222222222222
Search Protocols
Search our protocols for your specific technique.
Submit Your Protocols
BioTechniques is always looking for author-submitted protocols related to peer-reviewed papers, so submit your protocols today to our editorial team. Suppliers or advertisers interested in publishing their protocols should also submit.
Click here for more information
Popular Immunofluorescence Protocols