Human IFNg / IL-17A Dual ELISpot Set

ELISpot is a highly specific immunoassay method for analyzing the production of cytokines and other soluble molecules and the secretion from T cells at the single cell level, with minimal cell manipulation under conditions similar to the in vivo environment.

ELISpot is an ideal tool for studying Th1/Th2 response, vaccine development, viral infection monitoring and treatment, oncology, infectious diseases, autoimmune diseases and transplantation. Using sandwich immunoenzyme technology, LISpot can detect both secreted cytokines and single cells that produce multiple cytokines at the same time. Cytokines or soluble molecules secreted by cells are captured by coating antibodies to avoid diffusion in the supernatant, protease degradation or binding to soluble membrane receptors. After the cells are removed, the captured cytokines are displayed by the tracer antibody and the appropriate conjugate.

ELISpot (ES)

Small volumes of IFNg / IL-17A ELISpot set vial(s) may occasionally become entrapped in the seal of the product vial during shipment and storage. If necessary, briefly centrifuge the vial on a tabletop centrifuge to dislodge any liquid in the container's cap. Certain products may require to ship with dry ice and additional dry ice fee may apply.

Capture antibodies highly specific for the analytes of interest are coated to the wells of a PVDF bottomed 96 well microtitre plate either during kit manufacture or in the laboratory. The plate is then blocked to minimise any non-antibody dependent unspecific binding and finally washed before adding the cells to be investigated. Cell suspension and stimulant are added to the coated and blocked microtitre plate and the plate incubated allowing the specific antibodies to bind any analytes produced. Biotinylated and FITC detection antibodies are then added which bind to the previously captured analyte. HRP conjugated anti- FITC antibodies and Streptavidin Alkaline Phosphatase are added binding to the detection antibodies. Any excess unbound analyte and antibodies are removed by careful washing. Colour substrate is then applied to the wells resulting in coloured spots which can be quantified using appropriate analysis software or manually using microscopes.

IFN-g / IL-17A

Human

Sandwich

Sandwich

cell suspension

Recognizes natural human IL-17A and IFN-g

No cross reactivity with other human cytokines. Cross reactivity with simian IFN-g.

1x96 Wells

1.96 well PVDF bottomed plates (5 if ordered).
2.Capture Antibody for human IFNγ (0.5ml supplied sterile).
3.Capture Antibody for human IL-17A (0.5ml supplied sterile).
4.FITC conjugated detection antibody for IFNγ (lyophilised, resuspend in 0.55ml).
5.Biotinylated detection antibody for IL-17A (lyophilised, resuspend in 0.55ml).
6.Anti-FITC antibody HRP conjugated (100µl).
7.Streptavidin-Alkaline Phosphatase conjugate (50µl).
8.Bovine Serum Albumin (BSA).
9.50x Concentrated AEC substrate buffer (1ml).
10.10x Concentrated dilution buffer for AEC substrate (5ml).
11.Ready to use BCIP/NBT substrate buffer (50ml).

1.Miscellaneous laboratory plastic and/or glass, if possible sterile.
2.Ethanol.
3.Cell culture reagents.
4.Cell stimulation reagents.
5.CO2 incubator.
6.Tween 20.
7.Phosphate Buffered Saline (PBS).
8.96 well PVDF bottomed plates if not ordered.

100 µL

1.5h

Plate-not-included

1.1X Phosphate Buffered Saline (PBS) (Coating Buffer).
2.Cell culture media + 10% Serum (Blocking Buffer):For one plate add 1ml Serum (e.g. FCS) to 9ml of culture media (use same cell culture medium as used to derive the cell suspension).
3.1% BSA PBS Solution (Dilution Buffer):For one plate dissolve 0.2 g of BSA in 20 ml of 1X PBS.
4.0.05% PBS-T Solution (Wash Buffer):For one plate dissolve 50μl of Tween 20 in 100 ml of 1X PBS.
5.35% Ethanol (PVDF Membrane Activation Buffer):For one plate mix 3.5 ml of ethanol with 6.5 ml of distilled water.
6.Capture Antibody:Dilute 100μl of capture antibody in 10 mL of 1X PBS and mix well.
7.Detection Antibody:Reconstitute the lyophilised antibody with 0.55mL of distilled water. Gently mix the solution and wait until all the lyophilised material is back into solution.
8.Streptavidin - AP conjugate:Dilute in Dilution buffer according to the instructions of the supplier.
9.AEC Buffer:For one plate mix 1 ml of AEC buffer A 10X with 9 ml of distilled water. Then add 200µl of AEC buffer B 50X .

1.Add 25μl of 35% ethanol to every well.
2.Incubate plate at room temperature (RT) for 30 seconds.
3.Empty the wells by flicking the plate over a sink & gently tapping on absorbent paper. Thoroughly wash the plate 3x with 100μl of 1X PBS per well.
4.Add 100μl of diluted capture antibody to every well.
5.Cover the plate and incubate at 4°C overnight.
6.Empty the wells as previous and wash the plate once with 100μl of 1X PBS per well.
7.Add 100μl of culture media with 10% serum to every well.
8.Cover the plate and incubate at RT for 2 hours.
9.Empty the wells as previous and thoroughly wash once with 100μl of 1X PBS per well.
10.Add 100μl of sample, positive and negative controls cell suspension to appropriate wells providing the required concentration of cells and stimulant.
11.Cover the plate and incubate at 37°C in a CO2 incubator for an appropriate length of time (15-20 hours).
12.Empty the wells and remove excess solution then add 100μl of PBS-T to every well.
13.Incubate the plate at 4°C for 10 min.
14.Empty the wells as previous and wash the plate 3x with 100μl of PBS-T.
15.Add 100μl of diluted detection antibody to every well.
16.Cover the plate and incubate at RT for 1 hour 30 min.
17.Empty the wells as previous and wash the plate 3x with 100μl of PBS-T.
18.Add 100μl of diluted Streptavidin-AP conjugate to every well.
19.Cover the plate and incubate at RT.
20.Empty the wells and wash the plate 3x with 100μl of PBS-T.
21.Peel of the plate bottom and wash both sides of the membrane 3x under running distilled water, once washing complete remove any excess solution by repeated tapping on absorbent paper.
22.Add 100µl of prepared AEC buffer to every well.
23.Incubate the plate for 5-15 min monitoring spot formation visually throughout the incubation period to assess sufficient colour development.
24.Empty the wells and rinse both sides of the membrane 3x under running distilled water. Completely remove any excess solution by gentle repeated tapping on absorbent paper.
25.Add 100µl of ready to use BCIP/NBT buffer to every well.
26.Empty the wells and rinse both sides of the membrane 3x under running distilled water. Completely remove any excess solution by gentle repeated tapping on absorbent paper.

Human

Liquid

1.For research use only not to be used as a diagnostic test.
2.Do not eat, drink, smoke or apply cosmetics where kit reagents are used.
3.Cover or cap all reagents when not in use.
4.Do not mix or interchange reagents between different lots.
5.Do not use reagents beyond the expiration date of the kit.
6.When pipetting reagents, maintain a consistent order of addition from well-to-well. This will ensure equal incubation times for all wells.
7.BCIP/NBT buffer is potentially carcinogenic and should be disposed of appropriately, caution should be taken when handling this reagent, always wear gloves.

1.Handling of reagents, serum or plasma specimens should be in accordance with local safety procedures.
2.When not in use, kit components should be stored refrigerated or frozen as indicated on vials or bottles labels.
3.All reagents should be warmed to room temperature before use.
4.Use a clean disposable plastic pipette tip for each reagent, standard, or specimen addition in order to avoid cross contamination.
5.Use a clean plastic container to prepare the washing solution.
6.Thoroughly mix the reagents and samples before use by agitation or swirling.

Store kit reagents between 2 and 8°C.

Immediately after use remaining reagents should be returned to cold storage (2 to 8°C). The expiry of the kit components can only be guaranteed if the components are stored properly, and if in the case of repeated use of one component, the reagent is not contaminated by the first handling.

Expiry of the kit and reagents is stated on box front labels.

Select online data sheet information is drawn from bioinformatics databases, occasionally resulting in ambiguous or non-relevant product information. It is the responsibility of the customer to review, verify, and evaluate the information to make sure it matches their requirements before purchasing the kit. Our ELISA Kit assays are dynamic research tools and sometimes they may be updated and improved.

For Research Use Only. Not for use in diagnostic procedures.

IFN-g / IL-17A

1.96 PVDF-bottomed-well plates are first treated with 35% ethanol and then coated with anti-IFNγ and anti-IL17A capture antibodies.
2.Cells are incubated in the presence of the antigen. Upon stimulation they release cytokines which bind to the capture antibodies.
3.Anti-IFNγ-FITC and anti-IL-17A biotin detection antibodies are added and bind to the captured cytokines.
4.Detection antibodies are in turn bound by anti-FITCHRP for IFNγ and Streptavidin-AP for IL-17A.
5.Finally coloured spots are developed by separate incubations with first AEC and then BCIP/NBT substrate buffers. Cells producing IFNγ give red/brownish spots while those producing IL-17A give blue/purple spots.