Human IFN-gamma/IL-13 Dual-Color ELISpot Kit

The ELISpot kit is a highly sensitive, microplate-based method for detecting cytokine secreting cells. The kit is designed for simultaneous detection and enumeration of IFN-gamma/IL-13. The complete ELISpot kit is ready to run, no test development or improvement is required.

Pipette the appropriately stimulated cells directly into the microplate supported by pvdf. The PVDF membrane has been pre-coated with capture antibody against IFN-gamma/IL-13, and then the microplate is placed in a humidified CO2 incubator at 37°C for the required time. During this incubation period, the immobilized capture antibody near the secreting cell binds to the secreted IFN-gamma/IL-13.
At the desired time point, an analyte is detected using a biotinylated detection antibody and alkaline phosphatase (AP) bound to streptavidin. Horseradish peroxidase (HRP)-conjugated detection antibody is used to detect the second analyte. After incubating with BCIP/NBT (AP substrate, blue-black precipitate) and AEC (HRP substrate, red precipitate), spots formed, indicating the presence of IFN-gamma/IL-13 secreting cells. Each dot represents a single secreting cell. Spots can be counted using the ELISpot reading system or using a stereo microscope.

1.Detect and quantitate individual cells secreting IFN-gamma/IL-13.
2.High sensitivity - ELISpot assays can measure responses with frequencies well below 1 in 100,000 cells.
3.No in vitro expansion of cells required.
4.High-throughput - ELISpot assays use only a small number of primary cells.

ELISpot

A monoclonal antibody against human IFN-γ and a polyclonal antibody against human IL-13 are pre-coated on polyvinylidene fluoride (PVDF) supported microplates. Pipette appropriately stimulated cells into the wells, and place the microplate in a humidified CO2 incubator at 37°C for a period of time. During this incubation period, immobilized antibodies near the secreting cells bind to secreted IFN-γ and IL-13. After washing away all cells and unbound materials, biotinylated human IL-13 polyclonal antibody and horseradish peroxidase-labeled human IFN-γ polyclonal antibody were added to the wells. After washing to remove any unbound antibody, alkaline phosphatase bound to streptavidin is added. Then the unbound enzyme is removed by washing, and the substrate solution (BCIP/NBT) is added. After flushing the BCIP/NBT in the well with deionized water, add the AEC developer solution to the well. At the location of the cytokine, a blue-black precipitate formed and appeared in the form of spots, each spot representing an IL-13 secreting cell. A red precipitate will also form and appear in the form of spots, each of which represents a single IFN-γ secreting cell. Spots can be counted using the ELISpot reading system or using a stereo microscope.

IFN-gamma/IL-13

Human

Sandwich

Quantitative Sandwich ELISA

Whole Cells

1 Kit

1.Human IFN-gamma/IL-13 Microplate.
2.Biotin-conjugated Detection Antibody.
3.HRP-conjugated Detection Antibody.
4.Streptavidin-conjugated to Alkaline Phosphatase.
5.Dilution Buffers.
6.Wash Buffer Concentrate.
7.BCIP/NBT Chromogen.
8.AEC Chromogen.
9.Human IFN-gamma/IL-13 Positive Controls.

1.Pipettes and pipette tips.
2.Deionized or distilled water.
3.Squirt bottle, manifold dispenser, or automated microplate washer.
4.500 mL graduated cylinder.
5.37 °C CO2 incubator.
6.Sterile culture media.
7.Dissection microscope or an automated ELISpot reader.

100 µL

3 hours 35 mins to 4 hours 50 mins**

Pre-coated

1.Wash Buffer - If crystals have formed in the concentrate, warm to room temperature and mix gently until the crystals have completely dissolved. To prepare Wash Buffer, add 50 mL of Wash Buffer Concentrate to 450 mL of deionized water and mix well.
2.Positive Controls - Reconstitute the lyophilized Human IFN-γ Positive Control and Human IL-13 Positive Control with 250μL of culture medium that is used to incubate cells.
3.Detection Antibody Mixture (Concentrate A + Concentrate B) - Tap or vortex each vial to release reagent collected in the cap. Transfer 100μL of Detection Antibody Concentrate A and 200μL of Detection Antibody Concentrate B into the vial labeled Dilution Buffer 1 and mix well.
4.Streptavidin-AP Concentrate A - Tap or vortex the vial to release reagent collected in the cap. Transfer 100μL of Streptavidin-AP Concentrate A into the vial labeled Dilution Buffer 2 and mix well.
5.AEC Chromogen Solution - Transfer 250μL of AEC Chromogen to the vial labeled AEC Chromogen Buffer and mix well.

1.Fill all wells in the microplate with 200μL of sterile culture media and incubate for approximately 20 minutes at room temperature.
2.When cells are ready to be plated, aspirate the culture media from the wells. Immediately add 100μL of the appropriate cells or controls to each well.
3.Incubate cells in a humidified 37°C CO2 incubator.
4.Aspirate each well and wash, repeating the process three times for a total of four washes.
5.Add 100μL of the diluted Detection Antibody Mixture into each well, and incubate overnight at 2-8°C. Alternatively, incubation with detection antibodies can be done for 2 hours at room temperature on a rocking platform.
6.Repeat the wash procedure described in step 4.
7.Add 100 μL of diluted Streptavidin-AP Concentrate A into each well, and incubate for 2 hours at room temperature.
8.Repeat the wash procedure described in step 4.
9.Add 100μL of the BCIP/NBT Substrate into each well, and incubate for 1 hour at room temperature. Protect from light.
10.Decant the BCIP/NBT Substrate from the microplate and rinse the microplate with deionized water. Invert the microplate and tap to remove excess water.
11.Add 100 μL of AEC Chromogen Solution into each well, and incubate for 20 minutes at room temperature. Protect from light.
12.Decant the AEC Chromogen Solution from the microplate, and rinse the microplate with deionized water. Invert the microplate, and tap to remove excess water. Remove the flexible plastic underdrain from the bottom of the microplate, wipe the bottom of the microplate thoroughly with paper towels, and dry completely either at room temperature (60-90 minutes) or 37°C (15-30 minutes).

96-well PVDF-backed microplate

Some components of this kit contain sodium azide, which may react with lead and copper plumbing to form explosive metallic azides. Flush with large volumes of water during disposal.
AEC Chromogen may cause skin, eye, and respiratory irritation. Avoid breathing fumes.
BCIP/NBT is toxic if swallowed, in contact with skin, or if inhaled. It is a highly flammable liquid and vapor may cause serious irritation and damage to organs. Do not eat, drink, or smoke when using this product. Do not breathe fumes. Use only in a well-ventilated area. Keep away from heat, sparks, open flames, and hot surfaces. Keep the container tightly closed.

Some components in this kit contain a preservative which may cause an allergic skin reaction. Avoid breathing mist.
Wear protective gloves, clothing, eye, and face protection. Wash hands thoroughly after handling. Refer to the SDS on our website prior to use.

Store the unopened product at 2 - 8 °C.

Do not use past kit expiration date. This kit is validated for single use only.

This kit is validated for single use only. Results obtained using previously opened or reconstituted reagents may not be reliable.

For Research Grade Use Only.

1.Two different proteins secreted by cultured cells bind to the capture antibodies coated on the well.
2.Remove cells by washing. Add the biotinylated detection antibody which binds to the FIRST captured protein and the HRP-conjugated detection antibody which binds to the SECOND captured protein.
3.Wash to remove any unbound antibodies.
4.Add the alkaline phosphatase-conjugated streptavidin
5.Wash to remove unbound enzyme.
6.Add BCIP/NBT Substrate to develop the blue spots representing the FIRST protein. After finishing incubation with the BCIP/NBT Substrate, wash the wells with deionized water and add AEC Chromogen to develop the red spots representing SECOND protein. Analyze using either an ELISpot reader capable of detectiong blue and red spots or a dissection mircoscope.