Primate IFN-gamma ELISpot Kit

The ELISpot kit is a highly sensitive, microplate-based method for detecting cytokine secreting cells. This kit is used for the detection and counting of primate ifn-γ. The complete ELISpot kit is ready to run and does not require analytical development or improvement.

The ELISpot test uses an antibody that captures primate IFN-gamma, which is pre-coated on a pvdf-supported microtiter plate. The appropriately stimulated cells are pipetted directly into the wells, and the immobilized antibodies near the secreting cells bind to the secreted primate ifn-γ. Following washing steps and incubating with biotinylated detection antibody, alkaline phosphatase bound to streptavidin is added. Then the unbound enzyme is removed by washing, and the substrate solution (BCIP/NBT) is added. A blue-black precipitate is formed at the location where the cytokine is located, and appears in the form of spots. Each spot represents a single primate ifn-γ secreting cell. These spots can be counted manually with an automatic ELISpot reading system or with a stereo microscope.

1.Detect and quantitate individual cells secreting primate IFN-gamma.
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

The enzyme-linked immunospot (ELISpot) assay was originally developed for the detection of individual B cells secreting antigen-specific antibodies. This method has since been adapted for the detection of individual cells secreting specific cytokines or other antigens. ELISpot assays employ the quantitative sandwich enzyme-linked immunosorbent assay(ELISA) technique. An anti-human IFN-γ monoclonal antibody has been pre-coated onto a PVDF (polyvinylidene difluoride)-backed microplate. Appropriately stimulated cells are pipetted into the wells and the microplate is placed into a humidified 37°C CO₂ incubator for a specified period of time. During this incubation period, the immobilized antibody in the immediate vicinity of the secreting cells binds secreted IFN-γ. After washing away any cells and unbound substances, a biotinylated, anti-rhesus macaque IFN- polyclonal antibody is added to the wells. Following a wash to remove any unbound biotinylated antibody, alkalinephosphatase conjugated to streptavidin is added. Unbound enzyme is subsequently removed by washing and a substrate solution (BCIP/NBT) is added. A blue-black colored precipitate forms and appears as spots at the sites of cytokine localization, with each individual spot representing an individual IFN-γ secreting cell. The spots can be counted with an automated ELISpot reader system or manually using a stereomicroscope.

IFN-gamma

Primate

Sandwich

Quantitative Sandwich ELISA

Whole Cells

1 Kit

1.Primate IFN-gamma Microplate.
2.Biotinylated Detection Antibody.
3.Streptavidin conjugated to Alkaline Phosphatase.
4.Dilution Buffers.
5.Wash Buffer Concentrate.
6.BCIP/NBT Chromogen.
7.Primate IFN-gamma Positive Control.

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 15 mins to 4 hours 30 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.Primate Positive Control - Reconstitute the Primate IFN-γ positive Control with 250 μL of culture medium that is used to incubate cells. Mix well.
3.Detection Antibody Mixture - Tap or vortex the vial to release reagent collected in the cap. Transfer 100 μL of Detection Antibody Concentrate 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 100 μL of Streptavidin-AP Concentrate A into the vial labeled Dilution Buffer 2 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. Optimal incubation time for each stimulus should be determined by the investigator.
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 the 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. Remove the flexible plastic underdrain from the bottom of the microplate, wipe the bottom of the plate thoroughly with paper towels and dry completely either at room temperature (60-90 minutes) or 37 °C (15-30 minutes).

Primate

96-well PVDF-backed microplate

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 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.
Some components in this kit contain a preservative which may cause an allergic skin reaction. Avoid breathing mist.

Do not use reagents from this kit with components from ELISpot or ELISA kits and/or components manufactured by other vendors.
Do not remove the flexible plastic underdrain on the bottom of the microplate before or during incubation and development since it may damage the PVDF membrane filters. The underdrain cover may be removed only after completing the incubation with BCIP/NBT chromogen.
Wear protective gloves, clothing, eye, and face protection. Wash hands thoroughly after handling. Refer to the MSDS on our website prior to use.

Store the unopened kit at 2-8 °C.

Do not use past kit expiration date. 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.

IFN-gamma; Interferon gamma; Immune interferon; IFG; IFI

IFNG2

IFN-gamma (Interferon-gamma) is the prototype proinflammatory cytokine and is produced by a variety of immune cells under inflammatory conditions, notably by T cells and NK cells. It plays a key role in host defense by promoting the development and activation of Th1 cells, chemoattraction and activation of monocytes and macrophages, upregulation of antigen presentation molecules, and immunoglobulin class switching in B cells. It also exhibits antiviral, antiproliferative, and apoptotic effects. In addition, IFN-gamma functions as an anti-inflammatory mediator by promoting the development of regulatory T cells and inhibiting Th17 cell differentiation. IFN-gamma dimers signal through a receptor complex of two IFN-gamma R1 and two IFN-gamma R2 subunits.

102128291

P63309

Among its related pathways are Allograft rejection and IL1 and megakaryocytes in obesity.

1.Incubate IFN-γ-secreting cells in an antibody-coated well.
2.Remove cells by washing. Secreted IFN-γis captured by the immobilized antibody.
3.Incubate with biotinylated anti-IFN-γantibody.
4.Incubate with alkaline phosphatase conjugated streptavidin.
5.Add substrate and monitor the formation of colored spots.