Human IL-12/IL-23 p40 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 human IL-12/IL-23 p40. The complete ELISpot kit is ready to run and does not require analytical development or improvement.

The ELISpot detection uses a capture antibody against human IL-12/IL-23 p40, which is pre-coated on a pvdf-supported microplate. The appropriately stimulated cells are pipetted directly into the wells, and the immobilized antibody near the secreting cells binds to the secreted human IL-12/IL-23 p40. 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 cytokine location site and appears in the form of spots. Each spot represents a human IL-12/IL-23 p40 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 IL-12/IL-23 p40.
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 specific for human IL-12/IL-23 p40 has been pre-coated onto a polyvinylidene difluoride (PDVF)-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 bind secreted IL-12/IL-23 p40.After washing away any cells and unbound substances, a biotinylated polyclonal antibody specific for human IL-12/IL-23 p40 is added to the wells. Following a wash to remove any unbound biotinylated antibody, alkaline-phosphatase 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 IL-12/IL-23 p40 secreting cell. The spots can be counted with an ELISpot reader system or using a stereomicroscope.

IL-12/IL-23 p40

Human

Sandwich

Quantitative Sandwich ELISA

Whole Cells

1 Kit

1.Human IL-12/IL-23 p40 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 IL-12/IL-23 p40 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 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.Human IL-12/IL-23 p40 Positive Control - Reconstitute the lyophilized Human IL-12/IL-23 p40 Positive Control with 250 μL of culture medium that is used to incubate cells.
3.Detection Antibody - Tap or vortex the vial to release reagent collected in the cap. Transfer 100μL of Human IL-12/IL-23 p40 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 100μL 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.
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.

CLMF p40; CLMF; CLMF2; Cytotoxic lymphocyte maturation factor 40 kDa subunit; IL12 p40; IL-12 p40; IL-12 subunit p40; IL12B; IL-12B; IL-12BNK cell stimulatory factor chain 2; interleukin 12, p40; interleukin 12B (natural killer cell stimulatory factor 2, cytotoxic lymphocytematuration factor 2, p40); interleukin-12 beta chain; interleukin-12 subunit beta; natural killer cell stimulatory factor, 40 kD subunit; NKSF; NKSF2; NKSF2IL12, subunit p40

CLMF, CLMF2, IL-12B, IMD28, IMD29, NKSF, NKSF2

Interleukin 12 (IL-12), also known as natural killer cell stimulatory factor (NKSF) or cytotoxic lymphocyte maturation factor (CLMF), is a heterodimeric pleiotropic cytokine made up of a 40 kDa (p40) subunit and a 35 kDa (p35) subunit. The IL-12 p40 subunit is shared by IL-23, another heterodimeric cytokine that has biological activities similar to, as well as distinct from, IL-12. IL-12 is produced by macrophages and B cells and has been shown to have multiple effects on T cells and natural killer (NK) cells. While mouse IL-12 is active on both human and mouse cells, human IL-12 is not active on mouse cells.

3593

P29460

Among its related pathways are Tuberculosis and Proteoglycans in cancer.

1.Incubate IL-12/IL-23 p40 secreting cells in an antibody-coated well.
2.Remove cells by washing. Secreted IL-12/IL-23 p40 is captured by the immobilized antibody.
3.Incubate with biotinylated anti-IL-12/IL-23 p40 antibody.
4.Incubate with alkaline phosphatase conjugated streptavidin.
5.Add substrate and monitor the formation of colored spots.
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.