Human IL-17F ELISPOT Kit (with un-coated plates)

Product details

ab105242 is a highly specific immunoassay for the analysis of cytokine and other soluble molecule production and secretion from T-cells at a single cell level in conditions closely comparable to the in-vivo environment with minimal cell manipulation. This technique is designed to determine the frequency of cytokine producing cells under a given stimulation and the comparison of such frequency against a specific treatment or pathological state. The ELISPOT assay constitutes an ideal tool in the investigation of Th1 / Th2 responses, vaccine development, viral infection monitoring and treatment, oncology, infectious disease, autoimmune diseases and transplantation.

Utilising sandwich immune-enzyme technology, ELISPOT assays can detect both secreted cytokines and single cells that simultaneously produce multiple cytokines. Cell secreted cytokines or soluble molecules are captured by coated antibodies avoiding diffusion in supernatant, protease degradation or binding on soluble membrane receptors. After cell removal, the captured cytokines are revealed by tracer antibodies and appropriate conjugates.

A capture antibody highly specific for the molecule of interest is coated to the wells of a PVDF bottomed 96 well microtiter plate either during kit manufacture or in the laboratory. The plate is then blocked to minimise any non-antibody dependent unspecific binding and washed. Cell suspension and stimulant are added and the plate incubated allowing the specific antibodies to bind any molecules produced. Cells are then removed by washing prior to the addition of Biotinylated detection antibodies which bind to the previously captured molecule. Enzyme conjugated streptavidin is then added binding to the detection antibodies. Following incubation and washing substrate is then applied to the wells resulting in coloured spots which can be quantified using appropriate analysis software or manually using a microscope. See Figure 1 for visual scheme.

The assay recognizes natural human IL-17F. After testing, no cross reactivity was observed for IL17A, IL17B, IL17D, IL17E, IL5, IL23 and Perforin. The antibody pair shows cross reactivity with the human IL17A + IL-17F heterodimer.

• Store kit reagents between 4°C.

• Uncoated plates should be stored at RT.

• Immediately after use remaining reagents should be returned to cold storage (4°C).

• All reagents should be warmed to room temperature before use.

• BCIP/NBT buffer is potentially carcinogenic and should be disposed of appropriately, caution should be taken when handling this reagent, always wear gloves.

• Capture Antibody is supplied sterile: once opened keep the vial sterile or aliquot and store at -20^oC. For optimal performance prepare the Capture Antibody dilution immediately before use.

If not used within a short period of time, reconstituted Detection Antibody should be aliquoted and stored at -20°C. In these conditions the reagent is stable for at least one year.

• For optimal performance prepare the Streptavidin-AP dilution immediately prior to use. Do not keep this solution for further experiments.