Description
Human IL-13 ELISpot Kit
Assay Genie ELISpot is a highly specific immunoassay for the analysis of IL-13 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 IL-13 producing cells under a given stimulation and the comparison of such frequency against a specific treatment or pathological state. Utilising sandwich immuno-enzyme technology, Assay Genie ELISpot assays can detect both secreted IL-13 (qualitative analysis) and single cells that produce IL-13 (quantitative analysis). Cell secreted IL-13 is captured by coated antibodies avoiding diffusion in supernatant, protease degradation or binding on soluble membrane receptors. After cell removal, the captured IL-13 is revealed by tracer antibodies and appropriate conjugates.
| Product type: | ELISpot Kit |
| Size: | 10 x 96 Assays |
| Target species: | Human |
| Specificity: | Recognizes natural human IL-13 |
| Incubation: | 3h after cell stimulation |
| Kit content: | Assay Genie Pre-coated ELISpot kits include precoated PVDF plates, Detection antibody, Alkaline phosphatase conjugate, BSA, BCIP/NBT ready-to-use substrate buffer. |
| Synonyms: | N/A |
| Uniprot: | P35225 |
A capture antibody highly specific for IL-13 is 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 washed. Cell suspension and stimulant are added and the plate incubated allowing the specific antibodies to bind any IL-13 produced. Cells are then removed by washing prior to the addition of Biotinylated detection antibodies which bind to the previously captured IL-13. 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.
| Step | Procedure |
| 1. | For PVDF membrane activation, add 25 µl 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 100 µl of PBS 1X per well. |
| 4. | Add 100 µl 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 100 µl of PBS 1X per well. |
| 7. | Add 100 µl of blocking buffer 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 100 µl of PBS 1X per well. |
| 10. | Add 100 µl 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). Note: do not agitate or move the plate during this incubation. |
| 12. | Empty the wells and remove excess solution then add 100 µl of Wash Buffer 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 100 µl of Wash Buffer. |
| 15. | Add 100 µl 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 100 µl of Wash Buffer. |
| 18. | Add 100 µl of diluted Streptavidin-conjugate to every well. |
| 19. | Cover the plate and incubate at RT following the supplier's instructions. |
| 20. | Empty the wells and wash the plate 3x with 100 µl of Wash Buffer. |
| 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 100 µl of ready-to-use substrate buffer to every well. |
| 23. | Following the supplier's instructions, 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 Read Spots: allow the wells to dry and then read results. The frequency of the resulting coloured spots. |
| UniProt Protein Function: | IL13: Cytokine. Inhibits inflammatory cytokine production. Synergizes with IL2 in regulating interferon-gamma synthesis. May be critical in regulating inflammatory and immune responses. Defects in IL13 may be a cause of susceptibility to allergic rhinitis (ALRH). Allergic rhinitis is a common disease of complex inheritance and is characterized by mucosal inflammation caused by allergen exposure. Belongs to the IL-4/IL-13 family. |
| UniProt Protein Details: | Protein type:Motility/polarity/chemotaxis; Secreted, signal peptide; Secreted Chromosomal Location of Human Ortholog: 5q31 Cellular Component: extracellular region; extracellular space Molecular Function:interleukin-13 receptor binding; protein binding Biological Process: cell motility; cell-cell signaling; positive regulation of B cell proliferation; positive regulation of immunoglobulin production; positive regulation of macrophage activation; positive regulation of mast cell degranulation Disease: Allergic Rhinitis; Asthma, Susceptibility To |
| NCBI Summary: | This gene encodes an immunoregulatory cytokine produced primarily by activated Th2 cells. This cytokine is involved in several stages of B-cell maturation and differentiation. It up-regulates CD23 and MHC class II expression, and promotes IgE isotype switching of B cells. This cytokine down-regulates macrophage activity, thereby inhibits the production of pro-inflammatory cytokines and chemokines. This cytokine is found to be critical to the pathogenesis of allergen-induced asthma but operates through mechanisms independent of IgE and eosinophils. This gene, IL3, IL5, IL4, and CSF2 form a cytokine gene cluster on chromosome 5q, with this gene particularly close to IL4. [provided by RefSeq, Jul 2008] |
| UniProt Code: | P35225 |
| NCBI GenInfo Identifier: | 239938644 |
| NCBI Gene ID: | 3596 |
| NCBI Accession: | P35225.2 |
| UniProt Secondary Accession: | P35225,O43644, Q4VB52, Q9UDC7, |
| UniProt Related Accession: | P35225 |
| Molecular Weight: | 15,816 Da |
| NCBI Full Name: | Interleukin-13 |
| NCBI Synonym Full Names: | interleukin 13 |
| NCBI Official Symbol: | IL13‚ ‚ |
| NCBI Official Synonym Symbols: | P600; IL-13‚ ‚ |
| NCBI Protein Information: | interleukin-13 |
| UniProt Protein Name: | Interleukin-13 |
| Protein Family: | Interleukin |
| UniProt Gene Name: | IL13‚ ‚ |
| UniProt Entry Name: | IL13_HUMAN |
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