Human IL-10 ELISpot Pair



ELISA Kit Technical ManualMSDS

Human IL-10 ELISpot Pair

Assay Genie ELISpot is a highly specific immunoassay for the analysis of IL-10 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-10 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-10 (qualitative analysis) and single cells that produce IL-10 (quantitative analysis). Cell secreted IL-10 is captured by coated antibodies avoiding diffusion in supernatant, protease degradation or binding on soluble membrane receptors. After cell removal, the captured IL-10 is revealed by tracer antibodies and appropriate conjugates.

Human IL-10 ELISpot Pair - Kit Principle

A capture antibody highly specific for IL-10 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-10 produced. Cells are then removed by washing prior to the addition of Biotinylated detection antibodies which bind to the previously captured IL-10. 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.

Figure: Schematic representation of the principle of ELISpot. 1) Treat with ethanol, coat with capture antibody. 2) Incubate cells in the presence of stimuli, cytokines, are released, which bind to capture antibodies. 3) Wash plate. Incubate with biotinylated detection antibody. 4) Wash plate. Incubate with streptavidin-alkaline phosphatase conjugated. 5)Incubate with BCIP/NBT. Precipitated product forms, results in blue/purple spots.


Step Procedure
1. Add 100 µl of PBS 1X to every well.
2. Incubate plate at room temperature (RT) for 10 min.
2. Incubate plate at room temperature (RT) for 10 min.
3. Empty the wells by flicking the plate over a sink & gently tapping on absorbent paper.
3. Empty the wells by flicking the plate over a sink & gently tapping on absorbent paper.
4. Add 100 µl of sample, positive and negative controls cell suspension to appropriate wells providing the required concentration of cells and stimulant.
5. 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.
6. Empty the wells and remove excess solution then add 100 µl of Wash Buffer to every well.
7. Incubate the plate at 4°C for 10 min.
8. Empty the wells as previous and wash the plate 3x with 100 µl of Wash Buffer.
9. Add 100 µl of diluted detection antibody to every well.
10. Cover the plate and incubate at RT for 1 hour 30 min.
11. Empty the wells as previous and wash the plate 3x with 100 µl of Wash Buffer.
12. Add 100 µl of diluted Streptavidin-AP conjugate to every well.
13. Cover the plate and incubate at RT for 1 hour.
14. Empty the wells and wash the plate 3x with 100 µl of Wash Buffer.
15. Peel of the plate bottom and wash both sides of the membrane 3x under running distilled water, once washing is complete remove any excess solution by repeated tapping on absorbent paper.
16. Add 100 µl of ready-to-use BCIP/NBT buffer to every well.
17. Incubate the plate for 5-15 min monitoring spot formation visually throughout the incubation period to assess sufficient colour development.
18. 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.
19. Read Spots: allow the wells to dry and then read results. The frequency of the resulting coloured spots corresponding to the cytokine producing cells can be determined using an appropriate ELISpot reader and analysis software or manually using a microscope. Note: spots may become sharper after overnight incubation at 4°C in the dark.

Human IL-10 ELISpot Pair - Information

Product type: ELISpot Pairs
Target species: Human
Specificity: Recognizes natural human IL-10
Kit content: Assay Genie ELISpot matched antibody pairs are extensively validated and include pre-titrated capture antibody and biotinylated detection antibody. Antibodies are supplied in quantities sufficient for 10 x 96 samples.
Synonyms: N/A
Uniprot: P22301

IL-10 - Protein Information

UniProt Protein Function: IL10: Inhibits the synthesis of a number of cytokines, including IFN-gamma, IL-2, IL-3, TNF and GM-CSF produced by activated macrophages and by helper T-cells. Belongs to the IL-10 family.
UniProt Protein Details:

Protein type:Motility/polarity/chemotaxis; Cytokine; Secreted, signal peptide; Secreted

Chromosomal Location of Human Ortholog: 1q31-q32

Cellular Component: extracellular space

Molecular Function:growth factor activity; cytokine activity; interleukin-10 receptor binding

Biological Process: B cell proliferation; negative regulation of chronic inflammatory response to antigenic stimulus; regulation of isotype switching; positive regulation of transcription, DNA-dependent; leukocyte chemotaxis; response to glucocorticoid stimulus; positive regulation of JAK-STAT cascade; negative regulation of B cell proliferation; negative regulation of membrane protein ectodomain proteolysis; response to insulin stimulus; negative regulation of interferon-alpha biosynthetic process; negative regulation of T cell proliferation; cytoplasmic sequestering of NF-kappaB; cell-cell signaling; positive regulation of MHC class II biosynthetic process; negative regulation of interferon-gamma production; response to molecule of bacterial origin; negative regulation of interleukin-6 production; hemopoiesis; negative regulation of tumor necrosis factor biosynthetic process; inflammatory response; aging; response to drug; response to inactivity; negative regulation of interleukin-12 production; positive regulation of B cell apoptosis; T-helper 2 type immune response; negative regulation of myeloid dendritic cell activation; negative regulation of nitric oxide biosynthetic process; negative regulation of cytokine secretion during immune response; regulation of gene expression; B cell differentiation; defense response to bacterium; negative regulation of MHC class II biosynthetic process; positive regulation of transcription from RNA polymerase II promoter; positive regulation of transcription factor activity; receptor biosynthetic process; response to activity; regulation of sensory perception of pain; negative regulation of apoptosis; positive regulation of cytokine secretion

Disease: Graft-versus-host Disease, Susceptibility To; Rheumatoid Arthritis; Human Immunodeficiency Virus Type 1, Susceptibility To

NCBI Summary: The protein encoded by this gene is a cytokine produced primarily by monocytes and to a lesser extent by lymphocytes. This cytokine has pleiotropic effects in immunoregulation and inflammation. It down-regulates the expression of Th1 cytokines, MHC class II Ags, and costimulatory molecules on macrophages. It also enhances B cell survival, proliferation, and antibody production. This cytokine can block NF-kappa B activity, and is involved in the regulation of the JAK-STAT signaling pathway. Knockout studies in mice suggested the function of this cytokine as an essential immunoregulator in the intestinal tract. Mutations in this gene are associated with an increased susceptibility to HIV-1 infection and rheumatoid arthritis.[provided by RefSeq, May 2011]
UniProt Code: P22301
NCBI GenInfo Identifier: 124292
NCBI Gene ID: 3586
NCBI Accession: P22301.1
UniProt Related Accession: P22301
Molecular Weight: 20,517 Da
NCBI Full Name: Interleukin-10
NCBI Synonym Full Names: interleukin 10
NCBI Official Symbol: IL10  
NCBI Official Synonym Symbols: CSIF; TGIF; GVHDS; IL-10; IL10A  
NCBI Protein Information: interleukin-10; T-cell growth inhibitory factor; cytokine synthesis inhibitory factor
UniProt Protein Name: Interleukin-10
UniProt Synonym Protein Names: Cytokine synthesis inhibitory factor; CSIF
Protein Family: Interleukin
UniProt Gene Name: IL10  
UniProt Entry Name: IL10_HUMAN
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