Human IL-13 ELISpot Pair

SKU:
HUDC0166
€670

Description

ELISA Kit Technical ManualMSDS

Human IL-13 ELISpot Pair

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.

Human IL-13 ELISpot Pair - Kit Principle

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.

ELISpot Protocol
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.

Protocol

StepProcedure
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-13 ELISpot Pair - Information

Product type: ELISpot Pairs
Target species: Human
Specificity: Recognizes natural human IL-13
Incubation:
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: P35225

IL-13 - Protein Information

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:IL13Â Â
NCBI Official Synonym Symbols:P600; IL-13Â Â
NCBI Protein Information:interleukin-13
UniProt Protein Name:Interleukin-13
Protein Family:Interleukin
UniProt Gene Name:IL13Â Â
UniProt Entry Name:IL13_HUMAN
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Additional Information

Product type:
ELISpot
Reactivity:
Human
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