Human IL-1 beta ELISpot Pair

SKU:
HUDC0170
€670

Description

ELISA Kit Technical ManualMSDS

Human IL-1β ELISpot Pair

Assay Genie ELISpot is a highly specific immunoassay for the analysis of IL-1β 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-1β 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-1β (qualitative analysis) and single cells that produce IL-1β (quantitative analysis). Cell secreted IL-1β is captured by coated antibodies avoiding diffusion in supernatant, protease degradation or binding on soluble membrane receptors. After cell removal, the captured IL-1β is revealed by tracer antibodies and appropriate conjugates.

Human IL-1β ELISpot Pair - Kit Principle

A capture antibody highly specific for IL-1β 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-1β produced. Cells are then removed by washing prior to the addition of Biotinylated detection antibodies which bind to the previously captured IL-1β. 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-1β ELISpot Pair - Information

Product type: ELISpot Pairs
Target species: Human
Specificity: Recognizes natural human IL-1β
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: IL-1b, IL-1beta
Uniprot: P01584

IL-1β - Protein Information

UniProt Protein Function:IL1B: Produced by activated macrophages, IL-1 stimulates thymocyte proliferation by inducing IL-2 release, B-cell maturation and proliferation, and fibroblast growth factor activity. IL-1 proteins are involved in the inflammatory response, being identified as endogenous pyrogens, and are reported to stimulate the release of prostaglandin and collagenase from synovial cells. Monomer. Belongs to the IL-1 family.
UniProt Protein Details:

Protein type:Cytokine

Chromosomal Location of Human Ortholog: 2q14

Cellular Component: extracellular space; extracellular region; cytosol; secretory granule

Molecular Function:protein domain specific binding; interleukin-1 receptor binding; cytokine activity

Biological Process: positive regulation of granulocyte macrophage colony-stimulating factor production; negative regulation of MAP kinase activity; positive regulation of nitric oxide biosynthetic process; activation of MAPK activity; positive regulation of transcription, DNA-dependent; positive regulation of interleukin-2 biosynthetic process; germ cell programmed cell death; negative regulation of insulin receptor signaling pathway; positive regulation of lipid catabolic process; positive regulation of NF-kappaB import into nucleus; fever; positive regulation of membrane protein ectodomain proteolysis; response to carbohydrate stimulus; activation of NF-kappaB transcription factor; cell-cell signaling; positive regulation of phagocytosis; positive regulation of T cell mediated immunity; positive regulation of T cell proliferation; neutrophil chemotaxis; positive regulation of I-kappaB kinase/NF-kappaB cascade; positive regulation of heterotypic cell-cell adhesion; positive regulation of mitosis; smooth muscle adaptation; interleukin-1 beta production; positive regulation of interleukin-6 production; positive regulation of angiogenesis; positive regulation of transcription from RNA polymerase II promoter; positive regulation of transcription factor activity; negative regulation of lipid metabolic process; sequestering of triacylglycerol; positive regulation of histone phosphorylation; apoptosis; positive regulation of interleukin-6 biosynthetic process; positive regulation of JNK cascade; signal transduction; positive regulation of vascular endothelial growth factor receptor signaling pathway; positive regulation of protein export from nucleus; positive regulation of interleukin-8 production; negative regulation of cell proliferation; negative regulation of lipid catabolic process; hyaluronan biosynthetic process; protein kinase B signaling cascade; lipopolysaccharide-mediated signaling pathway; inflammatory response; regulation of I-kappaB kinase/NF-kappaB cascade; cytokine and chemokine mediated signaling pathway; MAPKKK cascade; response to ATP; positive regulation of interferon-gamma production; positive regulation of chemokine biosynthetic process; positive regulation of prostaglandin secretion; positive regulation of fever; positive regulation of histone acetylation; immune response; positive regulation of protein amino acid phosphorylation; regulation of insulin secretion; embryo implantation

Disease: Gastric Cancer, Hereditary Diffuse

NCBI Summary:The protein encoded by this gene is a member of the interleukin 1 cytokine family. This cytokine is produced by activated macrophages as a proprotein, which is proteolytically processed to its active form by caspase 1 (CASP1/ICE). This cytokine is an important mediator of the inflammatory response, and is involved in a variety of cellular activities, including cell proliferation, differentiation, and apoptosis. The induction of cyclooxygenase-2 (PTGS2/COX2) by this cytokine in the central nervous system (CNS) is found to contribute to inflammatory pain hypersensitivity. This gene and eight other interleukin 1 family genes form a cytokine gene cluster on chromosome 2. [provided by RefSeq, Jul 2008]
UniProt Code:P01584
NCBI GenInfo Identifier:62906858
NCBI Gene ID:3553
NCBI Accession:P01584.2
UniProt Secondary Accession:P01584,Q53X59, Q53XX2, Q7M4S7, Q7RU01, Q96HE5, Q9UCT6
UniProt Related Accession:P01584
Molecular Weight:30,748 Da
NCBI Full Name:Interleukin-1 beta
NCBI Synonym Full Names:interleukin 1, beta
NCBI Official Symbol:IL1BÂ Â
NCBI Official Synonym Symbols:IL-1; IL1F2; IL1-BETAÂ Â
NCBI Protein Information:interleukin-1 beta; IL-1 beta; catabolin; preinterleukin 1 beta; pro-interleukin-1-beta
UniProt Protein Name:Interleukin-1 beta
UniProt Synonym Protein Names:Catabolin
Protein Family:Interleukin
UniProt Gene Name:IL1BÂ Â
UniProt Entry Name:IL1B_HUMAN
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Additional Information

Product type:
ELISpot
Reactivity:
Human
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