IL-6 ELISpot Assay Kit

$350.00$1,550.00

This IL-6 ELISpot Assay Kit is designed to determine the frequency of IL-6 producing cells under a given stimulation and the comparison of such frequency against a specific treatment or pathological state. The Eagle Biosciences IL-6 ELISpot Assay Kit is for research use only and not to be used for diagnostic procedures.

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IL-6 ELISpot Assay Kit

The IL-6 ELISpot Assay Kit is for research use only.

Incubation Time: 18hrs – 23hrs
Sample Size: 100 µl


Assay Principle

The ELISpot 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, cancerology, infectious disease, autoimmune diseases and transplantation. Utilizing sandwich immuno-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 analyte 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 minimize 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 analytes produced. Cells are then removed by washing prior to the addition of Biotinylated detection antibodies which bind to the previously captured analyte. Enzyme conjugated streptavidin is then added binding to the detection antibodies. Following incubation and washing substrate is then applied to the wells resulting in colored spots which can be quantified using appropriate analysis software or manually using a microscope.


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Additional Information

Assay Background


Interleukin-6 (IL-6) is a multi-functional cytokine that regulates immune responses, acute phase reactions and hematopoiesis and may play a central role in host defense mechanisms. The gene for human IL-6 has been localized to chromosome 7p21. The genomic sequence has been determined. IL-6 is usually not produced constitutively by normal cells, but its expression is readily induced by a variety of cytokines, lipopolysaccharide or viral infections. The IL-6 gene product is a single chain protein with a molecular mass ranging from 21 to 28 kDa, depending on the cellular source. Extensive posttranslational modifications like N- and O-linked glycosylation as well as phosphorylation seem to account for this heterogeneity. The cDNA for IL-6 predicts a precursor protein of 212 amino acids. IL-6 is a pleiotropic cytokine produced by a variety of cells. It acts on a wide range of tissues, exerting growth-induction, growth-inhibition, and differentiation respectively, depending on the nature of the target cells.
IL-6 is involved in – the induction of B-cell differentiation,
– the induction of acute phase proteins in liver cells,
– growth promotion of myeloma / plasmacytoma / hybridoma cells,
– induction of IL-2 and IL-2 receptor expression,
– proliferation and differentiation of T cells,
– inhibition of cell growth of certain myeloid leukemic cell lines and induction of their differentiation to macrophages,
– enhancement of IL-3-induced multipotential colony cell formation in hematopoietic stem cells and induction of maturation of megakaryocytes as a thrombopoietic factor,
– induction of mesangial cell growth,
– induction of neural differentiation of PC 12 cells and
– induction of keratinocyte growth. The abnormal production of IL-6 was first suggested to be related to polyclonal B-cell activation with autoantibody production in patients with cardiac myxoma. Since then, IL-6 has been suggested to be involved in the pathogenesis of a variety of diseases. Measurement of IL-6 levels in serum and other body fluids thus provides more detailed insights into various pathological situations. For Example:
Infections:
Body fluids of patients with acute local bacterial or viral infections and serum of patients with gram-negative or positive bacteremia contain elevated levels of biologically active IL-6.
Obstetric Infections:
IL-6 has emerged as a reporter cytokine for intraamniotic infection.
Diseases associated with an altered immune system (polyclonal B-cell abnormalities or autoimmune diseases): Elevated levels of circulating IL-6 have been detected in patients with cardiac myxoma, Castleman’s disease, rheumatoid arthritis, IgM gammopathy and in those with acquired immunodeficiency syndrome as well as alcoholic liver cirrhosis.
Proliferative diseases:
Elevated plasma levels of IL-6 are observed in patients with psoriasis and mesangial proliferative glomerulonephritis.
Neoplastic Diseases:
Increased systemic levels of IL-6 have been detected in patients with multiple myeloma, other B-cell dyscrasias, Lennert’s T lymphoma, Castleman’s disease, renal cell carcinoma and various other solid tumors.
Inflammatory responses:
IL-6 is involved in the induction of acute phase proteins and induction of fever. Elevated serum levels of IL-6 are also found in patients with severe burns (24, 34), in serum and plasma as a marker for predicting postoperative complications, in serum and urine of recipients of kidney transplants before rejection (35), in the serum of septic shock patients and in patients with inflammatory arthritis and traumatic arthritis.

ELISpot Assay Kit Procedure

Documents

Product Manual