Human IL-6 ELISA Kit

Principle of the Assay

This assay employs the quantitative sandwich enzyme immunoassay technique. A monoclonal antibody specific for IL-6 has been pre-coated onto a microplate. Standards and samples are pipetted into the wells and any IL-6 present is bound by the immobilized antibody. Following incubation unbound samples are removed during a wash step, and then a detection antibody specific for IL-6 is added to the wells and binds to the combination of capture antibody- IL-6 in sample. Following a wash to remove any unbound combination, and enzyme conjugate is added to the wells. Following incubation and wash steps a substrate is added. A coloured product is formed in proportion to the amount of IL-6 present in the sample. The reaction is terminated by addition of acid and absorbance is measured at 450nm. A standard curve is prepared from seven IL-6 standard dilutions and IL-6 sample concentration determined.

For Use with serum, plasma and cell culture supernatants. For Research Use Only. Not for use in diagnostic procedures.

Target Information

Interleukin 6(IL-6) is a multifunctional protein produced by lymphoid and non-lymphoid cells and by normal and transformed cells, including T cells, monocyte/macrophages, fibroblasts, hepatocytes, vascular endothelial cells, cardiac myxomas, bladder cell carcinomas, myelomas, astrogliomas and glioblastomas. The production of IL-6 in these cells is regulated, either positively or negatively, by a variety of signals including mitogens, antigenic stimulation, lipopolysaccharides, IL-1, TNF, PDGF and viruses. For reviews on IL-6.

The human IL-6 cDNA sequence predicts a protein of 212 amino acid (aa) residues in length with two potential N-glycosylation sites. The hydrophobic N-terminal 28 aa residue signal peptide is cleaved to produce a mature protein of 184 amino acids with four cysteine residues and a predicted molecular mass of 21 kDa. The mouse IL-6 cDNA sequence shows a homology of 42% at the aa level when compared with the human sequence. On the basis of sequence similarity and gene structural motif similarity, IL-6 can be grouped in a family of cytokines that also includes OSM, G-CSF, LIF, and CNTF. All of these cytokines are predicted to have a four helix bundle structure similar to that found for growth hormone, suggesting that they all evolved from a common ancestral gene.

The effects of IL-6 on different cells are numerous and varied. The effect on B cells is stimulation of differentiation and antibody secretion. IL-6 also affects T cells, acting as a co-stimulant with sub-optimal concentrations of PHA or Con A to stimulate IL-2 production and IL-2 receptor expression.

IL-6 exhibits growth factor activity for mature thymic or peripheral T-cells and reportedly enhances the differentiation of cytotoxic T-cells in the presence of IL-2 or IFN-γ. IL-6 stimulates production of acute phase proteins by hepatocytes and has colony-stimulating activity on hematopoietic stem cells. IL-6 has growth factor activities and will stimulate the growth of myeloma/hybridoma/ plasmacytoma cells, EBV-transformed B cells, keratinocytes and mesangial cells.

GENE ID 3569
SWISS PROT P05231
SYNONYMS CDF; HGF; HSF; BSF2; IL-6; BSF-2; IFNB2; IFN-beta-2

Materials Supplied

Kit Components 96 Wells Quantity/Size
Aluminium pouches with a Microwell Plate coated with monoclonal antibody to human IL-6 (8﹡12) 1 plate
Human IL-6 Standard lyophilized, 1000 pg/ml upon reconstitution 2 vials
Concentrated Biotin-Conjugate anti-human IL-6 monoclonal antibody 2 vials
Streptavidin-HRP solution 2 vials
Standard /sample Diluent 1 bottle
Biotin-Conjugate antibody Diluent 1 bottle
Streptavidin-HRP Diluent 1 bottle
Wash Buffer Concentrate 20x (PBS with 1% Tween-20) 1 bottle
Substrate Solution 1 vial
Stop Solution 1 vial
Adhesive Films 4 pieces
Product data sheet 1 copy

Storage

Storage Store at 2 - 8°C

Performance Characteristics

REPEATABILITY The coefficient of variation of both intra-assay and inter-assay were less than 10%.
SENSITIVITY The minimum detectable dose was 2pg/mL.
ASSAY RANGE 3.90 - 250 pg/mL
SPECIFICITY This assay recognizes both natural and recombinant human IL-6. The factors listed below were prepared at 50ng/ml in Standard /sample Diluent and assayed for cross-reactivity and no significant cross-reactivity or interference was observed.
Factors assayed for cross-reactivity
Recombinant human CD40 Ligand, CT-1, CTLA-4, G-CSF, sgp130, IL-6 sR, IL-6 sR/sgp130, IL-9, IL-10, IL-12, IL-12/23 p40, LIF, LIF R, OSM
Recombinant mouse IL-6, IL-10, IL-11, IL-12, IL-12/23 p40, IL-13
Recombinant rat CNTF, G-CSF, IL-6, OSM