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.

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.

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.

Figure 1:Schematic diagram of the assay

1. Aluminium pouches with a Microwell Plate coated with antibody to human IL-6 (8 x 12)

2. 2 vials human IL-6 Standard lyophilized, 1000 pg/ml upon reconstitution

3. 2 vials concentrated Biotin-Conjugate anti-human IL-6 antibody

4. 2 vials Streptavidin-HRP solution

5. 1 bottle Standard /sample Diluent

6. 1 bottle Biotin-Conjugate antibody Diluent

7. 1 bottle Streptavidin-HRP Diluent

8. 1 bottle Wash Buffer Concentrate 20x (PBS with 1% Tween-20)

9. 1 vial Substrate Solution

10. 1 vial Stop Solution

11. 4 pieces Adhesive Films

12. package insert

NOTE: [96 Tests]

Unopened Kit：Store at 2 -8° C. Do not use past kit expiration date. opened/ReconstitutedReagents：Please refer to the datasheets for detail information.

1. Kishimoto, T. et al. (1992) Science 258:5593.

2. Kishimoto, T. (1992) Int. Arch. Allergy Immunol. 99:172.

3. Hirano, T. et al. (1990) Immunol. Today 11:443.

4. Hirano, T. (1992) Clin. Immunol. Immunopathol. 62:S60.

5. Hirano, T. et al. (1986) Nature 324:73.

6. Haegeman, G. et al. (1986) Eur. J. Biochem. 159:625.

7. Van Snick, J. et al. (1988) Eur. J. Immunol. 18:193.

8. Okada, M. et al. (1983) J. Exp. Med. 157:583.

9. Lotz, M. et al. (1988) J. Exp. Med. 167:1253.

10. Tosato, G. and S.E. Pike (1988) J. Immunol. 141:1556.

11. Wong, G.G. et al. (1988) J. Immunol. 140:3040.

12. Leary, A.G. et al. (1988) Blood 71:1759.

13. Bauman, H. et al. (1984) J. Biol. Chem. 259:7331.

14. Satoh, T. et al. (1988) Mol. Cell. Biol. 8:3546.

15. Hama, T. et al. (1989) Neurosci. Letts. 104:340