Interleukin-6 (IL-6) is a multifunctional cytokine produced by a wide variety of cell types including monocyte/macrophages, T cells, fibroblasts, hepatocytes, vascular endothelial cells, cardiac myxomas, bladder cell carcinomas, myelomas, astrogliomas, and glioblastomas. The effects of IL-6 on different cell types are numerous and varied. These activities include: stimulation of B cell differentiation and antibody secretion; action as a co-stimulant with PHA or Con A to increase IL-2 production and IL-2 receptor expression by T cells; enhancement of differentiation of cytotoxic T cells; action as a growth factor for mature thymic or peripheral T cells, myelomas, hybridomas, plasmacytomas, keratinocytes, and mesangial cells; colony-stimulating activity on hematopoetic cells; induction of neuronal cell differentiation; induction of maturation of megakaryocytes; and stimulation of production of acute phase response proteins by hepatocytes. These various activities indicate that IL-6 plays a major role in the mediation of the inflammatory and immune responses initiated by infection or injury. Elevated IL-6 levels have been reported to be associated with a variety of diseases, including autoimmune diseases, mesangial proliferative glomerulonephritis, psoriasis, and malignancies such as plasmacytomas and myelomas. For reviews of the properties and activities of IL-6, see references 1 to 3.
The biological activities of IL-6 are initiated by binding of the cytokine to a high affinity receptor complex consisting of two membrane glycoproteins; an 80kD component receptor that binds IL-6 with low affinity (IL-6R) and a signal-transducing component of 130kD (gp130) that does not bind IL-6 by itself, but is required for high affinity binding of IL-6 by the complex. IL-6R and gp130 have been cloned, sequenced and expressed (4-7).
A soluble form of the IL-6 R with a molecular weight of approximately 50kD has been found in the urine of healthy adult humans (8), in culture medium conditionned by the growth of a human myeloma cell line (9), in culture supernatants from PHA-stimulated human PBMC and HTLV-1 positive T cell lines (10) and in the serum of HIV-seropositive blood donors (10). This soluble form of the receptor apparently arises from proteolytic cleavage of membrane-bound IL-6R. Soluble forms of human and mouse IL-6 have also been constructed by insertion of termination codons into the regions of the IL-6R cDNAs encoding the external portions of the receptors and prior to the transmembrane domains (11, 12). These soluble receptors have been expressed in COS7 and CHO cells (11, 12) and have been shown to bind IL-6 in solution and to augment the activity of IL-6 as a result of the binding of the IL-6/IL-6 sR complex to membrane-bound gp130 (11, 12). The regulation in vivo of the shedding of the soluble IL-6Rs and the function and significance of these soluble receptors in biological fluids is not currently understood. It has been suggested, however, that pathogical states involving elevated levels to IL-6 might also be associated with increased production of soluble IL-6Rs (10).
This IL-6 sR ELISA is a 4.5 hour solid phase immunoassay readily applicable to measure
IL-6sR levels in serum, plasma, cell culture supernatant, and other biological fluids in the range of 0 to 2000pg/ml. It showed no cross reactivity with other cytokines tested. This IL-6 sR ELISA is expected to be effectively used for further investigations into the relationship between IL-6 sR and the various conditions mentioned.
Principle:
This IL-6 sR enzyme linked immunosorbent assay (ELISA) applies a technique called a quantitative sandwich immunoassay. The microtiter plate provided in this kit has been pre-coated with a monoclonal antibody specific to IL-6 sR. Standards or samples are then added to the appropriate microtiter plate wells with a biotin-conjugated polyclonal antibody preparation specific for IL-6 sR and incubated. IL-6 sR if present, will bind and become immobilized by the antibody pre-coated on the wells and then be "sandwiched" by biotin conjugate. The microtiter plate wells are thoroughly washed to remove unbound IL-6 sR and other components of the sample. In order to quantitate the amount of IL-6 sR present in the sample, Avidin conjugated to Horseradish Peroxidase (HRP) is added to each microplate well and incubated. Avidin is a tetramer containing four identical subunits that each have a high affinity binding site for biotin. The wells are thoroughly washed to remove all unbound HRP-conjugated Avidin and a TMB (3,3'5,5' tetramethyl-benzidine) substrate solution is added to each well. The enzyme (HRP) and substrate are allowed to react over a short incubation period. Only those wells that contain IL-6 sR, biotin-conjugated antibody, and enzyme-conjugated Avidin will exhibit a change in color. The enzyme-substrate reaction is terminated by the addition of a sulphuric acid solution and the color change is measured spectrophotometrically at a wavelength of 450nm � 2nm.
In order to measure the concentration of IL-6 sR in the samples, this kit includes two calibration diluents (Calibrator Diluent I for serum/plasma testing and Calibrator Diluent II for cell culture supernatant testing.) According to the testing system, the provided standard is diluted (2-fold) with the appropriate Calibrator Diluent and assayed at the same time as the samples. This allows the operator to produce a standard curve of Optical Density (O.D) versus IL-6 sR concentration (pg/ml). The concentration of IL-6sR in the samples is then determined by comparing the O.D. of the samples to the standard curve.
Kit Components:
I8428-39A1: IL-6sR Microtiter Plate 1x96 wells
I8428-39A2:. IL-6sR Pab (Biotin) 1x7ml
I8428-39A3: Avidin (HRP) 1x14ml
I8428-39A4: IL-6sR Standard 2x1 vial
I8428-39A5: Calibrator Diluent I (5X) 1x22ml
I8428-39A6: Calibrator Diluent II 1x22ml
I8428-39A7: Wash Buffer (20X) 1x60ml
I8428-39A8: Substrate A 1x10ml
I8428-39A9: Substrate B 1x10ml
I8428-39A10: Stop Solution 1x14ml, 2N Sulphuric Acid (H2SO4).
Sensitivity: <7pg/ml
Range: 7-2000pg/ml
Storage and Stability:
Store all kit components except I8428-39A4 at 4 degrees C. Stable for at least 6 months. Store I8428-39A4 powder at 4 degrees C liquid at -20 degrees C (30 days). For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap.
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