Mouse IL-6 ELISA Kit

INTENDED USE

This Mouse IL-6 ELISA kit is to be used for the in vitro quantitative determination of mouse interleukin 6 (IL-6) concentrations in serum, cell culture supernatant, and other biological fluids.  This kit is intended for LABORATORY RESEARCH USE ONLY.

INTRODUCTION

Interleukin 6 (IL-6) is a multifunctional cytokine secreted locally by a variety of cell types including T cells, B cells, monocytes/macrophages, endothelial cells, fibroblasts, hepatocytes, keratinocytes and astrocytes etc.  IL-6 is up-regulated upon the binding of Toll-like receptors to antigen pattern ligands and by mitogenic stimulation during infection, acute phase reaction, trauma (especially burns) and malignancies.  IL-6 secretion is significantly increased during physical excises.  Also known as B-cell stimulatory factor-2 (BSF-2), IL-6 is an important cytokine for the differentiation of B-cells into immunoglobulin-secreting cells.  In addition, IL-6 has been found to increase the number of platelets, influences cytotoxic T cell differentiation and activation, stimulate the differentiation and survival of neuronal cells, stimulate osteoclast formation, and induce terminal differentiation of M1 myeloid leukemic cells.  IL-6 also stimulates the growth of hybridomas, plasmacytomas, myelomas, sarcomas, carcinomas, EBV-transformed B cells, keratinocytes, and mesangial cells.  To melanoma cells and a few other cancer cells, IL-6 seemed to have an inhibitory effect.   

In acute inflammatory phase, IL-6 causes increased body temperature by initiating synthesis of Prostaglandin E2 in hypothalamus and IL-6 stimulates the synthesis of acute phase proteins.   IL-6 plays an anti-inflammatory role in acute reaction by decreasing the secretion of TNF-a and IL-1.  The anti-inflammatory activity is regulated via the classic signal pathway by the direct binding of IL-6 to its membrane receptor.  In chronic inflammation, IL-6 plays a pro-inflammatory role.  The transition is mediated by soluble IL-6 receptor alpha (sIL-6Ra) via the Tran-signaling pathway.  

Human IL-6 and murine IL-6 exhibit approximately 65% sequence homology at the nucleotide level, and 42% homology at the amino acid level. 

PRINCIPLE OF THE ASSAY

This mouse IL-6 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 for mouse IL-6.  Standards or samples are then added to the appropriate microtiter plate wells and incubated.  Mouse IL-6, if present, will bind and become immobilized by the antibody pre-coated on the wells.  The microtiter plate wells are thoroughly washed to remove unbound mouse IL-6 and other components of the sample.  In order to quantify the amount of mouse IL-6 present in the sample, a standardized preparation of biotin conjugated anti-mouse IL-6 detection antibody is added to each well.  The detection antibody will bind to mouse IL-6 and be immobilized during the incubation.  The plate is washed again and Avidin conjugated   horseradish peroxidase (HRP) is added to each wells and incubated.  Since Avidin has a very affinity to biotin,HRPwill be linked indirectly to the detection antibody on the plate via the binding with of avidin to biotin.  The microtiter plate is thoroughly washed to remove all unboundHRPand aTMB(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 mouse IL-6 and will exhibit a change in colour.  The enzyme-substrate reaction is terminated by the addition of a sulphuric acid solution and the colour change is measured spectrophotometrically at a wavelength of 450 nm ± 2 nm.

In order to measure the concentration of mouse IL-6 in the samples, this kit contains two calibration diluents (Calibrator Diluent I for serum/plasma testing and Calibrator Diluent II for cell culture supernatant/ urine 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 concentration (pg/mL).  The concentration of IL-6 in the samples is then determined by comparing the O.D. of the samples to the standard curve. 

This IL-6 ELISA is a 3 hour solid-phase immunoassay readily applicable to measure IL-6 levels in serum, plasma, cell culture supernatant, and other biological fluids in the range of 12.5 to 800 pg/mL.