Mouse IL-5 ELISA Kit

INTENDED USE

This Mouse IL-5 ELISA kit is to be used for the in vitro quantitative determination of mouse interleukin 5 (IL-5) concentrations in serum, cell culture supernatant, and other biological fluids.  This kit is intended for LABORATORY RESEARCH USE ONLY.

INTRODUCTION

Mouse IL-5 is a heamotapoietic cytokine, mainly produced by T-helper 2 cells and mast cells.  The cytokine is comprised of two identical 113 amino acid units linked by disulfide bond.  The gene for mouse IL-5 is located at chromosome 11 in conjunction with genes for IL4 and other T-helper 2 cytokines such as IL-13, GM-CSF, IL-3.  IL-5 regulates B cell proliferation, eosinophil maturation and activation.  IL-5 receptor is a heterodimer with a cytokine specific alpha unit, and a signal transduction beta subunit that is identical to the beta subunit of receptors for IL-3 and GM-CSF.  IL-5 receptor alpha (IL-5Ra) is expressed by eosinophils, B cells and basophils in member-bound form and soluble form.  While the membrane bound form participates in the activation of eosinophils and B cells, the soluble form of IL-5Ra has an antagonist effect to IL-5.  Increased IL-5 expression causes chronic IgE response and hypereosinophilic syndromes.  Elevated IL-5 level is found in allergic diseases such as allergic rhinitis, asthma and allergic conjunctivitis.

This ELISA kit provides a tool for studying IL-5 expression and regulation in animal model. 

PRINCIPLE OF THE ASSAY

This mouse IL-5 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-5.  When standards or samples are added to the appropriate microtiter plate wells, mouse IL-5 in the standards or samples will be immobilized by the precoated antibody during incubation.  Then, a biotin-conjugated antibody preparation specific for mouse IL-5 is added to each well and incubated.  The biotin labelled antibody attaches to the wells by binding to mouse IL-5.  After plate washing, other proteins, components and unattached biotin labelled antibody is removed.  After that, avidin-horseradish peroxidase (HRP) conjugate is added to each well.  Avidin has a very high affinity for biotin, thus, it links the tracer (HRP) sturdily to the biotin labelled antibody.  The wells are thoroughly washed to remove all unbound avidin-HRPconjugate and 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 wells that contain mouse IL-5 will exhibit a change in colour.  The extent of colour change is proportional to the quantity of mouse IL-5 present in the standards/samples.  The enzyme-substrate reaction is terminated by the addition of a sulphuric acid solution and the colour change is measured spectrophotometrically at a wave length of 450 nm ± 2 nm.

In order to measure the concentration of mouse IL-5 in the samples, this kit contains 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-5 concentration (pg/mL).  The concentration of mouse IL-5 in the samples is then determined by comparing the O.D. of the samples to the standard curve.

This mouse IL-5 ELISA is a 3.5-hour solid-phase immunoassay readily applicable to measure mouse IL-5 levels in serum, cell culture supernatant, and other biological fluids in the range of 0 to 200pg/mL.