Mouse IL-4 ELISA Kit

Description

INTENDED USE

This Mouse IL-4 ELISA kit is to be used for the in vitro quantitative determination of mouse interleukin 4 (IL-4) concentrations in serum, cell culture supernatant, and other biological fluids.  This kit is intended for LABORATORY RESEARCH USE ONLY. 

INTRODUCTION

IL-4 is a very important B cell stimulation and differentiation factor.  In addition, IL-4 modulates the immune function of other cell types including T cells, monocytes, macrophages, mast cells, fibroblasts, endothelial cells, osteoblasts, keratinocytes, hepatocytes, and astrocytes.  IL-4 is mainly produced by CD4+ TH0 and TH2 cells, CD8+ T cells, and mast cells.  IL-4 is the polarizing cytokine for TH2 phenotype.

The gene for mouse IL-4 is mapped to chromosome 11 in close conjunction with genes for other T-helper 2 cytokines such as IL-13, GM-CSF, IL-5.  Mouse IL-4 is a disulfide bond linked homodimer containing 113 amino acids in each subunit.  The receptor for IL-4 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-13 and GM-CSF.  IL-4 receptor alpha (IL-4Ra) 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-4Ra has an antagonist effect to IL-4.  Increased IL-4 expression causes chronic IgE response and hypereosinophilic syndromes.  Elevated IL-4 level is found in allergic diseases such as allergic rhinitis, asthma and allergic conjunctivitis.

This ELISA kit provides a tool for studying IL-4 expression and regulation in animal model.

PRINCIPLE OF THE ASSAY

This mouse IL-4 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-4.  When standards or samples are added to the appropriate microtiter plate wells, mouse IL-4 in the standards or samples will be immobilized by the precoated antibody during incubation.  Then, a biotin-conjugated antibody preparation specific for mouse IL-4 is added to each well and incubated.  The biotin labelled antibody attaches to the wells by binding to mouse IL-4.  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-4 will exhibit a change in colour.  The extent of colour change is proportional to the quantity of mouse IL-4 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-4 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-4 concentration (pg/mL).  The concentration of mouse IL-4 in the samples is then determined by comparing the O.D. of the samples to the standard curve.

This mouse IL-4 ELISA is a 3.5-hour solid-phase immunoassay readily applicable to measure mouse IL-4 levels in serum, cell culture supernatant, and other biological fluids in the range of 7.8 to 500pg/mL.

CITATIONS  

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