Mouse IL-2 ELISA Kit

Description

INTENDED USE

This Mouse IL-2 ELISA kit is to be used for the in vitro quantitative determination of mouse interleukin 2 (IL-2) concentrations in serum, cell culture supernatant, and other biological fluids.  This kit is intended for LABORATORY RESEARCH USE ONLY.

INTRODUCTION

Mouse IL-2 is a 17kDa protein produced by activated CD4+ T helper cells immediately after foreign antigens bind with the T-cell receptor.  IL-2 is an initiator of the acquired immunity and a major clone expansion stimulator of activated T cells.  The cytokine stimulates the growth, proliferation and differentiation of naïve T-cells into “effecter T-cells”, promotes the survival of antigen specific “memory T-cell”, and stimulates the maturation of “regulatory T-cells”.  The biological activity of IL-2 is mediated by receptor that is expressed primarily on the cell membrane of activated T-cells.  IL-2 receptor is comprised of a specific alpha subunit with high affinity to IL-2, a beta subunit, and a common gamma subunit that is also involved with the biological functions of a number of other cytokines such as IL-4, IL-7, IL-9, IL-15 and IL-21.

In addition to T cell clone expansion and haematopoietic effect, IL-2 also acts on activated B cells, monocytes, NK cells and LAK cells, and plays a role in tumor surveillance, anti-inflammatory reaction and self antigen-tolerance.

This ELISA kit provides a tool for studying IL-2 expression and regulation in animal model.

PRINCIPLE OF THE ASSAY

This mouse IL-2 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-2.  Standards or samples are then added to the appropriate microtiter plate wells and incubated.  Mouse IL-2, 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-2 and other components in the samples.  A biotin-conjugated antibody preparation specific for mouse IL-2 is added to each well and incubated.  The biotin labelled antibody will attach to the wells by binding to mouse IL-2 present in the standards/samples.  After plate washing, other proteins, components and unattached biotin labelled antibody are removed.  Then, 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 conjugated 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 the wells that contain mouse IL-2 will exhibit a change in colour.  The extent of colour change is proportional to the quantity of mouse IL-2 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 wavelength of 450 nm ± 2 nm.

In order to measure the concentration of mouse IL-2 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-2 concentration (pg/mL).  The concentration of IL-2 in the samples is then determined by comparing the O.D. of the samples to the standard curve.

This IL-2 ELISA is a 3.5-hour solid-phase immunoassay readily applicable to measure IL-2 levels in serum, cell culture supernatant, and other biological fluids in the range of 0 to 500pg/mL.