Mouse TGF-β1 ELISA Kit

INTENDED USE

This Mouse TGF-β1 ELISA kit is to be used for the in vitro quantitative determination of mouse transforming growth factor beta 1 (TGF-β1) concentrations in serum, plasma, cell culture supernatant, and other biological fluids.  This kit is intended for LABORATORY RESEARCH USE ONLY and is not for use in diagnostic or therapeutic procedures.

INTRODUCTION

Transforming growth factor beta (TGF-β1) is a stable, multifunctional polypeptide growth factor.  While specific receptors for this protein have been found on almost all mammalian cells examined, the effect of the molecule varies depending on the cell type and growth conditions.  TGF-β is an anti-proliferation factor in normal cells.  It increases the synthesis of p15 and p21, which can block the cyclin:CDK complex, and causes cells to stop at G1 phase.  In cancer cells, TGF-β signaling is altered and TGF-β no longer stops cell proliferation. TGF-β1, is only one member of a family of regulatory proteins consisting of a number of factors distantly related to TGF-β1 (30-40% sequence identity), including the activins, inhibins, and bone morphogenetic proteins (BMPs), and a number of more closely related proteins (70-80% sequence identity) designated TGF-β1, TGF-β2, TGF-β3, TGF-β4, and TGF-β5.  Human and mouse TGF-β1 share 99% gene homology and the mature form of TGF1beta in the two species are identical.

TGF-β1 is a homodimer linked by disulfide bind.  Inside cells, the cytokine forms a small latent complex with latent associated peptide (LAP).  This small complex binds to latent TGF-β1 binding protein (LTBP) to be secreted to extracellular matrix.  Dissociation of the latent proteins from TGF-β1 results in the release of the cytokine to its receptor.  The process is called activation, which can be influenced by various factors, including proteases, metalloproteases, extreme pH, mild acidic condition, reactive oxygen species and integrins.

Most of the currently published reports on the activities of TGF-β have dealt only with TGF-β1.  In general, TGF-β1, TGF-β2 and TGF-β3 appear to be functionally equivalent in biological activity in vitro, although there seem to be differences in potency on different cell types.  TGF-β1 has been found in the highest concentration in platelets and mammalian bone, but it is produced in smaller amounts by many cells.  TGF-β is an important modulator of the growth, differentiation, and activities of a number of the different types of cells involved in both cellular and humoral immune responses.  TGF-β1 enhances the deposition of extracellular matrix through promotion of synthesis and inhibition of degradation.  TGF-β1 is also immunosuppressive through a variety of mechanisms.  The specific action of TGF-β1 on a particular cell depends on the exact circumstances of that cell's environment.

The mouse TGF-β1 ELISA Kit is a 3 hours solid phase ELISA designed to measure TGF-β1 in cell culture supernatant, serum, plasma and other biological fluids in the range of 0 to 2000pg/mL.   Since TGF-β1 is not glycosylated, it is virtually certain that the Mouse TGF-β1 ELISA Kit will provide accurate quantitation for both recombinant and natural TGF-β.  It showed no cross reactivity with various other cytokines.  This TGF-β1 ELISA is expected to be effectively used for further investigations into the relationship between TGF-β1 and various disease models.

PRINCIPLE OF THE ASSAY

This TGF-β1 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 TGF-β1.  Standards or samples are then added to the appropriate microtiter plate wells and incubated. After washing plate, biotin-conjugated polyclonal antibody specific to TGF-β1 is added and incubated. TGF-β1 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 TGF-β1 antibody and other components.  In order to quantitatively determine the amount of TGF-β1 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 has a high affinity-binding site for biotin.  The wells are thoroughly washed to remove all unboundHRP-conjugated Avidin 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 those wells that contain TGF-β1, biotin-conjugated antibody, and enzyme-conjugated Avidin 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 TGF-β1 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 TGF-β1 concentration (pg/mL).  The concentration of TGF-β1 in the samples is then determined by comparing the O.D. of the samples to the standard curve.