This human Transforming growth factor beta (TNF-β) ELISA kit applies a technique called a quantitative sandwich immunoassay. This human TNF-β ELISA kit is a ready-to-use 3-hour solid phase immunoassay readily capable of measuring hTNF-β in the range of 0 to 4000 pg/mL in cell culture supernatant, serum and plasma. However, data collection for proliferation testing for hTNF-β detection will require additional days for completion. The human TNF-β ELISA kit assay has shown a specific reaction with hTNF-β and no cross-reactivity with various other cytokine superfamily proteins. Transforming growth factor beta (TGF-β) is a protein that comes in three isoforms called TGF-β1, TGF-β2 and TGF-β3; it was also the original name for the founding member of this family that is now called TGF-β1. The TGF-β family is part of a superfamily of proteins known as the transforming growth factor beta superfamily, which includes inhibins, activin, anti-müllerian hormone, bone morphogenetic protein, decapentaplegic and Vg-1. TGF beta controls proliferation, differentiation, and other functions in most cell types. It can also act as a negative autocrine growth factor.
The microtiter plate provided in this human TNF-β ELISA kit has been pre-coated with a monoclonal antibody specific to TNF-β. Standards or samples supplied with this human TNF-β ELISA kit are then added to the appropriate microtiter plate wells and incubated. After washing to remove unbound TNF-β and other components of the sample, biotin-conjugated polyclonal antibody specific to TNF-β is added and incubated. If present, TNF-β will bind and become immobilized by the antibody pre-coated on the wells and then become “sandwiched” by the biotin conjugate. In order to quantitatively determine the amount of TNF-β 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, each having a high affinity-binding site for biotin. The wells are thoroughly washed to remove all unbound HRP-conjugated Avidin and a TMB (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 TNF-β, 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.
This human TNF-β ELISA kit includes two calibration diluents (Calibrator Diluent I for serum/plasma testing and Calibrator Diluent II for cell culture supernatant testing) in order to measure the concentration of TNF-β in the samples. According to the testing system, the standard provided 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 TNF-β concentration (pg/mL). The concentration of TNF-β in the samples is then determined by comparing the O.D. of the samples to the standard curve. The minimum detectable quantities of human TNF-β using this human TNF-β ELISA kit as observed by the standard curve generated for both Calibrator Diluent I and Calibrator Diluent II are 22.0 pg/mL and 11.0 pg/mL respectively. The two standard deviations above the mean optical density of the 16 replicates of the zero standard were defined as the minimum detectable quantities.
TGF-β is a multifunctional peptide that controls proliferation, differentiation, and other functions in many cell types. TGF-β acts synergistically with TGF-α in inducing cellular transformation (MIM 190170). It also acts as a negative autocrine growth factor. Specific receptors for TGF-β activation trigger apoptosis when activated. Many cells synthesize TGF-β and almost all of them have specific receptors for this peptide. TGF-β1, TGF-β2, and TGF-β3 all function through the same receptor signaling systems.
This human TNF-β ELISA kit is to be used for the in vitro quantitative determination of human Tumour Necrosis Factor Beta (TNF-β) concentrations in serum, plasma, and cell culture supernatant. This human TNF-β ELISA kit assay can detect both natural and recombinant human TNF-β. The following factors were assayed at 10 ng/mL in Calibrator Diluent I for cross-reactivity. No significant cross-reactivity was observed using this human TNF-β ELISA kit. Recombinant human: IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-6, IL-8, TNF-α, GM-CSF. Other: bFGF acidic, hPDGF, pPDGF, hTGF-β1, pTGF-β1, pTGF-β1.2, pTGF-β2. This human TNF-β ELISA kit immunoassay is calibrated against NIBSC Standard (Reference preparation) Code No. 87/640. Fifty serum samples were tested in this assay and all had levels which fell below the lowest standard, 125 pg/mL. This human TNF-β ELISA kit is intended for LABORATORY RESEARCH USE ONLY and is not for use in diagnostic or therapeutic procedures.
The peptide structures of the three members of the TGF-β family are highly similar. They are all encoded as large protein precursors; TGF-β1 contains 390 amino acids and TGF-β2 and TGF-β3 each contain 412 amino acids. They each have an N-terminal signal peptide of 20-30 amino acids that they require for secretion from a cell, a pro-region (called latency associated peptide or LAP), and a 112-114 amino acid C-terminal region that becomes the mature TGF-β molecule following its release from the pro-region by proteolytic cleavage. The mature TGF-β protein dimerizes to produce a 25 KDa active molecule with many conserved structural motifs. TGF-β has nine cysteine residues that are conserved among its family; eight form disulphide bonds within the molecule to create a cysteine knot structure characteristic of the TGF-β superfamily while the ninth cysteine forms a bond with the ninth cysteine of another TGF-β molecule to produce the dimer. Many other conserved residues in TGF-β are thought to form secondary structure through hydrophobic interactions. The region between the fifth and sixth conserved cysteines houses the most divergent area of TGF-β molecules that is exposed at the surface of the molecule and is implicated in receptor binding and specificity of TGF-β.
Download Product Data Sheet