The transforming growth factor beta (TGF-b) family is a major and potent regulator of immune response; this cytokine family also has diverse effects on hemopoietic cells. TGF-b1 is produced by several cell lineages such as lymphocytes, macrophages, and dendritic cells. Maintaining immune tolerance via the regulation of cell proliferation, differentiation, migration, and survival is the critical function of TGF-b. Increased synthesis of TGF-b has been linked to immune defects associated with malignancy and autoimmune disorders.
The Human TGF-b1 ELISA Kit from R&D Systems is designed for the quantitative determination of activated human TGF-b1 concentrations in cell culture supernatant, serum, plasma, and urine. This kit employs the quantitative sandwich enzyme immunoassay method. I normally use this kit for measuring TGF-b1 in cell culture supernatants. Samples should be prepared immediately prior to running the assay by activating latent TGF-b1 to the immunoreactive form, which can be detected by the Quantikine TGF-b1 immunoassay. This can be achieved by acid activation and neutralization through treating samples in polypropylene test tubes with 1 N HCL and 1.2 N NaOH/0.5 M HEPES, respectively.
All required reagents are provided with this kit except hydrochloric acid, sodium hydroxide, and HEPES. A 96-well polystyrene microplate coated with a monoclonal antibody specific for TGF-b1 is provided with this kit. The whole microplate or any subset of wells can be used as required. Standards (provided with the kit), controls, and activated samples are added to the wells and any TGF-b1 is present is allowed to bind to the immobilized monoclonal antibody for 2 hours at room temperature. After washing away any unbound material, an antibody-enzyme conjugate (horseradish peroxidase-linked polyclonal antibody against TGF-b1 is added to each well and then incubates for 2 hours at room temperature in order to sandwich the TGF-b1 immobilized during the first incubation. After washing away any unbound conjugate, a substrate solution (prepared within 15 minutes of use by mixing equal volumes of hydrogen peroxide and chromogen) is added to each well and then incubated for 30 minutes at room temperature, protected from light. The color development is stopped with a diluted hydrochloric acid solution, and the optical density of each well is determined within 30 minutes using a microplate reader set to 450 nm.
I have used this kit and it worked from the first time. It is a reliable tool for measuring the important immuno-regulatory TGF-b1 cytokine in cell culture supernatant, serum, plasma, and urine. The procedures are straightforward and require 5 hours to complete. I would definitely recommend this kit for quantitative determination of TGF-b1.
Post-Doctoral Fellow
Paterson Institute for Cancer Research
University of Manchester