1. Quantification of transforming growth factor-beta in biological material using cells transfected with a plasminogen activator inhibitor-1 promoter-luciferase construct.
- Author
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van Waarde MA, van Assen AJ, Kampinga HH, Konings AW, and Vujaskovic Z
- Subjects
- Animals, Biological Assay standards, Biological Assay statistics & numerical data, Blood Chemical Analysis methods, Blood Chemical Analysis standards, Blood Chemical Analysis statistics & numerical data, Cells, Cultured, Enzyme-Linked Immunosorbent Assay statistics & numerical data, Evaluation Studies as Topic, Female, Gene Expression, Humans, Luciferases biosynthesis, Luciferases genetics, Lung chemistry, Male, Mink, Promoter Regions, Genetic, Rats, Reference Standards, Reproducibility of Results, Saliva chemistry, Sensitivity and Specificity, Transforming Growth Factor beta isolation & purification, Transforming Growth Factor beta metabolism, Biological Assay methods, Plasminogen Activator Inhibitor 1 genetics, Transfection, Transforming Growth Factor beta analysis
- Abstract
Transforming growth factor-beta (TGF-beta), a multifunctional cytokine, can be quantified by a variety of bioassays or immunoassays. One of the disadvantages of these techniques is that they require sample purification to remove components that interfere with the TGF-beta signal. In the current study the feasibility of quantifying TGF-beta in complex biological fluids directly with a recently developed bioassay was examined. This assay is based on the ability of TGF-beta to induce plasminogen activator inhibitor-1 (PAI-1) expression. Mature TGF-beta binds to the receptors of mink lung epithelial cells transfected with a plasminogen activator inhibitor-1 promoter-luciferase construct (PAI/L), resulting in a dose-dependent increase of luciferase activity. Specificity for TGF-beta was proven by treatment of the samples with neutralizing antibodies. The sensitivity and the intraassay precision are comparable to the ELISA. It is demonstrated, however, that, unlike the ELISA, a purification step by, e.g., acid-ethanol extraction prior to the PAI/L assay, is not required. This not only simplifies the assay but also reduces the minimal sample volume and allows to discriminate between latent and mature TGF-beta. The present study furthermore provides insight in the critical steps for accurate TGF-beta determination, which include careful blood collection and sample handling (storage and preparation). With this protocol TGF-beta has been quantified in human plasma, rat plasma, rat saliva, tissue extracts from rat lung, and in culture medium of TGF-beta-producing cells.
- Published
- 1997
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