Quantification of Transforming Growth Factor-β in Biological Material Using Cells Transfected with a Plasminogen Activator Inhibitor-1 Promoter–Luciferase Construct

doi:10.1006/abio.1997.2026

Analytical Biochemistry

Volume 247, Issue 1, 5 April 1997, Pages 45-51

https://doi.org/10.1006/abio.1997.2026 Get rights and content

Abstract

Transforming growth factor-β (TGF-β), 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-β signal. In the current study the feasibility of quantifying TGF-β in complex biological fluids directly with a recently developed bioassay was examined. This assay is based on the ability of TGF-β to induce plasminogen activator inhibitor-1 (PAI-1) expression. Mature TGF-β 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-β 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-β. The present study furthermore provides insight in the critical steps for accurate TGF-β determination, which include careful blood collection and sample handling (storage and preparation). With this protocol TGF-β has been quantified in human plasma, rat plasma, rat saliva, tissue extracts from rat lung, and in culture medium of TGF-β-producing cells.

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Citation Excerpt :
Another method of measuring active TGF-β is using cell-based biosensors. It employs cells with TGF-β receptors and signaling elements as the primary sensor, and translate the TGF-β signal into specific increases of gene products, e.g. luciferase, regulated by a TGF-β responsive promoter (Abe et al., 1994; Dennler et al., 1998; Dong et al., 2017; Duan et al., 2022; Tesseur et al., 2006; van Waarde et al., 1997). Changes in luciferase activities can then be fitted using a standard curve of recombinant active TGF-β and translated to quantitative levels of TGF-β.
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^☆: J. H. Clark, Ed.

¹: To whom correspondence should be addressed. Fax: 31-50-3632913. E-mail: [email protected].

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Regular ArticleQuantification of Transforming Growth Factor-β in Biological Material Using Cells Transfected with a Plasminogen Activator Inhibitor-1 Promoter–Luciferase Construct☆

Abstract

J. Biol. Chem.

Biochem. Biophys. Res. Commun.

J. Immunol. Methods

J. Biol. Chem.

J. Biol. Chem.

J. Immunol. Methods

Anal. Biochem.

Physiol. Behav.

Arch. Oral Biol.

Int. J. Rad. Onc. Biol. Phys.

Proc. Natl. Acad. Sci. USA

Recent Progress in Hormone Research

J. Clin. Invest.

Regular Article
Quantification of Transforming Growth Factor-β in Biological Material Using Cells Transfected with a Plasminogen Activator Inhibitor-1 Promoter–Luciferase Construct☆