Quantification of transforming growth factor-beta in biological material using cells transfected with a plasminogen activator inhibitor-1 promoter-luciferase construct.
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.
van Waarde
M A
MA
Department of Radiobiology, University of Groningen, The Netherlands.
van Assen
A J
AJ
Kampinga
H H
HH
Konings
A W
AW
Vujaskovic
Z
Z
D000818:Animals; D001681:Biological Assay / Q000379:methods* / Q000592:standards / Q000706:statistics & numerical data; D001774:Blood Chemical Analysis / Q000379:methods / Q000592:standards / Q000706:statistics & numerical data; D002478:Cells, Cultured; D004797:Enzyme-Linked Immunosorbent Assay / Q000706:statistics & numerical data; D005069:Evaluation Studies as Topic; D005260:Female; D015870:Gene Expression; D006801:Humans; D008156:Luciferases / Q000096:biosynthesis / Q000235:genetics; D008168:Lung / Q000737:chemistry; D008297:Male; D008907:Mink; D017395:Plasminogen Activator Inhibitor 1 / Q000235:genetics*; D011401:Promoter Regions, Genetic; D051381:Rats; D012015:Reference Standards; D015203:Reproducibility of Results; D012463:Saliva / Q000737:chemistry; D012680:Sensitivity and Specificity; D014162:Transfection; D016212:Transforming Growth Factor beta / Q000032:analysis* / Q000302:isolation & purification / Q000378:metabolism
D017395:Plasminogen Activator Inhibitor 1; D016212:Transforming Growth Factor beta; D008156:Luciferases