title: Quantification of active and total transforming growth factor-β levels in serum and solid organ tissues by bioassay.
abstract: BACKGROUND
  Transforming growth factor-β (TGF-β) is a multi-factorial peptide growth factor that has a vital role in the regulation of cell growth, differentiation, inflammation, and tissue repair. Quantification of biologically active TGF-β levels in tissues is crucial to illustrate mechanisms involved in various physiological and pathological processes, but direct measurement of bioactive TGF-β level in the tissue has been hampered by lack of reliable methods. Here, we introduced mink lung epithelial cell bioassay to quantify both active and total TGF-β levels in serum and protein lysates from solid organs in the mouse model.
  
  
  RESULTS
  Mink lung epithelial cells were stably transfected with plasminogen activator inhibitor-1 promoter/luciferase construct, in which bioactive TGF-β level was represented by luciferase activity. Serum total TGF-β levels were comparable between the bioassay and enzyme-linked immunosorbent assay (ELISA), but active TGF-β levels measured by ELISA were significantly lower than those obtained by the bioassay. Active and total TGF-β levels in the solid organs including heart, liver, and kidney were also measured. Total TGF-β levels were relatively comparable among these organs, but active TGF-β levels were slightly higher in hearts and kidneys than in livers. Positive luciferase activities in the bioassay were almost completely inhibited by adding pan-TGF-β neutralizing antibodies, suggesting its high specificity to bioactive TGF-β. We also measured myocardial TGF-β levels after myocardial infarction and sham control by the bioassay, and compared the values with those obtained by ELISA. The bioassay demonstrated that both active and total tissue TGF-β levels were significantly higher in post-myocardial infarction than in sham myocardium. ELISA was markedly less sensitive in detecting both active and total TGF-β levels than our bioassay and failed to show any statistically significant difference in TGF-β levels between myocardial infarction and sham myocardium.
  
  
  CONCLUSIONS
  Our data suggested that the bioassay was significantly more sensitive than ELISA in detecting active TGF-β in serum and both active and total TGF-β in solid organ tissues. The bioassay will be useful in investigating TGF-β profile in various solid organs in physiological and pathological conditions.
authors.affiliation: Nemours Biomedical Research, Alfred I, duPont Hospital for Children, 1600 Rockland Rd, Wilmington, DE 19803, USA.
authors.forename: Shaukat A
authors.initials: SA
authors.lastname: Khan
authors.forename: Jennifer
authors.initials: J
authors.lastname: Joyce
authors.forename: Takeshi
authors.initials: T
authors.lastname: Tsuda
mesh_terms: D000818:Animals; D001681:Biological Assay / Q000592:standards*; D002138:Calibration; D004797:Enzyme-Linked Immunosorbent Assay; D004847:Epithelial Cells / Q000166:cytology / Q000378:metabolism; D017930:Genes, Reporter; D007668:Kidney / Q000166:cytology / Q000378:metabolism; D008099:Liver / Q000166:cytology / Q000378:metabolism; D008156:Luciferases; D008168:Lung / Q000166:cytology / Q000378:metabolism; D051379:Mice; D008907:Mink; D009203:Myocardial Infarction / Q000097:blood* / Q000473:pathology; D009206:Myocardium / Q000378:metabolism* / Q000473:pathology; D017395:Plasminogen Activator Inhibitor 1 / Q000235:genetics / Q000378:metabolism; D012680:Sensitivity and Specificity; D014162:Transfection; D016212:Transforming Growth Factor beta / Q000032:analysis* / Q000096:biosynthesis
chemical_list: D017395:Plasminogen Activator Inhibitor 1; D016212:Transforming Growth Factor beta; D008156:Luciferases