A fast and sensitive colorimetric assay for IL-6 in hepatoma cells based on the production of a secreted form of alkaline phosphatase (SEAP).
Structure-function studies of cytokines require that simple, sensitive and reliable biological assays are available. A well known property of interleukin-6 (IL-6) is that of being able to induce transcription from several liver-specific promoters in human hepatoma cells. However, the available assays of IL-6 in hepatoma cells, which are either based on the detection of increased expression of endogenous acute phase response genes or on the activation of reporter genes transfected under the control of IL-6 responsive promoters, are not very sensitive and are time consuming. We have established a new assay for IL-6 in hepatoma cells which is based on the transfection of an IL-6 inducible promoter/secreted alkaline phosphatase (SEAP) gene fusion and which measures the inducible production and release of SEAP in the culture medium. SEAP activity is measured with a simple colorimetric assay that requires no cell manipulation, thus allowing a large set of samples to be analysed simultaneously. The CRP/SEAP assay can be used in studies on the structure-function relationships of human IL-6.
Gregory
B
B
Department of Genetics, Istituto di Ricerche di Biologia Molecolare P. Angeletti (IRBM), Rome, Italy.
Savino
R
R
Ciliberto
G
G
D000469:Alkaline Phosphatase / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D002097:C-Reactive Protein / Q000235:genetics; D002460:Cell Line; D002478:Cells, Cultured; D015500:Chloramphenicol O-Acetyltransferase / Q000032:analysis; D003124:Colorimetry / Q000379:methods*; D003470:Culture Media; D017930:Genes, Reporter / Q000235:genetics; D006801:Humans; D015850:Interleukin-6 / Q000032:analysis*; D008099:Liver / Q000166:cytology / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics; D012680:Sensitivity and Specificity; D013329:Structure-Activity Relationship; D014162:Transfection / Q000235:genetics
D003470:Culture Media; D015850:Interleukin-6; D002097:C-Reactive Protein; D015500:Chloramphenicol O-Acetyltransferase; D000469:Alkaline Phosphatase