Kinase Receptor Activation (KIRA): a rapid and accurate alternative to endpoint bioassays.
We have developed a novel strategy for a rapid bioassay that is accurate, precise, sensitive, and high capacity. It is capable of quantifying ligand bioactivity by measuring ligand-induced receptor tyrosine kinase activation in terms of receptor-phosphorylation. The assay, termed << Kinase Receptor Activation>> or KIRA, uses two separate microtiter plates, one for ligand stimulation of intact cells, and the other for receptor capture and phosphotyrosine ELISA. The assay makes use of either endogenously expressed receptors or stably transfected receptors with a polypeptide flag. KIRA assays for the ligands IGF-I and NGF were compared to their corresponding endpoint bioassays (3T3 cell proliferation for IGF-I and PC12 cell survival for NGF). The KIRA assays showed excellent correlation with the more classical endpoint bioassays. Further, they were highly reproducible, minimizing the requirement for repeat assays. The KIRA assay format has great potential as a rapid, accurate and precise bioassay, both for potency determination as well as stability-indicating analyses.
Sadick
M D
MD
Department of BioAnalytical Technology, Genentech, Inc., South San Francisco, CA 94080, USA.
D016475:3T3 Cells; D000818:Animals; D001681:Biological Assay / Q000379:methods* / Q000706:statistics & numerical data; D016466:CHO Cells; D002455:Cell Division; D002460:Cell Line; D006224:Cricetinae; D004789:Enzyme Activation; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D008024:Ligands; D051379:Mice; D016716:PC12 Cells; D010766:Phosphorylation; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism; D051381:Rats; D020794:Receptor Protein-Tyrosine Kinases / Q000235:genetics / Q000378:metabolism*; D017526:Receptor, IGF Type 1 / Q000378:metabolism; D020917:Receptor, trkA; D017475:Receptors, Nerve Growth Factor / Q000235:genetics / Q000378:metabolism; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D015398:Signal Transduction; D014162:Transfection
D008024:Ligands; D011518:Proto-Oncogene Proteins; D017475:Receptors, Nerve Growth Factor; D020794:Receptor Protein-Tyrosine Kinases; D017526:Receptor, IGF Type 1; D020917:Receptor, trkA