Kinase receptor activation (KIRA): a rapid and accurate alternative to end-point 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 a `kinase receptor...

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Bibliographic Details
Published in:Journal of pharmaceutical and biomedical analysis 1999-05, Vol.19 (6), p.883-891
Main Authors: Sadick, Michael D, Intintoli, Anthony, Quarmby, Valerie, McCoy, April, Canova-Davis, Eleanor, Ling, Victor
Format: Article
Language:English
Subjects:
Analysis
Biological and medical sciences
Biological Assay - methods
Enzyme Activation
Enzyme-Linked Immunosorbent Assay - methods
General pharmacology
Humans
Insulin-Like Growth Factor I - analysis
Kinase receptor activation (KIRA)
Ligand bioactivity
Medical sciences
Pharmacology. Drug treatments
Phosphorylation
Rapid bioassay
Receptor phosphorylation
Receptor Protein-Tyrosine Kinases - analysis
Receptor Protein-Tyrosine Kinases - metabolism
Receptor, trkA - analysis
Tumor Cells, Cultured
Viral Envelope Proteins - analysis
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Summary: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 a `kinase receptor activation' or KIRA, utilizes 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.
ISSN:0731-7085
1873-264X
DOI:10.1016/S0731-7085(98)00144-7