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

• 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
• ISSN: 0731-7085; 1873-264X
• DOI: 10.1016/S0731-7085(98)00144-7

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.