Development of a high throughput receptor binding assay using time-resolved fluorescent europium chelate

摘要

The structure-activity study of insulin-like peptide 3 (INSL3) requires the design and synthesis of various analogues. In order to test the designed analogues of INSL3 for their receptor binding affinity, previously, we have been using either radioactive-labelled ~(125)I-INSL3 or ~(33)P-relaxin with pK_i values of 9.68 ± 0.09 and 9.34 ± 0.24 respectively. The use of radio labelled peptides ha drawbacks including their short half life, high cost, and the need for prior preparation and their toxicity. To overcome these problems, we have decided to develop a high throughput receptor binding assay using time-resolved fluorescent lanthanide europium chelate. This has been used as probe in various biological assays such as kinase assays [1], immuno-fluorometric detection assays [2] and ligand-receptor interaction assays [3]. The emitted fluorescent light from this lanthanide probe has unique characteristics such as a very long luminescence halftime large Stoke's shift and sharp emission peak which allow measurement of signal to be made after the decay o short-lived background fluorescence [4]. These factor contribute to the low background and high sensitivity o this fluorescent probe which is becoming an increasingly common useful tool in clinical chemistry and molecula biology.^