Problem
Paclitaxel, better known by its trade name Taxol, has demonstrated anticancer activity against several classically refractory tumors. The Food and Drug Administration approves the use of paclitaxel for the treatment of breast, ovarian, and non-small-cell lung cancers and AIDS-related Kaposi's sarcoma. The mechanism of action of paclitaxel is based on the inhibition of human tumor cell replication by arresting the division of microtubules, which are minute cellular organelles vital to cell division that are the result of tubulin polymerization. Despite its success, paclitaxel has problems. It has a narrow therapeutic range, a broad elimination half time in patients, and many side effects that have severe implications for patients undergoing treatment. To respond to both clinical and research demands, there is a need to determine paclitaxel in a rapid, sensitive, and selective way.
The analysis of paclitaxel and other taxanes is performed mainly by reversed-phase high-performance liquid chromatography (HPLC), although other methods such as multimodal thin-layer chromatography, tandem mass spectrometry, and gas chromatography have been reported. Chromatography-based methods, however, are time consuming, tedious (requiring pretreatment and concentration), and relatively insensitive. Therefore, there is a great need for sensing techniques that would enable more sensitive and rapid measurement.
The specific interaction of paclitaxel or its analogues with tubulin has been reported. Therefore, like many receptor-based assays, the binding of paclitaxel to tubulin can be exploited for the development of a specific and sensitive bioassay for determining paclitaxel at the receptor level.
Your objective is to design an in vitro assay to study the binding interaction between the drug paclitaxel (or its analogues) and tubulin by applying an FP-based assay. You have been provided with the purified tubulin, fluorescent-labeled paclitaxel and native paclitaxel.
Discuss the following points in your answer:
a) Describe the Principle of the assay design.
b) Factors that you think need to be considered for optimization of the assay.
c) Positive and Negative controls.
d) Practical application of the assay design to determine the concentration of the paclitaxel in human plasma.