| 主权项 |
1. An assay for assessing the endosome release ability of a test molecule,
wherein said test molecule is a clostridial neurotoxin or a re-targeted non-cytotoxic protease (Targeted Secretion Inhibitor) in which the natural binding ability of the non-cytotoxic protease has been modified by the introduction of a binding ligand or Targeting Moiety, and wherein said test molecule has the ability to proteolytically cleave and inactivate a SNARE protein; said assay comprising: i) contacting a eukaryotic cell with a test molecule that is to be assessed for endosome release ability, wherein said eukaryotic cell comprises a cell membrane including a Binding Site present on the outer surface of the cell membrane of said cell; ii) incubating the test molecule with said eukaryotic cell, and thereby allowing
a) the test molecule to bind to and form a bound complex with the Binding Site present on the eukaryotic cell, thereby permitting said bound complex to enter the eukaryotic cell by endocytosis;b) one or more endosomes to form within said cell, wherein the one or more endosomes contain the test molecule; andc) said test molecule to enter the cytosol of the eukaryotic cell by crossing the endosomal membrane of the one or more endosomes; iii) removing excess test molecule that is not bound to the Binding Sites present on the eukaryotic cells; iv) after a predetermined period of time, detecting the amount of test molecule present in the one or more endosomes, or detecting the amount of test molecule present in the cytosol of said eukaryotic cell; v) comparing the amount of test molecule detected in step iv) with a control value, wherein said control value represents the amount of test molecule present in the one or more endosomes or the amount of test molecule present in the cytosol prior to step iv); vi) calculating an endosome release value for the test molecule by determining the relative change in the amount of test molecule that is present within the one or more endosomes, or by determining the relative change in the amount of test molecule present in the cytosol of said eukaryotic cell; wherein step (iv) comprises detecting the test molecule using a fluorescent label. |
