ASSAY FOR ASSESSMENT OF ENDOSOMAL TRANSPORT Russian patent published in 2018 - IPC G01N33/50 

FIELD: microbiology.

SUBSTANCE: invention relates to a method for assessing the ability of an endosome to release a test molecule, said test molecule being a clostridial neurotoxin or a redirected non-cytotoxic protease and being capable of proteolytic cleavage and inactivation of the SNARE protein, wherein the method comprises: I) contacting the eukaryotic cell with the test molecule, which is to be assessed for endosome release ability, wherein said eukaryotic cell comprises a cell membrane comprising a binding site present on the outer surface of the cell membrane of said cell; II) incubating the test molecule with said eukaryotic cell, which thus allows: a) the test molecule to bind to and form a bound complex with the binding site present on the eukaryotic cell, thereby allowing said bound complex to enter the eukaryotic cell by endocytosis; b) one or more endosomes to form in said cell, wherein one or more endosomes comprise the test molecule; and c) said test molecule to enter the cytosol of the eukaryotic cell through the endosomal membrane of one or more endosomes; III) removing an excess test molecule that is not bound to binding sites present on the eukaryotic cells; IV) detecting the amount of test molecules present in one or more endosomes, or detecting the number of test molecules present in the cytosol of said eukaryotic cell; V) comparing the number of test molecules detected in step IV), with a control value, wherein said control value indicates the number of test molecules present in one or more endosomes or the number of test molecules present in the cytosol prior to performing step IV); VI) calculating the endosome release parameter for the test molecule by determining the relative change in the number of test molecules that are present in one or more endosomes, or by determining the relative change in the amount of test molecules present in the cytosol of said eukaryotic cell; wherein step (IV) comprises detecting a test molecule using a fluorescent label. Also, the invention relates to a method for assessing the ability of a test molecule of an inhibitor to inhibit endosomal release of a control molecule with one or more eukaryotic cell endosomes.

EFFECT: assay for assessment of endosomal transport is proposed.

9 cl, 12 ex, 8 dwg