Luciferase-based assays
View Patent ↗A method and kit is provided for enhancing the tolerance of an assay reagent to compounds in an assay sample, the assay reagent including a luciferase enzyme. The method includes contacting the luciferase with a tolerance enhancement agent in an amount sufficient to substantially protect luciferase enzyme activity from interference of the compound and minimize interference by at least about 10% relative to an assay not having tolerance enhancement agent.
1. A method for protecting activity of a luciferase enzyme from an interfering effect of a test compound in a cell free test compound screening environment, said method comprising steps of:
contacting the luciferase enzyme with a detergent to form a first composition; and
contacting the first composition with a second composition, said second composition comprising the test compound,
wherein the detergent is present in an amount sufficient to substantially protect the activity of the luciferase enzyme and reduce the likelihood of a false positive resulting from the interfering effect of the test compound, relative to the absence of detergent, in a test compound screening environment.
2. The method according to claim 1 , wherein the detergent comprises a cationic, Anionic, non-ionic, or zwitterionic detergent.
3. The method according to claim 1 , wherein the detergent comprises a nonionic polyglycol ether, polyoxyethylene 23 lauryl ether, polyoxyethylene 20 cetyl ether (HO(CH 2 CH 2 O) 20 C 16 H 33 ), 4-(1,1,3,3-tetramethylbutyl)phenyl-polyethylene glycol (t- 1 )ct-C 6 H 4 —(OCH 2 ,CH 2 ) x OH, x=9-10), 4-(1,1,3,3-tetramethylbutyl)phenyl-polyethylene glycol, polyoxyethylene 9, branched nonylphenyl ether, 3-([3-cholamido propyl)dimethylammonio]-1-propanesulfonate, 3-([3-cholamido propyl)dimethylammonio)-2-hydroxy-1-propanesalfonate, N,N-bis(3-D-gluconamidopropropyl)cholamide, polyethylene glycol 400 dodecyl ether (HO(CH 2 CH 2 O) 6 (CH 2 ) 11 CH 3 ), poly(ethyleneglycol)-block-poly(propylene glycol)-block-poly(ethylene glycol), polyethoxylal :d (20) oleyl alcohol, polyoxyethylene 9 lauryl alcohol, 2,4,7,9-tetramethyl-5-decyne-4,7-diol ethoxylate 10, deoxycholate, cetyl-trimethyl ammonium bromide (CTAB), C8=Octyl-β-D-glucopyranoside, or n-decyl-β-D-maltoside (C10 alkyl side chain).
4. The method of claim 1 , wherein the test compound is one of a plurality of test compounds.
5. The method of claim 1 , wherein the test compound is part of a high throughput screening of test compound libraries.
6. The method of claim 1 , wherein the second composition further comprises a luciferase substrate.
7. The method according to claim 3 , wherein the nonionic polyglycol ether is nonylphenol polyethylene glycol ether (C 9 H 19 C 6 H 4 (OCH 2 CH 2 ) 9 OH).
8. The method of claim 6 , wherein the luciferase substrate is luciferin.