Luciferase-based assays
View Patent ↗A method and kit are 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 of determining the effect of a compound on an ATP-generating enzyme activity in a sample not containing living cells, comprising:
(a) contacting a compound, ADP, a sample containing or suspected of containing an ATP-generating enzyme, and a substrate for the ATP-generating enzyme, so as to produce a first reaction mixture;
(b) contacting the first reaction mixture with a reagent composition comprising luciferase, luciferin, and a tolerance enhancement agent so as to produce a second reaction mixture, wherein the tolerance enhancement agent is present in an amount effective to substantially protect the activity of the luciferase from interference from the compound, thereby reducing the likelihood of a false positive resulting from the interfering effect of the compound;
(c) detecting luminescence in the second reaction mixture; and
(d) determining the effect of the compound, if any, on the ATP-generating enzyme activity by comparing the luminescence of the second reaction mixture to a control reaction mixture.
2. The method according to claim 1 , wherein the steps are conducted consecutively.
3. The method according to claim 1 , wherein the tolerance enhancement agent comprises a detergent or a non-detergent.
4. The method according to claim 3 , wherein the tolerance enhancement agent comprises a cationic, anionic, non-ionic or zwitterionic detergent.
5. The method according to claim 4 , wherein the detergent comprises 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 ,CH2) 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-propanesulfonate, 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), polyethoxylated (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) or dodecyltrimethylammonium bromide (DTAB) detergent.
6. The method according to claim 4 , wherein the reaction composition comprises two or more detergents.
7. The method according to claim 3 , wherein the tolerance enhancement agent comprises a non-detergent.
8. The method according to claim 7 , wherein the non-detergent is selected from the group consisting of polyethylene glycol, polyvinyl pyridine, crown ether, and cyclodextrin.
9. The method according to claim 1 , wherein the ATP-generating enzyme is a kinase or phosphatase.
10. The method according to claim 9 , wherein the tolerance enhancement agent is selected from the group consisting of nonionic polyglycol ether, polyethylene glycol 400 dodecyl ether (HO(CH 2 CH 2 O) 6 (CH 2 ) 11 CH 3 ), or 3-([3-cholamidopropyl)dimethylammonio)-2-hydroxy-1-propanesalfonate detergent.
11. A method of determining the effect of a compound on an ATP-generating enzyme activity in a sample not containing living cells, comprising:
(a) contacting a compound, ADP, a sample containing or suspected of containing an ATP-generating enzyme, a substrate for the ATP-generating enzyme, and a reagent composition comprising luciferase, luciferin, and a tolerance enhancement agent so as to produce a reaction mixture, wherein the tolerance enhancement agent is present in an amount effective to substantially protect the activity of the luciferase from interference from the compound, thereby reducing the likelihood of a false positive resulting from the interfering effect of the compound;
(c) detecting luminescence in the reaction mixture; and
(d) determining the effect of the compound, if any, on the ATP-generating enzyme activity by comparing the luminescence of the reaction mixture to a control reaction mixture.
12. The method according to claim 11 , wherein the tolerance enhancement agent comprises a detergent or a non-detergent.
13. The method according to claim 12 , wherein the tolerance enhancement agent comprises a cationic, anionic, non-ionic or zwitterionic detergent.
14. The method according to claim 13 , wherein the detergent comprises 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 ,CH2) 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-propanesulfonate, 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), polyethoxylated (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) or dodecyltrimethylammonium bromide (DTAB) detergent.
15. The method according to claim 13 , wherein the reaction composition comprises two or more detergents.
16. The method according to claim 12 , wherein the tolerance enhancement agent comprises a non-detergent.
17. The method according to claim 16 , wherein the non-detergent is selected from the group consisting of polyethylene glycol, polyvinyl pyridine, crown ether, and cyclodextrin.
18. The method according to claim 11 , wherein the ATP-generating enzyme is a kinase or phosphatase.
19. The method according to claim 18 , wherein the tolerance enhancement agent is selected from the group consisting of nonionic polyglycol ether, polyethylene glycol 400 dodecyl ether (HO(CH 2 CH 2 O) 6 (CH 2 ) 11 CH 3 ), and 3-([3-cholamidopropyl)dimethylammonio)-2-hydroxy-1-propanesalfonate detergent.