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 a non-luminogenic enzyme activity in an assay sample not containing living cells, comprising:
(a) contacting a compound for testing, a non-luminogenic enzyme, wherein the non-luminogenic enzyme utilizes or produces ATP, a substrate for the non-luminogenic enzyme, and a nucleotide selected from ATP and ADP, 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 comprises at least one of a detergent or a non-detergent, and 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, wherein the non-detergent comprises polyethylene glycol, polyvinyl pyridine, or crown ether;
(c) detecting luminescence in the second reaction mixture; and
(d) determining the effect of the compound, if any, on the non-luminescent 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 steps (a) and (b) are conducted simultaneously to produce a reaction mixture, wherein luminescence is detected in the reaction mixture.
4. The method according to claim 1 where 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 ,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-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 1 wherein the tolerance enhancement agent comprises a non-detergent.
7. The method according to claim 1 wherein the non-luminogenic enzyme is a kinase.
8. The method according to claim 7 wherein the tolerance enhancement agent is a detergent selected from nonionic polyglycol ether, polyoxyethylene 23 lauryl ether, polyethylene glycol 400 dodecyl ether (HO(CH 2 CH 2 O) 6 (CH 2 ) 11 CH 3 ), dodecyltrimethylammonium bromide (DTAB) and 3-([3-cholamido propyl)dimethylammonio)-2-hydroxy-1-propanesulfonate detergent.
9. The method according to claim 1 wherein the reaction composition comprises two or more detergents.
10. A method of determining the effect of a compound on a kinase enzyme activity in an assay sample not containing living cells, comprising:
(a) contacting a compound for testing, a kinase enzyme, a substrate for the kinase enzyme, and a nucleotide selected from ATP and ADP, 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 said tolerance enhancement agent comprises at least one of a detergent, polyethylene glycol, polyvinyl pyridine, or crown ether, and 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 kinase enzyme activity by comparing the luminescence of the second reaction mixture to a control reaction mixture.
11. The method according to claim 10 wherein the kinase enzyme is a protein kinase.
12. The method according to claim 10 wherein the detergent comprises nonionic polyglycol ether, polyoxyethylene 23 lauryl ether, polyethylene glycol 400 dodecyl ether (HO(CH2CH2O)6(CH2)11CH3), dodecyltrimethylammonium bromide (DTAB) or 3-([3-cholamido propyl)dimethylammonio)-2-hydroxy-1-propanesulfonate.
13. The method according to claim 10 wherein the reaction composition comprises two or more detergents.
14. The method according to claim 10 wherein the steps are carried out sequentially.
15. The method according to claim 10 wherein step (b) and step(c) are carried out simultaneously.