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 test compound on a non-luminogenic enzyme activity in an assay sample not containing living cells, comprising:
(a) contacting a test compound and a non-luminogenic enzyme so as to produce a first mixture;
(b) contacting the first mixture with a luminogenic molecule so as to produce a first reaction mixture, wherein the luminogenic molecule is both a substrate of the non-luminogenic enzyme and a pro-substrate of a luciferase enzyme;
(c) contacting the first reaction mixture with a reagent composition comprising luciferase and a tolerance enhancement agent so as to produce a second reaction mixture, wherein the tolerance enhancement agent is present in an amount sufficient to substantially protect the activity of the luciferase from interference from the test compound and thereby reduce the likelihood of a false positive resulting from the interfering effect of the test compound relative to the absence of the tolerance enhancement agent;
(d) detecting luminescence in the second reaction mixture; and
(e) determining the effect of the test compound, if any, on the non-luminescent enzyme activity by comparing the detected luminescence of step (d) to
1) the luminescence of a control reaction mixture similar to the first reaction mixture but not containing the test compound, or
2) the luminescence of another first reaction mixture containing the test compound at a different concentration.
2. The method of claim 1 wherein the steps are conducted consecutively.
3. The method of claim 1 wherein the tolerance enhancement agent comprises a detergent.
4. The method of claim 3 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 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]-l-propanesulfonate, 3-([3-cholamido propyl)dimethylammonio)-2-hydroxy-1-propanesalfonate, N,N-bis(3D-gluconamidopropropyl)cholamide, polyethylene glycol 400 dodecyl ether (HO(CH 2 CH 20 ) 6 (CH 2 ) 11 CH 3 ), poly(ethyleneglycol)-block-poly(propvlene glvcol)-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, cetvl-trimethyl ammonium bromide (CTAB), C8= Octyl-beta-D-glucopyranoside, or n-decyl-beta-D-maltoside (C10 alkyl side chain).
6. The method of claim 1 wherein the tolerance enhancement agent comprises a non-detergent.
7. The method according to claim 1 wherein the non-detergent comprises, polyethylene glycol, polyvinyl pyridine, crown ether, or cyclodextrin.
8. The method of claim 1 wherein the reaction composition comprises two or more detergents.
9. The method of claim 1 wherein the non-luminogenic enzyme is a protease.
10. The method of claim 9 wherein the protease is trypsin, trypsinase, or caspase.
11. The method of claim 10 wherein caspase comprises caspase-3, caspase-7, caspase 8, or caspase-9.
12. The method of claim 11 wherein the luminogenic substrate comprises an aminoluciferin amino modified with an amino acid or peptide or a carboxyl protected derivative thereof.
13. The method of claim 1 wherein the non-luminogenic enzyme is a cytochrome P-450 enzyme.
14. The method of claim 13 wherein the luminogenic substrate is a D-luciferin derivative.
15. The method of claim 14 wherein the tolerance enhancement agent is a polyglycol ether detergent.
16. The method of claim 15 wherein the polyglycol ether detergent is nonylphenol polyethylene glycol ether.
17. A method of determining the effect of a test compound on a non-luminogenic enzyme activity in an assay sample not containing living cells, comprising:
(a) contacting a test compound, a luminogenic molecule and a non-luminogenic enzyme so as to produce a first reaction mixture, wherein the luminogenic molecule is both a substrate for the non-luminogenic enzyme and a pro-substrate for luciferase;
(b) contacting the first reaction mixture with a reagent composition comprising luciferase, and a tolerance enhancement agent so as to produce a second reaction mixture, wherein the tolerance enhancement agent is present in an amount sufficient to at least substantially protect the activity of the luciferase from interference from the test compound and thereby reduce the likelihood of a false positive resulting from the interfering effect of the test compound on luciferase relative to the absence of the tolerance enhancement agent;
(c) detecting luminescence in the second reaction mixture; and
(d) determining the effect of the test compound, if any, on the non-luminogenic enzyme activity by comparing the detected luminescence of step (c) to
1) the luminescence of a control reaction mixture similar to the first reaction mixture but not containing the test compound, or
2) the luminescence of another first reaction mixture containing the test compound at a different concentration.