Claims for Patent: 6,602,657

Summary for Patent: 6,602,657

Title:	Multiple reporter gene assay
Abstract:	A method of measuring the activity of at least two reporter gene products in an aliquot of a sample extract is disclosed. The activities of a first and second reporter enzyme are quantified by measuring the light signal produced by degradation of a first substrate by the first reporter enzyme and the light signal produced by the degradation of a second substrate by a second reporter enzyme. Both quantifications are sequentially performed on the same aliquot of sample extract.
Inventor(s):	Bronstein; Irena Y. (Newton, MA), Fortin; John J. (Georgetown, MA), Martin; Chris S. (Belmont, MA), Voyta; John C. (Sudbury, MA)
Assignee:	Tropix, Inc. (Bedford, MA)
Application Number:	08/579,787
Patent Claims:	1. A method of measuring the activity of at least two reporter gene products in an aliquot of a sample extract comprising quantifying the activity of a first reporter enzyme by measuring the light signal produced by degradation of a first substrate by the first reporter enzyme, and quantifying the activity of a second reporter enzyme by measuring the light signal produced by the degradation of a second substrate by a second reporter enzyme, wherein both quantifications are sequentially performed on the same aliquot of sample extract. 2. The method according to claim 1, wherein the presence of the first substrate enhances the light signal produced by degradation of the second substrate. 3. The method of claim 1, wherein the first and second substrates are added simultaneously. 4. The method of claim 1, wherein the second substrate is added subsequent to the first enzyme. 5. The method according to claim 1, further comprising decreasing the activity of the first reporter enzyme prior to quantifying the activity of the second reporter enzyme. 6. The method according to claim 5, wherein decreasing the activity of the first reporter enzyme comprises substantially inactivating the first reporter enzyme or decreasing the amount of the first substrate. 7. The method according to claim 6, wherein substantially inactivating the first reporter enzyme comprises altering the pH of the aliquot, heating the aliquot to degrade the first enzymes adding inactivating chemicals or substrate analogs. 8. The method according to claim 7, wherein substantially inactivating the first reporter enzyme comprises altering the pH of the reaction. 9. The method according to claim 6, wherein decreasing the amount of the first substrate comprises adding an additional amount of the first enzyme sufficient to degrade the first substrate. 10. The method according to claim 6, wherein decreasing the amount of the first substrate comprises heating the aliquot to degrade the first substrate. 11. The method according to claim 1, wherein quantifying the activity of the second reporter enzyme comprises inducing production of light from the degradation of the second substrate by the second reporter enzyme prior to measuring the light signal produced by the degradation of a second substrate by a second reporter enzyme. 12. The method according to claim 11, wherein the production of light is induced by increasing the pH of the aliquot. 13. The method according to claim 1, further comprising inducing the activity of the second reporter enzyme by adding the second substrate or activating the second reporter enzyme. 14. The method according to claim 1, wherein at least one of the first or second reporter enzymes is a hydrolytic enzyme capable of reacting with a dioxetane. 15. The method according to claim 14, wherein the first reporter enzyme is selected from the group consisting of luciferase, .beta.-galactosidase, .beta.-glucuronidase, alkaline phosphatase and carboxyl esterase. 16. The method according to claim 15, wherein the first reporter enzyme is luciferase. 17. The method according to claim 14, wherein the second reporter enzyme comprises .beta.-galactosidase, .beta.-glucuronidase, alkaline phosphatase or carboxyl esterase. 18. The method according to claim 17, wherein the second reporter enzyme comprises .beta.-galactosidase. 19. The method according to claim 1, wherein at least one of the first or second substrates is a dioxetane. 20. The method according to claim 19, wherein the dioxetane comprises a dioxetane containing a substituted or unsubstituted adamantyl group, a Y group which may be substituted or unsubstituted and an enzyme cleavable group. 21. The method according to claim 19, wherein the dioxetane substrate comprises 3-(4-methoxyspiro[1,2-dioxetane-3,2'-(5'-chloro)tricyclo[3.3.1.1.sup.3,7 ]-decan]-4-yl-phenyl-.beta.-D-galactopyranoside (Galacton.TM.), and 5-chloro-3-(methoxyspiro[1,2-dioxetane-3,2'-(5'-chloro)tricyclo[3.3.1.sup. 3,7 ]decan-4-yl-phenyl-.beta.-D-galactopyranoside (Galacton-Plus.TM.). 22. The method according to claim 1, further comprising adding a water soluble polymeric enhancer molecule to enhance the light signal produced by enzymatic degradation of the dioxetane. 23. The method according to claim 22, wherein the polymeric enhancer comprises bovine serum albumin, human serum albumin or polymeric quaternary onium salts. 24. The method according to claim 23, wherein the polymeric quaternary onium salts comprise polyvinylbenzyltrimethyl-ammonium chloride (TMQ), polyvinyl benzyl tributyl ammonium chloride (TBQ) or polyvinylbenzyl benzyldimethylammonium chloride (BDMQ), polyvinylbenzyltributylphosphonium chloride, or polyvinyl tributyl sulfonium chloride. 25. The method according to claim 1, further comprising adding an accelerator solution prior to quantifying the activity of the second enzyme. 26. The method according to claim 25, wherein the accelerator solution comprises a water soluble polymeric enhancer molecule at a pH from about 9 to about 14 which is capable of activating the signal production from the degradation of the second substrate by the second reporter enzyme and inactivate the first reporter enzyme. 27. The method according to claim 26, wherein the polymeric enhancer comprises bovine serum albumin, human serum albumin or polymeric quaternary onium salts. 28. The method according to claim 27, wherein the polymeric quaternary onium salts comprise poly vinylbenzyltrimethyl-ammonium chloride (TMQ), polyvinyl benzyl tributyl ammonium chloride (TBQ) or polyvinylbenzyl benzyldimethylammonium chloride (BDMQ), polyvinylbenzyltributylphosphonium chloride, or polyvinyl tributyl sulfonium chloride. 29. A method of measuring the activity of more than two reporter genes products in a single aliquot of cell extract comprising quantifying the activity of a first reporter enzyme in an aliquot of the cell extract by measuring the light signal produced by degradation of a first substrate, decreasing the activity of the first reporter enzyme, quantifying the activity of a second reporter enzyme in the aliquot of the cell extract by measuring the light signal produced by degradation of a second substrate, decreasing the activity of the second reporter enzyme, and quantifying the activity of a third reporter enzyme in the aliquot of the cell extract by measuring the light signal produced by degradation of a third substrate, wherein all quantifications are sequentially performed on the same aliquot of sample extract. 30. The method according to claim 29, wherein the first, second and third substrates are different and at least one of the substrates is a dioxetane. 31. The method according to claim 29, further comprising inducing the signal produced by the degradation of the second substrate by the second reporter enzyme. 32. The method according to claim 29, further comprising inducing the activity of the third reporter enzyme. 33. The method of measuring the activity of more than one reporter gene products in an aliquot of a sample extract comprising adding a first substrate which is the substrate of a first reporter enzyme and a second substrate which is the substrate of a second reporter enzyme to an aliquot of the cell extract, the first substrate comprising luciferin and the second substrate comprising a dioxetane and the second enzyme being a hydrolytic enzyme, measuring the activity of the first reporter enzyme, adding an accelerator solution which substantially inactivates the first reporter enzyme and simultaneously increases the chemiluminescent signal of the second substrate, and includes an enhancer molecule, and measuring the activity of the second reporter enzyme in the same aliquot of the cell extract, wherein the presence of the first substrate enhances the light signal produced by degradation of a second substrate. 34. The method according to claim 33, wherein the first reporter enzyme is luciferase and the second reporter enzyme is .beta.-galactosidase. 35. A kit for detecting the activity of two or more reporter gene products by quantifying each of two or more reporter enzymes in an aliquot of a sample extract, wherein the kit comprises: a first substrate which is acted on by a first reporter enzyme to produce a signal; a second substrate which is acted on by a second reporter enzyme to produce a signal, wherein the first substrate enhances the signal produced by the second reporter enzyme; and an accelerator solution containing a water soluble polymeric enhancer molecule. 36. The kit according to claim 35, wherein the first substrate comprises luciferin. 37. The kit according to claim 35, wherein the second substrate comprises a dioxetane. 38. The kit according to claim 35, wherein the accelerator solution comprises a water soluble polymeric enhancer molecule at a pH from about 9 to about 14, to induce signal production from the action of the second reporter enzyme on the second substrate and inactivate the first reporter enzyme. 39. The kit according to claim 38, wherein the polymeric enhancer comprises bovine serum albumin, human serum albumin or polymeric quaternary onium salts. 40. The kit according to claim 39, wherein the polymeric quaternary onium salts comprise poly vinylbenzyltrimethyl-ammonium chloride (TMQ), polyvinyl benzyl tributyl ammonium chloride (TBQ) or polyvinylbenzyl benzyldimethyl ammonium chloride (BDMQ), polyvinylbenzyl-tributylphosphonium chloride, or polyvinyl tributyl sulfonium chloride.

Details for Patent 6,602,657

Applicant	Tradename	Biologic Ingredient	Dosage Form	BLA	Approval Date	Patent No.	Expiredate
Grifols Therapeutics Llc	ALBUKED, PLASBUMIN-20, PLASBUMIN-25, PLASBUMIN-5	albumin (human)	For Injection	101138	10/21/1942		2040-01-28
Baxalta Us Inc.	BUMINATE, FLEXBUMIN	albumin (human)	Injection	101452	03/03/1954		2040-01-28
Csl Behring Ag	ALBURX	albumin (human)	Injection	102366	07/23/1976		2040-01-28