Claims
- 1. A method of detecting the presence of a target substance in a sample, wherein said target substance is a ligand which is bound by a second member of a ligand binding pair, comprising:combining said sample with a complex of said second member of said ligand binding pair and an enzyme, under conditions which permit binding of said ligand binding pair if said target is present in said sample, removing from said sample all said complex which is not bound, adding a dioxetane of the formula I: wherein Y1 and Y2 are independently H, a hydroxyl group, a halogen, an unsubstituted lower alkyl group, a hydroxy lower alkyl group, a halo lower alkyl group, a phenyl group, a halo phenyl group, an alkoxy phenyl group, an alkoxy phenoxy group, a hydroxy alkoxy group, a cyano group, an amide group, an alkoxy group or a carboxyl group, wherein R is C1-12 alkyl, aryl or aralkyl, wherein X is an enzyme-labile group selected from the group consisting of a phosphate, galactoside, acetate, 1-phospho-2,3-diacylglyceride, 1-thio-D-glucoside, adenosine triphosphate, adenosine diphosphate, adenosine monophosphate, adenosine, α-D-glucoside,β-D-glucoside, β-D-glucuronide, α-D-mannoside, β-D-mannoside, β-D-fructofuranoside, β-glucosiduronate, P-toluenesulfonyl-L-arginine ester, and P-toluenesulfonyl-L-arginine amide, and wherein Z is an electron-active group selected from the group consisting of electron withdrawing groups and electrondonating groups and occupies the four or five position on the phenyl ring, to said sample, and monitoring said sample for chemiluminescence emission, wherein said enzyme cleaves said enzyme-labile group and said emission is indicative of the presence of said target compound in said sample.
- 2. The method of claim 1, wherein said chemiluminescence emission is quantified, and wherein the amount of chemiluminescence emission is directly related to the amount of said target in said sample.
Parent Case Info
This application is a continuation of Ser. No. 09/296,539, Apr. 22, 1999, U.S. Pat. No. 6,140,495, which is a continuation of Ser. No. 08/904,847, Aug. 1, 1997, U.S. Pat. No. 6,022,964, which is a continuation of Ser. No. 08/588,810, Jan. 19, 1996, U.S. Pat. No. 5,851,771, which is a continuation of Ser. No. 08/057,903, May 7, 1993, U.S. Pat. No. 5,538,847, which is a continuation-in-part of Ser. No. 07/806,928, Dec. 12, 1991, U.S. Pat. No. 5,330,900, which is a division of Ser. No. 07/574,786, Aug. 30, 1990, U.S. Pat. No. 5,112,960, which is a continuation-in-part of Ser. No. 07/559,152, Jul. 25, 1990, abandoned, which is a division of Ser. No. 07/367,772, Jul. 17, 1989, abandoned, and a division of Ser. No. 07/140,197, Dec. 31, 1987, abandoned.
US Referenced Citations (57)
Foreign Referenced Citations (3)
Number |
Date |
Country |
8906226 |
Jul 1989 |
WO |
9204341 |
Mar 1992 |
WO |
92044341 |
Mar 1992 |
WO |
Non-Patent Literature Citations (2)
Entry |
Edwards, B., Sparks, A., Voyta, J.C., Strong, R., Murphy, O., Bronstein, I., “Naphthyl Dioxetane Phosphates: Synthesis of Novel Substrates for Enzymatic Chemiluminescent Assays”, J. Org. Chem. 55(25):6225-6229 (1990). |
Hann & Spencer, “The Preparation of Chlorovanillin and Some of Its Derivatives”, J. Am. Chem. Soc., 49:535-537 (1927). |
Continuations (4)
|
Number |
Date |
Country |
Parent |
09/296539 |
Apr 1999 |
US |
Child |
09/656213 |
|
US |
Parent |
08/904847 |
Aug 1997 |
US |
Child |
09/296539 |
|
US |
Parent |
08/588810 |
Jan 1996 |
US |
Child |
08/904847 |
|
US |
Parent |
08/057903 |
May 1993 |
US |
Child |
08/588810 |
|
US |
Continuation in Parts (2)
|
Number |
Date |
Country |
Parent |
07/806928 |
Dec 1991 |
US |
Child |
08/057903 |
|
US |
Parent |
07/559152 |
Jul 1990 |
US |
Child |
07/574786 |
|
US |