USOO5936O7OA United States Patent (19) 11 Patent Number: 5,936,070 Singh et al. (45) Date of Patent: *Aug. 10, 1999 54 CHEMILUMINESCENT COMPOUNDS AND FOREIGN PATENT DOCUMENTS METHODS OF USE O 270946 A2 6/1988 European Pat. Off.. 270946A2 6/1988 European Pat. Off.. 75 Inventors: Sharat Singh, San Jose; Rajendra 0.322 926 A2 7/1989 European Pat. Off.. Singh, Mountain View, both of Calif; 0.324 202 A1 7/1989 European Pat. Off.. Frank Meneghini, Keene, N.H.; Edwin 322926A2 7/1989 European Pat. Off.. F. Ullman, Atherton, Calif. 324202A1 7/1989 European Pat. Off.. 352713B1 7/1989 European Pat. Off.. 73 Assignee: Dade Behring Marburg GmbH, 330433A2 8/1989 European Pat. Off.. Marburg, Germany 34.5776A3 12/1989 European Pat. Off.. O 421 788 A2 4/1991 European Pat. Off.. * Notice: This patent issued on a continued pros- WO 88/OO695 1/1988 WIPO. ecution application filed under 37 CFR WO 89.12232 12/1989 WIPO. 1.53(d), and is subject to the twenty year WO 9007511 7/1990 WIPO. patent term provisions of 35 U.S.C. WO 9506877 3/1995 WIPO. 154(a)(2). OTHER PUBLICATIONS Hammond et al; J Biolum and Chemilum; Nucleophilic 21 Appl. No.: 08/664,269 Addition tO the 9 Position of 22 Filed: Jun. 11, 1996 9-Phenylcarboxylate-10-methylacridinium Protects against Hydrolysis of the Ester; 1990. Related U.S. Application Data Zomer et al; Analytica Chimica Acta. 227. 11-19; Chemi luminogenic Labels, Old and New; 1989. 62 Division of application No. 08/373,678, Jan. 17, 1995, Pat. No. 5,545,834, which is a continuation of application No. (List continued on next page.) 07/916,453, Jul. 20, 1992, abandoned. Primary Examiner Mary E. Ceperley 51) Int. Cl. ...................... G01N 33/531; G01N 33/576; Attorney, Agent, or Firm Theodore J Leitereg GO1N 33/76 52 U.S. Cl. ........................... 530/391.3; 435/6; 435/805; 57 ABSTRACT 435/815; 435/818; 436/544; 530/391.5 Methods are disclosed for determining an analyte in a 58 Field of Search ................................. 530/391.3, 405, medium Suspected of containing the analyte. One method 530/391.5; 435/6, 805, 815, 818; 436/544 comprises providing (1) combining a medium Suspected of containing the analyte and a novel chemiluminescent 56) References Cited compound, (2) combining a means for chemically activating the chemiluminescent compound; and (3) detecting the U.S. PATENT DOCUMENTS amount of luminescence generated by the chemiluminescent- 0 3,876,659 4/1975 Houlihan et al.. compound. The amount of luminescence generated is related 4,380,580 4/1983 Boguslaski et al. .. ... 436/532 to the amount of analyte in the medium. The chemilumi 4,383,031 5/1983 Boguslaski et al. .. ... 436/536 neScent compound can be chemically activated by hydrogen 4,729,950 3/1988 Kricka et al. ..... ... 436/800 peroxide. Compositions and kits are also disclosed. 4,857,652 8/1989 Schaap .......... ... 549/516 4,891,324 1/1990 Pease et al. ....... ... 436/519 4,931,223 6/1990 Bronstein et al. ... 436/537 The chemiluminiscent compound is a Spiro-acridan and has 4,950,588 8/1990 Dattagupta ... ... 435/6 4.959,182 9/1990 Schaap ...... ... 435/6 Z. 4,962,192 10/1990 Schaap . ... 536/18.1 5,013,827 5/1991 Schaap ................................... 536/17.3 Y 5,055,414 10/1991 Babb et al. ............................. 436/501 O X 5,068,339 11/1991 Schaap et al. ... 548/110 5,154,887 10/1992 Babb et al. ....... ... 435/794 5,225,584 7/1993 Brooks et al. ... 558/189 5,283,334 2/1994 McCapra ................................. 54.6/104 5,284.951 2/1994 McCapra et al. ....................... 54.6/107 N 5,290.936 3/1994 Beheshti et al. .. ... 54.6/104 5,338,847 8/1994 McCapra ........... ... 54.6/104 CH 5,438,146 8/1995 Schaap et al. ... 548/110 5,516,636 5/1996 McCapra ....... ... 435/6 5,536,834 7/1996 Singh et al. .. ... 544/98 where X and Y are independently O, S, Se or NH; and Z is 5,545,834 8/1996 Singh et al. ...... - - - - - - - 544/6 a chain 1-5 atoms in length; 0 to 8 hydrogens of the 5,578.498 11/1996 Singh et al. ............. ... 436/518 compound alone or taken together, may be replaced an alkyl, 5,593,845 1/1997 Akhavan-Tafiti et al. ... 435/7.92 alkylidine, aryl aralkyl, or an alkyl, aryl or aralkyl Substi 5,616,729 4/1997 Schaap et al. ........... ... 549/223 tuted with one or more radicals of functional groups; 1 to 4 E. 5.9. SNE s al - - - - - - - - of the aromatic carbon atoms may be replaced by nitrogen 5,670,6442 - - 12 9/1997 Akhavan-TafitiChaapel al. ...........et al. - ...- - - 546/103 alOmS,toms; anand 0 toO 1 hvidydrogens may beb replacedlaced bby a SpecIIIcifi 5,672,478 9/1997 Singh et al. ............. - - - - - - - 435/6 binding pair member or fluorescent group. 5,698,728 12/1997 Schaap et al. ........... ... 556/448 5,750,698 5/1998 Akhavan-Tafiti et al. - - - - - - - - - - - - - - 54.6/102 9 Claims, 3 Drawing Sheets 5,936,070 Page 2 OTHER PUBLICATIONS Lee et al; J of Organic Chem; 41:16, pp. 2685–2688; Chemiluminescence from the Reaction of Singlet Oxygen Lee et al; J of Amer Chem Soc; 102:11; pp. 3823–3829; with 10,10'-dimethyl-9,9'-biacridylidene. A Reactive Structural Effects on the Intramolecular Electron Transfer Induced Decomposition of a Series of 1,2-Dioxetanes 1,2-dioxethan; 1976. Derived from 9-Alkylidene-10-methylacridans; May 21, Weeks et al; Clin. Chem.; 29/8; 1474–1479; Acridinium 1980. Esters as High-Specific-Activity Labels in Immunoassay; McCapra et al; Tetrahedron Letters; 23:49, pp. 5225-5229; 1983. Metal Catalysed Light Emission from a Dioxetan; 1982. Heller, et al., Rapid Detection and Identification of Infec McCapra, et al.; J Biolum and Chemilum; 4:51-58; Lumi tious Agents, 1985, Academic Press, Inc., pp. 245-257, neScent Labels for Immunoassay-From Concept to Practice; “Chemiluminescent and Fluorescent Probes for DNA 1989. Hybridization Systems”. Turro et al; J Amer Chem Soc; 100:22; Generation, Diffu Zomer, et al., Analytica Chimica Acta, 1989, vol. 227, pp. sivity, and Reactivity of Singlet Oxygen in Polymer Matri 11-19, “Chemiluminogenic Labels, Old and New”. ces. A Convenient and Sensitive Chemiluminescent Tech Grant and Hackh's Chemical Dictionary, Fifth Edition nique; Oct. 25, 1978. (1987), pp. 30, 115, 220,323, 444-445, 560–561, 589. U.S. Patent Aug. 10, 1999 Sheet 1 of 3 5,936,070 2OOOO ESIGNAL BACKGROUND RELATIVE UNSLIGHT 10000 FIG.1 TIME (SECONDS) 60000 50000 - E. ESIGNAL BACKGROUND 40000 RELATIVE LIGHT 30000 UNITS 20000 1 OOOO FIG.2 10 2O 3O 40 50 TIME (SECONDS) U.S. Patent Aug. 10, 1999 Sheet 2 of 3 5,936,070 40OOO ESIGNAL 30000 BACKGROUND RELATIVE LIGHT UNTS 20000 1 OOOO FI G 3 O SS :lists:...as a passenge:Sea Eesa Easter s 1O 20 30 40 50 60 70 TIME (SECONDS) 3000 RELATIVE LIGHT UNITS 2000 1000 O 1OO 200 300 400 TIME (SECONDS) U.S. Patent Aug. 10, 1999 Sheet 3 of 3 5,936,070 1500 N-500 nM (IVa) WITH HO + HRP 1OOO RELATIVE LIGHT UNITS 500 FIG.5 O 100 200 300 4OO TIME (SECONDS) 5,936,070 1 2 CHEMILUMNESCENT COMPOUNDS AND There is a continuing need for new and accurate tech METHODS OF USE niques that can be adapted for a wide spectrum of different ligands or be used in Specific cases where other methods This application is a division of patent application U.S. may not be readily adaptable. Ser. No. 08/373,678 filed Jan. 17, 1995, now U.S. Pat. No. 5 Homogeneous immunoassays have previously been 5,545,834, which is a continuation of patent application U.S. described for Small molecules. These assays include Syva Ser. No. 07/916,453 filed Jul. 20, 1992, now abandoned. Company’s FRATOR assay, EMITOR assay, enzyme channel ing immunoassay, and fluorescence energy transfer immu BACKGROUND OF THE INVENTION noassay (FETI); enzyme inhibitor immunoassays (Hoffman LaRoche and Abbott Laboratories); and fluorescence polar Field of the Invention ization immunoassay (Dandlicker), among others. All of This invention relates to compositions, methods and kits these methods have limited Sensitivity, and only a few for determining an analyte in a Sample. including FETI and enzyme channeling, are Suitable for Chemiluminescent labels for immunoassays and nucleic large multiepitopic analytes. acid probe assays provide a high degree of Sensitivity when 15 Chemiluminescent compounds find wide application in compared to other commonly used labels. An excellent the assay field because of their ability to emit light. For this Overview of the Subject is discussed in McCapra, et al., reason, luminescers have been utilized as labels in assays Journal of Bioluminescence and Chemiluminescence Such as nucleic acid assays and immunoassayS. For example, 4:51–58 (1989). a member of a specific binding pair is conjugated to a Particularly important chemiluminescent compounds are lumineScer and various protocols are employed. The lumi acridinium esters and amides, which must be stored in water neScer conjugate can be partitioned between a Solid phase at a pH that is compatible with protein Stability require and a liquid phase in relation to the amount of analyte in a ments. This results in the formation of a pseudobase, which Sample Suspected of containing the analyte. By measuring is not chemiluminescent. Chemiluminescence is initiated by the luminescence of either of the phases, one can relate the adding acid to form the acridinium salt, followed by the 25 level of luminescence observed to a concentration of the addition of Strongly alkaline hydrogen peroxide.