Chromophores in the preparation of novel tandem conjugates

Details Chromophores in the preparation of novel tandem conjugates Chromophores in the preparation of novel tandem conjugates

2000-03-07

2002-04-16

Ceperley, Mary E. (Department: 1641)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving antigen-antibody binding, specific binding protein...

C435S006120, C435S007500, C435S040500, C436S501000, C436S532000, C436S546000, C436S800000, C530S391300, C530S391500, C530S391900, C530S402000, C530S403000

Reexamination Certificate

active

06372445

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to novel bifunctional chromophores useful for labeling materials such as proteins or cells, novel complexes containing bifunctional chromophores, and methods of using them.
BACKGROUND OF THE INVENTION
Fluorescent labeling reagents have become increasingly useful investigative tools. The wider use of fluorescently labeled probes has resulted partly from advances in instrumentation and partly from the availability of new and improved fluorescent dyes. The cyanine dyes have received particular interest since relatively minor alterations in their chemical structure allows for variation in their excitation and emission wavelengths, an advantage for designing multicolor systems useful for simultaneous detection of more than one fluorescent probe.
More recently the cyanine dyes have become widely used as one component of a tandem conjugate with a second fluor, often proteinaceous fluorophores such as phycoerythrin (PE) or Peridinin-chlorophyll a-protein (PerCP). When the emission spectrum of one fluor overlaps the excitation spectrum of another, and they are sufficiently close to each other (<10 nm), it is possible for the excitation energy of the first fluor to be transferred to the second through a fluorescent resonance energy transfer process (Glazer and Stryer,
Biophys. J.
43:383-386, 1983).
A significant drawback to the use of these reagents, particularly for labeling antibodies used in analysis and sorting of blood cells, is the tendency for the fluor complex to bind to components of the system in an indiscriminate manner (van Vugt et al.,
Blood
88:2358-2359, 1996; Beavis and Pennline,
Cytometry
24:390-394, 1996; Shapiro,
Practical Flow Cytometry,
3rd ed., p. 282, 1995).
A need, therefore, still exists for new labeling reagents that are sensitive, easily detected, and exhibit little or no undesirable fluor-mediated binding.
BRIEF DESCRIPTION OF THE INVENTION
The inventors herein disclose new heterobifunctional chromophores that are capable of coupling with two distinct moieties. One moiety may be either a signal-enhancing agent or a blocking agent. The second moiety may be one member of a specific binding pair. The invention is based in part on the surprising result that when a chromophore is used as a “cross-linker” between a signal-enhancing agent and a member of a binding pair (essentially being buried between the two), the signal of the chromophore is not quenched. This arrangement, wherein the chromophore acts simultaneously as a cross-linker and a detectable compound, provides significant advantages over previously known compounds since the chromophore is sterically hindered from interacting non-specifically with substances present in the analytical system. Moreover, the chromophore can be used as a cross-linker with little or no loss of detectable signal.
Also disclosed are complexes formed between a bifunctional chromophore and a signal-enhancing agent. The signal-enhancing agent is capable of participating in resonance energy transfer reactions. The interaction between the chromophore and the signal-enhancing agent may be covalent or non-covalent. Alternatively, the bifunctional chromophore may form a complex with a blocking agent.
Invention complexes can be used in assays involving non-covalent binding to the complementary member of the specific binding pair. Thus, the present invention should find widespread application since a wide variety of methods involve competitive or non-competitive binding of one member of a binding pair to another for detection, analysis or measurement of the presence of the complementary member of the binding pair.
Similar chromophore complexes known in the art have been widely used but exhibit significant drawbacks including relative dimness (making them unsuitable for detection of complementary binding pair members present at low levels), photobleaching, signal spillover into neighboring detection channels (a critical problem when detecting multiple colors simultaneously), instability, and non-specific binding to irrelevant or inappropriate components of the analytical system. The chromophore and methods of the invention, however, overcome these problems, particularly the problem of non-specific binding.
Also disclosed are methods of making the heterobifunctional chromophores of the invention and methods for making novel complexes having bifunctional chromophores as one of their components. The complexes are suitable for a variety of uses such as the labeling of antibodies for use as immunologic reagents and the labeling of DNA for use as a probe. Tandem complexes available in the art do not employ bifunctional chromophores, wherein the chromophore is buried between the signal enhancing agent and the member of the specific binding pair (Waggoner et al., EP 747700, published Dec. 11, 1996; Stryer et al., U.S. Pat. No. 4,542,105, issued Sep. 17, 1985).


REFERENCES:
patent: 4520110 (1985-05-01), Stryer et al.
patent: 4542104 (1985-09-01), Stryer et al.
patent: 4876190 (1989-10-01), Recktenwald
patent: 4981977 (1991-01-01), Southwick et al.
patent: 5145772 (1992-09-01), Voyta et al.
patent: 5171846 (1992-12-01), Gupta
patent: 5597696 (1997-01-01), Linn et al.
patent: 5622821 (1997-04-01), Selvin et al.
patent: 5648213 (1997-07-01), Reddy et al.
patent: 5714386 (1998-02-01), Roederer
patent: 5861256 (1999-01-01), Glass et al.
patent: 0 314 406 (1993-08-01), None
patent: 0 747 700 (1996-12-01), None
patent: 0 800 083 (1997-04-01), None
S. Mujumdar et al, Bioconjugate Chem., 7, 356-362 (1996).*
H. Gruber et al, Bioconjugate Chem., 8, 552-559 (1997).*
Ballou, et al., “Tumor labeling in vivo using cyanine-conjugated monoclonal antibodies,”Cancer Immunol. Immunother. 41:257-263 (1995).
Beavis and Pennline, “ALLO-7: A New Fluorescent Tandem Dye for Use in Flow Cytometry,”Cytometry24:390-394 (1996).
Chen and Evangelista, “Feasibility Studies for Simultaneous Immunochemical Multianalyte Drug Assay by Capillary Electrophoresis with Laser-Induced Fluorescence,”Clin. Chem. 40(9):1819-1822 (1994).
Daubner, et al., “Yellow light emission ofVibrio fischeristrain Y-1: Purification and characterization of the energy-accepting yellow fluorescent protein,”Proc. Natl. Acad. Sci. USA84:8912-8916 (1987).
Ernst, et al., “Cyanine Dye Labeling Reagents for Sulfhydryl Groups,”Cytometry10:3-10 (1989).
Feeney, R.E., “Chemical modification of proteins: comments and perspectives,”Int. J. Peptide Protein Res. 29:145-161 (1987).
Glazer and Stryer, “Fluorescent Tandem Phycobiliprotein Conjugates,”Biophys. J. 43:383-386 (1983).
Han, et al., “Chemical Cross-Links of Proteins By Using Bifunctional Reagents,”Int. J. Biochem. 16(2):129-145 (1984).
Hung, et al., “Cyanine Dyes with High Absorption Cross Section as Donor Chromophores in Energy Transfer Primers,”Analytical Biochemistry243:15-27 (1996).
Laso, F.,Cytometry26(4):275-280 (1996).
Mujumdar, et al., “Cyanine Dye Labeling Reagents Containing Isothiocyanate Groups,”Cytometry10:11-19 (1989).
Mujumdar, et al., “Cyanine Dye Labeling Reagents: Sulfoindocyanine Succinimidyl Esters,”Bioconjugate Chemistry4(2):105-111 (1993).
Prasher, et al., “Primary structure of theAequorea victoriagreen-fluorescent protein,”Gene111:229-233 (1992).
Robins, R., “Methods Mol. Biol.,”Immunochemical Protocols 2nd Edition80:337-346 (1998).
Rye, et al., “Stable fluorescent complexes of double-stranded DNA with bis-intercalating asymmetric cyanine dyes: properties and applications,”Nucleic Acids Research20(11):2803-2812 (1992).
Shapiro, M.D., H.M., “cyanine Dye labels: From Cy-Fi to Hi5 for Cy5,”Practical Flow Cytometry, Third Edition281-282 (1995).
Southwick, et al., “Cyanine Dye Labeling Reagents-Carboxymethlindocyanine Succinimidyl Esters,”Cytometry11:418-430 (1990).
van Vugt, et al., “Binding of PE-CY5 Conjugates to the Human High-Affinity Receptor for IgG (CD64),”Blood88:2358-2359 (1996).
Waggoner, et al., “PE-CY5 A New Fluorecent Antibody Label for Three-Color Flow Cytometry with a Single Laser,”Ann. N.Y. Acad. Sci. 677:185-193 (1993).
Yu, et al., “Cyanine dye dUTP analogs for enzyma

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