Phycobiliprotein fluorescent conjugates

• US 4,542,104 A
• Filed: 04/06/1983
• Issued: 09/17/1985
• Est. Priority Date: 04/06/1983
• Status: Expired due to Term

• Current Assignee: Board of Trustees of the Leland Stanford Junior University (Stanford Management Co.)
• Original Assignee: Board of Trustees of the Leland Stanford Junior University (Stanford Management Co.)
• Inventors: Glazer, Alexander N., Stryer, Lubert
• Primary Examiner(s): Nucker, Christine M.
• Application Number:
  US06/483,006
• Time in Patent Office: 895 Days
• Field of Search: 436/536, 436/537, 436/543, 436/544, 436/546, 436/800, 250/461.2
• US Class Current: 436/536
• CPC Class Codes: G01N 33/533 with fluorescent label
• CPC Class Codes: G01N 33/542 with steric inhibition or signal modification, e.g. fluorescent quenching
• CPC Class Codes: Y10S 436/80 Fluorescent dyes, e.g. rhodamine

Abstract

Fluorescent conjugates are employed providing combinations of a fluorescent sensitizer and a fluorescent phycobiliprotein. The conjugates find use in applications where large Stokes shifts, high absorption coefficients and high fluorescence quantum yields are desired. Particularly, combinations of phycobiliproteins are employed where the wavelength of excitation may be 50 nm or more different from the emission wavelength.

Claims

1. A conjugate comprising an energy donor as a first component and a fluorescer as a second component joined together at an energy exchanging distance, wherein one of said first and second components is a phycobiliprotein and the two components have a spectral overlap integral of at least about 10^-16 cm³ M^-1.

2. A conjugate according to claim 1, wherein said first and second components are phycobiliproteins.

3. A conjugate according to claim 2, wherein said phycobiliproteins are covalently joined by a linking group wherein said first and second components are separated by not more than 10 nm.

4. A conjugate according to claim 3, wherein a portion of said linking group is a disulfide linkage.

5. A conjugate according to claim 1, wherein said fluorescer fluoresces at a wavelength in the range of about 550 to 800 nm.

6. A conjugate according to claim 1, wherein said first and second components are covalently joined.

10. A conjugate comprising an energy donor-acceptor pair according to claim 1 covalently joined to a ligand or receptor.

11. In a cell staining, fluorescence-activated cell sorting or competitive protein binding diagnostic assay method employing a fluorescent label, where a labeled ligand and/or receptor are brought together and fluorescence is detected in relation to the binding of said ligand and receptor, the improvement comprising employing the conjugate composition according to claim 1 as the fluorescent label.

12. A method according to claim 11, wherein said method is fluorescence activated cell sorting.

13. A method according to claim 11, wherein said method is a diagnostic assay employing a fluorescent label.

7. A conjugate comprising two phycobiliproteins joined within energy exchanging distance, wherein one of said phycobiliproteins absorbs light in the range of about 300 to 600 nm and emits light in the range of about 400 to 700 nm and the other of said phycobiliproteins absorbs light in the range of about 400 to 650 nm and emits light in the range of about 500 to 800 nm, wherein there is substantial overlap between the emission spectrum of one and the absorption spectrum of the other to provide a spectral overlap integral of at least about 10^-16 cm ³ M^-1.

8. A conjugate according to claim 7, wherein one of said phycobiliproteins is phycoerythrin and the other of said phycobiliproteins is allophycocyanin.

9. A conjugate according to claim 8, wherein said phycobiliproteins are joined by a linking group containing a disulfide linkage.