Fluorescent antibody composition for immunofluorometric assay

• US 4,341,957 A
• Filed: 11/26/1975
• Issued: 07/27/1982
• Est. Priority Date: 11/26/1975
• Status: Expired due to Term

• Current Assignee: Irwin Wieder
• Original Assignee: Analytical Radiation Corporation
• Inventors: Wieder, Irwin
• Primary Examiner(s): Dixon, Harold A.
• Application Number:
  US05/635,411
• Time in Patent Office: 2,435 Days
• Field of Search: 250/461, 250/365, 250/302, 250/461 B, 424/2, 424/12
• US Class Current: 250/461.2
• CPC Class Codes: G01N 21/6408 with measurement of decay time, time resolved fluorescence
• CPC Class Codes: G01N 21/6428 Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" in vivo A61B5/00; immunoassay G01N33/53
• CPC Class Codes: G01N 33/533 with fluorescent label

Abstract

A new family of fluorescent antibodies (FAB) and a method for their synthesis is disclosed for use in an apparatus and method wherein the effects of background fluorescence are virtually eliminated from fluorescence measurements. The novel fluorescent antibodies consist of antibodies conjugated with rare-earth chelates which have unique fluorescent properties. These fluorescent antibodies are useful because when excited with short pulses of light, they exhibit fluorescent decay times which are much longer than typical background fluorescence at room temperature and with a spectral width far narrower than typical background fluorescence.

Claims

1. A fluorescent antibody comprising,

an antibody specific to an antigen to be detected, and

a fluorescent rare-earth chelate conjugated with said antibody, said rare-earth chelate consisting of a rare-earth ion coordinated by chelating ligands.

2. The fluorescent antibody of claim 1 where the rare-earth ion is Eu(III).

3. The fluorescent antibody of claim 1 where the rare-earth ion is Tb(III).

4. The fluorescent antibody of claim 2 where the chelating ligands are thenolytrifluoroacetonates and thenolytrifluoroacetonates modified for conjugation with said antibody.

5. The fluorescent antibody of claim 2 where the chelating ligands are hexafluoroacetylactonates and hexafluoroacetylacetonates modified for conjugation with said antibody.

6. The fluorescent antibody of claim 2 where the chelating ligands are benzoyltrifluoroacetonates and benzoyltrifluoroacetonates modified for conjugation with said antibody.

7. The fluorescent antibody of claim 3 where the chelating ligands are acetylacetonates and acetylacetonates modified for conjugation with said antibody.

8. A method of fluorescent detection of a target substance comprising:

tagging a target substance with a fluorescent antibody, said fluorescent antibody comprising an antibody specific to the target substance and a fluorescent rare-earth chelate conjugated with said antibody, said rare-earth chelate consisting of a rare-earth ion coordinated by chelating ligands, said rare-earth chelate having sharp spectral emission and lifetime long compared with the longest of the decay lifetimes of competing untagged ambient substances,

exciting said tagged target substance with pulses of radiation, such pulses having a duration which is short compared with the fluorescent decay lifetime of said fluorescent antibody, and

gating on a fluorescence detection system only after the fluorescence of said ambient substances has substantially decayed but while fluorescent antibody fluorescence is actively decaying.

9. The method of claim 8 where the rare-earth ion is Eu(III).

10. The method of claim 8 where the rare-earth ion is Tb(III).

11. The method of claim 9 where the chelating ligands are hexafluoroacetylacetonates and hexafluoroacetylacetonates modified for conjugation with said antibody.

12. The method of claim 9 where the chelating ligands are benzoyltrifluoroacetonates and benzoyltrifluoroacetonates modified for conjugation with said antibody.

13. The method of claim 9 where the chelating ligands are thenolytrifluoroacetonates and thenolytrifluoroacetonates modified for conjugation with said antibody.

14. The method of claim 10 where the chelating ligands are acetylacetonates and acetylacetonates modified for conjugation with said antibody.

15. A method of fluorescent detection of target substance distribution, wherein said target substance has at least one antibody combining site, the method comprising:

complexing said target substance with a fluorescent antibody, said fluorescent antibody comprising an antibody specific to the target substance and a fluorescent rare-earth chelate conjugated with said antibody, said rare-earth chelate consisting of a rare-earth ion coordinated by chelating ligands, said rare-earth chelate having sharp spectral emission and lifetime long compared with the longest of the decay lifetimes of competing untagged ambient substances,

exciting said target substance-fluorescent antibody complex with pulses of radiation, each pulse having a duration which is short compared with the fluorescent decay lifetime of said fluorescent antibody, and

gating on an image converter tube only after the fluorescence of said ambient substances has decayed but while fluorescent antibody fluorescence is actively decaying.

16. The method of claim 15 where the rare-earth ion is Eu(III).

17. The method of claim 15 where the rare-earth ion is Tb(III).

18. The method of claim 16 where the chelating ligands are hexafluoroacetylacetonates and hexafluoroacetylacetonates modified for conjugation with said antibody.

19. The method of claim 16 where the chelating ligands are benzoyltrifluoroacetonates and benzoyltrifluoroacetonates modified for conjugation with said antibody.

20. The method of claim 16 where the chelating ligands are thenolytrifluoroacetonates and thenolytrifluoroacetonates modified for conjugation with said antibody.

21. The method of claim 17 where the chelating ligands are acetylacetonates and acetylacetonates modified for conjugation with said antibody.

22. In fluorometry, for determining the amount of target substance present after tagging said target substance with antibody specific to the target substance, said antibody conjugated with a fluorophore, the improvement wherein said fluorophore is a fluorescent rare-earth chelate comprising a rare-earth ion coordinated by chelating ligands, said rare-earth chelate having sharp spectral emission and a fluorescent lifetime long compared with the longest of the decay lifetimes of competing untagged ambient substances.