Process for the determination of a specifically bindable substance

• US 5,126,241 A
• Filed: 10/10/1989
• Issued: 06/30/1992
• Est. Priority Date: 10/12/1988
• Status: Expired due to Term

• Current Assignee: Boehringer Mannheim GmbH (Roche Holding AG)
• Original Assignee: Boehringer Mannheim GmbH (Roche Holding AG)
• Inventors: Schenk, Roland
• Primary Examiner(s): Kepplinger, Esther L.
• Assistant Examiner(s): SCHEINER, TONI R
• Application Number:
  US07/418,516
• Time in Patent Office: 994 Days
• Field of Search: 435/7.1, 435/7.9, 435/975, 435/7.92, 435/7.93, 435/7.5, 436/501, 436/504, 436/512, 436/518, 436/536, 436/538
• US Class Current: 435/7.1
• CPC Class Codes: G01N 33/54306 Solid-phase reaction mechanisms
• CPC Class Codes: G01N 33/78 Thyroid gland hormones , e.g. T3, T4, TBH, TBG or their receptors

Abstract

The present invention provides a process for the determination of a specifically bindable substance by incubation of a sample solution with at least three receptors R₁, R₂ and R₃, of which R₁ is specifically bindable with R₂ and R₃, as well as with the substance to be determined, R₂ brings about the binding to a solid phase and R₃ carries a labelling, separation of bound labelling from unbound labelling and measurement of the labelling in one of the two phases, wherein, as receptor R₁, a receptor is used which has at least two binding positions which bind specifically with an epitope of the substance to be determined, as R₂ a conjugate of a partner P₁ of a specifically binding pair and of a substance S which corresponds to the substance to be determined or is a derivative thereof and has at least one epitope of the substance to be determined, the partner P₁ thereby either being bound to a solid phase or being immobilized, and as R₃ a complex is used which contains at least the substance S and a labelling.

Claims

1. Method for determination of a specifically bindable substance comprising:

incubating a liquid sample with at least three receptors R₁, R₂ and R₃, wherein

(a) R₁ specifically binds with R₂, R₃ and said specifically bindable substance and is an antibody or an Fab₂ fragment;

(b) R₂ comprises a conjugate of (i) a substance S which is identical to the specifically bindable substance to be determined and (ii) a first partner P₁ of a binding pair,

(c) R₃ comprises a conjugate of S and a label, to form complexes containing R₁, R₂ and R₃,

(d) adding a second partner of a specific binding pair P₂ to said liquid sample together with a solid phase, wherein P₂ binds to said solid phase and to P₁ to immobilize said complexes containing R₁, R₂ and R₃, and,

determining R₃ bound to solid phase or in liquid phase as a determination of said specifically bindable substance.

5. Method of claim 1, wherein P₁ is biotin and P₂ is streptavidin, avidin or a biotin specific antibody.

7. Method of claim 1, wherein R₃ consists of S and a label.

9. Method of claim 1, wherein said label is radioactive chemiluminescent, or fluorescent.

11. Method of claim 1, wherein R₃ comprises a first member complex consisting of S and P₁ and a second member complex consisting of P₂ and a label, wherein P₁ and P₂ are bound to each other.

13. Method of claim 1, wherein P₁ and P₂ are selected from the group consisting of biotin-streptavidin, biotin-avidin, biotin-biotin specific antibody, antigen-antigen specific antibody, hapten-hapten binding protein and oligopeptide-oligopeptide specific antibody.

15. Method of claim 1, wherein said solid phase is a matrix consisting of a synthetic resin and P₂ is bound thereto via a spacer.

17. Method of claim 1, wherein P₁ is biotin and P₂ is avidin or streptavidin.

2. Method for determination of a specifically bindable substance, comprising:

incubating a liquid sample with at least three receptors R₁, R₂ and R₃, and with a solid phase bound partner P₂ of a binding pair, wherein;

a) R₁ specifically binds with R₂, R₃ and said specifically bindable substance and is an antibody or an Fab₂ fragment;

(b) R₂ comprises a conjugate of (i) a substance S which is identical to the specifically bindable substance to be determined and (ii) a first partner P₁ of a binding pair;

(c) R₃ comprises a conjugate of S and a label,

(d) P₂ binds to P₁, to form immobilized complexes containing R₁, R₂ and R₃, and;

determining R₃ bound to solid phase or in liquid phase as a determination of said specifically bindable substance.

3. Method of claim 2, wherein P₂ is bound to said solid phase via solid phase bound P₁.

4. Method of claim 2, comprising adding said solid phase bound member of a specific binding pair P₂ after addition of R₁, R₂ and R₃ to said sample solution.

6. Method of claim 2, wherein P₁ is biotin and P₂ is streptavidin, avidin or a biotin specific antibody.

8. Method of claim 2, wherein R₃ consists of S and a label.

10. Method of claim 2, wherein said label is radioactive chemiluminescent, or fluorescent.

12. Method of claim 2, wherein R₃ comprises a first member complex consisting of S and P₁ and a second member complex consisting of P₂ and a label, wherein P₁ and P₂ are bound to each other.

14. Method of claim 2, wherein P₁ and P₂ are selected from the group consisting of biotin-streptavidin, biotin-avidin, biotin-biotin specific antibody, antigen-antigen specific antibody, hapten-hapten binding protein and oligopeptide-oligopeptide specific antibody.

16. Method of claim 2, wherein said solid phase is a matrix consisting of a synthetic resin and P₂ is bound thereto via a spacer.

18. Method of claim 2, wherein P₁ is biotin and P₂ is avidin or streptavidin.