Nucleic acid hybridization based assay for determining a substance of interest

• US 5,792,606 A
• Filed: 05/05/1995
• Issued: 08/11/1998
• Est. Priority Date: 02/26/1990
• Status: Expired due to Term

• Current Assignee: Roche Diagnostics GmbH (Roche Holding AG)
• Original Assignee: Boehringer Mannheim GmbH (Roche Holding AG)
• Inventors: Deger, Arno, Schenk, Roland
• Primary Examiner(s): Knode, Marian C.
• Assistant Examiner(s): Wortman, Donna C.
• Application Number:
  US08/435,808
• Time in Patent Office: 1,194 Days
• Field of Search: 435/6, 435/7.1, 435/7.5, 435/7.9, 435/7.92, 435/7.93, 435/7.94, 436/500, 436/501, 436/512, 436/518, 436/532, 436/536, 436/538, 436/543, 436/547
• US Class Current: 435/6.16
• CPC Class Codes: G01N 33/538 by sorbent column, particles or resin strip , i.e. sorbent materials

Abstract

The application describes homogeneous assays for determining ligands. A sample is contacted with a labeled substance P₂ and receptor R. The receptor R consists of one molecule of P₁, which monovalently and specifically binds to P₂, and one molecule for R₁, which binds to the ligand of interest. Complexes of P₁, P₂, R₁ and ligand form, and are determined. R₁ must be a nucleic acid molecule.

Claims

1. Method for determining a ligand via a homogeneous assay, comprising:

contacting a sample with an amount of a labelled substance P₂, and a receptor R which consists of one molecule of a binding partner P₁, which monovalently and specifically binds to P₂, and one molecule of R₁, which specifically binds to said ligand, to form complexes comprising P₁, P₂, R₁ and said ligand, and determining said complexes as a determination of said ligand in said sample, wherein R₁ is a nucleic acid molecule.

2. The method of claim 1, wherein said homogeneous assay is a competitive assay and further comprises adding to said sample a conjugate C which consists of (a) a member of the group consisting of a molecule of ligand and a molecule of ligand analogue and (b) a molecule of P₁, wherein said conjugate C competes with any ligand in said sample for binding to R₁.

3. The method of claim 1, wherein P₁ is biotin, a hapten, or an epitope.

4. The method of claim 1, wherein P₂ is a member selected from the group consisting of avidin, streptavidin, a streptavidin polymer, an antibody, an Fab antibody fragment, and an F(ab'"'"')₂ antibody fragment.

5. Method for determining a ligand in a heterogenous assay, comprising:

(i) incubating a liquid sample with two receptors R'"'"' and R" and an unlabeled substance P₂ immobilized on a solid phase, wherein said R'"'"' consists of one molecule P₁ '"'"' and one molecule R₁ '"'"', and said R" consists of one molecule P₁ " and one molecule R₁ ", wherein P₁ '"'"' specifically and monovalently binds said P₂, each of R₁ '"'"' and R₁ " specifically bind to said ligand, to immobilize said ligand on said solid phase to form a complex comprising P₂ -P₁ '"'"'-R₁ '"'"'-ligand --R₁ "-P₂ "--,

(ii) contacting said immobilized complex with labeled P₂, wherein P₂ " specifically and monovalently binds labeled P₂ to form a complex comprising P₂ -P₁ '"'"'-ligand --R₁ "-P₂ "-P₂, and

(iii) measuring a level of labeled P₂ bound to said labeled phase as a determination of said ligand in said sample, wherein R₁ '"'"' and R₁ " are different nucleic acid molecules.

6. The method of claim 5, wherein said heterogeneous assay is a competitive assay and further comprises adding to said sample a conjugate C which consists of (a) a member of the group consisting of a molecule of ligand and a molecule of ligand analogue and (b) a molecule of P₁, wherein said conjugate C competes with any ligand in said sample for binding to R₁.

7. The method of claim 5, wherein P₁ is biotin, a hapten, or an epitope.

8. The method of claim 5, wherein P₂ is a member selected from the group consisting of avidin, streptavidin, a streptavidin polymer, an antibody, an Fab antibody fragment, and an F(ab'"'"')₂ antibody fragment.