Homogenous protection assay

• US 5,283,174 A
• Filed: 11/08/1990
• Issued: 02/01/1994
• Est. Priority Date: 09/21/1987
• Status: Expired due to Term

• Current Assignee: Gen-Probe Incorporated (Hologic Incorporated)
• Original Assignee: Gen-Probe Incorporated (Hologic Incorporated)
• Inventors: Nelson, Norman C., Arnold, Lyle J. Jr.
• Primary Examiner(s): Wax, Robert A.
• Assistant Examiner(s): KIM, HYO
• Application Number:
  US07/613,603
• Time in Patent Office: 1,181 Days
• Field of Search: 435/6, 436/546, 436/501, 536/25.32
• US Class Current: 435/6.12
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6886 for cancer immunoassay for cancer G01N33/574
• CPC Class Codes: C12Q 1/689 for bacteria
• CPC Class Codes: C12Q 2565/107 Alteration in the property of hybridised versus free label oligonucleotides
• CPC Class Codes: C12Q 2600/158 Expression markers
• CPC Class Codes: G01N 33/5306 Improving reaction conditions, e.g. reduction of non-specific binding, promotion of specific binding
• CPC Class Codes: G01N 33/532 Production of labelled immunochemicals
• 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: G01N 33/58 involving labelled substances G01N33/53 takes precedence
• CPC Class Codes: G01N 33/582 with fluorescent label

Abstract

Improved homogenous diagnostic assay methods and labels for detecting an analyte in a medium when the analyte is a member of a specific binding pair. The methods and labels provide procedures for reducing background and increasing sensitivity. The binding partner of the analyte is labeled with a substance, the stability of which detectably changes whenever said analyte is bound as a member of the specific binding pair. In a closely related system, the analyte is labeled with a substance susceptible to differential degradation depending on whether or not the analyte is bound as a member of its specific binding pair. After incubation and selective degradation or chemical or biochemical alteration, the amount of analyte bound is detected by measuring either the stability change or the extent of degradation of the label. In a particular system, chemiluminescent acridinium ester labeled probes are used in a homogenous hybridization assay format for sensitively detecting the presence of complement any target polynucleotide sequences.

Claims

1. A homogeneous assay method for determining the presence or amount of a first nucleic acid in a medium, comprising the steps of:

contacting a free second nucleic acid with said medium to form a hybridization mixture, wherein said free second nucleic acid is able to hybridize with said first nucleic acid under hybridizing conditions to form a binding pair, and wherein said second nucleic acid is chemically bonded to an N-acridinium ester which is selectively susceptible to chemical degradation by a chemical selected from the group consisting of an acid, a base, and an oxidizing agent, to form an altered N-acridinium ester dependent upon whether said second nucleic acid is free or forms part of said binding pair;

treating said hybridization mixture with said chemical such that N-acridinium ester chemically bonded to free second nucleic acid is degraded to form altered N-acridinium ester and N-acridinium ester present in a binding pair is undegraded; and

determining the amount of undegraded N-acridinium ester remaining after said treating step, without physically separating any said free second nucleic acid from any said binding pair, as a measure of the presence or amount of said first nucleic acid.

2. The method of claim 1 wherein determining the amount of label comprises the addition of H₂ O₂.

3. The method of claim 1 wherein said chemical is an acid.

4. The method of claim 1 wherein said chemical is a base.

5. The method of claim 1 wherein said chemical is an oxidizing agent.

6. The method of claim 3 wherein said acid is HNO₃.

7. The method of claim 4 wherein said base is NaOH.

8. The method of claim 1 wherein said method allows detection of 10^-16 Mol of said first nucleic acid.

9. The method of claim 1, in which said N-acridinium ester comprises the chemical formula ##STR11## wherein is selected from the group consisting of O, N, S, halogen, phosphorus, boron, and arsenic;

Y is selected from the group consisting of O, S, or NH;

R₁ is selected from the group consisting of alkyl, alkenyl, aryl, alkoxy, and aryloxy, or wherein R₁ is absent when X is a halogen;

R₂ is selected from the group consisting of H, alkyl, alkenyl, aryl, alkoxy and aryloxy when X is N, or wherein R₂ is absent when X is not N;

R₃ is selected from the group consisting of H, amino, hydroxy, thiol, halogen, nitro, amido, acetyl, alkyl, aryl, alkenyl, alkoxy, and aryloxy;

R₄ is selected from the group consisting of alkyl, alkenyl, and aryl; and

wherein at least one of R₁, R₂, R₃, or R₄ contains a reactive site which allows chemical conjugation.

10. The method of claim 9 wherein said chemical is an acid.

11. The method of claim 9 wherein said chemical is a base.

12. The method of claim 9 wherein said chemical is an oxidizing agent.