Nucleic acid hybridization assay employing detectable anti-hybrid antibodies

• US 5,200,313 A
• Filed: 04/25/1988
• Issued: 04/06/1993
• Est. Priority Date: 08/05/1983
• Status: Expired due to Fees

• Current Assignee: Miles, Inc. (Bayer AG)
• Original Assignee: Miles, Inc. (Bayer AG)
• Inventors: Carrico, Robert J.
• Primary Examiner(s): Yarbrough, Amelia Burgess
• Assistant Examiner(s): Marschel, Ardin H.
• Application Number:
  US07/188,114
• Time in Patent Office: 1,807 Days
• Field of Search: 435/6, 435/7, 435/803, 435/810, 935/2, 935/78, 536/27, 530/387, 436/501, 436/508, 436/512, 436/808
• US Class Current: 435/6.11
• CPC Class Codes: C12Q 1/68 involving nucleic acids
• CPC Class Codes: C12Q 1/6804 Nucleic acid analysis using immunogens immunoassay G01N33/53
• CPC Class Codes: C12Q 1/689 for bacteria
• CPC Class Codes: G01N 33/5308 for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
• CPC Class Codes: Y10S 435/803 Physical recovery methods, e.g. chromatography, grinding
• CPC Class Codes: Y10S 435/81 Packaged device or kit
• CPC Class Codes: Y10S 436/808 Automated or kit

Abstract

A nucleic acid hybridization assay employing an immobilized or immobilizable polynucleotide probe selected to form DNA.RNA or RNA.RNA hybrids with the particular polynucleotide sequence to be determined. Resulting hybrids are detected by binding of an antibody reagent, preferably labeled with a detectable chemical group, selective for binding the hybrids in the presence of the single stranded sample and probe nucleic acids. No immobilization or labeling of sample nucleic acids is necessary and hybridization can be performed entirely in solution.

Claims

1. A nucleic acid hybridization method for determining a polynucleotide having a particular base sequence in a test medium containing single stranded nucleic acids, comprising the steps of:

(a) combining the test medium with an immobilized polynucleotide probe under hybridization conditions, said immobilized probe comprising at least one single stranded base sequence which is substantially complementary to the sequence in the polynucleotide to be determined and which is (i) substantially composed of RNA when the polynucleotide to be determined is RNA or DNA, or (ii) is substantially composed of DNA or RNA when the polynucleotide to be determined is RNA,

whereby hybridization of the polynucleotide to be determined with said probe results in the formation of hybrid duplexes that are either DNA.RNA or RAN.RNA, and

(b) detecting immobilized hybridized probe resulting from step (a) by adding an antibody reagent that binds to either of said DNA.RNA or RNA.RNA duplexes but not both duplex types when formed between the polynculeotide to be determined and the complementary probe and determining the antibody reagent that becomes bound to such duplexes.

2. The method of claim 1 wherein before step(b) the resulting immobilized hybridized nucleic acids from the test medium are separated from the remainder of the reaction mixture.

3. The method of claim 1 wherein the antibody reagent is labeled with a detectable chemical group.

4. The method of claim 3 wherein the detectable chemical group is an enzymatically active group, a fluorescer, a chromophore, a luminescer, a specifically bindable ligand, or a radioisotope.

5. The method of claim 3 wherein the detectable chemical group is an enzyme.

6. The method of claim 3 wherein the labeled antibody reagent which becomes bound to said duplexes is separated from that which does not become so bound and wherein the detectable chemical group is measured in one of the separated fractions.

7. The method of claim 1 wherein the probe is immobilized by being fixed to a solid support.

8. The method of claim 1 wherein the particular polynucleotide to be determined is RNA or DNA and said probe is substantially composed of RNA.

9. The method of claim 1 wherein the particular polynucleotide to be determined is RNA and said probe is substantially composed of DNA.