Detection of synthetic oligonucleotides extracted from body fluids or tissues

• US 5,693,466 A
• Filed: 01/03/1995
• Issued: 12/02/1997
• Est. Priority Date: 01/08/1993
• Status: Expired due to Term

• Current Assignee: Idera Pharmaceuticals, Inc. (Aceragen Incorporated)
• Original Assignee: Hybridon, Inc. (Aceragen Incorporated)
• Inventors: Temsamani, Jamal, Agrawal, Sudhir
• Primary Examiner(s): Horlick, Kenneth R.
• Assistant Examiner(s): Tung, Joyce
• Application Number:
  US08/368,243
• Time in Patent Office: 1,064 Days
• Field of Search: 435/6, 536/24.3
• US Class Current: 435/6.16
• CPC Class Codes: C07H 1/06 Separation; Purification
• CPC Class Codes: C12N 15/1017 by filtration, e.g. using filters, frits, membranes
• CPC Class Codes: C12Q 1/6834 Enzymatic or biochemical coupling of nucleic acids to a solid phase

Abstract

The invention provides a method for detecting specific synthetic oligonucleotides that are present in body fluid or tissue samples taken from an animal or human patient to whom oligonucleotides have been administered.

Claims

1. A method of detecting synthetic oligonucleotides that are present in body fluids or tissues, said oligonucleotides being extractable with isobutanol and ethyl ether, the method comprising the steps of:

(a) proteolytically digesting a body fluid or tissue sample;

(b) extracting the proteolytically digested sample with a partitioning agent;

(c) extracting the aqueous phase in the extracted sample with isobutanol and ethyl ether,

(d) drying the remaining solution containing nucleic acids to a pellet and resuspending the nucleic acids into a solution;

(e) binding the nucleic acids to a hybridization membrane;

(f) treating the membrane having bound nucleic acids with a labeled oligonucleotide probe that is complementary to and specifically binds to the synthetic oligonucleotide to be detected;

(g) washing the membrane to remove any labeled oligonucleotide that is not specifically hybridized to nucleic acids bound to the membrane; and

(h) detecting the presence of the probe on the membrane.

2. The method according to claim 1, wherein the body fluid is blood.

3. The method according to claim 1, wherein the body fluid is urine.

4. The method according to claim 1, wherein the tissue is lymphatic tissue.

5. The method according to claim 1, wherein the labelled oligonucleotide probe is a radiolabeled probe.

6. The method according to claim 1, wherein the labeled oligonucleotide probe comprises an oligonucleotide labeled with an antigen to which an antigen-binding antibody or antibody fragment can bind or with a ligand to which a ligand-binding receptor can bind, and wherein the detection of the probe comprises the steps of:

(a) blocking the membrane with a suitable blocking agent to prevent non-specific attachment to the membrane of the antigen-binding antibody or antibody fragment or ligand-binding receptor;

(b) incubating the membrane with an antigen-binding antibody, antibody fragment or receptor that specifically binds to the antigen or ligand and is conjugated to an enzyme that acts upon a substrate to produce chemiluminescence;

(c) washing the membrane to remove any non-specifically bound antibody, antigen-binding antibody fragment or receptor;

(d) adding the substrate upon which the enzyme acts; and

(e) exposing the membrane to photographic film.

7. A method of detecting the presence in body fluids or tissues of a synthetic oligonucleotide, the method comprising detecting the presence on a membrane of an oligonucleotide duplex formed by a labeled oligonucleotide probe hybridized to the synthetic oligonucleotide to be detected, said synthetic oligonucleotide capable of being extracted by isobutanol and ethyl ether, the oligonucleotide duplex on the membrane having been prepared by:

(a) proteolytically digesting a body fluid or tissue sample;

(b) extracting the proteolytically digested sample with a partitioning agent;

(c) extracting the aqueous phase in the extracted sample with isobutanol and ethyl ether;

(d) drying the remaining solution containing nucleic acids to a pellet and resuspending the nucleic acids into a solution;

(e) binding the nucleic acids to a hybridization membrane;

(f) treating the membrane having bound nucleic acids with a labeled oligonucleotide probe that is complementary to and specifically binds to the synthetic oligonucleotide to be detected;

(g) washing the membrane to remove any labeled oligonucleotide that is not specifically hybridized to nucleic acids bound to the membrane.

8. The method according to claim 7, wherein the body fluid is blood.

9. The method according to claim 7, wherein the body fluid is urine.

10. The method according to claim 7, wherein the tissue is lymphatic tissue.

11. The method according to claim 7, wherein the labeled oligonucleotide probe is a radiolabeled probe.

12. A method according to claim 7, wherein the labeled oligonucleotide probe comprises an oligonucleotide labeled with an antigen to which an antigen-binding antibody or antibody fragment can bind or with a ligand to which a ligand-binding receptor can bind, and wherein the detection of the oligonucleotide duplex further comprises exposing to photographic film the membrane of claim 7 that has further been prepared by:

(a) blocking the membrane having with a suitable blocking agent to prevent non-specific attachment to the membrane of the antigen-binding antibody or antibody fragment or ligand-binding receptor;

(b) incubating the membrane with an antigen-binding antibody, antibody fragment or receptor that specifically binds to the antigen or ligand and is conjugated to an enzyme that acts upon a substrate to produce chemiluminescence;

(c) washing the membrane to remove any non-specifically bound antibody, antigen-binding antibody fragment or receptor;

(d) adding the substrate upon which the enzyme acts.