Macromolecular conjugate made using unsaturated aldehydes

• US 6,326,136 B1
• Filed: 12/23/1994
• Issued: 12/04/2001
• Est. Priority Date: 04/01/1988
• Status: Expired due to Term

• Current Assignee: Digene Corporation (Qiagen NV)
• Original Assignee: Digene Corporation (Qiagen NV)
• Inventors: Lazar, James G., Taub, Floyd E.
• Primary Examiner(s): HOUSEL, JAMES C
• Application Number:
  US08/372,810
• Time in Patent Office: 2,538 Days
• Field of Search: 435/5, 435/6, 435/7--, 435/180, 435/810, 435/972, 436/531, 436/536, 436/808, 935/77.78, 536/104, 530/350, 530/402, 530/403
• US Class Current: 435/5
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: G01N 33/532 Production of labelled immunochemicals
• CPC Class Codes: Y10S 435/972 Modified antibody, e.g. hybrid, bifunctional

Abstract

Conjugate probes are prepared in a one step process by incubating a macromolecule and a labeling group with an unsaturated polyaldehyde as the conjugating agent. The conjugating agent is capable of bonding virtually any labeling group to a macromolecule. Conjugate probes have been shown to have a high degree of specificity and exhibit a strong signal with minimal background.

Claims

1. A labeled conjugate probe comprising:

a labeling group conjugated to

a water-soluble polyglutaraldehyde prepared by polymerization at a pH of between 8 and 12 for up to about 48 hours, then acidifying to a pH of between 2 and 7, then separating the water-soluble polyglutaraldehyde from any water-insoluble polyglutaraldehyde,

wherein the water-soluble polyglutaraldehyde is bound to a probe molecule selected from the group consisting of proteins, nucleic acids, carbohydrates, and lipids.

2. The conjugate probe of claim 1 wherein the proteins are selected from the group consisting of antigens, antibodies, receptors, and ligands.

3. The conjugate probe of claim 1 wherein the nucleic acid is selected from the group consisting of single and double stranded DNA and RNA.

4. The conjugate probe of claim 3 having a labeling group to nucleic acid mass ratio from 1:

2 to 15;

1.

5. The conjugate probe of claim 4 wherein the ratio is from 2:

1 to 10;

1.

6. The conjugate probe of claim 5 wherein the ratio is from 4:

1 to 8;

1.

7. The conjugate probe of claim 1 wherein the labeling group is selected from the group consisting of antibodies, zymogen activators, antigens, colored or fluorescent dyes, enzymes, chemiluminescent labels, phycobilins, ferritin, nucleic acid binding proteins, and radiolabels.

8. The conjugate probe of claim 1 wherein the probe specifically binds to a target molecule is selected from the group consisting of antigens, antibodies, receptor molecules, receptor ligands, single and double stranded DNA and RNA.

9. The conjugate probe of claim 8 wherein the target molecule is single or double stranded viral DNA or RNA.

10. The conjugate probe of claim 1 bound to a solid support.

11. A conjugate probe comprising

a labeling group conjugate to

a water-soluble polymeric unsaturated aldehyde selected from the group consisting of alpha-beta unsaturated aldehydes other than polyglutaraldehyde, aryl aldehydes, and aldehydes containing both alpha-beta unsaturated and aryl aldehyde groups, and unsaturated aldehydes thereof containing additional unsaturation,

wherein the aldehyde is bound to a probe molecule selected from the group consisting of proteins, nucleic acids, carbohydrates, and lipids.

12. The conjugate probe of claim 11 wherein the alpha-beta unsaturated aldehyde is selected from the group consisting of polyadipaldehyde, 2,4-biformyl-1,5-pentadiene, and 3,5-biformyl-2,6-heptadiene-1,7-dial and wherein the aldehyde containing both alpha-beta unsaturated and aryl unsaturated groups is 4-(1,5-pentadiene-5-al) benzaldehyde.

13. The conjugate probe of claim 11 bound to a solid support.

14. A method of preparing a labelled conjugate probe comprising

attaching a water-soluble polyglutaraldehyde prepared by polymerization at a pH of between 8 and 12 for up to about 48 hours, then acidifying to a pH of between 2 and 7, then separating the water-soluble polyglutaraldehyde from any water-insoluble polyglutaraldehyde, to

a labeling group and

a probe molecule selected from the group consisting of proteins, nucleic acids, carbohydrate, and lipid.

15. The method of claim 14 wherein the labeling group is selected from the group consisting of antibodies, zymogen activators, antigens, colored or fluorescent dyes, enzymes, chemiluminescent labels, phycobilins, ferritin, nucleic acid binding proteins, and radiolabels.

16. The method of claim 14 wherein the proteins are selected from the group consisting of antigens, antibodies, receptors, and ligands.

17. The method of claim 14 wherein the nucleic acid is selected from the group consisting of single and double stranded DNA and RNA.

18. A method of preparing a labelled conjugate probe comprising attaching a water-soluble polymeric unsaturated aldehyde selected from the group consisting of alpha-beta unsaturated aldehydes other than polyglutaraldehyde, aryl aldehydes, and aldehydes containing both alpha-beta unsaturated and aryl aldehyde groups, and unsaturated aldehydes thereof containing additional unsaturation, to

a labeling group and

a probe molecule selected from the group consisting of proteins, nucleic acids, carbohydrate, and lipid.

19. The method of claim 18 wherein the alpha beta unsaturated aldehyde is selected from the group consisting of volvadivaldehyde, 2,4-biformyl-1,5-pentadiene, and 3,5-biformyl-2,6-heptadiene-1,7-dial and wherein the aldehyde containing both alpha-beta unsaturated and aryl unsaturated groups is 4-(1,5-pentadiene-5-al) benzaldehyde.

20. A method of detecting a target molecule in a biological sample comprising the steps of:

a) reacting a labeled conjugate probe with the sample;

b) binding the labeled conjugate to the target molecule in the sample to form a complex; and

c) detecting the label of the bound conjugate, wherein detection of the label indicates the presence of the target molecule,

wherein the target molecule is selected from the group consisting of antigens, antibodies, receptor molecules, receptor ligands, single and double stranded DNA and RNA, and the labeled conjugate probe comprises;

a labeling group conjugated to

a water-soluble polyglutaraldehyde prepared by polymerization at a pH of between 8 and 12 for up to about 48 hours, then acidifying to a pH of between 2 and 7, then separating the water-soluble polyglutaraldehyde from any water-insoluble polyglutaraldehyde,

and wherein the water-soluble polyglutaraldehyde is bound to a probe molecule selected from the group consisting of proteins, nucleic acids, carbohydrates, and lipids.

21. The method of claim 20 wherein the probe molecule specifically reacts with the target molecule by binding thereto.

22. The method of claim 21 wherein the probe molecule is first incubated with the water-soluble polyglutaraldehyde to form a conjugate which is then incubated with the labeling group to form the labeled conjugate probe.

23. The method of claim 22 wherein either or both incubations occur in the presence of a cationic detergent.

24. The method of claim 21 wherein the labeling group is first incubated with the water-soluble polyglutaraldehyde to form a labeled conjugate which is then incubated with the probe molecule to form the labeled conjugate probe.

25. The method of claim 24 wherein either or both incubations occur in the presence of a cationic detergent.

26. The method of claim 20 wherein the target molecule is viral nucleic acid.

27. The method of claim 20 further comprising removing unbound sample and detecting the labeled conjugate probe bound to the target molecule.

28. The method of claim 20 wherein the labeled conjugate probe is a labeled nucleic acid, comprising the additional step of mixing the probe with a hybridization medium prior to the step of reacting the probe with the target molecule.

29. The method of claim 20 wherein the target molecule is a double stranded nucleic acid molecule comprising the additional step of denaturing the target molecule with a denaturation medium prior to the step of reacting the target molecule with the labeled conjugate probe.

30. The method of claim 29 wherein a concentration of from 0.1 μ

g/ml to 50 μ

g/ml of labeled conjugate probe in a buffer solution is reacted with the denatured target molecule.

31. The method of claim 30 wherein the concentration of labeled conjugate probe is from 0.2 μ

g/ml to 30 μ

g/ml.