Diagnostic methods and probes

• US 5,824,478 A
• Filed: 04/30/1996
• Issued: 10/20/1998
• Est. Priority Date: 04/30/1996
• Status: Expired due to Term

• Current Assignee: Vysis, Inc. (Abbott Laboratories Incorporated)
• Original Assignee: Vysis, Inc. (Abbott Laboratories Incorporated)
• Inventors: Muller, Uwe Richard
• Primary Examiner(s): Jones, W. Gary
• Assistant Examiner(s): WHISENANT, ETHAN C
• Application Number:
  US08/643,109
• Time in Patent Office: 903 Days
• Field of Search: 435/6, 435/7.1, 435/7.5, 435/7.72, 435/7.9, 435/7.94, 435/183, 436/94, 436/501, 436/800, 536/24.3, 536/24.31, 536/24.32, 536/23.1, 935/78, 935/76, 935/77
• US Class Current: 435/6.12
• CPC Class Codes: G01N 33/54306 Solid-phase reaction mechanisms
• CPC Class Codes: G01N 33/58 involving labelled substances G01N33/53 takes precedence

Abstract

The invention features methods for detecting a target analyte in a sample. In these methods, a sample is contacted with a detector probe and a capture probe to form a detector probe-analyte-capture probe complex. The detector probe includes a new moiety, having a predetermined pI and containing a detectable label. The complex is isolated from detector probe that is not bound in the complex, the moiety is released from the complex, and the released moiety is concentrated, e.g., by isoelectric focusing or other methods. Detection of the moiety, e.g., at a position in a pH gradient corresponding to its pI, can be used as a measure of the presence and concentration of the analyte in the sample.

Claims

1. A method for detecting an analyte in a sample, said method comprising the steps of:

contacting said sample with a detector probe and a capture probe to form a detector probe-analyte-capture probe complex, wherein said detector probe comprises a moiety having a predetermined pI and comprising a detectable label;

separating said complex from any detector probe that is not bound in said complex;

releasing said moiety from said complex;

isolating said moiety released from said complex and concentrating said moiety by isoelectric focusing; and

detecting said moiety as a measure of the presence of said analyte in said sample.

2. The method of claim 1, wherein

said detecting of said moiety as a measure of the presence of said analyte in said sample is carried out by detecting said concentrated moiety at a position in a pH gradient corresponding to said pI of said moiety.

3. The method of claim 1, wherein said isolating is carried out by the use of column chromatography or a molecular sieve.

4. The method of claim 1, wherein said capture probe is immobilized in the lumen of a fractionation chamber and said isolating, releasing, and concentrating steps are carried out in said lumen.

5. The method of claim 4, wherein said sample is contacted with said detector probe prior to its contact with said immobilized capture probe in said lumen.

6. The method of claim 4, wherein said sample and said detector probe are independently contacted with said capture probe in said lumen.

7. The method of claim 6, wherein said sample is contacted with said capture probe in said lumen prior to said detector probe.

8. The method of claim 1, wherein said capture probe comprises a first member of a specific binding pair and said separation step is carried out by contacting said complex with a second member of said specific binding pair, wherein said second member of said specific binding pair is immobilized on a solid support.

9. The method of claim 8, wherein said solid support is in the lumen of a fractionation chamber.

10. The method of claim 8, wherein said specific binding pair comprises a pair of complementary nucleic acids.

11. The method of claim 8, wherein said specific binding pair comprises a pair of polypeptides that specifically bind to one another.

12. The method of claim 11, wherein said first member of said specific binding pair comprises an antigen binding site of an antibody and said second member of said specific binding pair comprises an antigen to which said antigen binding site binds.

13. The method of claim 8, wherein said first member of said specific binding pair comprises avidin, and said second member of said specific binding pair comprises biotin.

14. The method of claim 1, wherein said isoelectric focusing is carried out in the lumen of a capillary tube.

15. The method of claim 1, wherein said isoelectric focusing is carried out in a groove in the surface of a plate.

16. The method of claim 1, wherein said analyte comprises a nucleic acid.

17. The method of claim 16, wherein said analyte comprises a chromosome comprising a translocation, or a pre-mRNA or mRNA transcript thereof.

18. The method of claim 1, wherein said detector probe comprises a nucleic acid.

19. The method of claim 1, wherein said capture probe comprises a nucleic acid.

20. The method of claim 1, wherein said analyte comprises a polypeptide.

21. The method of claim 1, wherein said analyte comprises a carbohydrate, lipid, metabolite, or drug.

22. The method of claim 1, wherein said detector probe comprises a polypeptide.

23. The method of claim 22, wherein said polypeptide comprises an antigen binding site of an antibody.

24. The method of claim 23, wherein said polypeptide comprises a single chain antibody.

25. The method of claim 1, wherein said capture probe comprises a polypeptide.

26. The method of claim 25, wherein said polypeptide comprises an antigen binding site of an antibody.

27. The method of claim 26, wherein said detector probe comprises a single chain antibody.

28. The method of claim 1, wherein said releasing step is carried out by chemical cleavage.

29. The method of claim 28, wherein said moiety is linked to said detector probe by a disulfide bond and said releasing step is carried out by reduction of said disulfide bond.

30. The method of claim 1, wherein said releasing step is carried out by enzymatic cleavage.

31. The method of claim 1, wherein said moiety comprises phycoerythrin, or a modified version thereof.

32. A method for detecting the presence of a chromosome comprising a chromosomal translocation in a sample, wherein said chromosome comprises a portion of a first chromosome and a portion of a second chromosome, said method comprising the steps of:

contacting said sample with a capture probe and a detector probe to form a detector probe-analyte-capture probe complex, wherein

said capture probe binds to said portion of said first chromosome, or a corresponding RNA transcript thereof, and comprises the first member of a specific binding pair, and

said detector probe binds to said portion of said second chromosome, or a corresponding RNA transcript thereof, and comprises a moiety having a predetermined pI and a detectable label;

separating said complex from any detector probe that is not bound in said complex by contacting said complex with the second member of said specific binding pair, said second member being immobilized on a solid support;

releasing said moiety from said complex;

concentrating said moiety by isoelectric focusing; and

detecting the concentrated moiety as a measure of the presence of said chromosome containing said chromosomal translocation in said sample.