NOVEL ASSAY

• US 20190317087A1
• Filed: 05/21/2019
• Published: 10/17/2019
• Est. Priority Date: 04/05/2013
• Status: Abandoned Application

• Current Assignee: IDL Biotech AB
• Original Assignee: IDL Biotech AB
• Inventors: D'AMICO, Yiva, Magnusson, Gunnar
• Application Number:
  US16/418,272
• Publication Number:
  US 20190317087A1
• Time in Patent Office: Days
• CPC Class Codes: G01N 2333/4742 Keratin; Cytokeratin
• CPC Class Codes: G01N 33/54306 Solid-phase reaction mechanisms
• CPC Class Codes: G01N 33/57496 involving intracellular compounds
• CPC Class Codes: G01N 33/577 involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens; G01N33/53 - G01N33/576 take precedence
• CPC Class Codes: G01N 33/6887 from muscle, cartilage or connective tissue

Abstract

A method is described for the detection of at least two of cytokeratins 8, 18 and 19 in a sample. It is practiced by contacting the sample with a solid phase having a first antibody with specificity for cytokeratin 8, a second antibody with specificity for cytokeratin 18 and, optionally, a third antibody with specificity for a first epitope of cytokeratin 19 bound to it and allowing cytokeratins in the sample to bind to the bound antibodies to form complexes. The complexes are then contacted with a first labelled antibody with specificity for a dimer of cytokeratin 8 and 18 and optionally a second labelled antibody with specificity for a second epitope of cytokeratin 19 and allowing the labelled antibodies to bind to the complexes. The labelled antibodies bound to the complexes are then detected. A method for quantitative determination of soluble fragments of at least two of cytokeratin 8, 18 and 19 in a sample and a kit are also described.

Claims

1. A method for detecting soluble fragments of at least two cytokeratins selected from the group consisting of cytokeratin 8, 18 and 19 in a fluid sample of a human subject, comprising the steps of contacting said fluid sample with a solid phase having immobilized thereon a first antibody having specificity for cytokeratin 8, a second antibody having specificity for cytokeratin 18 and optionally a third antibody having specificity for a first epitope of cytokeratin 19;

allowing soluble fragments of cytokeratins to bind to said first, second and optionally third antibodies thereby forming complexes;

contacting said complexes with a first labelled antibody having specificity for a dimer of cytokeratin 8 and 18 and optionally a second labelled antibody having specificity for a second epitope of cytokeratin 19;

allowing said labelled antibodies to bind to said complexes; and

detecting said labelled antibodies bound to said complexes.

2. The method according to claim 1, wherein said antibodies are monoclonal antibodies.

3. The method according to claim 1, wherein said first labelled antibody has specificity for the α

-helix 2B 2 (aa 414-429 as set forth in SEQ ID NO;

1) of cytokeratin 18, and, if present, said second labelled antibody has specificity for the α

-helix 2B 2 (aa 311-335 as set forth in SEQ ID NO;

2) of cytokeratin 19.

5. The method according to claim 3, wherein said first labelled antibody is M21, and, if present, said second labelled antibody is A53-B/A2.

7. The method according to claim 1, wherein said first immobilized antibody has specificity for the α

-helix 2B (aa 340-365 as set forth in SEQ ID NO;

3), said second immobilized antibody has specificity for the α

-helix 2B (aa 320-350 as set forth in SEQ ID NO;

4), and, if present, said third immobilized antibody has specificity for the α

-helix 2B (aa 340-370 as set forth in SEQ ID NO;

5).

8. The method according to claim 7, wherein said first immobilized antibody is 6D7, said second immobilized antibody is 3F3, and, if present, said third immobilized antibody is IDLC4.

9. The method according to claim 1, wherein said first antibody having specificity for cytokeratin 8 constitutes 20-40% of total immobilized antibody, said second antibody having specificity for cytokeratin 18, constitutes 5-15% of total immobilized antibody, and said third antibody having specificity for cytokeratin 19 constitutes 50-70% of total immobilized antibody.

10. The method according to claim 9, wherein said first antibody having specificity for cytokeratin 8 constitutes 30% of total immobilized antibody, said second antibody having specificity for cytokeratin 18, constitutes 10% of total immobilized antibody, and said third antibody having specificity for cytokeratin 19 constitutes 60% of total immobilized antibody.

11. The method according to claim 1, wherein the soluble fragments of cytokeratin 8 and 18 in the fluid sample are detected by a method comprising the steps of:

contacting said fluid sample with a solid phase having immobilized thereon a first antibody having specificity for cytokeratin 8, and a second antibody having specificity for cytokeratin 18;

allowing soluble fragments of cytokeratins in said sample to bind to said first and second antibodies thereby forming complexes;

contacting said complexes with a first labelled antibody having specificity for a dimer of cytokeratin 8 and 18;

allowing said labelled antibodies to bind to said complexes; and

detecting said labelled antibody bound to said complexes.

12. The method according to claim 1, wherein said labelled antibodies are labelled with horse radish peroxidase and detection is performed by adding a substrate capable of being converted to a detectable substance by said horse radish peroxidase.

13. The method according to claim 12, wherein said substrate is converted to a chromogenic substance by said horse radish peroxidase.

14. The method according to claim 13, wherein said substrate is TMB (3,3′

,5,5′

-Tetramethylbenzidine).

15. The method for quantitative determination of soluble fragments of at least two cytokeratins selected from the group consisting of cytokeratin 8, 18 and 19 in a fluid sample of a human subject, comprising the method according to claim 1, and further comprising the step of:

quantitatively correlating the amount of bound first labelled antibody having specificity for a dimer of cytokeratin 8 and 18 and, if present, second labelled antibody having specificity for cytokeratin 19 with known amounts of soluble fragments of cytokeratins 8, 18 and/or 19.

16. The method for quantitative determination of soluble fragments of at least two cytokeratins selected from the group consisting of cytokeratin 8, 18 and 19 in a fluid sample of a human subject, comprising the method according to claim 11, and further comprising the step of:

quantitatively correlating the amount of bound first labelled antibody having specificity for a dimer of cytokeratin 8 and 18 and, if present, second labelled antibody having specificity for cytokeratin 19 with known amounts of soluble fragments of cytokeratins 8, 18 and/or 19.

4. (canceled)

6. (canceled)

17. A method for detecting at least two cytokeratins selected from the group consisting of cytokeratin 8, 18 and 19 and soluble fragments thereof in a fluid sample of a human subject, comprising the steps of

contacting said fluid sample with a solid phase having immobilized thereon a first antibody having specificity for cytokeratin 8, a second antibody having specificity for cytokeratin 18 and optionally a third antibody having specificity for a first epitope of cytokeratin 19;

allowing cytokeratins and soluble fragments thereof to bind to said first, second and optionally third antibodies thereby forming complexes;

contacting said complexes with a first labelled antibody having specificity for a dimer of cytokeratin 8 and 18 and optionally a second labelled antibody having specificity for a second epitope of cytokeratin 19;

allowing said labelled antibodies to bind to said complexes; and

detecting said labelled antibodies bound to said complexes.

18. The method according to claim 17, wherein said antibodies are monoclonal antibodies.

19. The method according to claim 17, wherein said first labelled antibody has specificity for the α

-helix 2B 2 (aa 414-429 as set forth in SEQ ID NO;

1) of cytokeratin 18, and, if present, said second labelled antibody has specificity for the α

-helix 2B 2 (aa 311-335 as set forth in SEQ ID NO;

2) of cytokeratin 19.

20. The method according to claim 19, wherein said first labelled antibody is M21, and, if present, said second labelled antibody is A53-B/A2.

21. The method according to claim 17, wherein said first immobilized antibody has specificity for the α

-helix 2B (aa 340-365 as set forth in SEQ ID NO;

3), said second immobilized antibody has specificity for the α

-helix 2B (aa 320-350 as set forth in SEQ ID NO;

4), and, if present, said third immobilized antibody has specificity for the α

-helix 2B (aa 340-370 as set forth in SEQ ID NO;

5).

22. The method according to claim 21, wherein said first immobilized antibody is 6D7, said second immobilized antibody is 3F3, and, if present, said third immobilized antibody is IDLC4.

23. The method according to claim 17, wherein said first antibody having specificity for cytokeratin 8 constitutes 20-40% of total immobilized antibody, said second antibody having specificity for cytokeratin 18, constitutes 5-15% of total immobilized antibody, and said third antibody having specificity for cytokeratin 19 constitutes 50-70% of total immobilized antibody.

24. The method according to claim 23, wherein said first antibody having specificity for cytokeratin 8 constitutes 30% of total immobilized antibody, said second antibody having specificity for cytokeratin 18, constitutes 10% of total immobilized antibody, and said third antibody having specificity for cytokeratin 19 constitutes 60% of total immobilized antibody.

25. The method according to claim 17, wherein the cytokeratin 8 and 18 and soluble fragments thereof in the fluid sample are detected by a method comprising the steps of:

contacting said fluid sample with a solid phase having immobilized thereon a first antibody having specificity for cytokeratin 8, and a second antibody having specificity for cytokeratin 18;

allowing cytokeratins 8 and 18 and soluble fragments thereof in said sample to bind to said first and second antibodies thereby forming complexes;

contacting said complexes with a first labelled antibody having specificity for a dimer of cytokeratin 8 and 18;

allowing said labelled antibodies to bind to said complexes; and

detecting said labelled antibody bound to said complexes.

26. The method according to claim 17, wherein said labelled antibodies are labelled with horse radish peroxidase and detection is performed by adding a substrate capable of being converted to a detectable substance by said horse radish peroxidase.

27. The method according to claim 26, wherein said substrate is converted to a chromogenic substance by said horse radish peroxidase.

28. The method according to claim 27, wherein said substrate is TMB (3,3′

,5,5′

-Tetramethylbenzidine).

29. A method for quantitative determination of at least two cytokeratins selected from the group consisting of cytokeratin 8, 18 and 19 and soluble fragments thereof in a fluid sample of a human subject, comprising the method according to claim 17, and further comprising the step of:

quantitatively correlating the amount of bound first labelled antibody having specificity for a dimer of cytokeratin 8 and 18 and, if present, second labelled antibody having specificity for cytokeratin 19 with known amounts of soluble fragments of cytokeratins 8, 18 and/or 19.

30. A method for quantitative determination of at least two cytokeratins selected from the group consisting of cytokeratin 8, 18 and 19 and soluble fragments thereof in a fluid sample of a human subject, comprising the method according to claim 25, and further comprising the step of:

quantitatively correlating the amount of bound first labelled antibody having specificity for a dimer of cytokeratin 8 and 18 and, if present, second labelled antibody having specificity for cytokeratin 19 with known amounts of soluble fragments of cytokeratins 8, 18 and/or 19.