Assay

• US 10,386,363 B2
• Filed: 03/28/2014
• Issued: 08/20/2019
• Est. Priority Date: 04/05/2013
• Status: Active Grant

• Current Assignee: IDL Biotech AB
• Original Assignee: IDL Biotech AB
• Inventors: D'Amico, Ylva, Magnusson, Gunnar
• Primary Examiner(s): Counts, Gary
• Application Number:
  US14/782,527
• Publication Number:
  US 20160025718A1
• Time in Patent Office: 1,971 Days
• Field of Search: None
• 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 at least two cytokeratins selected from the group consisting of cytokeratin 8, 18 and 19 in a sample, comprising the steps of

contacting said 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 in said sample 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 or 2, 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.

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

5. 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).

6. The method according to claim 5, wherein said first immobilized antibody is 6D7, said second immobilized antibody is 3F3, and, if present, said third immobilized antibody is IDLC4.

7. 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.

8. The method according to claim 1, wherein cytokeratin 8 and 18 in a sample are detected, comprising the steps of

contacting said 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 cytokeratin 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.

9. 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.

10. The method according to claim 9, wherein said substrate is converted to a chromogenic substance by said horse radish peroxidase.

11. A method for quantitative determination of at least two cytokeratins selected from the group consisting of cytokeratin 8, 18 and 19 in a sample, comprising the method according to claim 1 or 8 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 cytokeratins 8, 18 and/or 19.

19. The method according to claim 7, 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.

20. The method according to claim 10, wherein said substrate is TMB (3,3′

,5,5′

-Tetramethylbenzidine).

12. A kit for detecting at least two cytokeratins selected from the group consisting of cytokeratin 8, 18 and 19 in a sample , comprising

a solid phase substrate having immobilized thereon at least two cytokeratin-specific antibodies selected from the group consisting of a first antibody having specificity for cytokeratin 8, a second antibody having specificity for cytokeratin 18;

and a third antibody having specificity for cytokeratin 19; and

a first labelled antibody having specificity for a dimer of cytokeratin 8 and 18 and optionally a second labelled antibody having specificity for cytokeratin 19.

13. The kit according to claim 12, further comprising diluent means, standard means, control means, substrate means, stop means, wash means and/or instructions for use.

14. The kit according to claim 12 or 13, adapted for an Enzyme-Linked ImmunoSorbent Assay (ELISA).

15. The kit according to claim 12, wherein a first antibody having specificity for cytokeratin 8 and a second antibody having specificity for cytokeratin 18 have been immobilized to the solid substrate.

16. The kit according to claim 15, wherein said first labelled antibody is M21, and said optional second labelled antibody is C11.

17. The kit according to claim 12, which further comprises written instructions for the detection of lung cancer.

18. The kit according to claim 12, which further comprises written instructions for the detection of bladder cancer.

21. The kit of claim 12, wherein the labeled antibodies are in a buffered solution.

22. The kit according to claim 17, wherein the lung cancer is non-small cell lung cancer.