Novel assay

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.

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. 
     
     
         4 . (canceled) 
     
     
         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. 
     
     
         6 . (canceled) 
     
     
         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.   
     
     
         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.