Method for improved diagnosis of dysplasias

Abstract

The present invention relates to a method for improved diagnosis of dysplasias based on simultaneous detection of INK4a gene products and at least one marker for cell proliferation. Particularly the present invention provides a method for discriminating dysplastic cells over-expressing INK4a gene products from cells over-expressing INK4a gene products without being dysplastic by detection of a marker suitable for characterizing the proliferation properties of the respective cell. The characterization of the proliferation properties may comprise the detection of a marker or a set of markers characteristic for active cell proliferation and/or a marker or a set of markers characteristic for retarded or ceased cell proliferation. The method presented herein thus enables for a specific diagnosis of dysplasias in histological and cytological specimens.

1 . A kit consisting of (i) a pair of primary antibodies, and (ii) optionally detection agents for performing an immunoenzymatic assay, the pair of primary antibodies consisting of an anti-human p16 INK4a  monoclonal antibody and an anti-human Ki67 monoclonal antibody. 
     
     
         2 . The kit of  claim 1 , wherein the anti-human p16 INK4a  monoclonal antibody is a mouse monoclonal antibody, and wherein the anti-human Ki67 monoclonal antibody is a rabbit monoclonal antibody. 
     
     
         3 . The kit of  claim 1 , wherein the anti-human p16 INK4a  monoclonal antibody and the anti-human Ki-67 monoclonal antibody are provided as a cocktail. 
     
     
         3 . The kit of  claim 1 , further comprising a secondary antibody specific to the anti-human p16 INK4a  monoclonal antibody, wherein the secondary antibody specific to the anti-human p16 INK4a  monoclonal antibody is conjugated to a peroxidase enzyme. 
     
     
         4 . The kit of  claim 1 , further comprising a secondary antibody specific to the anti-human Ki-67 monoclonal antibody, wherein the secondary antibody specific to the anti-human Ki-67 monoclonal antibody is conjugated to an alkaline phosphatase enzyme. 
     
     
         5 . The kit of  claim 1 , further comprising a first chromogenic agent and a second chromogenic agent. 
     
     
         6 . The kit of  claim 5 , wherein the first chromogenic agent produces brown signals; and
 wherein the second chromogenic agent produces red signals.   
     
     
         7 . The kit of  claim 5 , wherein the first chromogenic agent is 3,3′-diaminobenzidine, and wherein the second chromogenic agent is FastRed. 
     
     
         8 . The kit of  claim 1 , wherein the kit consists of the pair of primary antibodies. 
     
     
         9 . The kit of  claim 1 , wherein the anti-human p16 INK4a  monoclonal antibody is specific to a human p16 INK4a  protein in a human cervical specimen; and wherein the anti-human Ki-67 monoclonal antibody is specific to a human Ki-67 protein in the human cervical specimen. 
     
     
         10 . The kit of  claim 9 , wherein the human cervical specimen is a cytological specimen derived from the cervix uteri. 
     
     
         11 . A kit comprising (i) a pair of primary antibodies, the pair of primary antibodies consisting of an anti-human p16 INK4a  monoclonal antibody and an anti-human Ki67 monoclonal antibody; (ii) 3,3′-diaminobenzidine; and (iii) a chromogenic agent which produces red signals. 
     
     
         12 . The kit of  claim 11 , wherein the chromogenic agent which produces the red signals is FastRed. 
     
     
         13 . The kit of  claim 11 , wherein the anti-human p16 INK4a  monoclonal antibody is a mouse monoclonal antibody, and wherein the anti-human Ki67 monoclonal antibody is a rabbit monoclonal antibody. 
     
     
         14 . The kit of  claim 11 , wherein the anti-human p16 INK4a  monoclonal antibody is specific to a human p16 INK4a  protein in a human cervical specimen; and wherein the anti-human Ki-67 monoclonal antibody is specific to a human Ki-67 protein in the human cervical specimen. 
     
     
         15 . The kit of  claim 14 , wherein the human cervical specimen is a cytological specimen derived from the cervix uteri. 
     
     
         16 . The kit of  claim 11 , further comprising a secondary antibody specific to the anti-human p16 INK4a  monoclonal antibody; and a secondary antibody specific to the anti-human Ki-67 monoclonal antibody. 
     
     
         17 . The kit of  claim 16 , wherein the secondary antibody specific to the anti-human p16 INK4a  monoclonal antibody is associated with a peroxidase enzyme; and wherein the secondary antibody specific to the anti-human Ki-67 monoclonal antibody is associated with an alkaline phosphatase enzyme. 
     
     
         18 . The kit of  claim 16 , wherein the secondary antibody specific to the anti-human p16 INK4a  monoclonal antibody is conjugated to a peroxidase enzyme; and wherein the secondary antibody specific to the anti-human Ki-67 monoclonal antibody is conjugated to an alkaline phosphatase enzyme. 
     
     
         19 . The kit of  claim 11 , wherein the kit consists of (i) the pair of primary antibodies, (ii) the 3,3′-diaminobenzidine; and (iii) the chromogenic agent which produces red signals. 
     
     
         20 . A kit comprising:
 a pair of primary antibodies, the pair of primary antibodies consisting of an anti-human p16 INK4a  monoclonal antibody and an anti-human Ki-67 monoclonal antibody;   a secondary antibody specific to the anti-human p16 INK4a  monoclonal antibody, wherein the secondary antibody specific to the anti-human p16 INK4a  monoclonal antibody is associated with a peroxidase enzyme; and   a secondary antibody specific to the anti-human Ki-67 monoclonal antibody, wherein the secondary antibody specific to the anti-human Ki-67 monoclonal antibody is associated with an alkaline phosphatase enzyme.   
     
     
         21 . The kit of  claim 20 , wherein the secondary antibody specific to the anti-human p16 INK4a  monoclonal antibody is conjugated to a peroxidase enzyme; and wherein the secondary antibody specific to the anti-human Ki-67 monoclonal antibody is conjugated to an alkaline phosphatase enzyme. 
     
     
         22 . The kit of  claim 20 , wherein the anti-human p16 INK4a  monoclonal antibody is a mouse monoclonal antibody, and wherein the anti-human Ki-67 monoclonal antibody is a rabbit monoclonal antibody. 
     
     
         23 . The kit of  claim 20 , further comprising a first chromogenic agent and a second chromogenic agent. 
     
     
         24 . The kit of  claim 23 , wherein the first chromogenic agent is 3,3′-diaminobenzidine. 
     
     
         25 . The kit of  claim 23 , wherein the second chromogenic agent is FastRed. 
     
     
         26 . The kit of  claim 20 , wherein the anti-human p16 INK4a  monoclonal antibody is specific to a human p16 INK4a  protein in a human cytological specimen; and wherein the anti-human Ki-67 monoclonal antibody is specific to a human Ki-67 protein in the human cytological specimen. 
     
     
         27 . The kit of  claim 20 , wherein the kit consists of the pair of primary antibodies, the secondary antibody specific to the anti-human p16 INK4a  monoclonal antibody, and the secondary antibody specific to the anti-human Ki-67 monoclonal antibody. 
     
     
         28 . A kit comprising:
 an antibody cocktail consisting of an anti-human p16 INK4a  monoclonal antibody and an anti-human Ki-67 monoclonal antibody;   a first chromogen that produces brown signals; and   a second chromogen that produces red signals.   
     
     
         29 . The kit of  claim 28 , wherein the anti-human p16 INK4a  monoclonal antibody is a mouse monoclonal antibody, and wherein the anti-human Ki-67 monoclonal antibody is a rabbit monoclonal antibody. 
     
     
         30 . The kit of  claim 28 , wherein the anti-human p16 INK4a  monoclonal antibody is specific to a human p p16 INK4a  protein in a human cervical specimen; and wherein the anti-human Ki-67 monoclonal antibody is specific to a human Ki-67 protein in the human cervical specimen; and wherein the human cervical specimen is a cytological specimen derived from the cervix uteri.