Monoclonal antibodies and methods for their use in the detection of cervical disease

Abstract

Compositions and methods for diagnosing high-grade cervical disease in a patient sample are provided. The compositions include novel monoclonal antibodies, and variants and fragments thereof, that specifically bind to MCM2. Monoclonal antibodies having the binding characteristics of an MCM2 antibody of the invention are further provided. Hybridoma cell lines that produce an MCM2 monoclonal antibody of the invention are also disclosed herein. The compositions find use in practicing methods for diagnosing high-grade cervical disease comprising detecting overexpression of MCM2 in a cervical sample from a patient. Kits for practicing the methods of the invention are further provided. Polypeptides comprising the amino acid sequence for an MCM2 epitope and methods of using these polypeptides in the production of antibodies are also encompassed by the present invention.

1 . A monoclonal antibody that is capable of specifically binding to MCM2, or a variant or fragment thereof, wherein the antibody is selected from the group consisting of: 
 (a) the monoclonal antibody produced by the hybridoma cell line 26H6.19, deposited with the ATCC as Patent Deposit No. PTA-6667;    (b) a monoclonal antibody having the binding characteristics of the monoclonal antibody produced by the hybridoma cell line 26H6.19;    (c) a monoclonal antibody that binds to an epitope capable of binding the monoclonal antibody produced by the hybridoma cell line 26H6.19;    (d) a monoclonal antibody that binds to an epitope comprising the amino acid sequence of SEQ ID NO:14    (e) a monoclonal antibody that competes in a competitive binding assay with the monoclonal antibody produced by the hybridoma cell line 26H6.19; and,    (f) a monoclonal antibody that is an antigen binding fragment of a monoclonal antibody of (a)-(e), wherein the fragment retains the capability of specifically binding to MCM2, or a variant or fragment thereof.    
     
     
         2 . The hybridoma cell line 26H6.19, deposited with the ATCC as Patent Deposit No. PTA-6667.  
     
     
         3 . A hybridoma cell line capable of producing a monoclonal antibody of  claim 1 .  
     
     
         4 . A kit for diagnosing high-grade cervical disease comprising at least one monoclonal antibody according to  claim 1 .  
     
     
         5 . The kit of  claim 4 , wherein the monoclonal antibody is the monoclonal antibody produced by the hybridoma cell line 26H6.19, deposited with the ATCC as Patent Deposit No. PTA-6667.  
     
     
         6 . The kit of  claim 4  comprising at least two antibodies, wherein a first antibody is the monoclonal antibody produced by the hybridoma cell line 26H6.19, deposited with the ATCC as Patent Deposit No. PTA-6667.  
     
     
         7 . The kit of  claim 6  further comprising an antibody that specifically binds to Topo2A.  
     
     
         8 . The kit of  claim 6 , wherein each antibody is provided as a separate antibody reagent.  
     
     
         9 . The kit of  claim 6 , wherein all of the antibodies are provided as an antibody cocktail.  
     
     
         10 . The kit of  claim 4 , wherein said kit further comprises a peroxidase blocking reagent, a protein blocking reagent, chemicals for the detection of antibody binding to said biomarker proteins, a counterstain, a bluing agent, and instructions for use.  
     
     
         11 . The kit of  claim 4  further comprising reagents for Pap staining.  
     
     
         12 . The kit of  claim 11 , wherein the reagents for Pap staining comprise EA50 and Orange G.  
     
     
         13 . A method for diagnosing high-grade cervical disease in a patient, the method comprising: 
 a) obtaining a cervical sample from the patient;    b) contacting the sample with at least one monoclonal antibody according to  claim 1  that specifically binds to MCM2; and,    c) detecting binding of the antibody to MCM2.    
     
     
         14 . The method of  claim 13 , wherein the monoclonal antibody is the monoclonal antibody produced by the hybridoma cell line 26H6.19, deposited with the ATCC as Patent Deposit No. PTA-6667.  
     
     
         15 . The method of  claim 13  comprising contacting the sample with at least two monoclonal antibodies that specifically bind to MCM2, wherein a first antibody is the monoclonal antibody produced by the hybridoma cell line 26H6.19, deposited with the ATCC as Patent Deposit No. PTA-6667.  
     
     
         16 . The method of  claim 15  further comprising contacting the sample with an antibody that specifically binds to Topo2A.  
     
     
         17 . The method of  claim 15 , wherein the antibodies are contacted with the sample sequentially as individual antibody reagents or simultaneously as an antibody cocktail.  
     
     
         18 . An isolated polypeptide comprising an epitope for binding an MCM2 monoclonal antibody, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of: 
 (a) a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:14; and,    (b) a polypeptide having at least 90% sequence identity to SEQ ID NO:14, wherein the polypeptide has antigenic activity.    
     
     
         19 . A method for producing an MCM2 antibody comprising immunizing an animal with a polypeptide according to  claim 19 .  
     
     
         20 . A method for producing an MCM2 monoclonal antibody comprising: 
 (a) immunizing an animal with a polypeptide according to  claim 19  under conditions to elicit an immune response;    (b) isolating antibody-producing cells from the animal;    (c) fusing the antibody-producing cells with immortalized cells in culture to form monoclonal antibody-producing hybridoma cells;    (d) culturing the hybridoma cells; and,    (e) isolating monoclonal antibodies from culture.