Detection method for monitoring beta tubulin isotype specific modification

Abstract

This invention relates to monoclonal antibodies that recognize modified β-tubulin isotypes, methods of using such antibodies to detect modified β-tubulin isotypes, methods of using such antibodies to monitor β-tubulin modifying agents administered to a patient, methods of using such antibodies to isolate modified β-tubulin, and methods of detecting the anti-modified β-tubulin antibodies.

What is claimed is:  
     
         1 . A method of detecting in a sample a β-tubulin isotype modified at cysteine residue 239, the method comprising the steps of: 
 (a) providing a sample treated with a β-tubulin modifying agent;  
 (b) contacting the sample with an antibody that specifically binds to a β-tubulin isotype modified at cysteine residue 239; and  
 (c) determining whether the sample contains a modified β-tubulin isotype by detecting the antibody.  
 
     
     
         2 . The method of  claim 1 , wherein the antibody is a monoclonal antibody.  
     
     
         3 . The method of  claim 2 , wherein the antibody is selected from the group consisting of 1F6D8, 1B2C11, 3A1C11, 2C1H7, 3F2A4, 5F5C11, and 6D4D11.  
     
     
         4 . The method of  claim 1 , further comprising the step of using a control antibody that recognizes both modified and unmodified β-tubulins.  
     
     
         5 . The method of  claim 4 , wherein the control antibody is a monoclonal antibody selected from the group consisting of 3D12D1, 4B6G6, 5F1D4, 6H8E3, AND 6H10C7.  
     
     
         6 . The method of  claim 1 , further comprising the step of using a control antibody that recognizes only unmodified β-tubulins.  
     
     
         7 . The method of  claim 6 , wherein the control antibody is a monoclonal antibody selected from the group consisting of 3E10A3, 6A7F9, and 6E7G1.  
     
     
         8 . The method of  claim 1 , wherein the step of determining whether the sample contains a modified β-tubulin isotype comprises detecting the antibody in an assay selected from the group consisting of an ELISA assay, a western blot, an immunohistochemical assay, an immunofluorescence assay, and a real time imaging assay.  
     
     
         9 . The method of  claim 1 , wherein the step of determining whether the sample contains a modified β-tubulin isotype further comprises quantitating the amount of modified β-tubulin isotype in the sample.  
     
     
         10 . The method of  claim 1 , wherein the antibody is bound to a solid substrate.  
     
     
         11 . The method of  claim 1 , wherein the sample is selected from the group consisting of an in vitro tubulin polymerization reaction sample, a cultured cell, and a patient sample.  
     
     
         12 . The method of  claim 11 , wherein the patient sample is a blood sample.  
     
     
         13 . The method of  claim 11 , wherein the patient sample is from a cancer patient receiving pentafluorobenzenesulfonamide chemotherapy.  
     
     
         14 . The method of  claim 11 , wherein the patient sample is from a cancer patient receiving 2-fluoro-1-methoxy-4-pentafluorophenylsulfonamidobenzene chemotherapy.  
     
     
         15 . The method of  claim 11 , wherein the patient sample is from a human patient.  
     
     
         16 . The method of  claim 1 , wherein the antibody is covalently linked to a detectable moiety.  
     
     
         17 . The method of  claim 16 , wherein the antibody is covalently linked to a biotin moiety, an iodine moiety, or an enzyme moiety.  
     
     
         18 . A monoclonal antibody that specifically binds to a β-tubulin isotype modified at cysteine residue 239, the antibody selected from the group consisting of 1F6D8, 1B2C11, 3A1C11, 2C1H7, 3F2A4, 5F5C11, and 6D4D11.  
     
     
         19 . The monoclonal antibody of  claim 18 , wherein the antibody is covalently linked to a detectable moiety.  
     
     
         20 . The monoclonal antibody of  claim 19 , wherein the antibody is covalently linked to a biotin moiety, an iodine moiety, or an enzyme moiety.  
     
     
         21 . A method of monitoring the amount of modified β-tubulin isotype in a patient treated with an agent that modifies cysteine residue 239 in a β-tubulin isotype, the method comprising the steps of: 
 (a) providing a sample from the patient treated with the β-tubulin modifying agent;  
 (b) contacting the sample with an antibody that specifically binds to a modified β-tubulin isotype; and  
 (c) determining the amount of modified β-tubulin isotype in the patient sample by detecting the antibody and comparing the amount of antibody detected in the patient sample to a standard curve, thereby monitoring the amount of modified β-tubulin isotype in the patient.  
 
     
     
         22 . The method of  claim 21 , further comprising the step of adjusting the dose of the β-tubulin modifying agent administered to the patient.  
     
     
         23 . The method of  claim 21 , wherein the agent is a pentafluorobenzenesulfonamide.  
     
     
         24 . The method of  claim 21 , wherein the agent is 2-fluoro-1-methoxy-4-pentafluorophenylsulfonamidobenzene.  
     
     
         25 . The method of  claim 21 , wherein the sample is a blood sample.  
     
     
         26 . The method of  claim 21 , wherein the antibody is a monoclonal antibody.  
     
     
         27 . The method of  claim 26 , wherein the monoclonal antibody is selected from the group consisting of 1F6D8, 1B2C11, 3A1C11, 2C1H7, 3F2A4, 5F5C11, and 6D4D1.  
     
     
         28 . The method of  claim 21 , wherein the antibody is covalently linked to a detectable moiety.  
     
     
         29 . The method of  claim 28 , wherein the antibody is covalently linked to a biotin moiety, an iodine moiety, or an enzyme moiety.  
     
     
         30 . The method of  claim 21 , wherein the antibody is bound to a solid substrate.  
     
     
         31 . A method of isolating a β-tubulin isotype modified at cysteine residue 239, the method comprising the steps of: 
 (a) providing a sample treated with a β-tubulin modifying agent;  
 (b) contacting the sample with an antibody that specifically binds to a modified β-tubulin isotype; and  
 (c) isolating the modified β-tubulin isotype by isolating the antibody.  
 
     
     
         32 . The method of  claim 31 , wherein the antibody is a monoclonal antibody.  
     
     
         33 . The method of  claim 32 , wherein the monoclonal antibody is selected from the group consisting of 1F6D8, 1B2C11, 3A1C11, 2C1H7, 3F2A4, 5F5C11, and 6D4D11.  
     
     
         34 . The method of  claim 31 , wherein the antibody is covalently linked to a biotin moiety, an iodine moiety, or an enzyme moiety.  
     
     
         35 . The method of  claim 33 , wherein the antibody is covalently linked to a biotin moiety, an iodine moiety, or an enzyme moiety.  
     
     
         36 . The method of  claim 31 , wherein the antibody is bound to a solid substrate.  
     
     
         37 . A method of detecting an antibody that specifically binds to β-tubulin modified at cysteine residue 239, the method comprising the steps of: 
 (a) providing a sample;  
 (b) contacting the sample with a peptide that specifically binds to the antibody; and  
 (c) detecting the antibody.  
 
     
     
         38 . The method of  claim 37 , wherein the peptide is ATMSGVTTCLRFPGQLNA, GTMECVTTCLRFPGQLNA, or KATMSGVTTCLRFPGQLNA.  
     
     
         39 . The method of  claim 37 , wherein the step of detecting the antibody comprises an ELISA assay.  
     
     
         40 . The method of  claim 37 , wherein the peptide is bound to a solid substrate.  
     
     
         41 . A method of detecting in a sample a modified tubulin, the method comprising the steps of: 
 (a) providing a sample treated with a tubulin modifying agent;    (b) contacting the sample with an antibody that specifically binds to a modified tubulin isotype; and    (c) determining whether the sample contains a modified tubulin by detecting the antibody.    
     
     
         42 . A method of monitoring the amount of modified tubulin in a patient treated with an agent that modifies tubulin, the method comprising the steps of: 
 (a) providing a sample from the patient treated with the tubulin modifying agent;    (b) contacting the sample with an antibody that specifically binds to a modified tubulin; and    (c) determining the amount of modified tubulin in the patient sample by detecting the antibody and comparing the amount of antibody detected in the patient sample to a standard curve, thereby monitoring the amount of modified tubulin in the patient.