Detection of histone modification in cell-free nucleosomes

Abstract

This invention relates to the diagnosis of disease conditions, such as cancer and autoimmune disease, by the analysis of cell-free nucleosomes in samples from individuals. Methods of the invention may include contacting cell-free nucleosomes from a biological fluid sample obtained from the individual with an antibody that binds specifically with a modified histone protein. Binding of the antibody to the nucleosomes is indicative that the individual has the disease condition.

1 - 22 . (canceled) 
     
     
         23 . The method of assessing a cell-free nucleosome comprising a histone modification in a sample, the method comprising:
 contacting a biological fluid sample with a first antibody that binds to cell-free nucleosomes, and;   contacting cell-free nucleosomes bound to said first antibody with a second antibody which binds to a histone modification.   
     
     
         24 . The method according to  claim 23  wherein the first antibody binds to a modified histone in said cell-free nucleosomes. 
     
     
         25 . The method according to  claim 23  wherein the histone modification is selected from the group consisting of acetylation, methylation, phosphorylation, ribosylation, citrullination, ubiquitination, hydroxylation, glycosylation, nitrosylation, glutamination and isomerisation. 
     
     
         26 . The method according to  claim 23  wherein the biological fluid sample is selected from the group consisting of blood, serum, plasma, lymph, blood fractions, urine, synovial fluid, spinal fluid and mucous. 
     
     
         27 . The method according to  claim 23  wherein said first antibody or said second antibody is immobilised. 
     
     
         28 . The method according to  claim 23  wherein the non-immobilised antibody of said first antibody and second antibody comprises a detectable label. 
     
     
         29 . A method of assessing a cell-free nucleosome comprising a histone modification in a sample, the method comprising:
 contacting a biological fluid sample with a first antibody that binds to a histone modification, and;   contacting cell-free nucleosomes bound to said first antibody with a second antibody which binds to cell-free nucleosomes.   
     
     
         30 . The method according to  claim 29  wherein the second antibody binds to a modified histone in said cell-free nucleosomes. 
     
     
         31 . The method according to  claim 29  wherein the histone modification is selected from the group consisting of acetylation, methylation, phosphorylation, ribosylation, citrullination, ubiquitination, hydroxylation, glycosylation, nitrosylation, glutamination and isomerisation. 
     
     
         32 . The method according to  claim 29  wherein the biological fluid sample is selected from the group consisting of blood, serum, plasma, lymph, blood fractions, urine, synovial fluid, spinal fluid and mucous. 
     
     
         33 . The method according to  claim 23  wherein said first antibody or said second antibody is immobilised. 
     
     
         34 . The method according to  claim 23  wherein the non-immobilised antibody of said first antibody and second antibody comprises a detectable label. 
     
     
         35 . A method of assessing a disease condition in an individual, the method comprising:
 contacting a biological fluid sample obtained from the individual with a first antibody that binds to cell-free nucleosomes, and;   contacting cell-free nucleosomes bound to said first antibody with a second antibody which binds to a histone modification,   wherein binding of said second antibody to cell-free nucleosomes bound to said first antibody is indicative that said individual has a disease condition.   
     
     
         36 . The method according to  claim 35  wherein the first antibody binds to a modified histone in said cell-free nucleosomes. 
     
     
         37 . The method according to  claim 35  wherein the histone modification is selected from the group consisting of acetylation, methylation, phosphorylation, ribosylation, citrullination, ubiquitination, hydroxylation, glycosylation, nitrosylation, glutamination and isomerisation. 
     
     
         38 . The method according to  claim 35  wherein the biological fluid sample is selected from the group consisting of blood, serum, plasma, lymph, blood fractions, urine, synovial fluid, spinal fluid and mucous. 
     
     
         39 . The method according to  claim 35  comprising determining a parameter selected from;
 the presence of one or more cell-free nucleosomes comprising histone modifications in the sample; an increase in the number of cell-free nucleosomes comprising histone modifications relative to normal levels; an alteration in the ratio of one or more histone modifications in cell-free nucleosomes in the sample relative to another histone modification; and the presence of a threshold number of cell-free nucleosomes in the sample which comprise a histone modification, 
 wherein said parameter is indicative that said individual has a disease condition. 
 
     
     
         40 . The method according to  claim 35  wherein said first antibody or said second antibody is immobilised. 
     
     
         41 . The method according to  claim 35  wherein the non-immobilised antibody of said first antibody and second antibody comprises a detectable label. 
     
     
         42 . A method of assessing a disease condition in an individual, the method comprising:
 contacting a biological fluid sample obtained from the individual with a first antibody which binds to a histone modification, and;   contacting cell-free nucleosomes bound to said first antibody with a second antibody that binds to cell free nucleosomes,   wherein binding of said second antibody to a nucleosome in said sample is indicative that said individual has a disease condition.   
     
     
         43 . The method according to  claim 42  wherein the second antibody binds to a modified histone in said cell-free nucleosomes. 
     
     
         44 . The method according to  claim 42  wherein the histone modification is selected from the group consisting of acetylation, methylation, phosphorylation, ribosylation, citrullination, ubiquitination, hydroxylation, glycosylation, nitrosylation, glutamination and isomerisation. 
     
     
         45 . The method according to  claim 42  wherein the biological fluid sample is selected from the group consisting of blood, serum, plasma, lymph, blood fractions, urine, synovial fluid, spinal fluid and mucous. 
     
     
         46 . The method according to  claim 42  comprising determining a parameter selected from;
 the presence of one or more cell-free nucleosomes comprising histone modifications in the sample; an increase in the number of cell-free nucleosomes comprising histone modifications relative to normal levels; an alteration in the ratio of one or more histone modifications in cell-free nucleosomes in the sample relative to another histone modification; and the presence of a threshold number of cell-free nucleosomes in the sample which comprise a histone modification, 
 wherein said parameter is indicative that said individual has a disease condition. 
 
     
     
         47 . The method according to  claim 42  wherein said first antibody or said second antibody is immobilised. 
     
     
         48 . The method according to  claim 42  wherein the non-immobilised antibody of said first antibody and second antibody comprises a detectable label.