Systems and methods of epitope binning and antibody profiling

Abstract

Methods for antibody profiling and epitope mapping are provided herein. More particularly, methods for screening and mapping epitopes of candidate antibodies and protein target identification are provided herein.

We claim: 
     
         1 . A method of identifying an epitope recognized by an antibody, the method comprising the steps of:
 (a) contacting a sample comprising the antibody to a plurality of peptides immobilized on an array;   (b) identifying peptides that bind to the antibody with a K d  of less than 10 −7  M; and   (c) screening the peptide sequences of the identified peptides for a consensus sequence motif, wherein the motif corresponds to an epitope of the antigen to which the antibody specifically binds.   
     
     
         2 . The method of  claim 1 , wherein screening the peptide sequences of the identified peptides for a consensus sequence motif comprises using a search algorithm. 
     
     
         3 . The method of  claim 1 , wherein the peptide sequences are from a database of amino acid sequences. 
     
     
         4 . The method of  claim 3 , wherein the peptides are randomly generated. 
     
     
         5 . The method of  claim 1 , wherein the array comprises at least 10,000 peptide features per 1 cm 2 . 
     
     
         6 . The method of  claim 1 , wherein the array comprises at least 300,000 peptide features per 0.5 cm 2 . 
     
     
         7 . The method of  claim 1 , wherein the peptides have a length of 1 to 25 amino acids. 
     
     
         8 . The method of  claim 1 , wherein identifying peptides that bind to the antibody comprises an immunofluorescence assay. 
     
     
         9 . The method of  claim 1 , wherein screening the peptide sequences of the identified peptides for a consensus sequence motif comprises aligning the peptide sequences using a search algorithm. 
     
     
         10 . The method of  claim 1 , wherein the number of peptide sequences screened is at least 500. 
     
     
         11 . The method of  claim 1 , wherein the antibody is a monoclonal antibody. 
     
     
         12 . The method of  claim 1 , wherein the sample is a hybridoma culture supernatant. 
     
     
         13 . The method of  claim 1 , wherein the sample is a serum sample. 
     
     
         14 . The method of  claim 13 , wherein the serum sample is from a vertebrate. 
     
     
         15 . The method of  claim 13 , wherein the serum sample is from a mammal. 
     
     
         16 . The method of  claim 13 , wherein the serum sample is from a human. 
     
     
         17 . The method of  claim 13 , wherein the serum sample comprises an antibody that recognizes an epitope in an antigen from an infectious organism. 
     
     
         18 . The method of  claim 17 , wherein the infectious organism is a pathogen. 
     
     
         19 . The method of  claim 17 , wherein the infectious organism is selected from the group consisting of viruses, bacteria, and protists. 
     
     
         20 . The method of  claim 18 , wherein the pathogen is  Borrelia, Bordetella , hepatitis B virus,  Plasmodium, Treponema , or dengue virus. 
     
     
         21 . The method of  claim 1 , further comprising identifying a protein target of the antibody, comprising
 (i) searching a protein sequence database for proteins that contain sequences homologous to the consensus sequence motif;   (ii) identifying proteins from step (i); and   (iii) verifying that the antibody binds to a protein retrieved from the database search.   
     
     
         22 . The method of  claim 21 , wherein homologous sequences show at least 80% identity. 
     
     
         23 . The method of  claim 21 , wherein the database comprises proteomes from bacteria, viruses, and eukaryotes. 
     
     
         24 . The method of  claim 23 , wherein the eukaryotes are protists. 
     
     
         25 . The method of  claim 23 , wherein the bacteria, viruses, and eukaryotes are pathogenic. 
     
     
         26 . The method of  claim 1 , wherein the identified peptide sequences binding to the antibody are hierarchically clustered and aligned. 
     
     
         27 . The method of  claim 1 , further comprising examining peptides on the array that are not bound to antibody. 
     
     
         28 . A method of characterizing the binding specificity of an antibody, the method comprising the steps of:
 (a) contacting a sample comprising the antibody to a plurality of peptides immobilized on an array;   (b) identifying peptides that bind to the antibody with a K d  of less than 10 −7  M;   (c) identifying peptides on the array that do not bind to the antibody; and   (d) clustering and aligning the identified peptides from (b) and (c) to determine the level of specific binding recognized by the antibody.   
     
     
         29 . The method of  claim 28 , wherein the antibody is a monoclonal antibody. 
     
     
         30 . The method of  claim 29 , wherein the identified peptides are clustered by similarity of the identified peptides in (b) and (c) to the eliciting peptide used to make the monoclonal antibody. 
     
     
         31 . The method of  claim 30 , wherein the identified peptides are hierarchically clustered and aligned. 
     
     
         32 . The method  claim 30 , wherein the level of similarity of identified peptides in steps (b) and (c) to the eliciting peptide is indicative of the degree of promiscuity of antibody binding. 
     
     
         33 . The method of  claim 28 , wherein the peptide sequences are from a database of amino acid sequences. 
     
     
         34 . The method of  claim 33 , wherein the peptides are randomly generated. 
     
     
         35 . The method of  claim 28 , wherein the array comprises at least 10,000 peptide features per 1 cm 2 . 
     
     
         36 . The method of  claim 28 , wherein the array comprises at least 300,000 peptide features per 0.5 cm 2 . 
     
     
         37 . The method of  claim 28 , wherein the peptides have a length of 1 to 25 amino acids. 
     
     
         38 . The method of  claim 28 , wherein identifying peptides that bind to the antibody comprises an immunofluorescence assay. 
     
     
         39 . The method of  claim 28 , wherein screening the peptide sequences of the identified peptides binding to an antibody in step (b) further comprises determining a consensus sequence motif comprises aligning the peptide sequences using a search algorithm. 
     
     
         40 . The method of  claim 39 , wherein determining the consensus sequence comprises aligning the identified peptide sequences using a search algorithm. 
     
     
         41 . The method of  claim 28 , wherein the number of peptide sequences screened is at least 500. 
     
     
         42 . The method of  claim 28 , wherein the sample is a hybridoma culture supernatant. 
     
     
         43 . The method of  claim 28 , wherein the sample is a serum sample. 
     
     
         44 . The method of  claim 43 , wherein the serum sample is from a vertebrate. 
     
     
         45 . The method of  claim 43 , wherein the serum sample is from a mammal. 
     
     
         46 . The method of  claim 43 , wherein the serum sample is from a human. 
     
     
         47 . The method of  claim 43 , wherein the serum sample comprises an antibody that recognizes an epitope in an antigen from an infectious organism. 
     
     
         48 . The method of  claim 47 , wherein the infectious organism is a pathogen. 
     
     
         49 . The method of  claim 47 , wherein the infectious organism is selected from the group consisting of viruses, bacteria, and protists. 
     
     
         50 . The method of  claim 48 , wherein the pathogen is  Borrelia, Bordetella , hepatitis B virus,  Plasmodium, Treponema , or dengue virus.