Method of obtaining a binder to prepro-vasopressin or fragments thereof

Abstract

Method of obtaining and/or verifying a binder to prepro-Vasopressin (SEQ ID NO. 1) or fragments thereof of at least 6 amino acids in length, including Copeptin (SEQ ID NO. 2), comprising at least one of the steps of: a) generating the binder using a developer comprising an amino acid sequence of at least 6 amino acids in length contained in an amino acid sequence corresponding to the C-terminal part but lacking amino acid 164 of prepro-Vasopressin (SEQ ID NO. 1); b) determining whether the binder is capable of binding to an amino acid sequence of at least 4 amino acids in length contained in an amino acid sequence corresponding to the C-terminal part but lacking amino acid 164 of prepro-Vasopressin (SEQ ID NO. 1); c) selecting and optionally isolating the binder from a plurality of binders which is capable of binding to an amino acid sequence contained in an amino acid sequence corresponding to the C-terminal part but lacking amino acid 164 of prepro-Vasopressin (SEQ ID NO. 1); d) carrying out binding assays with the binder in order to determine the ex vivo stability of prepro-Vasopressin or fragments thereof of at least 6 amino acids in length, including Copeptin, in a biological sample; e) carrying out binding assays with the binder and another binder for comparison purposes in order to determine the concentration of prepro-Vasopressin or fragments thereof of at least 6 amino acids in length, including Copeptin, in a biological sample; wherein the C-terminal part consists of amino acids 138 to 164 of prepro-Vasopressin (SEQ ID NO. 1), in order to obtain a binder or a mixture of binders capable of binding to an epitope contained in an amino acid sequence corresponding to amino acids 138 to 163 but lacking amino acid 164 of prepro-Vasopressin (SEQ ID NO. 1).

1 . Method of obtaining and/or verifying a binder to prepro-Vasopressin (SEQ ID NO. 1) or fragments thereof of at least 6 amino acids in length, including Copeptin (SEQ ID NO. 2), comprising at least one of the steps of:
 a) generating the binder using a developer comprising an amino acid sequence of at least 6 amino acids in length contained in an amino acid sequence corresponding to the C-terminal part but lacking amino acid 164 of prepro-Vasopressin (SEQ ID NO. 1);   b) determining whether the binder is capable of binding to an amino acid sequence of at least 4 amino acids in length contained in an amino acid sequence corresponding to the C-terminal part but lacking amino acid 164 of prepro-Vasopressin (SEQ ID NO. 1);   c) selecting and optionally isolating the binder from a plurality of binders which is capable of binding to an amino acid sequence contained in an amino acid sequence corresponding to the C-terminal part but lacking amino acid 164 of prepro-Vasopressin (SEQ ID NO. 1);   d) carrying out binding assays with the binder in order to determine the ex vivo stability of prepro-Vasopressin or fragments thereof of at least 6 amino acids in length, including Copeptin, in a biological sample;   e) carrying out binding assays with the binder and another binder for comparison purposes in order to determine the concentration of prepro-Vasopressin or fragments thereof of at least 6 amino acids in length, including Copeptin, in a biological sample;   wherein the C-terminal part consists of amino acids 138 to 164 of prepro-Vasopressin (SEQ ID NO. 1),   in order to obtain a binder or a mixture of binders capable of binding to an epitope contained in an amino acid sequence corresponding to amino acids 138 to 163 but lacking amino acid 164 of prepro-Vasopressin (SEQ ID NO. 1).   
     
     
         2 . The method of  claim 1 , wherein amino acids 163-164, preferably amino acids 162-164 and most preferably amino acids 161-164 are lacking from the amino acid sequence corresponding to the C-terminal part of prepro-Vasopressin (SEQ ID NO. 1). 
     
     
         3 . The method of  claim 1 , wherein the amino acid sequence corresponding to the C-terminal part of prepro-Vasopressin (SEQ ID NO. 1) corresponds to amino acids 140 to 163, preferably 142 to 163, especially preferably 144 to 163 and most preferably 146 to 163 of prepro-Vasopressin (SEQ ID NO. 1). 
     
     
         4 . The method of any one of  claim 1 , wherein the amino acid sequence comprised in the developer and/or the amino acid sequence contained in the amino acid sequence corresponding to the C-terminal part of prepro-Vasopressin (SEQ ID NO. 1) contains at least 8, preferably at least 10 and most preferably at least 12 consecutive amino acids contained in the amino acid sequence corresponding to amino acids 146 to 163 of prepro-Vasopressin (SEQ ID NO. 1) and is preferably selected from the group comprising the peptides consisting of amino acids 146-163 (SEQ ID NO. 7), 146-162 (SEQ ID NO. 8), 146-161 (SEQ ID NO. 9), 146-160 (SEQ ID NO. 10), 146-159 (SEQ ID NO. 11), 146-158 (SEQ ID NO. 12) and 146-157 (SEQ ID NO. 13) of prepro-Vasopressin (SEQ ID NO. 1). 
     
     
         5 . The method of  claim 1 , wherein the binder is capable of binding to an amino acid sequence of at least 6, preferably at least 8, more preferably at least 10 and most preferably at least 12 consecutive amino acids contained in the amino acid sequence corresponding to amino acids 146 to 163 of prepro-Vasopressin (SEQ ID NO. 1), preferably an amino acid sequence selected from the group consisting of amino acids 146-163 (SEQ ID NO. 7), 146-162 (SEQ ID NO. 8), 146-161 (SEQ ID NO. 9), 146-160 (SEQ ID NO. 10), 146-159 (SEQ ID NO. 11), 146-158 (SEQ ID NO. 12) and 146-157 (SEQ ID NO. 13) of prepro-Vasopressin (SEQ ID NO. 1). 
     
     
         6 . The method of  claim 1 , wherein the determining step b) comprises epitope mapping. 
     
     
         7 . The method of  claim 1 , wherein the specificity of binding of the binder to the said epitope is determined, particularly, in steps d) and/or e), by comparison with the epitope specificity of another binder for comparison purposes, preferably a binder known not to require the said amino acid 164 of prepro-Vasopressin (SEQ ID NO. 1) for binding to the said epitope. 
     
     
         8 . The method of  claim 1 , wherein the selecting step comprises affinity separation. 
     
     
         9 . The method of  claim 1 , comprising
 step a) but not steps b), c), d) and e), or   step b) but not steps a), c), d) and e), or   step c) but not steps a), b), d) and e), or   step d) but not steps a), b), c) and e), or   step e) but not steps a), b), c) and d), or   steps b) and c) but not steps a), d) and e).   
     
     
         10 . A peptide, consisting of or containing, in addition to a linking part, as a functional part at least 6, preferably at least 8, more preferably at least 10 and most preferably at least 12 consecutive amino acids contained in the amino acid sequence corresponding to amino acids 146 to 163 of prepro-Vasopressin (SEQ ID NO. 1), preferably peptide selected from the group comprising the peptides consisting of amino acids 146-163 (SEQ ID NO. 7), 146-162 (SEQ ID NO. 8), 146-161 (SEQ ID NO. 9), 146-160 (SEQ ID NO. 10), 146-159 (SEQ ID NO. 11), 146-158 (SEQ ID NO. 12) and 146-157 (SEQ ID NO. 13) of prepro-Vasopressin (SEQ ID NO. 1). 
     
     
         11 . The use of a peptide of  claim 10  as a developer in the method of  claim 1 , wherein the peptide is consisting of or containing, in addition to a linking part, as a functional part at least 6, preferably at least 8, more preferably at least 10 and most preferably at least 12 consecutive amino acids contained in the amino acid sequence corresponding to amino acids 146 to 163 of prepro-Vasopressin (SEQ ID NO. 1), preferably peptide selected from the group comprising the peptides consisting of amino acids 146-163 (SEQ ID NO. 7), 146-162 (SEQ ID NO. 8), 146-161 (SEQ ID NO. 9), 146-160 (SEQ ID NO. 10), 146-159 (SEQ ID NO. 11), 146-158 (SEQ ID NO. 12) and 146-157 (SEQ ID NO. 13) of prepro-Vasopressin (SEQ ID NO. 1). 
     
     
         12 . A binder, obtainable by the method of  claim 1 . 
     
     
         13 . A binder according to  claim 12 , namely antibody, preferably monoclonal antibody, polyclonal antiserum, enriched or purified polyclonal antibody, recombinant antibody, or a functional derivative thereof. 
     
     
         14 . A use of the binder of  claim 12  for qualitatively or quantitatively detecting prepro-Vasopressin or fragments thereof, including Copeptin, in a biological sample. 
     
     
         15 . The use of  claim 14 , wherein the biological sample is a bodily fluid selected from the group comprising blood, serum, plasma, urine, cerebrospinal fluid and saliva. 
     
     
         16 . The use of  claim 14 , wherein at least one additional binder is utilized which is capable of binding to an epitope fully or partially contained in an amino acid sequence corresponding to amino acids 126 to 164 of prepro-Vasopressin (SEQ ID NO. 1). 
     
     
         17 . The use of  claim 16 , wherein the at least one additional binder is an antibody, preferably monoclonal antibody, polyclonal antiserum, enriched or purified polyclonal antibody, recombinant antibody, or a functional derivative thereof. 
     
     
         18 . A kit for qualitatively or quantitatively detecting prepro-Vasopressin or fragments thereof, including Copeptin, in a biological sample, comprising the binder of  claim 12 .