METHOD OF DIAGNOSING A CLINICAL CONDITION BY DETECTION OF A PAPP-A/proMBP COMPLEX

Abstract

The present invention provides a method of diagnosing Down's syndrome, acute coronary syndrome or pre-eclampsia, or a predisposition to any of them, by a method comprising measuring the level of the human PAPP-A/proMBP complex in a body fluid sample.

1 . An antibody having specific binding affinity for a polypeptide which
 (a) consists of amino acid residues 234-1791 of SEQ ID NO:2; or   (b) is at least 95% identical to the polypeptide of (a), but differs from the polypeptide of (a) solely by
 (b1) deletion of 1-10 amino acid residues from, or addition of 1-10 amino acid residues to, the amino terminal, and/or 
 (b2) deletion of 1-10 amino acid residues from, or addition of 1-10 amino acid residues to, the carboxy terminal and/or 
 (b3) one or more conservative substitutions, each conservative substitution being the replacement of an amino acid with a different amino acid of the same class, the amino acid classes being defined as follows: 
   i) Amino acids having polar side chains (Asp, Glu, Lys, Arg, His, Asn, Gin, Ser, Thr, Tyr, and Cys)   ii) Amino acids having non-polar side chains (Gly, Ala, Val, Leu, Ile, Phe, Trp, Pro, and Met)   iii) Amino acids having aliphatic side chain (Gly, Ala, Val, Leu, Ile)   iv) Amino acids having cyclic side chains (Phe, Tyr, Trp, His, Pro)   v) Amino acids having aromatic side chains (Phe, Tyr, Trp)   vi) Amino acids having acidic side chains (Asp, Glu)   vii) Amino acids having basic side chains (Lys, Arg, His)   viii) Amino acids having amide side chains (Asn, Gin)   ix) Amino acids having hydroxy side chains (Ser, Thr)   x) Amino acids having sulphur-containing side chains (Cys, Met)   xi) Neutral, weakly hydrophobic amino acids (Pro, Ala, Gly, Ser, Thr)   xii) Hydrophilic, acidic amino acids (Gln, Asn, Glu, Asp), and   xiii) Hydrophobic amino acids (Leu, Ile, Val)   wherein said polypeptide has a proteolytic activity against Insulin Like Growth Factor Binding Protein 5 (IGFBP-5).   
     
     
         2 . The antibody according to  claim 1 , wherein said antibody has specific binding affinity for a polypeptide that differs from a polypeptide consisting of residues 234-1791 of SEQ ID NO:2, if at all, solely by one or more conservative substitutions. 
     
     
         3 . The antibody according to  claim 1 , wherein said antibody has specific binding affinity for a polypeptide consisting of amino acids 234 to 1791 of SEQ ID NO:2. 
     
     
         4 . An antibody having specific binding affinity for a polypeptide which is
 (1) a polypeptide consisting of an amino acid sequence which is (a) identical to amino acid residues 234-1791 of SEQ ID NO:2, or (b) a fragment, at least 5 amino acids in length, of mature PAPP-A2 (residues 234-1791 of SEQ ID NO:2), where said fragment   i) has a proteolytic activity against Insulin Like Growth Factor Binding Protein 5 (IGFBP-5); and/or   ii) is recognized by an antibody, or a binding fragment thereof, which recognizes mature PAPP-A2; and where said fragment comprises at least one of the following regions of SEQ ID NO:2:   Cys-403 to Cys-499   Cys-828 to Cys-881   Cys-1048 to Cys-1115   Cys-1390 to Cys-1396   Cys-1459 to Cys-1464   Cys-1521 to Cys-1525   Cys-1590 to Cys-1595   Cys-1646 to Cys-1653   Cys-1729 to Cys-1733   or   (2) a polypeptide which consists of a fusion of the polypeptide of (1) with an immunogenic carrier protein, or with a tag which may be used to facilitate the detection or purification of the fusion.   
     
     
         5 . The antibody according to  claim 4 , wherein said antibody has specific binding affinity for a fragment of at least 17 amino acids in length of mature PAPP-A2 (residues 234-1791 of SEQ ID NO:2). 
     
     
         6 . The antibody according to  claim 4 , wherein said antibody has specific binding affinity for a polypeptide comprising at least 1169 consecutive amino acids of the polypeptide (mature PAPP-A2) consisting of residues 234-1791 of the polypeptide of SEQ ID NO:2. 
     
     
         7 . The antibody according to  claim 4 , wherein said antibody has specific binding affinity for a polypeptide comprising the elongated zinc binding consensus sequence (amino acids 733 to 743 of SEQ ID NO:2), LNR 1 (amino acids 586 to 612 of SEQ ID NO:2), LNR 2 (amino acids 619 to 644 of SEQ ID NO:2), LNR 3 (amino acids 1733 to 1758 of SEQ ID NO:2), SCR1 (amino acids 1396 to 1459 of SEQ ID NO:2), SCR2 (amino acids 1464 to 1521 of SEQ ID NO:2), SCR3 (amino acids 1525 to 1590 of SEQ ID NO:2), SCR4 (amino acids 1595 to 1646 of SEQ ID NO:2), SCR5 (amino acids 1653 to 1729 of SEQ ID NO:2), and all cysteine residues of mature PAPP-A2. 
     
     
         8 . An antibody having specific binding affinity for a polypeptide comprising
 (I) amino acids 1-22 of SEQ ID NO:2, and   (II) an amino acid sequence selected from the group consisting of
 (a) an amino acid sequence consisting of residues 234-1791 of SEQ ID NO:2, and 
 (b) an amino acid sequence which is at least 95% identical to (a) above, 
   where said polypeptide, or a cleavable fragment, at least five amino acids in length, of said polypeptide, which comprises amino acid sequence (II) of said polypeptide, has a proteolytic activity against Insulin Like Growth Factor Binding Protein 5 (IGFBP-5).   
     
     
         9 . An antibody having specific binding affinity for a polypeptide comprising
 (I) amino acids 23 233 of SEQ ID NO:2, and   (II) an amino acid a sequence selected from the group consisting of
 (a) an amino acid sequence consisting of residues 234-1791 of SEQ ID NO:2, and 
 (b) an amino acid sequence which is at least 95% identical to (a) above, 
   where said polypeptide, or a cleavable fragment, at least five amino acids in length, of said polypeptide, which comprises amino acid sequence (II) of said polypeptide, has a proteolytic activity against Insulin Like Growth Factor Binding Protein 5 (IGFBP-5).   
     
     
         10 . An antibody having specific binding affinity for a polypeptide comprising
 (I) amino acids 1-233 of SEQ ID NO:2 and   (II) an amino acid sequence selected from the group consisting of
 (a) an amino acid sequence consisting of residues 234-1791 of SEQ ID NO:2, and 
 (b) an amino acid sequence which is at least 95% identical to (a) above 
   where said polypeptide, or a cleavable fragment, at least five amino acids in length, of said polypeptide, which comprises sequence (II) of said polypeptide, has a proteolytic activity against Insulin Like Growth Factor Binding Protein 5 (IGFBP-5).   
     
     
         11 . An antibody having specific binding affinity for a polypeptide which
 (a) consists of residues 234-1791 of SEQ ID NO:2; or   (b) differs by not more than 16 insertions and/or deletions and/or substitutions from the polypeptide of (a),   where said polypeptide has a proteolytic activity against Insulin Like Growth Factor Binding Protein 5 (IGFBP-5).   
     
     
         12 . An antibody having specific binding affinity for a polypeptide which
 (a) consists of residues 234-1791 of SEQ ID NO:2; or   (b) is at least 99% identical to the polypeptide of (a), and which consists of 1548-1568 amino acids.   
     
     
         13 . An antibody having specific binding affinity for an isolated polypeptide comprising or essentially consisting of the amino acid sequence of SEQ ID NO:2, or a fragment thereof, wherein said fragment
 i) has a proteolytic activity specific at least for Insulin Like Growth Factor Binding Protein 5 (IGFBP-5); and/or   ii) is recognized by an antibody, or a binding fragment thereof, which is capable of recognising a polypeptide having the amino acid sequence as shown in SEQ ID NO:2; and/or   iii) competes with a polypeptide having the amino acid sequence as shown in SEQ ID NO:2 for binding to a cell surface receptor with an affinity for said polypeptide.   
     
     
         14 . The antibody according to  claim 1 , wherein said antibody is monoclonal. 
     
     
         15 . The antibody according to  claim 1 , wherein said antibody is polyclonal. 
     
     
         16 . A method for detecting PAPP-A2, or measuring the level of PAPP-A2, in a biological sample obtained from an individual, said method comprising the steps of
 i) obtaining a biological sample from said individual,   ii) detecting or measuring the level of PAPP-A2 in said sample by detecting or measuring the level of
 a) an isolated polypeptide comprising the amino acid sequence of SEQ ID NO:2, or a fragment consisting of five or more amino acids, thereof, wherein said fragment 
   has a proteolytic activity specific at least for Insulin Like Growth Factor Binding Protein 5 (IGFBP-5); and/or   is recognised by an antibody, or a binding fragment thereof, which is capable of recognising a polypeptide having the amino acid sequence as shown in SEQ ID NO:2; and/or competes with a polypeptide having the amino acid sequence as shown in SEQ ID NO:2 for binding to a cell surface receptor with an affinity for said polypeptide.
 b) a polynucleotide in the form of mRNA originating from PAPP-A2 expression, and/or 
 c) PAPP-A2 specific protease activity. 
   
     
     
         17 . The method of  claim 16 , said method comprising the further step of comparing the PAPP-A2 or the level of PAPP-A2 detected in step ii) with a predetermined value selected from the group consisting of
 i) a predetermined amount and/or concentration of PAPP-A2; and/or   ii) a predetermined amount and/or concentration of PAPP-A2 mRNA; and/or   iii) a predetermined PAPP-A2 specific protease activity.   
     
     
         18 . The method of  claim 17 , wherein said predetermined value is indicative of a normal physiological condition of said individual. 
     
     
         19 . The method of  claim 16 , wherein said biological sample is selected from the group consisting of blood, urine, pleural fluid, oral washings, tissue biopsies, and follicular fluid. 
     
     
         20 . The method of  claim 16 , wherein said level of PAPP-A2 is measured as PAPP-A2 specific protease activity. 
     
     
         21 . The method of  claim 16 , wherein said level of PAPP-A2 is measured as amount of PAPP-A2 protein. 
     
     
         22 . The method of  claim 16 , wherein said level of PAPP-A2 is measured as amount of PAPP-A2 messenger RNA. 
     
     
         23 . The method of  claim 21 , wherein said amount of PAPP-A2 protein is measured by immunochemical analysis. 
     
     
         24 . The method of  claim 23 , wherein said amount of PAPP-A2 protein is detected by at least one monoclonal antibody. 
     
     
         25 . The method of  claim 16 , wherein said PAPP-A2 protein is detected in a complex comprising at least one additional component. 
     
     
         26 . The method of  claim 16 , wherein said PAPP-A2 is detected as a PAPP-A2 monomer. 
     
     
         27 . The method of  claim 16 , wherein said PAPP-A2 is detected as a PAPP-A2 dimer.