Determination of a midregional proadrenomedullin partial peptide in biological fluids for diagnostic purposes, and immunoassays for carrying out such a determination

Abstract

Method for the determination of adrenomedullin immunoreactivity in biological fluids for diagnostic purposes, in particular in sepsis, cardiac and cancer diagnosis, in which the midregional partial peptide (mid-proAM; SEQ ID NO:3) of proadrenomedullin, which comprises the amino acids (45-92) of the complete preproadrenomedullin (pre-proAM; SEQ ID NO:1), is measured in particular by means of an immunoassay which operates with at least one labelled antibody which specifically recognizes a sequence of mid-proAM.

1 .- 18 . (canceled) 
     
     
         19 . A method for detecting and quantitating in a biological fluid sample from a human the mid-regional partial peptide of proadrenomedullin (mid-proAM) which consists of the sequence of SEQ ID NO: 3, comprising
 (a) contacting the sample with a labeled monoclonal or polyclonal antibody which specifically binds to said mid-proAM partial peptide, and   (b) detecting and quantitating the resulting peptide:antibody complex using an immunoassay, wherein said immunoassay
 (i) is not a radioimmunoassay, and 
 (ii) has a limit of detection of about 50 pmol/l. 
   
     
     
         20 . The method of  claim 19 , wherein said immunoassay using said at least one labeled antibody is an assay further employing a solid phase-bound competitor for the mid-proAM or a sandwich assay further employing at least one additional antibody which specifically binds to a different partial sequence of mid-proAM (SEQ ID NO: 3) from that bound by said at least one labeled antibody. 
     
     
         21 . The method of  claim 19 , wherein the level of circulating mid-proAM (SEQ ID NO: 3) is determined and the biological fluid is plasma or serum. 
     
     
         22 . The method of  claim 20 , wherein both antibodies bind to a region of mid-proAM which extends from the amino acid 60 to the amino acid 92 of preproadrenomedullin (SEQ ID NO:1). 
     
     
         23 . The method of  claim 20 , wherein all said antibodies are monoclonal and/or polyclonal. 
     
     
         24 . The method of  claim 20 , wherein all said antibodies are affinity-purified polyclonal antibodies. 
     
     
         25 . The method of  claim 20 , wherein for said sandwich assay, one of the antibodies is labeled and the other antibody is bound to a solid phase or is not bound to a solid phase but can be subsequently bound thereto during the assay. 
     
     
         26 . The method of  claim 20 , wherein for said sandwich assay, both said at least one labeled antibody and said at least one additional antibody are present dispersed in a liquid reaction mixture and a first labeling component which is part of a labeling system based on fluorescence or chemiluminescence extinction or amplification is bound to said at least one labeled antibody, and a second labeling component of said labeling system is bound to said at least one additional antibody so that, after binding of both antibodies to the mid-proAM to be detected, a measurable signal which permits detection of the resulting sandwich complexes is generated. 
     
     
         27 . The method of  claim 26 , wherein the labeling system comprises cryptate emission in combination with a fluorescent or chemiluminescent dye. 
     
     
         28 . The method of  claim 19 , wherein at least one antibody is obtained by immunization with an antigen which contains a synthetic peptide sequence having a cysteine not present in mid-proAM at the N-terminus of the synthetic peptide. 
     
     
         29 . An immunoassay method for detecting and quantitating in a biological fluid sample the mid-regional partial peptide of proadrenomedullin (mid-proAM) which consists of the sequence of SEQ ID NO: 3, comprising contacting the sample with at least one labeled monoclonal or polyclonal antibody which specifically binds to said mid-proAM partial peptide, and detecting the thus-formed antibody:mid-proAM complex,
 wherein said immunoassay
 (a) is not a radioimmunoassay, and 
 (b) has a limit of detection of about 50 pmol/l. 
   
     
     
         30 . The immunoassay of  claim 29 , wherein at least one antibody is obtained by immunization with an antigen which contains a synthetic peptide sequence having a cysteine not present in mid-proAM at the N-terminus of the synthetic peptide. 
     
     
         31 . The immunoassay of  claim 29 , further employing a solid phase-bound competitor for the mid-proAM or a sandwich assay further employing at least one additional antibody which specifically binds to a different partial sequence of mid-proAM (SEQ ID NO: 3) from that bound by said at least one labeled antibody. 
     
     
         32 . The immunoassay of  claim 29 , wherein the level of circulating mid-proAM (SEQ ID NO: 3) is determined and the biological fluid is plasma or serum. 
     
     
         33 . The immunoassay of  claim 31 , wherein both antibodies bind to a region of mid-proAM which extends from the amino acid 60 to the amino acid 92 of preproadrenomedullin (SEQ ID NO:1). 
     
     
         34 . The immunoassay of  claim 31 , wherein all said antibodies are monoclonal and/or polyclonal. 
     
     
         35 . The immunoassay of  claim 31 , wherein all said antibodies are affinity-purified polyclonal antibodies. 
     
     
         36 . The immunoassay of  claim 31 , wherein for said sandwich assay, one of the antibodies is labeled and the other antibody is bound to a solid phase or is not bound to a solid phase but can be subsequently bound thereto during the assay. 
     
     
         37 . The immunoassay of  claim 31 , wherein for said sandwich assay, both said at least one labeled antibody and said at least one additional antibody are present dispersed in a liquid reaction mixture and a first labeling component which is part of a labeling system based on fluorescence or chemiluminescence extinction or amplification is bound to said at least one labeled antibody, and a second labeling component of said labeling system is bound to said at least one additional antibody so that, after binding of both antibodies to the mid-proAM to be detected, a measurable signal which permits detection of the resulting sandwich complexes is generated. 
     
     
         38 . The immunoassay of  claim 37 , wherein the labeling system comprises cryptate emission in combination with a fluorescent or chemiluminescent dye. 
     
     
         39 . The immunoassay of  claim 29 , further comprising removing from a human said sample to be measured. 
     
     
         40 . The immunoassay of  claim 19 , further comprising removing from a human said sample to be measured.