Immunoassay for determining the release of neurotensin into the circulation

Abstract

The invention relates to an immunodiagnostic determination method for determining the release of neurotensin into the circulation of mammals during which an immunoreactivity of the N-terminal portion of a mammal proneurotensin (PNT immunoreactivity) is selectively determined in a serum sample or plasma sample of a test mammal, this immunoreactivity not being a neurotensin or neuromedin immunoreactivity.

1 - 18 . (canceled) 
     
     
         19 . An immunodiagnostic assay method for determining the release of neurotensin (amino acids 151-163 of SEQ ID NO:1) into the circulation of a mammal, comprising
 a. contacting a plasma or serum sample obtained from said mammal with at least one antibody which specifically binds to an immunoreactive portion of a polypeptide having the amino acid sequence of amino acids 24-140 of SEQ ID NO:1, and   b. determining the amount of bound antibody.   
     
     
         20 . The method of  claim 19 , wherein neurotensin is released from a precursor polypeptide, and said precursor is preproneurotensin (SEQ ID NO:1) or proneurotensin (amino acids 24-170 of SEQ ID NO:1). 
     
     
         21 . The method of  claim 19 , wherein the antibody does not selectively bind to neurotensin (amino acids 151-163 of SEQ ID NO:1) or neuromedin (amino acids 143-148 of SEQ ID NO:1). 
     
     
         22 . The method of  claim 19 , wherein the antibody comprises a detectable label. 
     
     
         23 . The method of  claim 22 , wherein said detectable label is a radioisotope or a chemiluminescence, bioluminescence, fluorescence or enzyme label. 
     
     
         24 . The method of  claim 19 , wherein said antibody is a monoclonal antibody, an affinity-purified polyclonal antibody or a combination of monoclonal and affinity-purified polyclonal antibodies. 
     
     
         25 . The method of  claim 21 , further comprising a second antibody which specifically binds to an immunoreactive portion of a polypeptide having the amino acid sequence of amino acids 24-140 of SEQ ID NO:1 which is different from the immunoreactive portion to which the first antibody binds, and wherein at least one antibody comprises a detectable label. 
     
     
         26 . The method of  claim 25 , wherein said detectable label is a radioisotope or a chemiluminescence, bioluminescence, fluorescence or enzyme label. 
     
     
         27 . The method of  claim 25 , wherein said first and second antibodies are both monoclonal antibodies, both affinity-purified polyclonal antibodies or a combination of monoclonal and affinity-purified polyclonal antibodies. 
     
     
         28 . The method of  claim 19 , wherein the mammal is a human. 
     
     
         29 . The method of  claim 25 , wherein the determination is carried out using a sandwich assay. 
     
     
         30 . The method of  claim 25 , wherein one antibody selectively binds to the same region within amino acids 24-140 of SEQ ID NO:1 as an antibody obtained by immunization of an animal with an antigen consisting of a polypeptide having the amino acid sequence of 67-85 of SEQ ID NO:1. 
     
     
         31 . The method of  claim 25 , wherein one antibody selectively binds to the same region within amino acids 24-140 of SEQ ID NO:1 as an antibody obtained by immunization of an animal with an antigen consisting of a polypeptide having the amino acid sequence of 120-140 of SEQ ID NO:1. 
     
     
         32 . The method of  claim 30 , wherein the second antibody selectively binds to the same region within amino acids 24-140 of SEQ ID NO:1 as an antibody obtained by immunization of an animal with an antigen consisting of a polypeptide having the amino acid sequence of 120-140 of SEQ ID NO:1. 
     
     
         33 . The method of  claim 25 , wherein both of said first and second antibodies selectively bind to a polypeptide comprising amino acids 24 to 140 of SEQ ID NO:1, but do not bind to neurotensin or neuromedin. 
     
     
         34 . The method of  claim 25 , wherein one of the antibodies is bound to a solid phase, and the other antibody comprises a detectable label. 
     
     
         35 . The method of  claim 25 , wherein both first and second antibodies are used in dispersion in a liquid reaction mixture and both first and second antibodies comprise detectable labels that are part of a marker system based on fluorescence or chemiluminescence extinction or amplification wherein, after binding of both first and second antibodies to the polypeptide having the amino acid sequence of amino acids 24-140 of SEQ ID NO:1 to form polypeptide/antibody complexes, a measurable signal is produced which permits detection of polypeptide/antibody complexes in the liquid reaction mixture. 
     
     
         36 . The method of  claim 35 , wherein the marker system comprises rare earth cryptates or chelates in combination with a fluorescent or chemiluminescent dye. 
     
     
         37 . The method of  claim 25 , which is carried out as a point-of-care method with the use of an immunochromatographic measuring apparatus, and the detectable label is directly visually detectable. 
     
     
         38 . The method of  claim 37 , which is carried out for monitoring the nutritional status and/or the digestive functions of a human patient. 
     
     
         39 . The method of  claim 38 , wherein the patient is artificially fed. 
     
     
         40 . The method of  claim 25 , wherein said method is used for diagnosis of a disease, or monitoring disease progression or treatment in patients who are suffering from a disease selected from Parkinson's disease; celiac disease; a chronic inflammatory intestinal disease, including Crohn's disease or Colitis ulcerosa; intestinal cancer; cancer of the pancreas; pancreatitis; a thyroid disease; cystic fibrosis; or hepatitis, by comparing the amount of the polypeptide having the amino acid sequence of amino acids 24-140 of SEQ ID NO:1 in the patient sample with a standard amount in patients having or free of said disease, or with a prior sample from said patient. 
     
     
         41 . A kit for determining the release of neurotensin (amino acids 151-163 of SEQ ID NO:1) from a precursor of neurotensin into the circulation of a mammal, said kit comprising
 a. a first antibody that specifically binds to a first region within amino acids 24-140 of SEQ ID NO:1; and   b. a second antibody that specifically binds to one or more amino acids of a second region within amino acids 24 to 140 of SEQ ID NO:1,   
       wherein both of said first and second antibodies selectively bind to a polypeptide comprising amino acids 24 to 140 of SEQ ID NO:1, but do not bind to neurotensin or neuromedin, and wherein at least one of said first and second antibodies is labeled with a detectable marker. 
     
     
         42 . The kit of  claim 41 , wherein said first antibody selectively binds to the same region within amino acids 24-140 of SEQ ID NO:1 as an antibody generated by immunization of an animal with a peptide consisting of the amino acid sequence of SEQ ID NO:4, and the second antibody selectively binds to the same region within amino acids 24-140 of SEQ ID NO:1 as an antibody generated by immunization of an animal with a peptide consisting of the amino acid sequence of SEQ ID NO:5. 
     
     
         43 . The kit of  claim 42 , wherein said first antibody selectively binds to the amino acid sequence of SEQ ID NO:4, and the second antibody selectively binds to the amino acid sequence of SEQ ID NO:5 in a polypeptide comprising amino acids 24 to 140 of SEQ ID NO:1. 
     
     
         44 . The kit of  claim 42 , wherein said first antibody was generated by immunization of an animal with a peptide consisting of the amino acid sequence of SEQ ID NO:4, and the second antibody was generated by immunization of an animal with a peptide consisting of the amino acid sequence of SEQ ID NO:5. 
     
     
         45 . An immunodiagnostic assay method, comprising
 a. contacting a plasma or serum sample obtained from a mammal with at least one antibody which specifically binds to an immunoreactive portion of a polypeptide having the amino acid sequence of amino acids 24-140 of SEQ ID NO:1, and   b. determining the amount of bound antibody;   
       whereby the amount of said polypeptide is determined. 
     
     
         46 . The method of  claim 45 , wherein the amount of said polypeptide is used to determine the release of neurotensin (amino acids 151-163 of SEQ ID NO:1) into the circulation of a mammal. 
     
     
         47 . The method of  claim 45 , further comprising a second antibody which specifically binds to an immunoreactive portion of a polypeptide having the amino acid sequence of amino acids 24-140 of SEQ ID NO:1 which is different from the immunoreactive portion to which the first antibody binds, and wherein at least one antibody comprises a detectable label. 
     
     
         48 . The method of  claim 45 , wherein said method is used for diagnosis of a disease, or monitoring disease progression or treatment in patients who are suffering from a disease selected from Parkinson's disease; celiac disease; a chronic inflammatory intestinal disease, including Crohn's disease or Colitis ulcerosa; intestinal cancer; cancer of the pancreas; pancreatitis; a thyroid disease; cystic fibrosis; or hepatitis, by comparing the amount of the polypeptide having the amino acid sequence of amino acids 24-140 of SEQ ID NO:1 in the patient sample with a standard amount in patients having or free of said disease, or with a prior sample from said patient.