Biomarker fragments for the detection of human BNP

Abstract

The present invention relates among other things to a composition comprising at least two (2) human BNP fragments, wherein each of the human BNP fragments of the composition are cross-linked to at least one of the other human BNP fragments of the composition.

1 . A composition comprising:
 at least two human BNP fragments, wherein each of the human BNP fragments of said composition are cross-linked to at least one of the other human BNP fragments of said composition.   
     
     
         2 . The composition of  claim 1 , wherein said composition comprises a first human BNP fragment and a second human BNP fragment. 
     
     
         3 . The composition of  claim 2 , wherein the first human BNP fragment has an amino acid sequence comprising amino acid residues a-32, b-31 or c-30 of human BNP, wherein a is an integer selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 and 30, b is an integer selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 and 29, and c is an integer selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 and 28. 
     
     
         4 . The composition of  claim 2 , wherein the second human BNP fragment has an amino acid sequence comprising amino acid residues a-32, b-31 or c-30 of human BNP, wherein a is an integer selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 and 30, b is an integer selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 and 29, and c is an integer selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 and 28. 
     
     
         5 . The composition of  claim 2 , wherein at least one amino acid residue in the first human BNP fragment, at least one amino acid residue in the second human BNP fragment, or at least one amino acid residue in the first human BNP fragment and the second human BNP fragment is modified, changed, substituted or altered to allow for cross-linking of the human BNP fragments by a cross-linking agent, a cross-linker, or a combination of a cross-linking agent and a cross-linker. 
     
     
         6 . The composition of  claim 5 , wherein the first human BNP fragment and the second human BNP fragment are cross-linked by a cross-linking agent, a cross-linker, or a combination of a cross-linking agent and a cross-linker. 
     
     
         7 . A composition comprising:
 at least two human BNP fragments, wherein (a) each of the human BNP fragments has an amino acid sequence that includes a portion or all of amino acids 10 to 26 of human BNP; and (b) each of the human BNP fragments of said composition are cross-linked to at least one of the other human BNP fragments of said composition.   
     
     
         8 . The composition of  claim 7 , wherein said composition comprises a first human BNP fragment and a second human BNP fragment. 
     
     
         9 . The composition of  claim 8 , wherein the first human BNP fragment and the second human BNP fragment have an amino acid sequence comprising amino acids residues selected from the group consisting of 1-31, 1-30, 1-29, 1-28, 1-27, 1-26, 1-25, 1-24, 1-23, 1-22, 1-21, 1-20, 1-19, 1-18, 1-17, 1-16, 1-15, 1-14, 1-13, 1-12, 1-11, 1-10, 2-32, 2-31, 2-30, 2-29, 2-28, 2-27, 2-26, 2-25, 2-24, 2-23, 2-22, 2-21, 2-20, 2-19, 2-18, 2-17, 2-16, 2-15, 2-14, 2-13, 2-12, 2-11, 2-10, 3-32, 3-31, 3-30, 3-29, 3-28, 3-27, 3-26, 3-25, 3-24, 3-23, 3-32, 3-21, 3-20, 3-19, 3-18, 3-17, 3-16, 3-15, 3-14, 3-13, 3-12, 3-11, 3-10, 4-32, 4-31, 4-30, 4-29, 4-28, 4-27, 4-26, 4-25, 4-24, 4-23, 4-22, 4-21, 4-20, 4-19, 4-18, 4-17, 4-16, 4-15, 4-14, 4-13, 4-12, 4-11, 4-10, 5-32, 5-31, 5-30, 5-29, 5-28, 5-27, 5-26, 5-25, 5-24, 5-23, 5-22, 5-21, 5-20, 5-19, 5-18, 5-17, 5-16, 5-15, 5-14, 5-13, 5-12, 5-11, 5-10, 6-32, 6-31, 6-30, 6-29, 6-28, 6-27, 6-26, 6-25, 6-24, 6-23, 6-22, 6-21, 6-20, 6-19, 6-18, 6-17, 6-16, 6-15, 6-14, 6-13, 6-12, 6-11, 6-10, 7-32, 7-31, 7-30, 7-29, 7-28, 7-27, 7-26, 7-25, 7-24, 7-23, 7-22, 7-21, 7-20, 7-19, 7-18, 7-17, 7-16, 7-15, 7-14, 7-13, 7-12, 7-11, 7-10, 8-32, 8-31, 8-30, 8-29, 8-28, 8-27, 8-26, 8-25, 8-24, 8-23, 8-22, 8-21, 8-20, 8-19, 8-18, 8-17, 8-16, 8-15, 8-14, 8-13, 8-12, 8-11, 8-10, 9-32, 9-31, 9-30, 9-29, 9-28, 9-27, 9-26, 9-25, 9-24, 9-23, 9-22, 9-21, 9-20, 9-19, 9-18, 9-17, 9-16, 9-15, 9-14, 9-13, 9-12, 9-11, 10-32, 10-31, 10-30, 10-29, 10-28, 10-27, 10-26, 10-25, 10-24, 10-23, 10-22, 10-21, 10-20, 10-19, 10-18, 10-17, 10-16, 10-15, 10-14, 10-13, 10-12, 11-32, 11-31, 11-30, 11-29, 11-28, 11-27, 11-26, 11-25, 11-24, 11-23, 11-22, 11-21, 11-20, 11-19, 11-18, 11-17, 11-16, 11-15, 11-14, 11-13, 12-32, 12-31, 12-30, 12-29, 12-28, 12-27, 12-26, 12-25, 12-24, 12-23, 12-22, 12-21, 12-20, 12-19, 12-18, 12-17, 12-16, 12-15, 12-14, 13-32, 13-31, 13-30, 13-29, 13-28, 13-27, 13-26, 13-25, 13-24, 13-23, 13-22, 13-21, 13-20, 13-19, 13-18, 13-17, 13-16, 13-15, 14-32, 14-31, 14-30, 14-29, 14-28, 14-27, 14-26, 14-25, 14-24, 14-23, 14-22, 14-21, 14-20, 14-19, 14-18, 14-17, 14-16, 15-32, 15-31, 15-30, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 16-32, 16-31, 16-30, 16-29, 16-28, 16-27, 16-26, 16-25, 16-24, 16-23, 16-22, 16-21, 16-20, 16-19, 16-18, 17-32, 17-31, 17-30, 17-29, 17-28, 17-27, 17-26, 17-25, 17-24, 17-23, 17-22, 17-21, 17-20, 17-19, 18-32, 18-31, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-32, 19-31, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 20-32, 20-31, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 21-32, 21-31, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, 22-32, 22-31, 22-30, 22-29, 22-28, 22-27, 22-26, 22-25, 22-24, 23-32, 23-31, 23-30, 23-29, 23-28, 23-27, 23-26, 23-25, 24-32, 24-31, 24-30, 24-29, 24-28, 24-27, 24-26, 25-32, 25-31, 25-30, 26-29 and 26-28 of human BNP. 
     
     
         10 . The composition of  claim 8 , wherein at least one amino acid residue in the first human BNP fragment, at least one amino acid residue in the second human BNP fragment, or at least one amino acid residue in the first human BNP fragment and the second human BNP fragment is modified, changed, substituted or altered to allow for cross-linking of the human BNP fragments by a cross-linking agent, a cross-linker or a combination of a cross-linking agent and a cross-linker. 
     
     
         11 . The composition of  claim 10 , wherein the first human BNP fragment and the second human BNP fragment are cross-linked by a cross-linking agent, a cross-linker, or a combination of a cross-linking agent and a cross-linker. 
     
     
         12 . The composition of  claim 10 , wherein the first human BNP fragment has an amino acid sequence comprising amino acid residues selected from the group consisting of residues 1-13, 1-17, 3-13 and 3-17 of human BNP, and the second human BNP fragment has an amino acid sequence comprising residues 18-30 or 18-32 of human BNP. 
     
     
         13 . The composition of  claim 12 , wherein the first human BNP fragment and second human BNP fragment are cross-linked at a cysteine residue contained within the first human BNP fragment and the second human BNP fragment. 
     
     
         14 . An immunogen comprising a composition of  claim 1 . 
     
     
         15 . An antibody produced by the immunogen of  claim 14  which is capable of immunospecifically binding to an epitope of human BNP. 
     
     
         16 . An immunoassay for human BNP or a human BNP fragment, wherein said immunoassay comprises the antibody of  claim 15 . 
     
     
         17 . An immunoassay for a human BNP (hereinafter “hBNP”) composition, wherein said composition comprises at least two hBNP fragments, wherein each of the human BNP fragments of said composition are cross-linked to at least one of the other human BNP fragments of said composition, the immunoassay comprising the steps of:
 (a) contacting at least one antibody or functionally active fragment thereof with a test sample suspected of containing or known to contain said hBNP composition to form an antibody-hBNP complex; and   (b) detecting the formation of the antibody-hBNP complex.   
     
     
         18 . The immunoassay of  claim 17 , wherein the immunoassay further comprises contacting the antibody-hBNP complex with at least one detection antibody or functionally active fragment thereof to form a capture antibody-hBNP detection antibody complex, wherein said detection antibody or functionally active fragment thereof is conjugated to a detectable label. 
     
     
         19 . The immunoassay of  claim 17 , wherein the immunoassay relates the amount of the capture-hBNP-detection antibody complexes formed to the amount of the human BNP composition in the test sample via use of a standard curve for the human BNP composition. 
     
     
         20 . An immunoassay for a hBNP composition, wherein said composition comprises at least two hBNP fragments, wherein each of the human BNP fragments of said composition are cross-linked to at least one of the other human BNP fragments of said composition, the immunoassay comprising the steps of:
 (a) immobilizing at least one capture antibody or functionally active fragment thereof that binds to the hBNP composition onto a solid phase to produce an immobilized first antibody;   (b) contacting said immobilized capture antibody with a test sample suspected of containing the hBNP composition to form an immobilized capture antibody-hBNP complex;   (c) contacting said capture antibody-hBNP complex with at least one detection antibody or a functionally active fragment thereof to form an immobilized capture antibody-hBNP-detection antibody complex, wherein said detection antibody or functionally active fragment thereof is conjugated to a detectable label;   (d) removing the test sample and washing said immobilized capture antibody-hBNP-detection antibody complexes;   (e) determining the amount of capture antibody-hBNP-detection antibody complexes formed in step (c); and   (f) relating the amount of the capture antibody-hBNP-detection antibody complexes formed to the amount of the hBNP composition via use of a standard curve for hBNP or hBNP fragment.   
     
     
         21 . The immunoassay of  claim 20 , wherein the detection antibody is a monoclonal antibody or a functionally active fragment thereof. 
     
     
         22 . The immunoassay of  claim 20 , wherein the detectable label is a radioactive label, an enzymatic label, a chemiluminescent label, a fluorescence label, a thermometric label or an immuno-polymerase chain reaction label. 
     
     
         23 . An immunoassay for a hBNP composition, wherein said composition comprises at least two hBNP fragments, wherein each of the human BNP fragments of said composition are cross-linked to at least one of the other human BNP fragments of said composition, the immunoassay comprising the steps of:
 (a) immobilizing at least one capture antibody or functionally active fragment thereof that binds to the hBNP composition onto a solid phase to produce an immobilized first antibody;   (b) contacting said immobilized capture antibody with a test sample suspected of containing the hBNP composition to form an first immobilized antibody-hBNP complex;   (c) contacting said immobilized capture antibody with a hBNP peptide, hBNP fragment, or hBNP analogue thereof that has been conjugated to a detectable label to form an second immobilized antibody-hBNP complex;   (d) determining the amount of detectable label in second immobilized antibody-hBNP complexes formed in step (c); and   (e) relating the amount of the second antibody-hBNP-antibody complexes formed to the amount of the hBNP composition via use of a standard curve for hBNP or hBNP fragment.   
     
     
         24 . An immunoassay for a hBNP composition, wherein said composition comprises at least two hBNP fragments, wherein each of the human BNP fragments of said composition are cross-linked to at least one of the other human BNP fragments of said composition, the immunoassay comprising the steps of:
 (a) immobilizing a hBNP peptide, hBNP fragment, or hBNP analogue thereof onto a solid phase to produce an immobilized hBNP peptide, hBNP fragment, or hBNP analogue thereof;   (b) contacting said immobilized hBNP peptide, hBNP fragment, or hBNP analogue thereof with a test sample suspected of containing the hBNP composition;   (c) contacting said immobilized hBNP peptide, hBNP fragment, or hBNP analogue thereof and the test sample suspected of containing the hBNP composition with at least one antibody or functionally active fragment thereof that has been conjugated to a detectable label to form an immobilized hBNP-antibody complex and a non-immobilized hBNP-antibody complex;   (d) removing the non-immobilized hBNP-antibody complex;   (e) determining the amount of immobilized hBNP-antibody complex formed in step (c); and   (t) relating the amount of the immobilized hBNP-antibody complex formed to the amount of the hBNP composition via use of a standard curve for hBNP or hBNP fragment.   
     
     
         25 . An immunoassay for a hBNP composition, wherein said composition comprises at least two hBNP fragments, wherein (a) each of the hBNP fragments has an amino acid sequence that includes a portion or all of amino acids 10 to 26 of hBNP; and (b) each of the human BNP fragments of said composition are cross-linked to at least one of the other human BNP fragments of said composition, the immunoassay comprising the steps of:
 (a) contacting at least one antibody or functionally active fragment thereof with a test sample suspected of containing or known to contain said hBNP composition to form an antibody-hBNP complex; and   (b) detecting the formation of the antibody-hBNP complex.   
     
     
         26 . The immunoassay of  claim 25 , wherein the immunoassay further comprises contacting the antibody-hBNP complex with at least one detection antibody or functionally active fragment thereof to form a capture antibody-hBNP detection antibody complex, wherein said detection antibody or functionally active fragment thereof is conjugated to a detectable label. 
     
     
         27 . The immunoassay of  claim 25 , wherein the immunoassay relates the amount of the capture-hBNP-detection antibody complexes formed to the amount of the human BNP composition in the test sample via use of a standard curve for the human BNP composition. 
     
     
         28 . An immunoassay for a hBNP composition, wherein (a) each of the hBNP fragments has an amino acid sequence that includes a portion or all of amino acids 10 to 26 of hBNP; and (b) each of the human BNP fragments of said composition are cross-linked to at least one of the other human BNP fragments of said composition, the immunoassay comprising the steps of:
 (a) immobilizing at least one capture antibody or functionally active fragment thereof that binds to the hBNP composition onto a solid phase to produce an immobilized first antibody;   (b) contacting said immobilized capture antibody with a test sample suspected of containing the hBNP composition to form an immobilized capture antibody-hBNP complex;   (c) contacting said capture antibody-hBNP complex with at least one detection antibody or a functionally active fragment thereof to form an immobilized capture antibody-hBNP-detection antibody complex, wherein said detection antibody or functionally active fragment thereof is conjugated to a detectable label;   (d) removing the test sample and washing said immobilized capture antibody-hBNP-detection antibody complexes;   (e) determining the amount of capture antibody-hBNP-detection antibody complexes formed in step (c); and   (f) relating the amount of the capture antibody-hBNP-detection antibody complexes formed to the amount of the hBNP composition via use of a standard curve for hBNP or hBNP fragment.   
     
     
         29 . The immunoassay of  claim 28 , wherein the detection antibody is a monoclonal antibody or a functionally active fragment thereof. 
     
     
         30 . The immunoassay of  claim 28 , wherein the detectable label is a radioactive label, an enzymatic label, a chemiluminescent label, a fluorescence label, a thermometric label or an immuno-polymerase chain reaction label. 
     
     
         31 . An immunoassay for a hBNP composition, wherein said composition comprises at least two hBNP fragments, wherein (a) each of the hBNP fragments has an amino acid sequence that includes a portion or all of amino acids 10 to 26 of hBNP; and (b) each of the human BNP fragments of said composition are cross-linked to at least one of the other human BNP fragments of said composition, the immunoassay comprising the steps of:
 (a) immobilizing at least one capture antibody or functionally active fragment thereof that binds to the hBNP composition onto a solid phase to produce an immobilized first antibody;   (b) contacting said immobilized capture antibody with a test sample suspected of containing the hBNP composition to form an first immobilized antibody-hBNP complex;   (c) contacting said immobilized capture antibody with a hBNP peptide, hBNP fragment, or hBNP analogue thereof that has been conjugated to a detectable label to form an second immobilized antibody-hBNP complex;   (d) determining the amount of detectable label in second immobilized antibody-hBNP complexes formed in step (c); and   (e) relating the amount of the second antibody-hBNP-antibody complexes formed to the amount of the hBNP composition via use of a standard curve for hBNP or hBNP fragment.   
     
     
         32 . An immunoassay for a hBNP composition, wherein (a) each of the hBNP fragments has an amino acid sequence that includes a portion or all of amino acids 10 to 26 of hBNP; and (b) each of the human BNP fragments of said composition are cross-linked to at least one of the other human BNP fragments of said composition, the immunoassay comprising the steps of:
 (a) immobilizing a hBNP peptide, hBNP fragment, or hBNP analogue thereof onto a solid phase to produce an immobilized hBNP peptide, hBNP fragment, or hBNP analogue thereof;   (b) contacting said immobilized hBNP peptide, hBNP fragment, or hBNP analogue thereof with a test sample suspected of containing the hBNP composition;   (c) contacting said immobilized hBNP peptide, hBNP fragment, or hBNP analogue thereof and the test sample suspected of containing the hBNP composition with at least one antibody or functionally active fragment thereof that has been conjugated to a detectable label to form an immobilized hBNP-antibody complex and a non-immobilized hBNP-antibody complex;   (d) removing the non-immobilized hBNP-antibody complex;   (e) determining the amount of immobilized hBNP-antibody complex formed in step (c); and   (f) relating the amount of the immobilized hBNP-antibody complex formed to the amount of the hBNP composition via use of a standard curve for hBNP or hBNP fragment.