Form-specific antibodies for nag-1 (mic-1, gdf-15), h6d and other tgf-beta subfamily and heart disease and cancer diagnoses

Abstract

A method of producing form-specific anti-peptide antibodies for a wild type protein and its one amino acid mutated protein using a peptide antigen, by obtaining a protein sequence of the wild type protein and its one amino acid mutated protein, selecting a continuous amino acid sequence without any internal cysteine residues that includes the one amino acid mutated sequence and wild type sequence corresponding to the mutated site at the end of the sequence to obtain a synthetic mutation peptide and a synthetic wild type peptide, conjugating the synthetic peptides to a carrier protein, and immunizing an animal to produce antibodies. Methods of detecting cancer and methods of treating cancer.

What is claimed is: 
     
         1 . A method of producing form-specific anti-peptide antibodies for a wild type protein and its one amino acid mutated protein using a peptide antigen, including the steps of:
 obtaining a protein sequence of the wild type protein and its one amino acid mutation protein;   selecting a continuous amino acid sequence without any internal cysteine residues that includes the one amino acid mutated sequence and wild type sequence corresponding to the mutated site at the end of the sequence to obtain a synthetic mutation peptide and a synthetic wild type peptide;   conjugating the synthetic peptides to a carrier protein; and   immunizing an animal to produce antibodies.   
     
     
         2 . The method of  claim 1 , wherein said conjugating step is further defined as conjugating the synthetic mutation peptide to a carrier protein via an amino acid at the opposite end of an end having the mutated amino acid sequence. 
     
     
         3 . The method of  claim 1 , wherein the synthetic mutation peptide and synthetic wild type peptide are 4-mer-10-mer. 
     
     
         4 . The method of  claim 3 , wherein the synthetic mutation peptide and the synthetic wild type peptide are conjugated to the carrier molecule via a cysteine residue. 
     
     
         5 . The method of  claim 4 , wherein the protein sequence is a member of the transforming growth factor (TGF)-beta subfamily. 
     
     
         6 . The method of  claim 4 , wherein the wild type protein is NAG-1 and the one amino acid mutated protein is an H6D polymorphism, and the antibodies produced are form-specific to NAG-1 and the H6D polymorphism. 
     
     
         7 . The method of  claim 5 , wherein said selecting step is further defined as selecting an amino acid sequence corresponding to the peptide sequence SEQ ID NO: 28 and SEQ ID NO: 29. 
     
     
         8 . A method of detecting cancer, including the steps of:
 measuring an amount a wild type epitope and its one amino acid mutated epitope in a biological system;   comparing the amounts to a control sample; and   if the amount of wild type epitopes and one amino acid mutated epitopes are higher than amount of the control sample, detecting the presence of cancer.   
     
     
         9 . The method of  claim 8 , wherein the wild type epitope is from NAG-1 and the one amino acid mutated epitope is from an H6D polymorphism. 
     
     
         10 . The method of  claim 9 , wherein the wild type epitope is SEQ ID NO: 3 and the one amino acid mutation epitope is SEQ ID NO: 5. 
     
     
         11 . The method of  claim 9 , wherein the wild type epitope is SEQ ID NO: 28 and the one amino acid mutated epitope is SEQ ID NO: 29. 
     
     
         12 . The method of  claim 10 , wherein said detecting step further includes detecting the presence of cancer-associated inflammation. 
     
     
         13 . The method of  claim 8 , wherein the biological system is chosen from the group consisting of plasma, urine, cerebrospinal fluids, bile and joint fluids. 
     
     
         14 . The method of  claim 8 , wherein said measuring step further includes the step of an assay chosen from the group consisting of ELISA, radioimmunoassay, and fluoroimmunoassays. 
     
     
         15 . The method of  claim 8 , wherein the cancer is chosen from the group consisting of prostate, breast, colon, and pancreatic. 
     
     
         16 . A method of treating cancer, including the step of:
 administering an effective amount of anti-peptide form-specific antibodies that react with wild type NAG-1 and an H6D polymorphism.   
     
     
         17 . The method of  claim 16 , wherein the anti-peptide form-specific antibodies cross-react with SEQ ID NO: 3 and SEQ ID NO: 5. 
     
     
         18 . The method of  claim 16 , further including the step of reducing cancer-associated inflammation. 
     
     
         19 . A method of detecting the presence of cancer, including the steps of:
 measuring an amount of PSA or an amount of NAG-1 in a biological system; and   if the amount of PSA or NAG-1 is higher than control samples, detecting the presence of inflammation and cancer.   
     
     
         20 . The method of  claim 19 , wherein the cancer is chosen from the group consisting of prostate, breast, colon, and pancreatic. 
     
     
         21 . The method of  claim 20 , wherein the amount of PSA or NAG-1 is 10-fold higher than the control samples. 
     
     
         22 . The method of  claim 19 , wherein the PSA amount is 120 ng/ml or higher than 120 ng/ml. 
     
     
         23 . The method of  claim 19 , wherein the NAG-1 amount is 2.4 ng/ml or higher than 2.4 ng/ml. 
     
     
         24 . The method of  claim 21 , wherein the biological system is chosen from the group consisting of plasma, urine, cerebrospinal fluids, bile and joint fluids. 
     
     
         25 . The method of  claim 21 , wherein the NAG-1 amount is obtained by adding wild type NAG-1 and H6D protein levels obtained by measuring an amount of form-specific antibodies for wild type NAG-1 and H6D proteins.