Pathway specific assays for predicting irritable bowel syndrome diagnosis

Abstract

The present invention provides antibodies and methods for preparing antibodies to metabolites in the serotonin, tryptophan, kynurenine pathways. The prepared antibodies have low cross-reactivity to related metabolites, and are useful reagents for specific and sensitive immunoassays The present invention also provides stable derivatives of various metabolites and short chain fatty acids. The derivative can be conjugated to a biomolecule such as a carrier protein and combined with an adjuvant to stimulate an immune response. The derivatives can also be conjugated to other biomolecules.

What is claimed is: 
     
         1 . A compound of Formula III: 
       
         
           
           
               
               
           
         
         wherein R, R 1 , R 2 , R 3 , and R 4  are each a member independently selected from the group consisting of hydrogen, alkyl, halo, hydroxyl, alkoxy, amino, aroyl, alkanoyl, amido, substituted amido, cyano, carboxyl, alkoxycarbonyl, sulfonato, alkoxyalkyl, carboxy, carboxyalkyl, alkoxycarbonylalkyl, sulfonatoalkyl, L and R 11 B; 
         L is a linker; 
         R 11  is the resultant attachment between the compound and a biomolecule; and 
         B is a biomolecule. 
       
     
     
         2 . The compound of  claim 1 , wherein at least one member of the group of R, R 1 , R 2 , R 3 , and R 4  is R 11 B. 
     
     
         3 . The compound of  claim 2 , wherein B is a member selected from the group consisting of a protein, an immunogenic peptide or biotin. 
     
     
         4 . The compound of  claim 3 , wherein the compound has the formula: 
       
         
           
           
               
               
           
         
       
       wherein R, R 1 , R 2 , R 3 , and R 4  are each hydrogen. 
     
     
         5 . A compound of Formula IV: 
       
         
           
           
               
               
           
         
         wherein R 1 , R 2 , R 3 , R 4  and R 5  are each a member independently selected from the group consisting of hydrogen, alkyl, halo, hydroxyl, alkoxy, amino, aroyl, alkanoyl, amido, substituted amido, cyano, carboxyl, alkoxycarbonyl, sulfonato, alkoxyalkyl, carboxy, carboxyalkyl, alkoxycarbonylalkyl, sulfonatoalkyl L and R 11 B; 
         L is a linker; 
         R 11  is the resultant attachment between the compound and a biomolecule; and 
         B is a biomolecule. 
       
     
     
         6 . The compound of  claim 5 , wherein at least one member of the group of R 1 , R 2 , R 3 , R 4  and R 5  is R 11 B. 
     
     
         7 . The compound of  claim 6 , wherein B is a member selected from the group consisting of a protein, an immunogenic peptide or biotin. 
     
     
         8 . The compound of  claim 7 , wherein the compound as the formula: 
       
         
           
           
               
               
           
         
         L is a linker; 
         R 11  is the resultant attachment between the compound and a biomolecule; and 
         B is a biomolecule. 
       
     
     
         9 . An isolated antibody or antigen binding fragment thereof that specifically binds to kynurenine, wherein said antibody has less than 1% cross-reactivity to one or more members selected from the group consisting of kynurenic acid, 3-hydroxykynurenine, 3-hydroxyanthranilic acid, quinolinic acid, anthranilic acid, serotonin-O-sulfate and serotonin-O-phosphate. 
     
     
         10 . The antibody or fragment thereof according to  9 , wherein the antibody is a polyclonal or monoclonal antibody. 
     
     
         11 . The antibody or fragment thereof according to  claim 10 , wherein the polyclonal antibody is a rabbit antibody. 
     
     
         12 . The antibody or fragment thereof according to  claim 9 , wherein the antibody is a Fab or Fab2 fragment. 
     
     
         13 . The antibody or fragment thereof according to  claim 9 , wherein the antibody is a recombinant antibody. 
     
     
         14 . A method for assaying a metabolite selected from the group consisting of serotonin (5-HT), 5-hydroxyindole acetic acid (5-HIAA), 5-hydroxy tryptophan (5-HTP), kynurenine (K), kynurenic acid (KA), 3-hydroxykynurenine (3-HK), 3-hydroxyanthranilic acid (3-HAA), quinolinic acid, anthranilic acid and a combination thereof in a fluid or tissue sample from a mammal, the method comprising:
 admixing the sample with antibodies that specifically bind to a metabolite selected from the group consisting of serotonin (5-HT), 5-hydroxyindole acetic acid (5-HIAA), 5-hydroxy tryptophan (5-HTP), kynurenine (K), kynurenic acid (KA), 3-hydroxykynurenine (3-HK), 3-hydroxyanthranilic acid (3-HAA), quinolinic acid, anthranilic acid and a combination thereof; and   determining whether the antibodies specifically bind to the metabolite, wherein specific antibody binding indicates that the metabolite is present in the sample.