Conjugation of biomolecules using diels-alder cycloaddition

Abstract

A method is provided for covalently linking carbohydrates, proteins, nucleic acids, and other biomolecules under neutral conditions, using a Diels-Alder cycloaddition reaction. In an example, activated carbon-carbon double bonds were attached to free amino sites of a carrier protein, and a conjugated diene was attached to a carbohydrate hapten. Spontaneous coupling of the carbohydrate and the protein components under very mild conditions provided glycoconjugates containing up to 37 carbohydrate hapten units per carrier protein molecule. The method is also applicable to the immobilization of biomolecules on gel or solid supports. The conjugated products are useful as immunogens and as analytical and diagnostic reagents.

1 - 31 . (canceled) 
     
     
         32 . An antibody which immunoreacts with a polysaccharide, wherein said antibody is obtained from a mammal, and wherein the production of the antibody by the mammal has been induced by administering to said mammal a composition, wherein the composition comprises a conjugate of biomolecules selected from the group consisting of 
       
         
           
           
               
               
           
         
         wherein R and R′ are independently H or methyl, or together constitute CH 2 , CH 2 CH 2 , or O; X is CH or N; Y is N, CH═C, or NH—N; and B 1  and B 2  comprise biomolecules independently selected from the group consisting of polypeptides, carbohydrates, polysaccharides, and nucleic acids, and are optionally attached via a linker wherein one of the biomolecules is a polysaccharide. 
       
     
     
         33 - 36 . (canceled) 
     
     
         37 . An antibody, produced by a hybridoma, which immunoreacts with a polysaccharide, wherein nucleic acid sequences encoding said antibody in said hybridoma are obtained from a mammal in which the production of the antibody has been induced by administering to said mammal a composition, wherein the composition comprises a conjugate of biomolecules selected from the group consisting of 
       
         
           
           
               
               
           
         
         wherein R and R′ are independently H or methyl, or together constitute CH 2 , CH 2 CH 2 , or O; X is CH or N; Y is N, CH═C, or NH—N; and B 1  and B 2  comprise biomolecules independently selected from the group consisting of polypeptides, carbohydrates, polysaccharides, and nucleic acids, and are optionally attached via a linker wherein one of the biomolecules is a polysaccharide. 
       
     
     
         38 - 41 . (canceled) 
     
     
         42 . A method of inducing passive immunity in a mammal, comprising administering to said mammal an effective amount of an antibody according to  claim 32 . 
     
     
         43 . A method of inducing passive immunity in a mammal, comprising administering to said mammal an effective amount of an antibody according to  claim 37 . 
     
     
         44 - 47 . (canceled) 
     
     
         48 . A method of inducing, in a mammal, antibodies which immunoreact with a polysaccharide, comprising administering to said mammal a composition comprising a pharmaceutically acceptable carrier and a conjugate of biomolecules prepared by
 (a) covalently attaching a diene moiety to a first biomolecule to form a diene component;   (b) covalently attaching a dienophile to a second biomolecule to form a dienophile component; and   (c) contacting the diene component with the dienophile component under conditions that permit a cycloaddition reaction to occur between the components.   
     
     
         49 . A method according to  claim 48 , wherein the first biomolecule is a polysaccharide and the second biomolecule is a polypeptide. 
     
     
         50 . The method of  claim 49  wherein the polysaccharide is selected from the group consisting of bacterial capsular polysaccharides, fragments thereof, and synthetic analogues thereof. 
     
     
         51 . The method of  claim 49 , wherein the bacterial capsular polysaccharide is selected from the group consisting of capsular polysaccharides of  Haemophilus influenzae  type b,  Neisseria meningitides , Group B Streptococci,  Salmonella typhi, E. coli , and Pneumococci. 
     
     
         52 . The method of  claim 49  wherein the polypeptide is selected from the group consisting of bacterial toxins, bacterial toxoids, bacterial outer membrane proteins, keyhole limpet hemocyanin, horseshoe crab hemocyanin, edestin, mammalian serum albumins, mammalian gamma-globulins, and IgG-G. 
     
     
         53 . An antibody which immunoreacts with a polysaccharide, wherein said antibody is obtained from a mammal, and wherein the production of the antibody by the mammal has been induced by the method of  claim 48 . 
     
     
         54 . An antibody which immunoreacts with a polysaccharide, wherein said antibody is obtained from a mammal, and wherein the production of the antibody by the mammal has been induced by the method of  claim 49 . 
     
     
         55 . An antibody, produced by a hybridoma, which immunoreacts with a polysaccharide, wherein nucleic acid sequences encoding said antibody in said hybridoma are obtained from a mammal in which the production of the antibody has been induced by the method of  claim 48 . 
     
     
         56 . An antibody, produced by a hybridoma, which immunoreacts with a polysaccharide, wherein nucleic acid sequences encoding said antibody in said hybridoma are obtained from a mammal in which the production of the antibody has been induced by the method of  claim 49 .