Ultrasensitive immunoassays

Abstract

The present invention relates to an immunological test kit and immunoassay using a first immobilized antibody having affinity for a specific antigen. The invention is characterized by a second and third antibody being specific for different determinants of the antigen and modified with cross-linkable oligonucleotides. For detection, the oligonucleotides are amplified, whereby only such oligonucleotides will be amplified which have been cross-linked to each other. In this way unspecific background is avoided and detection is possible down to single molecules.

1 . Reagents for use in an assay for detection of a macromolecule comprising two affinity reagents, each reagent being specific for a different determinant of said macromolecule, and each reagent having attached thereto an oligonucleotide which is conjugatable to the oligonucleotide attached to the other of said two reagents, wherein said oligonucleotides are conjugatable by means of ligation or hybridization to each other only when said affinity reagents have became bound to the same said macromolecule and wherein said conjugated oligonucleotides may then serve as a template for a nucleic acid amplification reaction, for detection of said macromolecule.  
     
     
         2 . The reagents of  claim 1 , wherein said oligonucleotides conjugate through 
 i) hybridization of an oligonucleotide complementary to the conjugatable oligonucleotides;    ii) hybridization of the conjugatable oligonucleotides to each other; or    iii) ligation of the oligonucleotides.    
     
     
         3 . The reagents of  claim 1 , wherein said macromolecule is a protein.  
     
     
         4 . The reagents according to  claim 1 , wherein the reagents are antibodies.  
     
     
         5 . The reagents of  claim 1 , wherein said reagents are polyclonal antibodies, monoclonal antibodies or single chain antibodies.  
     
     
         6 . The reagents according to  claim 1 , wherein the oligonucleotides are complementary to each other.  
     
     
         7 . The reagents of  claim 1 , wherein said oligonucleotides have complementary 3′ ends.  
     
     
         8 . A method for detection of a macromolecule, which comprises binding said macromolecule to a first affinity reagent having affinity for said macromolecule, said first affinity reagent being specific for a first determinant of said macromolecule; 
 incubating said first affinity reagent-bound macromolecule with said detection reagents of  claim 1 , specific for second and third determinants of said macromolecule, wherein said oligonucleotides conjugate to each other by hybridization or ligation when said detection reagents are both bound to said macromolecule;    amplifying said conjugated oligonucleotides; and    detecting the amplified products.    
     
     
         9 . The method of  claim 8 , wherein said first affinity reagent is immobilized.  
     
     
         10 . The method of  claim 8 , wherein said oligonucleotides conjugate through 
 i) hybridization of an oligonucleotide complementary to the conjugatable oligonucleotides;    ii) hybridization of the conjugatable oligonucleotides to each other; or    iii) ligation of the oligonucleotides.    
     
     
         11 . The method according to  claim 8 , wherein the conjugation occurs through hybridization of an oligonucleotide complementary to the conjugatable oligonucleotides.  
     
     
         12 . The method according to  claim 8 , wherein the conjuation occurs through hybridization of the conjugatable oligonucleotides to each other.  
     
     
         13 . The method according to  claim 11 , wherein the conjugation occurs through ligation of the oligonucleotides.  
     
     
         14 . The method  claim 8 , wherein said affinity reagents are antibodies.  
     
     
         15 . The method of  claim 8 , wherein said affinity reagents are polyclonal antibodies, monoclonal antibodies or single chain antibodies.