Nucleic acid aptamer-based diagnostic methods with novel techniques for signal enhancement

Abstract

The present invention concerns methods for the detection of target molecules in a sample including several steps of signal amplification allowing the detection of a very low number of target molecules in the tested sample. The detection assay is based on the use of a universal probe which enables the signal amplification. The specific recognition of the target molecule is achieved by using a specific binding agent, preferably an aptamer. The invention further concerns kits and methods for the diagnosis of pathological conditions.

1 - 73 . (canceled) 
     
     
         74 . A method for the detection of a target molecule in a sample comprising:
 a. obtaining at least one aptamer capable of binding to said target molecule, wherein said at least one aptamer is bound to a matrix;   b. incubating said at least one aptamer which is bound to the matrix with the sample under conditions allowing the binding of the aptamer to the target molecule; thereby forming a matrix-aptamer-target molecule complex;   c. contacting the matrix-aptamer-target molecule complex formed in step (b) with a polymer associated with a member of an affinity couple wherein said polymer further comprising a reactive group; thereby forming a matrix-aptamer-target molecule-polymer complex; and   d. contacting said matrix-aptamer-target molecule-polymer complex with a complementary member of said member of an affinity couple, wherein said complementary member is associated with a detectable moiety,   wherein the amount of said detectable moiety is indicative, of the presence of said target molecule in the sample, and wherein said target molecule is a viral antigen, a cancer marker or a soluble antigen selected from the group consisting of soluble cancer markers, inflammation-associated markers, hormones, cytokines, drugs, viral derived soluble molecules, bacterial derived soluble molecules and fungal derived soluble molecules.   
     
     
         75 . The method of  claim 74  wherein the polymer is a nucleic acid molecule comprising a reactive group and further comprising a polymerase promoter sequence, thereby forming a matrix-aptamer-target molecule-nucleic acid molecule complex; and wherein the method further comprises
 a. adding a DNA or RNA polymerase enzyme and nucleotides associated with a member of an affinity couple under suitable conditions to affect DNA or RNA polymerization, thereby obtaining DNA or RNA molecules associated with a member of an affinity couple, and 
 b. contacting said DNA or RNA molecules associated with a member of an affinity couple with a complementary member of said member of an affinity couple associated with a detectable moiety; 
 wherein the amount of said detectable moiety is indicative of the presence of said target molecule in the sample. 
 
     
     
         76 . A method for the detection of a target molecule in a sample comprising:
 a. obtaining at least one first binding agent capable of binding to said target molecule, wherein said first binding agent is bound to a matrix;   b. incubating said at least one first binding agent which is bound to the matrix with the sample under conditions allowing the binding of the binding agent to the target molecule; thereby forming a matrix-binding agent-target molecule complex;   c. contacting the matrix-binding agent-target molecule complex formed in step (b) with a second binding agent-polymer complex, wherein said second binding agent-polymer complex is obtained by either
 i. obtaining at least one biotinylated second binding agent; 
 ii. incubating said at least one biotinylated second binding agent with streptavidin thereby a biotinylated second binding agent streptavidin (b-binding agent-SA) complex is formed; and 
 iii. incubating said b-binding agent-SA complex formed in step (ii) with a polymer associated with a member of an affinity couple wherein said polymer further having a reactive group thereby forming a second binding agent-polymer complex; or 
 iv. obtaining at least one second binding agent, wherein said at least one second binding agent comprises a reactive group; and 
 v. incubating said at least one second binding agent comprising a reactive group with a polymer associated with a member of an affinity couple wherein said polymer further having a reactive group thereby forming a second binding agent-polymer complex; 
   d. contacting the matrix-binding agent-target molecule complex formed in step (b) with the second binding agent-polymer complex formed in step (c), under conditions allowing the binding of the second binding agent to the target molecule, thereby obtaining a target molecule-polymer complex;   e. contacting said target molecule-polymer complex formed in step (d) with a complementary member of said member of an affinity couple associated with a detectable moiety,   wherein the amount of said detectable moiety is indicative of the presence of said target molecule in the sample.   
     
     
         77 . The method of  claim 76  wherein the second binding agent-polymer complex formed in step c is
 a second binding agent-nucleic acid complex, and wherein the polymer of step c (iii) or c (v) is
 a nucleic acid having an active group and further comprising a polymerase promoter sequence thereby forming a second binding agent-nucleic acid complex; and wherein the method further comprises 
 
 a. adding a DNA or RNA polymerase enzyme and nucleotides associated with a member of an affinity couple under suitable conditions to affect DNA or RNA polymerization, thereby obtaining DNA or RNA molecules associated with a member of an affinity couple, and 
 b. contacting said DNA or RNA molecules associated with a member of an affinity couple with a complementary member of said member of an affinity couple associated with a detectable moiety; 
 wherein the amount of said detectable moiety is indicative of the presence of said target molecule in the sample. 
 
     
     
         78 . A method according to  claim 74 , wherein said aptamer comprises a reactive group at the 3′ and/or 5′ termini, wherein said reactive group is a member of an affinity couple selected from the group consisting of biotin/avidin, antigen/antibody, Molecular Imprinted Polymers/target ligand, protein-A/IgG, ligand/receptor, and a nucleic acid molecule/complementary sequence. 
     
     
         79 . A method according to  claim 74 , wherein said aptamer comprises reactive groups and said matrix is pre-coated with a protein, and wherein said aptamer is bound to the matrix by adding a cross linking agent, thereby forming an aptamer-matrix complex. 
     
     
         80 . A method according to  claim 74 , wherein said matrix is precoated with a molecularly imprinted polymer (MIP), an antibody, or a ligand. 
     
     
         81 . A method according to  claim 80  wherein said aptamer-matrix complex is further incubated with a blocking agent, thereby blocking the remaining free active groups on the matrix and wherein said blocking agent is selected from a group consisting of DMP, citraconic anhydride, sulfo-NHS-Acetate, glutaraldehyde, photo-reactive groups, N-acetylcysteine and N,N′-Methanetetraylbis(2-propanamine) (DIPCDI) and 1-Ethyl-3-[ 3 -dimethylaminopropyl]carbodiimide hydrochloride (EDC). 
     
     
         82 . A method according to  claim 74  wherein said member of an affinity couple associated with the polymer is biotin. 
     
     
         83 . A method according to  claim 74  wherein said polymer is a nucleic acid molecule and wherein said nucleic acid molecule comprises a reactive group at its 5′ and/or 3′ termini and wherein said reactive group is reactive with primary NH 2 . 
     
     
         84 . A method according to  claim 83  wherein said nucleic acid molecule is a single stranded nucleic acid forming a complex with biotin during a branched nucleic acid process or as a pre-prepared branch unit. 
     
     
         85 . A method according to  claim 74  wherein said complementary member of said member of an affinity couple is a biotin-binding protein. 
     
     
         86 . A method according to  claim 74  wherein said detectable moiety is an enzyme capable of catalyzing a reaction producing a detectable signal and a suitable substrate for said enzyme, wherein said enzyme is Alkaline Phosphatase (AP) or Horse Radish Peroxidase (HRP). 
     
     
         87 . A kit comprising:
 (a) a first binding agent; and   (b) a second binding agent-polymer complex, wherein the polymer is associated with a member of an affinity couple and wherein said polymer is further associated with a reactive group.   
     
     
         88 . A kit according to  claim 87  wherein said first binding agent is at least one aptamer; and wherein said second binding agent-polymer complex is
 a biotinylated polymer and wherein said biotinylated polymer is further associated with a reactive group. 
 
     
     
         89 . A kit according to  claim 88  wherein said biotinylated polymer is a biotinylated nucleic acid molecule comprising a reactive group and further comprising a polymerase promoter sequence. 
     
     
         90 . A kit according to  claim 87 —wherein said reactive group is a succinimidyl ester group. 
     
     
         91 . A kit according to  claim 87 , further comprising at least one detection enzyme and optionally a substrate for said detection enzyme. 
     
     
         92 . A method for the diagnosis of a pathological condition in a subject comprising using a detection method in accordance with  claim 74 , wherein said target molecule is a target molecule associated with the pathological condition and wherein the amount of said detectable moiety is indicative of the presence of a pathological condition in the subject, and wherein said pathological condition is cancer, an autoimmune disease, or a viral, bacterial or fungal infection. 
     
     
         93 . A method for monitoring the efficiency of a therapeutic regimen in a subject suffering from a pathological condition comprising using a detection method in accordance with  claim 74 , wherein said target molecule is an antigen associated with the pathological condition and wherein the amount of said detectable moiety is indicative of the level of the pathological condition and thereby of the efficiency of the therapeutic regimen in the subject, and wherein said pathological condition is cancer, an autoimmune disease, or a viral, bacterial or fungal infection.