Method for the formation and use of an immunolabeling complex

Abstract

The present invention generally relates to method for forming an immunolabeling complex, the immunolabeling complex comprising a labeled monovalent biotin-binding composition. The invention also related to the use of the antibody-reporter molecule complex for detecting a target in a sample.

1 . A method for forming an immunolabeling complex, wherein the method comprises:
 selecting a first biotinylated primary antibody;   selecting a first monovalent biotin-binding composition labeled with a first reporter element, and   mixing, in a reaction vessel, the first biotinylated primary antibody with the first labeled monovalent biotin-binding composition.   
     
     
         2 . The method according to  claim 1 , wherein mixing the first biotinylated primary antibody with the first labeled monovalent biotin-binding composition attaches the first reporter element to the first biotinylated primary antibody. 
     
     
         3 . The method according to  claim 1 , wherein the immunolabeling complex is to be subsequently used in contacting with a sample. 
     
     
         4 . The method according to  claim 1 , further comprising:
 contacting the immunolabeling complex with the sample.   
     
     
         5 . The method according to  claim 1 , wherein the sample is different from the first biotinylated primary antibody. 
     
     
         6 . The method according to  claim 1 , wherein the immunolabeling complex is to be subsequently used in contacting with a sample, the sample being different from the first biotinylated primary antibody. 
     
     
         7 . The method according to  claim 1 , further comprising:
 providing an instruction to use the immunolabeling complex for immunolabeling of a sample.   
     
     
         8 . The method according to  claim 7 , wherein selecting the first biotinylated primary antibody and selecting the first monovalent biotin-binding composition is performed separated from mixing the first biotinylated primary antibody with the first labeled monovalent biotin-binding composition. 
     
     
         9 . The method according to  claim 1 , wherein first labeled monovalent biotin binding composition is based on at least one of streptavidin, avidin, traptavidin, captavidin, tamavidin, bradavidin, neutravidin, and rhizavidin. 
     
     
         10 . The method according to  claim 1 , wherein the method further comprises adding a blocking agent to block unbound portions of the monovalent biotin-binding composition after the first immunolabeling complex has been formed. 
     
     
         11 . The method according to  claim 1 , wherein the reporter element is selected from a group comprising a fluorochrome, an enzyme, a peptide, quantum dots, and a transition metal. 
     
     
         12 . The method according to  claim 11 , wherein the first monovalent biotin-binding composition is conjugated with the reporter element. 
     
     
         13 . The method according to  claim 1 , wherein the ratio between the first monovalent biotin-binding composition and the first biotinylated primary antibody is selected such that the risk of the first monovalent biotin-binding composition blocking active biding sites of the first biotinylated primary antibody is reduced. 
     
     
         14 . The method according to  claim 1 , wherein the reporter element is an oligonucleotide. 
     
     
         15 . The method according to  claim 1 , wherein the reporter element is an antigen. 
     
     
         16 . The method according to  claim 1 , wherein the monovalent biotin-binding composition is modified to increase the number of conjugation sites, allowing attachment of an increased number of reporter elements. 
     
     
         17 . The method according to  claim 1 , wherein the monovalent biotin-binding composition is conjugated with macromolecules containing a plurality of molecules of a reporter element. 
     
     
         18 . A method for detecting a target molecule in a sample, wherein the method comprises:
 contacting a first immunolabeling complex formed according to any one of the preceding claims with the sample;   incubating the sample for a time sufficient to permit the first immunolabeling complex to selectively bind to the target; and   detecting the target.   
     
     
         19 . The method according to  claim 18 , wherein the first reporter element is a fluorochrome and detecting the target comprises illuminating the immunolabeling complex. 
     
     
         20 . The method according to  claim 18 , further comprising:
 contacting a second immunolabeling complex formed according to  claim 1  with the sample, and   incubating the sample for a time sufficient to permit the second immunolabeling complex to selectively bind to the target,   wherein the second immunolabeling complex comprising a second biotinylated primary antibody mixed with a second monovalent biotin binding composition, the second biotinylated primary antibody being different from the first biotinylated primary antibody, and the second monovalent biotin binding composition being labeled with a second reporter molecule being different from the first reporter molecule.   
     
     
         21 . A kit of immunolabeling complexes, comprising:
 a first immunolabeling complex formed according to  claim 1 ; and   a second immunolabeling complex formed according to  claim 1 ,   wherein the second immunolabeling complex comprising a second biotinylated primary antibody mixed with a second monovalent biotin-binding composition, the second biotinylated primary antibody being different from the first biotinylated primary antibody, and the second monovalent biotin-binding composition being labeled with a second reporter molecule being different from the first reporter molecule.   
     
     
         22 . A kit of an immunolabeling complex and an enhancer step, comprising:
 an immunolabeling complex formed according to  claim 1 ; and   at least one first enhancer component.   wherein the monovalent biotin-binding composition comprises a first set of at least one of antigens and oligonucleotides that are recognized by the first enhancer component, and   the first enhancer component is conjugated with reporter elements, or   the first enhancer component is conjugated with a second set of at least one of antigens and oligonucleotide that are recognized by a second enhancer component that is conjugated with reporter elements, wherein the second set is different from the first set.