Antibody complexes and methods for immunolabeling

Abstract

The present invention provides labeling reagents and methods for labeling primary antibodies and for detecting a target in a sample using an immuno-labeled complex that comprises a target-binding antibody and one or more labeling reagents. The labeling reagents comprise monovalent antibody fragments or non-antibody monomeric proteins whereby the labeling proteins have affinity for a specific region of the target-binding antibody and are covalently attached to a label. Typically, the labeling reagent is an anti-Fc Fab or Fab′ fragment that was generated by immunizing a goat or rabbit with the Fc fragment of an antibody. The present invention provides for discrete subsets of labeling reagent and immuno-labeled complexes that facilitate the simultaneous detection of multiple targets in a sample wherein the immuno-labeled complexes are distinguished by i) a ratio of label to labeling reagent, or ii) a physical property of said label, or iii) a ratio of labeling reagent to said target-binding antibody, or iv) by said target-binding antibody. This is particularly useful for fluorophore labels that can be attached to labeling reagents and subsequently immuno-labeled complexes in ratios for the detection of multiple targets.

1 . A method of forming an immuno-labeled complex, wherein said method comprises the steps of: 
 b) contacting a solution of target-binding antibodies with a discreet labeling reagent subset, wherein said labeling reagent subsets are distinguished by i) ratio of label to labeling reagent or ii) a physical properties of said label;    b) incubating said target-binding antibodies and said labeling reagent subset for a time period sufficient for one or more labeling reagents to form an immuno-labeled complex with a target-binding antibody wherein a region of said target binding antibody is selectively bound by labeling reagent whereby a discreet immuno-labeled complex subset is formed;    c) optionally removing unbound labeling reagent by adding a capture reagent comprising immunoglobulin proteins or fragments thereof; and,    c) optionally repeating said steps a), b), and c) to form a panel of immuno-labeled complex subsets wherein each subset is distinguished from another subset by i) a ratio of label to labeling reagent, or ii) a physical property of said label, or iii) a ratio of labeling reagent to said target-binding antibody, or iv) by said target-binding antibody.    
     
     
         2 . The method according to  claim 1 , wherein said target binding antibody is a murine monoclonal antibody, a rabbit polyclonal antibody or a goat polyclonal antibody.  
     
     
         3 . The method according to  claim 2 , wherein said target-binding antibodies are in a solution comprising serum proteins or ascites proteins.  
     
     
         4 . The method according to  claim 1 , wherein said labeling reagent is a Fab or Fab′fragment and is selected from the group consisting of anti-Fc antibody fragment, anti-kappa light chain antibody fragment, anti-lambda light chain antibody fragment, and a single chain variable protein fragment; or is a non-antibody protein selected from the group consisting of protein G, protein A, protein L, lectin, and derivatives thereof.  
     
     
         5 . The method according to  claim 4 , wherein said label is selected from the group consisting of a chromophore, a fluorophore, a fluorescent protein, a phosphorescent dye, a tandem dye, a particle, a hapten, an enzyme and a radioisotope.  
     
     
         6 . The method according to  claim 5 , wherein said fluorophore is selected from the group consisting of a coumarin, a xanthene, a cyanine, a pyrene, a borapolyazaindacene, an oxazine, and derivatives thereof.  
     
     
         7 . The method according to  claim 5 , wherein said fluorescent protein is a phycobiliprotein.  
     
     
         8 . The method according to  claim 5 , wherein said tandem dye is selected from the group consisting of a cyanine-phycobiliprotein derivative and xanthene-phycobiliprotein derivative.  
     
     
         9 . The method according to  claim 5 , wherein said enzyme is selected from the group consisting of a peroxidase, a phosphatase, a glycosidase, and a luciferase.  
     
     
         10 . A method for detecting a target in a sample, wherein said method comprises the steps of: 
 a) contacting said sample with an immuno-labeled complex comprising a target-binding antibody and a labeling reagent;    b) incubating said sample of step a) for a time sufficient to permit said immuno-labeled complex to selectively bind to said target; and,    c) illuminating said immuno-labeled complex whereby said target is detected.    
     
     
         11 . The method according to  claim 11 , wherein said sample comprises a population of cells, cellular extract, subcellular component, proteins, peptides, tissue culture, tissue, a bodily fluid, or a portion or combination thereof.  
     
     
         12 . The method according to  claim 11 , wherein said sample is immobilized on a solid or semi-solid matrix.  
     
     
         13 . The method according to  claim 12 , wherein said matrix is a gel, a membrane, an array, a glass surface or a microparticle.  
     
     
         14 . The method according to  claim 10 , wherein said labeling reagent is a Fab or Fab′fragment and is selected from the group consisting of anti-Fc antibody fragment, anti-kappa light chain antibody fragment, anti-lambda light chain antibody fragment, and a single chain variable protein fragment; or is a non-antibody protein selected from the group consisting of protein G, protein A, protein L, lectin, and derivatives thereof.  
     
     
         15 . The method according to  claim 14 , wherein said label is selected from the group consisting of a chromophore, a fluorophore, a fluorescent protein, a phosphorescent dye, a tandem dye, a particle, a hapten, an enzyme and a radioisotope.  
     
     
         16 . The method according to  claim 15 , wherein said fluorophore is selected from the group consisting of a coumarin, a xanthene, a cyanine, a pyrene, a borapolyazaindacene, an oxazine, and derivatives thereof.  
     
     
         17 . The method according to  claim 15 , wherein said fluorescent protein is a phycobiliprotein.  
     
     
         18 . The method according to  claim 15 , wherein said tandem dye is selected from the group consisting of a cyanine-phycobiliprotein derivative and xanthene-phycobiliprotein derivative.  
     
     
         19 . The method according to  claim 15 , wherein said enzyme selected from the group consisting of a peroxidase, a phosphatase, a glycosidase, and a luciferase.  
     
     
         20 . The method according to  claim 19 , wherein said method further comprises adding a colorimetric, fluorescent or chemiluminescent enzyme substrate.  
     
     
         21 . The method according to  claim 10 , wherein said immuno-labeled complex comprises a labeling reagent that is a Fab or Fab′ anti-Fc fragment wherein said fragment is independently covalently bonded to one or more labels that are selected from the group consisting of a coumarin, a xanthene, a cyanine, a pyrene, a phycobiliprotein, a borapolyazaindacene, a peroxidase, a phosphatase, a tandem dye and derivatives thereof.  
     
     
         22 . A method for detecting multiple targets in a sample, said method comprising: 
 a) contacting said sample with a solution comprising A) a pooled subset of immuno-labeled complexes wherein each said complex comprises a target-binding antibody and a labeling reagent, wherein each said subset is distinguished from another subset by i) a ratio of label to labeling reagent, or ii) a physical property of said label, or iii) a ratio of labeling reagent to said target-binding antibody, or iv) by said target-binding antibody or B) an individual immuno-labeled complex subset wherein multiple individual subsets are added to said sample;    b) incubating said sample for a time sufficient to allow said immuno-labeled complex to selectively bind to said target; and,    c) illuminating said immuno-labeled complex whereby said multiple targets are detected.    
     
     
         23 . The method according to  claim 22 , wherein said sample comprises a population of cells, cellular extract, subcellular component, tissue culture, tissue, a bodily fluid, or a portion or combination thereof.  
     
     
         24 . The method according to  claim 23 , wherein said sample is immobilized on a solid or semi-solid matrix.  
     
     
         25 . The method according to  claim 24 , wherein said matrix is a gel, a membrane, an array, a glass surface or a microparticle.  
     
     
         26 . The method according to  claim 23 , wherein said labeling reagent is a Fab or Fab′fragment and is selected from the group consisting of anti-Fc antibody fragment, anti-kappa light chain antibody fragment, anti-lambda light chain antibody fragment, and a single chain variable protein fragment; or is a non-antibody protein selected from the group consisting of protein G, protein A, protein L, lectin, and derivatives thereof.  
     
     
         27 . The method according to  claim 26 , wherein said label is selected from the group consisting of a chromophore, a fluorophore, a fluorescent protein, a phosphorescent dye, a tandem dye, a particle, an electron transfer agent, a hapten, an enzyme and a radioisotope.  
     
     
         28 . The method according to  claim 27 , wherein said fluorophore is selected from the group consisting of a coumarin, a xanthene, a cyanine, a pyrene, a borapolyazaindacene, an oxazine and derivatives thereof.  
     
     
         29 . The method according to  claim 27 , wherein said fluorescent protein is a phycobiliprotein.  
     
     
         30 . The method according to  claim 27 , wherein said tandem dye is selected from the group consisting of a cyanine-phycobiliprotein derivative and xanthene-phycobiliprotein derivative.  
     
     
         31 . The method according to  claim 27 , wherein said enzyme is selected from the group consisting of a peroxidase, a phosphatase, a glycosidase, and a luciferase.  
     
     
         32 . The method according to  claim 31 , wherein said method further comprises adding a calorimetric, fluorescent or chemiluminescent enzyme substrate.  
     
     
         33 . A method for identifying and quantitating multiple targets in a sample, wherein said method comprises the steps of: 
 a) contacting a population of cells or fragments thereof in a sample with A) a solution comprising a pooled subset of immuno-labeled complexes wherein said complex comprises a target-binding antibody and a labeling reagent, wherein each subset is. distinguished from another subset i) a ratio of label to labeling reagent, or ii) a physical property of said label, or iii) a ratio of labeling reagent to said target-binding antibody, or iv) by said target-binding antibody or B) an individual subset of immuno-labeled complexes wherein multiple individual subsets are added to said sample;    b) incubating said cells or fragments thereof for a time period sufficient to allow said immuno-labeled complex to bind said targets;    c) passing said incubated population of cells or fragments thereof through an examination zone; and,    d) collecting data from said cells or fragments thereof passed through said examination zone whereby said identification and quantitation of multiple targets is determined.    
     
     
         34 . The method according to  claim 33 , wherein said labeling reagent is a Fab or Fab′fragment and is selected from the group consisting of anti-Fc antibody fragment, anti-kappa light chain antibody fragment, anti-lambda light chain antibody fragment, and a single chain variable protein fragment; or is a non-antibody protein selected from the group consisting of protein G, protein A, protein L, lectin, and derivatives thereof.  
     
     
         35 . The method according to  claim 34 , wherein said label is selected from the group consisting of a chromophore, a fluorophore, a fluorescent protein, a phosphorescent dye, a tandem dye, a particle, a radioisotope, and a hapten.  
     
     
         36 . The method according to  claim 35 , wherein said fluorophore is selected from the group consisting of a coumarin, a xanthene, a cyanine, a pyrene, a borapolyazaindacene, an oxazine and derivatives thereof.  
     
     
         37 . The method according to  claim 35 , wherein said fluorescent protein is a phycobiliprotein.  
     
     
         38 . The method according to  claim 35 , wherein said tandem dye is selected from the group consisting of cyanine-phycobiliprotein and xanthene-phycobiliprotein.  
     
     
         39 . A method of manufacturing an isolated labeling reagent, wherein said method comprises the steps of: 
 a) cleaving an intact anti-region antibody with an enzyme to generate Fab fragments;    b) isolating said anti-region Fab fragments of step a);    c) contacting a matrix comprising intact immunoglobulin proteins or fragments that selectively bind anti-region Fab fragments with a solution comprising said anti-region fragments of step b) wherein said anti-region Fab fragments are immobilized on said matrix;    d) contacting said matrix of step c) with a solution comprising a fluorophore label that contains a reactive group;    e) washing said matrix of step d) to remove unbound label, and;    f) eluting said labeling reagent from said matrix whereby said isolated labeling reagent is manufactured.    
     
     
         40 . The method according to  claim 39 , wherein said anti-region Fab fragment are selected from the group consisting of anti-Fc antibody fragment, anti-kappa light chain antibody fragment, anti-lambda light chain antibody fragment, and a single chain variable protein fragment.  
     
     
         41 . The method according to  claim 40 , wherein said fluorophore is selected from the group consisting of a coumarin, a xanthene, a cyanine, a pyrene, a borapolyazaindacene, an oxazine and derivatives thereof.  
     
     
         42 . A method of manufacturing an isolated labeling reagent, wherein said method comprises the steps of: 
 a) contacting a matrix comprising intact immunoglobulin proteins or fragments thereof that selectively bind non-antibody proteins with a solution comprising said non-antibody proteins wherein said non-antibody proteins are immobilized on said matrix;    b) contacting said matrix of step a) with a solution comprising a fluorophore label that contains a reactive group;    c) washing said matrix to remove unbound label, and;    d) eluting said labeling reagent from said matrix whereby said isolated labeling reagent is manufactured that comprises a fluorophore label.    
     
     
         43 . The method according to  claim 42 , wherein said non-antibody protein is selected from the group consisting of protein G, protein A, protein L, lectin, and derivatives thereof.  
     
     
         44 . The method according to  claim 43 , wherein said fluorophore is selected from the group consisting of a coumarin, a xanthene, a cyanine, a pyrene, a borapolyazaindacene, an oxazine and derivatives thereof.  
     
     
         45 . A method of manufacturing an isolated labeling reagent, wherein said method comprises the steps of: 
 a) cleaving an intact anti-region antibody with an enzyme resulting in Fab or Fab′fragments;    a) contacting said anti-region Fab or Fab′ fragments of step a) with a solution comprising a label that contains a reactive group; and,    c) isolating labeled anti-region Fab or Fab′ fragments by size exclusion or affinity chromatography whereby an isolated labeling reagent is manufactured.    
     
     
         46 . The method according to  claim 45 , wherein said anti-region Fab or Fab′ fragment is selected from the group consisting of anti-Fc antibody fragment, anti-kappa light chain antibody fragment, anti-lambda light chain antibody fragment, and a single chain variable protein fragment.  
     
     
         47 . The method according to  claim 46 , wherein said label is a fluorescent protein or a tandem dye.  
     
     
         48 . The method according to  claim 47 , wherein said fluorescent protein is a phycobiliprotein.  
     
     
         49 . The method according to  claim 47 , wherein said tandem dye is selected from the group consisting of cyanine-phycobiliprotein and xanthene-phycobiliprotein.  
     
     
         50 . An isolated labeling reagent made by a process comprising: 
 a) cleaving an intact anti-region antibody with an enzyme resulting in Fab or Fab′fragments;    b) contacting said Fab or Fab′ fragments of step a) with a solution comprising a label that contains a reactive group; and,    c) isolating labeled anti-region Fab or Fab′ fragments by size exclusion or affinity chromatography.    
     
     
         51 . The labeling reagent according to  claim 50 , wherein said anti-region Fab or Fab′fragment is selected from the group consisting of anti-Fc antibody fragment, anti-kappa light chain antibody fragment, anti-lambda light chain antibody fragment, and a single chain variable protein fragment.  
     
     
         52 . The labeling reagent according to  claim 51 , wherein said label is selected from the group consisting of a chromophore, a fluorophore, a fluorescent protein, a phosphorescent dye, a tandem dye, a particle, a radioisotope, and a hapten.  
     
     
         53 . The labeling reagent according to  claim 52 , wherein said fluorophore is selected from the group consisting of a coumarin, a xanthene, a cyanine, a pyrene, a borapolyazaindacene, an oxazine and derivatives thereof.  
     
     
         54 . An isolated labeling reagent made by a process comprising: 
 a) cleaving an intact anti-region antibody with an enzyme to generate F(ab′) 2  fragments;    b) contacting said anti-region F(ab′) 2  with a reducing agent to produce anti-region Fab′ fragments containing a thiol group;    c) contacting said Fab′ fragments with a solution comprising a label that contains a reactive group; and,    d) isolating said Fab′ fragments of step d) that are covalently attached to a label by size exclusion or affinity chromatography.    
     
     
         55 . The labeling reagent according to  claim 54 , wherein said anti-region Fab′ fragment is selected from the group consisting of anti-Fc antibody fragment, anti-kappa light chain antibody fragment, anti-lambda light chain antibody fragment, and a single chain variable protein fragment.  
     
     
         56 . The method according to  claim 55 , wherein said label is selected from the group consisting of a chromophore, a fluorophore, a fluorescent protein, a phosphorescent dye, a tandem dye, a particle, a radioisotope, and a hapten.  
     
     
         57 . An isolated labeling reagent made by a process comprising: 
 a) cleaving an intact anti-region antibody with an enzyme to generate Fab fragments;    b) isolating said anti-region Fab fragments of step a);    c) contacting a matrix comprising intact immunoglobulin proteins or fragments thereof that specifically bind anti-region Fab fragments with a solution comprising said anti-region Fab fragments of step b) wherein said anti-region Fab fragments are immobilized;    d) contacting said matrix of step c) with a solution comprising a fluorophore label that contains a reactive group;    e) washing said matrix to remove unbound label, and;    f) eluting said labeling reagent from said matrix whereby said labeling reagent is manufactured comprising a label and being isolated from other proteins and fragments thereof.    
     
     
         58 . The labeling reagent according to  claim 57 , wherein said anti-region Fab fragment is selected from the group consisting of anti-Fc antibody fragment, anti-kappa light chain antibody fragment, anti-lambda light chain antibody fragment, and a single chain variable protein fragment.  
     
     
         59 . The labeling reagent according to  claim 58 , wherein said fluorophore is selected from the group consisting of a coumarin, a xanthene, a cyanine, a pyrene, a borapolyazaindacene, an oxazine and derivatives thereof.  
     
     
         60 . An isolated labeling reagent made by a process comprising: 
 a) contacting a matrix comprising intact immunoglobulin proteins or fragments that selectively bind non-antibody proteins with a solution comprising said non-antibody proteins wherein said non-antibody proteins are immobilized;    b) contacting said matrix of step c) with a solution comprising a fluorophore label that contains a reactive group;    c) washing said matrix to remove unbound label, and;    d) eluting said labeling reagent from said matrix whereby said labeling reagent is manufactured comprising a label.    
     
     
         61 . The labeling reagent according to  claim 60 , wherein said non-antibody protein is selected from the group consisting of protein G, protein A, protein L, lectin, and derivatives thereof.  
     
     
         62 . The labeling reagent according to  claim 61 , wherein said fluorophore is selected from the group consisting of a coumarin, a xanthene, a cyanine, a pyrene, a borapolyazaindacene, an oxazine and derivatives thereof.  
     
     
         63 . An immuno-labeled complex generated by a process comprising: 
 a) contacting a solution of target-binding antibodies with a labeling reagent subset, wherein said labeling reagent subset is distinguished from another labeling reagent subset by i) ratio of label to labeling reagent or ii) a physical properties of said label;    b) incubating said target-binding antibodies and said labeling reagent subset for a time period sufficient for one or more labeling reagents to form an immuno-labeled complex with a target-binding antibody wherein a region of said target binding antibody is selectively bound by labeling reagent; and,    c) optionally removing unbound labeling reagent by adding a capture reagent comprising immunoglobulin proteins or fragments thereof whereby an immuno-labeled complex is produced.    
     
     
         64 . The immuno-labeled complex according to  claim 63 , wherein said process further comprises repeating said steps a), b), and c) to form subsets of immuno-labeled complexes wherein each subset is distinguished from another subset by i) ratio of label to labeling reagent or ii) a physical properties of said label, or iii) a ratio of labeling reagent to said target-binding antibody or iv) by said target-binding antibody.  
     
     
         65 . The immuno-labeled complex according to  claim 63 , wherein said target-binding antibody is a murine monoclonal antibody, a rabbit polyclonal antibody or a goat polyclonal antibody.  
     
     
         66 . The immuno-labeled complex according to  claim 65 , wherein said labeling reagent is a Fab or Fab′ fragment and is selected from the group consisting of anti-Fc antibody fragment, anti-kappa light chain antibody fragment, anti-lambda light chain antibody fragment, and a single chain variable protein fragment; or is a non-antibody protein selected from the group consisting of protein G, protein A, protein L, lectin, and derivatives thereof.  
     
     
         67 . The immuno-labeled complex according to  claim 66 , wherein said label is selected from the group consisting of a chromophore, a fluorophore, a fluorescent protein, a phosphorescent dye, a tandem dye, a particle, a hapten, an enzyme and a radioisotope.  
     
     
         68 . The immuno-labeled complex according to  claim 67 , wherein said fluorophore is selected from the group consisting of a coumarin, a xanthene, a cyanine, a pyrene, a borapolyazaindacene, an oxazine, and derivatives thereof.  
     
     
         69 . A labeling solution comprising a buffer and either an individual labeling reagent subset or a pooled subset of labeling reagents, wherein each subset is distinguished from another subset by i) a ratio of label to labeling reagent, or ii) by a physical property of said label.  
     
     
         70 . The solution according to  claim 69 , wherein said labeling reagent is a Fab or Fab′fragment and is selected from the group consisting of anti-Fc antibody fragment, anti-kappa light chain antibody fragment, anti-lambda light chain antibody fragment, and a single chain variable protein fragment; or is non-antibody protein is selected from the group consisting of protein G, protein A, protein L, lectin, and derivatives thereof.  
     
     
         71 . The solution according to  claim 70 , wherein said label is selected from the group consisting of a chromophore, a fluorophore, a fluorescent protein, a phosphorescent dye, a tandem dye, a particle, an enzyme, a hapten and a radioisotope.  
     
     
         72 . The solution according to  claim 71 , wherein said fluorophore is selected from the group consisting of a coumarin, a xanthene, a cyanine, a pyrene, a borapolyazaindacene, an oxazine, and derivatives thereof.  
     
     
         73 . A kit for preparing an immuno-labeling complex, said kit comprising: 
 a) a labeling solution comprising a labeling reagent that is independently covalently bonded to one or more labels; or    b) a panel of labeling solutions wherein each labeling solution comprises a subset of a labeling reagent and each subset is distinguished from another subset by i) a ratio of label to labeling reagent, or ii) by a physical property of said label; and,    c) a solution comprising a capture reagent.    
     
     
         74 . The kit according to  claim 73 , wherein said labeling reagent is a Fab or Fab′fragment and is selected from the group consisting of anti-Fc antibody fragment, anti-kappa light chain antibody fragment, anti-lambda light chain antibody fragment, and a single chain variable protein fragment; or is a non-antibody protein selected from the group consisting of protein G, protein A, protein L, lectin, and derivatives thereof.  
     
     
         75 . The kit according to  claim 74 , wherein said label is selected from the group consisting of a chromophore, a fluorophore, a fluorescent protein, a phosphorescent dye, a tandem dye, a particle, an enzyme, a hapten and a radioisotope.  
     
     
         76 . The kit according to  claim 75 , wherein said fluorophore is selected from the group consisting of a coumarin, a xanthene, a cyanine, a pyrene, a borapolyazaindacene, an oxazine and derivatives thereof.  
     
     
         77 . The kit according to  claim 76 , wherein the kit further comprises an addition component selected from the group consisting of (a) stains for characterization of cellular organelles, cell viability, or cell proliferation state, (b) enzyme substrates and (c) enzyme conjugates.