Antibody-linked immuno-sedimentation agent and method of isolating a target from a sample using same

Abstract

The present disclosure is directed to antibody-linked immuno-sedimentation agent, the antibody being linked to a sedimentation agent by a non-antigen binding region of the antibody, and a method of isolating a target from a sample using the antibody-linked immuno-sedimentation agent. The methods involve forming a mixture including a sample with an antibody linked immuno-sedimentation agent and red blood cells under conditions sufficient to form red blood cell rouleaux and allow antibody-antigen binding.

1 . A method of isolating a target from a sample, the method comprising:
 a) forming a mixture comprising a sample, an antibody-linked immuno-sedimentation agent and red blood cells, wherein the antibody-linked immuno-sedimentation agent comprises
 1) at least one sedimentation agent and 
 2) at least one antibody linked to the at least one sedimentation agent by a non-antigen binding region of the antibody; 
   b) incubating the mixture under conditions sufficient to form a rouleaux and allow the antibody to bind to an antigen present in the mixture; and   c) recovering a target from the mixture.   
     
     
         2 . The method of  claim 1 , wherein the rouleaux is formed by adsorption of the at least one sedimentation agent onto the red blood cells of the rouleaux. 
     
     
         3 . The method of  claim 1 , wherein the at least one antibody is linked to the at least one sedimentation agent prior to forming the mixture. 
     
     
         4 . The method of  claim 1 , wherein the antibody binds a target antigen or a non-target antigen in the sample. 
     
     
         5 . The method of  claim 1 , further comprising incubating the mixture under conditions sufficient to form a rouleaux network. 
     
     
         6 . The method of  claim 1 , wherein the sample is selected from the group consisting of whole blood, blood plasma, blood stem cell isolates, platelet-rich plasma, fractionated blood, packed red blood cells, umbilical cord blood, bone marrow, bone-marrow aspirates, a buffy-coat layer, a leukapheresis isolate, a plasmapheresis isolate, a cell suspension, a cell culture medium, a cell mixture, a cell homogenate, a pancreas isolate, an organ homogenate, a saline solution, a Ringer's solution, a lactated Ringer's solution, a phosphate buffered saline solution, a viral culture medium, an oocyte mixture, a sperm, a gamete mixture, and combinations thereof. 
     
     
         7 . The method of  claim 1 , wherein the mixture comprises red blood cells selected from the group consisting of endogenous red blood cells, exogenous red blood cells, and combinations thereof. 
     
     
         8 . The method of  claim 7 , wherein the exogenous red blood cells are selected from the group consisting of homologous red blood cells, autologous red blood cells, heterologous red blood cells, and combinations thereof. 
     
     
         9 . The method of  claim 1 , wherein the target comprises a multicellular organism, a single-cell organism, a cell, a subcellular component, a cellular fragment, a virus, a viroid, a virus-like target, a membrane-bound particle, a lipid particle, a macromolecule, a DNA, a RNA, a nucleic acid-like macromolecule, a peptide, a polypeptide, and a protein. 
     
     
         10 . The method of  claim 1 , wherein the at least one sedimentation agent is selected from the group consisting of a soluble sedimentation agent, a partially soluble sedimentation agent, and combinations thereof. 
     
     
         11 . The method of  claim 1 , wherein the at least one sedimentation agent comprises an insoluble sedimentation agent. 
     
     
         12 . The method of  claim 1 , wherein the at least one sedimentation agent comprises a polymer. 
     
     
         13 . The method of  claim 12 , wherein the polymer is selected from the group consisting of a biopolymer, a synthetic polymer, a modified biopolymer, and combinations thereof. 
     
     
         14 . The method of  claim 13 , wherein the biopolymer is selected from the group consisting of a polysaccharide, a polypeptide, and combinations thereof. 
     
     
         15 . The method of  claim 14 , wherein the polysaccharide is selected from the group consisting of a starch, dextran, cellulose, chitin, xanthum gum, a glycosaminoglycan, and combinations thereof. 
     
     
         16 . The method of  claim 15 , wherein the glycosaminoglycan is selected from the group consisting of heparin, heparin sulfate, chondroitin sulfate, and combinations thereof. 
     
     
         17 . The method of  claim 14 , wherein the polypeptide is selected from the group consisting of albumin, collagen, fibrinogen, immunoglobulin, and combinations thereof. 
     
     
         18 . The method of  claim 13 , wherein the synthetic polymer is selected from the group consisting of polyoxyethylene, polyoxyethylene glycol, polyoxyethylene oxide, polyvinylpyrrolidone, polylactic acid, polyglycolic acid, polydioxanone, and combinations thereof. 
     
     
         19 . The method of  claim 13 , wherein the modified biopolymer is selected from the group consisting of hydrolyzed collagen-gelatin, FICOLL, ethylene oxide modified starch, and combinations thereof. 
     
     
         20 . The method claim of  claim 13 , wherein the modified biopolymer comprises an iodinated contrast media, wherein the iodinated contrast media is selected from the group consisting of diatrizoate, iopamidol, iohexol, ioxilan, iopromide, iodixanol, and ioxaglate. 
     
     
         21 . The method of  claim 19 , wherein the ethylene oxide modified starch is selected from the group consisting of hydroxyethyl starch, pentastarch, and combinations thereof. 
     
     
         22 . The method of  claim 1 , wherein the antibody is selected from the group consisting of a monoclonal antibody, a polyclonal antibody, and combinations thereof. 
     
     
         23 . The method of  claim 1 , wherein the antibody comprises at least one of an antibody fragment, a chimeric antibody, a bifunctional antibody, and a bispecific antibody. 
     
     
         24 . The method of  claim 23 , wherein the antibody fragment comprises an antigen-binding domain, an Fab, an F(ab′) 2 , an F(ab′). 
     
     
         25 . The method of  claim 1 , wherein the sample comprises blood. 
     
     
         26 . The method of  claim 25 , wherein the blood comprises whole blood, blood plasma, a blood stem cell isolate, platelet-rich plasma, fractionated blood, packed red blood cells, umbilical cord blood, bone marrow, a bone-marrow aspirate, and a buffy-coat layer. 
     
     
         27 . The method of  claim 1 , wherein the incubating step is from about 10 minutes to about 120 minutes. 
     
     
         28 . The method of  claim 1 , wherein the method is performed at a temperature between about 0° C. to about 45° C. 
     
     
         29 . The method of  claim 1 , wherein the method is performed at a pH of between about 5.4 to about 9.4. 
     
     
         30 . The method of  claim 1 , wherein recovering a target from the mixture comprises separating the rouleaux from the mixture. 
     
     
         31 . The method of  claim 5 , wherein recovering a target from the mixture comprises separating the rouleaux network from the mixture. 
     
     
         32 . The method of  claim 1 , wherein recovering a target from the mixture comprises subjecting the mixture to at least one of sedimentation, centrifugation, density gradient centrifugation, a magnetic field, an electrical field, cell lysis, chemical crosslinking, extraction, a phase transition, freeze-thaw cycling, evaporation, boiling, subliming, melting, crystallizing, plasticizing, filtration, and combinations thereof. 
     
     
         33 . The method of  claim 1 , wherein the target is isolated by positive selection or negative selection. 
     
     
         34 . The method of  claim 1 , wherein the target comprises at least one of a multicellular organism, a single-cell organism, a cell, a subcellular component, a cellular fragment, a virus, a virus-like target, a membrane-bound particle, a lipid particle, a macromolecule, a DNA, a RNA, and a protein. 
     
     
         35 . The method of  claim 1 , wherein the antibody-linked immuno-sedimentation agent comprises antibodies directed to at least two different antigens and wherein the antibodies are linked to the sedimentation agent by a non-antigen binding region. 
     
     
         36 . The method of  claim 1 , wherein the antibody-linked immuno-sedimentation agent comprises at least two different sedimentation agents. 
     
     
         37 . The method of  claim 1 , further comprising adding a blocking agent to the mixture. 
     
     
         38 . The method of  claim 37 , wherein the blocking agent is selected from the group consisting of human albumin, bovine serum albumin, fetal bovine serum, immunoglobulins, anti-CD16 antibodies, anti-Human CD32 (FcγRII), Fc receptor antibodies, and combinations thereof. 
     
     
         39 . A method of isolating a target from a sample, the method comprising:
 a) forming a mixture comprising a sample, an antibody-linked immuno-sedimentation agent and red blood cells, wherein the antibody-linked immuno-sedimentation agent comprises
 1) at least one sedimentation agent selected from the group consisting of polyethylene glycol, dextran, and hydroxyethyl starch; and 
 2) at least one antibody linked to the at least one sedimentation agent by a non-antigen binding region of the antibody, wherein the antibody is selected from the group consisting of an anti-CD3 antibody, an anti-CD19 antibody, an anti-insulin antibody, an anti-CD34 antibody, an anti-CD4 antibody, an anti-CD14 antibody, an anti-CD16 antibody, an anti-CD19 antibody, an anti-CD20 antibody, an anti-CD36 antibody, an anti-CD56 antibody, an anti-CD123 antibody, an anti-TCRγ/δ antibody, an anti-glycophorin A antibody, and an anti-GP120 antibody; 
   b) incubating the mixture under conditions sufficient to form a rouleaux and allow the antibody to bind to an antigen present in the mixture; and   c) recovering a target from the mixture.   
     
     
         40 . An antibody-linked immuno-sedimentation agent comprising at least one sedimentation agent and at least one antibody linked to the at least one sedimentation agent, wherein the at least one antibody is linked to the at least one sedimentation agent by a non-antigen binding region. 
     
     
         41 . The antibody-linked immuno-sedimentation agent of  claim 40 , wherein the at least one antibody linked to the at least one sedimentation agent is selected from the group consisting of a monoclonal antibody, a polyclonal antibody, and combinations thereof. 
     
     
         42 . The antibody-linked immuno-sedimentation agent of  claim 40 , wherein the at least one antibody comprises at least one of a chimeric antibody, a bifunctional antibody, a bispecific antibody, an IgA, an IgD, an IgE, an IgG, an IgM, an intact antibody, and an antibody fragment. 
     
     
         43 . The antibody-linked immuno-sedimentation agent of  claim 42 , wherein the antibody fragment comprises at least one of an antigen binding region, an antibody hypervariable region, an antibody paratope, an antibody light chain, an antibody heavy chain, a paired antibody heavy and light chain, an Fab, an F(ab′) 2 , and an F(ab′). 
     
     
         44 . The antibody-linked immuno-sedimentation agent of  claim 40 , wherein the at least one antibody linked to the at least one sedimentation agent is linked to the at least one sedimentation agent by an Fc region, a hinge region, a light chain constant (C L ) region, a heavy chain (C H   1 ) region, a light chain (C L ) region and heavy chain (C H   1 ) region, a heavy chain (C H   2 ) region, and a heavy chain (C H   3 ) region. 
     
     
         45 . The antibody-linked immuno-sedimentation agent of  claim 40 , wherein the at least one antibody linked to the at least one sedimentation agent is directly linked to the at least one sedimentation agent. 
     
     
         46 . The antibody-linked immuno-sedimentation agent of  claim 40 , wherein the at least one antibody linked to the at least one sedimentation agent is indirectly linked to the at least one sedimentation agent. 
     
     
         47 . The antibody-linked immuno-sedimentation agent of  claim 40 , wherein the at least one sedimentation agent is selected from the group consisting of a soluble sedimentation agent, a partially soluble sedimentation agent, and combinations thereof. 
     
     
         48 . The antibody-linked immuno-sedimentation agent of  claim 40 , wherein the at least one sedimentation agent comprises an insoluble sedimentation agent. 
     
     
         49 . The antibody-linked immuno-sedimentation agent of  claim 40 , wherein the at least one sedimentation agent comprises a polymer. 
     
     
         50 . The antibody-linked immuno-sedimentation agent of  claim 49 , wherein the polymer is selected from the group consisting of a biopolymer, a synthetic polymer, a modified biopolymer, and combinations thereof. 
     
     
         51 . The antibody-linked immuno-sedimentation agent of  claim 50 , wherein the biopolymer comprises at least one of a polysaccharide and a polypeptide. 
     
     
         52 . The antibody-linked immuno-sedimentation agent of  claim 50 , wherein the biopolymer comprises a polysaccharide. 
     
     
         53 . The antibody-linked immuno-sedimentation agent of  claim 51 , wherein the polysaccharide comprises at least one of a starch, dextran, cellulose, chitin, xanthum gum, and a glycosaminoglycan. 
     
     
         54 . The antibody-linked immuno-sedimentation agent of  claim 53 , wherein the glycosaminoglycan comprises at least one of heparin, heparin sulfate, and chondroitin sulfate. 
     
     
         55 . The antibody-linked immuno-sedimentation agent of  claim 51 , wherein the polypeptide comprises at least one of albumin, collagen, fibrinogen, and immunoglobulin. 
     
     
         56 . The antibody-linked immuno-sedimentation agent of  claim 50 , wherein the polymer comprises a synthetic polymer. 
     
     
         57 . The antibody-linked immuno-sedimentation agent of  claim 50 , wherein the synthetic polymer comprises at least one of polyoxyethylene, polyoxyethylene glycol, polyoxyethylene oxide, polyvinylpyrrolidone, polylactic acid, polyglycolic acid, and polydioxanone. 
     
     
         58 . The antibody-linked immuno-sedimentation agent of  claim 50 , wherein the modified biopolymer comprises at least one of hydrolyzed collagen-gelatin, FICOLL, and ethylene oxide modified starch. 
     
     
         59 . The antibody-linked immuno-sedimentation agent of  claim 50 , wherein the modified biopolymer comprises an iodinated contrast media, wherein the iodated contrast media is selected from the group consisting of diatrizoate, iopamidol, iohexol, ioxilan, iopromide, iodixanol, and ioxaglate. 
     
     
         60 . The antibody-linked immuno-sedimentation agent of  claim 58 , wherein the ethylene oxide modified starch comprises at least one of hydroxyethyl starch and pentastarch. 
     
     
         61 . The antibody-linked immuno-sedimentation agent of  claim 40 , comprising antibodies directed to at least two different antigens and wherein the at least two different antibodies are linked to the sedimentation agent by a non-antigen binding region. 
     
     
         62 . The antibody-linked immuno-sedimentation agent of  claim 61 , wherein the antibodies directed to at least two different antigens are linked to at least two different sedimentation agents by a non-antigen binding region. 
     
     
         63 . The antibody-linked immuno-sedimentation agent of  claim 40 , comprising at least two different sedimentation agents. 
     
     
         64 . An antibody-linked immuno-sedimentation agent comprising at least one sedimentation agent selected from the group consisting of polyethylene glycol, dextran, and hydroxyethyl starch and at least one antibody linked to the at least one sedimentation agent, wherein the at least one antibody is linked to the at least one sedimentation agent by a non-antigen binding region and wherein the at least one antibody is selected from the group consisting of an anti-CD3 antibody, an anti-CD19 antibody, an anti-insulin antibody, an anti-CD34 antibody, an anti-CD4 antibody, an anti-CD14 antibody, an anti-CD16 antibody, an anti-CD19 antibody, an anti-CD20 antibody, an anti-CD36 antibody, an anti-CD56 antibody, an anti-CD123 antibody, an anti-TCRγ/δ antibody, an anti-glycophorin A antibody, and an anti-GP120 antibody.