Immunogenicity Assay

Abstract

Assays for detecting antibodies to pharmaceutical preparations, food allergens and environmental allergens are described.

1 . An immunoassay method for detecting the presence of an antibody against a drug of interest (DOI), a food antigen (FA) or an environmental antigen (EA) comprising:
 contacting at least one immunochromatographic solid phase with a sample on a proximal region thereof, wherein said sample comprises at least one antibody in a mobile phase;   allowing a mobilized antibody to flow from said proximal region to a distal region of said solid phase, wherein said distal region comprises an immobilized capture agent, and wherein said capture agent immobilizes an antibody complex, wherein said antibody complex comprises a first DOI, FA or EA moiety bound to a first antigen binding region of said antibody and said first DOI, FA or EA moiety comprises a reporter moiety; and   detecting said reporter binding to said immobilized complex, wherein said detecting correlates with the presence of an antibody against said DOI, FA or EA.   
     
     
         2 . The method of  claim 1 , wherein said first DOI, FA or EA moiety and said reporter are connected through a first binding pair, and wherein the DOI, FA or EA moiety comprises a first cognate tag and the reporter comprises a second cognate tag for the first binding pair. 
     
     
         3 . The method of  claim 2 , wherein the capture agent is the DOI, FA or EA; or a first member of a second binding pair that binds to second member of the second binding pair that is attached to the DOI, FA or EA. 
     
     
         4 . The method of  claim 3 , wherein the capture agent is an antibody or the first member of the second binding pair. 
     
     
         5 . The method of  claim 4 , wherein the antibody complex further comprises a second DOI, FA or EA moiety bound to a second antigen binding region, and wherein said second DOI, FA or EA moiety comprises a first cognate tag for a second binding pair, wherein said capture antibody specifically binds to said first cognate tag for said second binding pair. 
     
     
         6 . The method of  claim 5 , wherein the first and/or second cognate tag further comprises a symmetrically or an asymmetrically branched polymer. 
     
     
         7 . The method of  claim 6 , wherein the symmetrically branched polymer is selected from the group consisting of a star-shaped polymer, a comb-shaped polymer, a dendrimer, a starburst dendrimer, a combburst dendrigraft, and a hypercombbranched polymer. 
     
     
         8 . The method of  claim 6 , wherein the asymmetrically branched polymer is selected from a polylysine dendrimer or a randomly branched polymer. 
     
     
         9 . The method of  claim 5 , further comprising mixing said sample with the first and second DOI, FA or EA moiety prior to contacting said solid phase. 
     
     
         10 . (canceled) 
     
     
         11 . The method of  claim 1 , further comprising prior to contacting;
 mixing said sample with the reporter, wherein the reporter comprises a first cognate tag of a first binding pair, and wherein said DOI, FA or EA moiety comprises a second cognate tag of a first binding pair.   
     
     
         12 . The method of  claim 11 , wherein the first and/or second cognate tags further comprise symmetrically or asymmetrically branched polymers. 
     
     
         13 - 16 . (canceled) 
     
     
         17 . The method of  claim 2 , wherein the binding pair is selected from the group consisting of an antibody or an antigen-binding portion thereof and an antigen, an avidin/streptavidin/neutravidin and a biotin, a dinitrophenol (DNP) and an anti-DNP antibody, a digoxin and an anti-digoxin antibody, a digoxigenin and an anti-digoxigenin antibody, a hapten and an anti-hapten, a polysaccharide and a polysaccharide binding moiety, a lectin and a receptor, a ligand and a receptor, a fluorescein and an anti-fluorescein antibody, and complementary nucleic acids. 
     
     
         18 . The method of  claim 1 , wherein the reporter is selected from the group consisting of an enzyme, a ferritin, a fluorescent or colored microparticle/bead or nanoparticle/bead, a colloid metal, a quantum dot, a magnetic particle, an up-converting phosphorescent particle, an electrochemiluminescent molecule, compounds containing a transition metal, and compound containing a lanthanide metal. 
     
     
         19 . The method of  claim 1 , wherein the DOI is selected from the group consisting of organic compounds, polypeptides, antibodies, monoclonal antibodies, antigen binding immunoglobulins, polynucleotides, siRNA, RNAi, miRNA, lipids, saccharides, polysaccharides, and combinations thereof. 
     
     
         20 . The method of  claim 1 , wherein the antibody binds to a drug substituent. 
     
     
         21 . The method of  claim 20 , wherein the drug substituent is selected from the group consisting of a charged group, an ester group, a glycan, a carbohydrate, a water soluble polymer and a synthetic polymer. 
     
     
         22 . The method of  claim 21 , wherein the water soluble polymer is polyethylene glycol (PEG) or polyethyleneoxide (PEO). 
     
     
         23 . The method of  claim 1 , wherein the solid phase comprises a membrane, a glass micro/nanochannel, a plastic micro/nanochannel, a natural wick, a synthetic wick, microbead particles, nanobead particles or a combination thereof. 
     
     
         24 . The method of  claim 23 , wherein said membrane comprises nitrocellulose, glass fiber, cotton or polyamide. 
     
     
         25 . An immunoassay method for determining the isotype of an antibody against a drug of interest (DOI), food antigen (FA) or environmental antigen (EA) comprising:
 contacting at least one immunochromatographic solid phase with a sample in a proximal region of a first strip, wherein said sample comprises at least one antibody in a mobile phase;   allowing a mobilized antibody to flow from said proximal region into a distal region of said solid phase, wherein said distal region comprises an immobilized capture agent, and wherein said capture agent immobilizes an antibody complex comprising a first DOI, FA or EA moiety bound to a first antigen binding region of said antibody and said first DOI, FA or EA moiety comprises a hapten specifically bound by said capture agent; a subtype reagent which specifically binds to the antibody subtype, and a reporter which binds to said subtype reagent; and   determining whether said reporter forms a complex with the capture agent, wherein said determining correlates with the presence of an antibody isotype in said sample.   
     
     
         26 . The method of  claim 25 , wherein said isotype is IgA, IgD, IgE, IgG or IgM. 
     
     
         27 - 40 . (canceled) 
     
     
         41 . A method of detecting an antibody to a drug of interest (DOI), food antigen (FA) or environmental antigen (EA) comprising:
 contacting a sample comprising at least one antibody to a solid phase containing an immobilized capture agent under conditions which allow for binding of an antibody complex to said capture agent, wherein the capture agent comprises a member of a first binding pair;   contacting at least one antibody with a first DOI, FA or EA moiety comprising a first cognate tag of a first binding pair and a second DOI, FA or EA moiety comprising a first cognate tag of a second binding pair;   contacting the second moiety with a reporter, wherein said reporter comprises a second cognate tag of the second binding pair, and wherein one or more of the first and/or second cognate tags of the first and/or second binding pairs comprise a symmetrically branched polymer or an asymmetrically branched polymer; and   detecting a captured antibody complex comprising the reporter bound through the second binding pair to said capture agent,   
       wherein detecting correlates with the presence of an antibody to said DOI, FA or EA. 
     
     
         42 . The method of  claim 41 , wherein the capture agent is a DOI, FA or EA, or a binding partner thereof. 
     
     
         43 . The method of  claim 41 , wherein the first and second binding pair is selected from the group consisting of an antibody or an antigen-binding portion thereof and an antigen, an avidin/streptavidin/neutravidin and a biotin, a dinitrophenol (DNP) and an anti-DNP antibody, a digoxin and an anti-digoxin antibody, a digoxigenin and an anti-digoxigenin antibody, a hapten and an anti-hapten, a polysaccharide and a polysaccharide binding moiety, a lectin and a receptor, a ligand and a receptor, a fluorescein and an anti-fluorescein antibody, and complementary nucleic acids. 
     
     
         44 . The method of  claim 41 , wherein the symmetrically branched polymers are selected from the group consisting of star-shaped polymers, comb-shaped polymers, dendrimers, starburst dendrimers, combburst dendrigrafts, and hypercombbranched polymers. 
     
     
         45 . The method of  claim 41 , wherein the asymmetrically branched polymers are selected from polylysine dendrimers or randomly branched polymers. 
     
     
         46 . The method of  claim 41 , wherein said solid phase comprises a tissue culture plate, a multi-well plate or a combination thereof. 
     
     
         47 . The method of  claim 41 , further comprising detecting the reporter with a reader. 
     
     
         48 . The method of  claim 41 , wherein the solid phase comprises beads.