Binding surfaces for affinity assays

Abstract

Non-saturated or non-saturated and orientated binding surfaces are provided, and methods and compositions for making non-saturated or non-saturated and orientated binding surfaces. Methods of making binding surfaces for preparing a support surface for an affinity assay are provided, including using non-saturated or non-saturated and orientated binding surfaces on paramagnetic microparticles. The methods include methods for making ligand::support coupler-based complexes by a process optionally employing a low input ratio of ligand to support coupler, by dilution, and by methods employing a dispersion and/or coating step using a block copolymer. Specific examples employing biotin-BSA and biotin-ovalbumin binding surfaces are provided, as well as strepavidin-coated microparticles and microparticles coated with capture moieties such as, for example, biotinylated immunoglobulins or fragments thereof. Other examples couple a ligand to the solid surface. Use of the methods and compositions in connection with a wide variety of analytes and capture moieties is provided. Non-saturated or non-saturated and orientated binding surfaces for immunoassays are provided. Further provided are dispersed microparticles and methods for making them.

1 . A non-saturated binding surface for an affinity assay, comprising:
 a) an assay support; and,   b) at least one support coupler, wherein less than a saturating amount of the support coupler is coupled with the assay support.   
     
     
         2 . The non-saturated assay binding surface of  claim 1 , further comprising at least one ligand coupled with the support coupler. 
     
     
         3 . The non-saturated assay binding surface of  claim 2 , wherein the non-saturated assay binding surface has about 0.5×10 −4  to about 10×10 −4  micromoles of ligand per milligram of the assay support, or about 0.5×10 −2  to about 2.0×10 −1  micromoles of ligand per square meter of the assay support. 
     
     
         4 . The non-saturated assay binding surface of  claim 1 , wherein the assay binding surface has about 1.0×10 −4  to about 5.5×10 −4  micromoles of ligand per milligram of the assay support, or about 1.0×10 −2  to about 2.0×10 −1  micromoles of ligand per square meter of the assay support. 
     
     
         5 . The non-saturated assay binding surface of  claim 2 , further comprising at least one ligand binder associated with the ligand. 
     
     
         6 . The non-saturated assay binding surface of  claim 5 , wherein the ligand binder is at least bivalent, the bivalent ligand binder comprising streptavidin, avidin, neutravidin, a fragment of streptavidin, a fragment of avidin, a fragment of neutravidin, or combinations thereof. 
     
     
         7 . The non-saturated assay binding surface of  claim 5  further comprising a capture moiety associated with the ligand binder. 
     
     
         8 . The non-saturated assay binding surface of  claim 7 , wherein the capture moiety is present on the assay support at a density of about 1.0×10 −4  micromoles per square meter to about 2.0×10 −2  micromoles per square meter of the assay support. 
     
     
         9 . The non-saturated assay binding surface of  claim 7 , wherein the capture moiety is spatially orientated as to the assay support. 
     
     
         10 . The non-saturated assay binding surface of  claim 7 , wherein the capture moiety is spatially orientated and physically orientated as to the assay support. 
     
     
         11 . The non-saturated assay binding surface of  claim 1 , further comprising a block copolymer, wherein the block copolymer comprises a hydrophobic head group flanked by at least two hydrophilic tail groups, and wherein the hydrophobic head group contacts the assay support. 
     
     
         12 . The non-saturated assay binding surface of  claim 1 , wherein the support coupler comprises a protein or a fusion protein. 
     
     
         13 . The non-saturated assay binding surface of  claim 1 , wherein the support coupler is covalently coupled with the assay support. 
     
     
         14 . The non-saturated assay binding surface of  claim 2 , wherein the ligand is covalently coupled with the support coupler. 
     
     
         15 . The non-saturated assay binding surface of  claim 1 , wherein the support coupler is bovine serum albumin, a fragment of bovine serum albumin, ovalbumin, a fragment of ovalbumin, or mixtures thereof. 
     
     
         16 . The non-saturated assay binding surface of  claim 2 , wherein the ligand comprises biotin. 
     
     
         17 . The non-saturated assay binding surface of  claim 16 , wherein the support coupler is biotinylated so as to yield a molar incorporation ratio of biotin to support coupler of less than or equal to 5:1. 
     
     
         18 . The non-saturated assay binding surface of  claim 17 , wherein the support coupler is biotinylated so as to yield a molar incorporation ratio of biotin to support coupler of less than or equal to 3:1. 
     
     
         19 . The non-saturated assay binding surface of  claim 16 , further comprising a ligand binder associated with the biotin. 
     
     
         20 . The non-saturated assay binding surface of  claim 19 , wherein the ligand binder is at least bivalent, the at least bivalent ligand binder comprising streptavidin, avidin, neutravidin, a fragment of streptavidin, a fragment of avidin, a fragment of neutravidin, or combinations thereof. 
     
     
         21 . The non-saturated assay binding surface of  claim 19 , further comprising a capture moiety associated with the ligand binder. 
     
     
         22 . The non-saturated assay binding surface of  claim 21 , wherein the capture moiety is biotinylated. 
     
     
         23 . The non-saturated assay binding surface of  claim 22 , wherein the capture moiety that is biotinylated is selected from the group consisting of at least one of an antibody, a binding fragment of an antibody, a receptor, a ligand of a receptor, a hormone, a receptor of a hormone, an enzyme, a substrate of an enzyme, a single stranded oligonucleotide, a double stranded oligonucleotide, a single stranded polynucleotide, a double stranded polynucleotide, an antigen, a peptide, and a protein. 
     
     
         24 . The non-saturated assay binding surface of  claim 22 , wherein the biotinylated capture moiety comprises a spacer. 
     
     
         25 . The non-saturated assay binding surface of  claim 1 , wherein the assay support comprises a microparticle, a bead, a microtiter plate, a coated tube, a mylar-backed nitrocellulose support, a nylon support, a microtubule, a nanoparticle, a nanotube, or a paramagnetic or superparamagnetic material. 
     
     
         26 . The non-saturated assay binding surface of  claim 7 , further comprising at least a second capture moiety associated with the ligand binder. 
     
     
         27 . The non-saturated assay binding surface of  claim 5 , wherein the ligand binders are present on the assay support at a density of about 1.0×10 −2  to about 5.0×10 −2  micromoles per square meter of the assay support. 
     
     
         28 . The non-saturated assay binding surface of  claim 27 , wherein the ligand binders are strepavidin, avidin, neutravidin, a fragment of streptavidin, a fragment of avidin, a fragment of neutravidin, or combinations thereof. 
     
     
         29 . The non-saturated assay binding surface of  claim 1 , wherein the at least one support coupler is present on the assay support at a density of about 1.2×10 −2  micromoles per square meter to about 7.5×10 −2  micromoles per square meter of the assay support. 
     
     
         30 . The non-saturated assay binding surface of  claim 1 , further comprising a ligand coupled with the support coupler, wherein the ligand is biotin, or a derivative thereof, and wherein the biotin, or a derivative thereof, is present at a density of about 1.6×10 −2  to about 2.0×10 −1  micromoles per square meter of the assay support. 
     
     
         31 . The non-saturated assay binding surface of  claim 1 , wherein the assay binding surface comprises a plurality of microparticles, and wherein the plurality of microparticles have a capacity for at least about 25 picomoles of biotin, or a derivative thereof, per milligram of microparticles but no more than about 425 picomoles of biotin, or a derivative thereof, per milligram of microparticles. 
     
     
         32 . A non-saturated and orientated binding surface for an affinity assay, comprising:
 a) a support;   b) a protein or a fusion protein covalently coupled with the support, wherein the protein or fusion protein is biotinylated, wherein there are less than  5  moles of biotin coupled per mole of protein or fusion protein;   c) a biotin-binding moiety associated with the biotin, wherein the biotin-binding moiety is at least bivalent;   d) a biotinylated capture moiety associated with the biotin-binding moiety; and,   e) a block copolymer contacting the support, wherein the block copolymer comprises a polypropylene oxide head group flanked by at least two polyethylene oxide tail groups, wherein the polypropylene oxide head group contacts the support.   
     
     
         33 . A method of preparing a non-saturated assay binding surface, comprising:
 a) combining support couplers and ligands at a ratio of ligand to support coupler selected so as to result in a mixture of ligand::support coupler complexes; and,   b) covalently attaching the ligand::support coupler complexes to an assay support so as to yield a non-saturating density of ligand::support coupler complexes.   
     
     
         34 . The method of preparing a non-saturated assay binding surface of  claim 33 , wherein after covalently attaching the ligand::support coupler complexes with the assay support, the assay support comprises about 1.0×10 −2  micromoles of ligand per square meter of assay support to about 2.0×10 −1  micromoles of ligand per square meter of assay support. 
     
     
         35 . The method of preparing a non-saturated assay binding surface of  claim 33 , wherein the ligand is covalently coupled with the support coupler. 
     
     
         36 . The method of preparing a non-saturated assay binding surface of  claim 33 , wherein the support coupler is bovine serum albumin, a fragment of bovine serum albumin, ovalbumin, a fragment of ovalbumin, or mixtures thereof, and wherein the ligand is biotin, and wherein the bovine serum albumin, fragment of bovine serum albumin, ovalbumin, fragment of ovalbumin, or mixtures thereof, are biotinylated so as to yield a molar incorporation ratio of biotin:support coupler of less than or equal to 5:1. 
     
     
         37 . The method of preparing a non-saturated assay binding surface of  claim 33 , further comprising prearing a non-saturated and orientated assay binding surface, comprising contacting the assay binding surface with a a plurality of block copolymer molecules, wherein the block copolymer molecules comprise a hydrophobic head group flanked by at least two hydrophilic tail groups. 
     
     
         38 . The method of preparing a non-saturated and orientated assay binding surface of  claim 34 , wherein the ligand binder is physically orientated as to the ligand::support coupler complex. 
     
     
         39 . A blocked assay binding surface, comprising
 a) an assay support,   b) at least one support coupler; and,   c) a plurality of block copolymer molecules contacting the assay support, wherein the block copolymer molecules comprise a hydrophobic head group flanked by at least two hydrophilic tail groups.   
     
     
         40 . The blocked assay binding surface of  claim 39 , wherein the length of the two or more hydrophilic tails can each be independently about 2 to about 2.5 times the length of the hydrophobic head group. 
     
     
         41 . The blocked assay binding surface of  claim 39 , wherein the block copolymer comprises a structure of formula I,
   HO(C 2 H 4 O) x (C 3 H 6 O) y (C 2 H 4 O) z H   (I),   
       wherein x is about 100 to about 135, y is about 40 to about 75, and z is about 100 to about 135. 
     
     
         42 . The blocked assay binding surface of  claim 39 , wherein the block copolymer has an average molecular weight of about 9,000 Daltons to about 18,000 Daltons. 
     
     
         43 . A population of dispersed microparticles, wherein the microparticles are exposed to a dispersant, the dispersant comprising a block copolymer having a hydrophobic head group flanked by at least two hydrophilic tail groups. 
     
     
         44 . The population of dispersed microparticles of  claim 43 , wherein the microparticles comprise
 a) at least one support coupler; and,   b) a plurality of block copolymer molecules contacting the microparticles, wherein the block copolymer molecules comprise a hydrophobic head group flanked by at least two hydrophilic tail groups.   
     
     
         45 . The population of dispersed microparticles of  claim 44 , further comprising a ligand coupled with the support coupler. 
     
     
         46 . The population of dispersed microparticles of  claim 44 , wherein the population is substantially monodispersed. 
     
     
         47 . A method of preparing a population of coated and dispersed microparticles, comprising:
 a) exposing microparticles to a dispersant to form a population of dispersed microparticles, wherein:
 i) the microparticles comprise support couplers and ligands that are coupled to yield ligand::support coupler complexes; 
 ii) the dispersant comprises a block copolymer having a hydrophobic head group flanked by at least two hydrophilic tail groups; and 
   b) coating the dispersed microparticles with ligand binder by exposing the population of dispersed microparticles to a ligand binder that associates with the ligand of the ligand::support coupler complexes.   
     
     
         48 . A method of preparing a non-saturated assay binding surface, comprising:
 a) preparing a mixture of analyte-associating-moieties and space-filling moieties, wherein the analyte-associating-moieties associate, either directly or indirectly, with an analyte of interest, and the space-filling moieties do not associate, either directly or indirectly, with an analyte of interest; and,   b) exposing the mixture to an assay support such that the analyte-associating-moieties and the space-filling moieties couple with the assay support and form a non-saturated assay binding surface that is non-saturated with respect to the amount of analyte-associating-moieties coupled with the assay support.   
     
     
         49 . The method of  claim 48 , wherein the analyte-associating-moieties comprise ligand::support coupler complexes and the space-filling moieties comprise support coupler that lacks ligand. 
     
     
         50 . The method of  claim 48 , wherein the non-saturated assay binding surface further comprises a block copolymer, wherein the block copolymer comprises a hydrophobic head group flanked by at least two hydrophilic tail groups. 
     
     
         51 . A method of making a blocked assay binding surface, comprising
 coupling an assay support with a support coupler to yield a binding surface,   contacting the binding surface With a block copolymer, wherein the block copolymer comprises a hydrophobic head group flanked by at least two hydrophilic tail groups.   
     
     
         52 . An affinity assay employing a non-saturated assay binding surface, the affinity assay comprising
 contacting a non-saturated assay binding surface with an analyte, wherein the non-saturated assay binding surface comprises a support coupler, a ligand, a ligand binder, and an analyte-specific capture moiety, and   detecting the binding of the analyte with the non-saturated assay binding surface.   
     
     
         53 . The affinity assay of  claim 52 , wherein the analyte-specific capture moiety is biotinylated. 
     
     
         54 . The affinity assay of  claim 52 , wherein the affinity assay is an immunoassay. 
     
     
         55 . A non-saturated assay binding surface, comprising:
 a) an assay support; and   b) at least one ligand, wherein less than a saturating amount of the ligand is coupled with the assay support.   
     
     
         56 . The non-saturated assay binding surface of  claim 55 , further comprising at least one ligand binder associated with the ligand, wherein the ligand binder is at least bivalent. 
     
     
         57 . The non-saturated assay binding surface of  claim 56 , wherein the ligand binder is streptavidin, avidin, neutravidin, a fragment of streptavidin, a fragment of avidin, a fragment of neutravidin, or combinations thereof. 
     
     
         58 . The non-saturated assay binding surface of  claim 57 , further comprising a capture moiety associated with the ligand binder. 
     
     
         59 . The non-saturated assay binding surface of  claim 55 , wherein the ligand is biotin or a derivative thereof.