Site News  |   Monitor Keywords  |   Monitor Archive  |   Organizer  |   Account Info  |

Binding surfaces for affinity assays

Binding surfaces for affinity assays description/claims

This application claims priority to U.S. Provisional Patent Application No. 60/863,820, filed Nov. 1, 2006.

The present invention relates to supports having binding surfaces that are non-saturated or non-saturated and orientated, including supports having binding surfaces comprising ligands for use in affinity assays. Particular aspects of the invention relate to binding surfaces comprising biotin, or biotin and a biotin-specific ligand binder such as streptavidin (SA). Binding surfaces for immunoassays are provided, including binding surfaces comprising antigens or antibodies, or fragments thereof. Further embodiments of the present invention relate to blocked solid support surfaces and dispersed microparticles. Methods for making and using such supports are provided.

Binding surfaces are used in a wide variety of applications including, for example, affinity assays. Conventional binding surfaces are typically prepared by maximizing the amount of ligand binders per unit surface area of the solid phase support surface. Although the conventional approach results in a support surface with a high density of ligand binders, simply maximizing the number of ligand binders on a support surface does not invariably improve performance of the binding surface. Some conventional binding surfaces used in affinity assays maximize binding capacity by direct coating of a ligand binder such as an antibody, or a biotin-specific ligand binder such as SA, onto a solid phase and blocking the solid phase with bovine serum albumin (BSA) or ovalbumin. Although this maximizes the antigen or biotin binding capacity of the binding surface, the respective antibodies or biotin-specific ligand binders are crowded on the binding surface without specific orientation, and traditional blocking strategies do not necessarily prevent or mitigate sloughing of antibodies or biotin-specific ligand binders from the binding surface. Further, sloughing can result in poor assay sensitivity (sub-optimal signal-to-noise ratio), accuracy, precision, stability, or manufacturability, or combinations thereof. Binding surface crowding and random orientation can decrease binding efficiency or capacity of the binding surface due to steric hindrance. Accordingly, there is a need for improved binding surfaces and compositions and methods for making improved binding surfaces that do not rely on simply maximizing the density of ligand binders on a binding surface.

Non-saturated or non-saturated and orientated binding surfaces, methods for making non-saturated or non-saturated and orientated binding surfaces, and methods for making components of non-saturated or non-saturated and orientated binding surfaces are provided. Methods and compositions for non-saturated or non-saturated and orientated binding surfaces for affinity assays, such as, for example, immunoassays, are also provided.

In one aspect, the invention provides methods and compositions for preparing a non-saturated or non-saturated and orientated capture moiety for an immunoassay, wherein the capture moiety is non-saturated or non-saturated and orientated by virtue of binding to a non-saturated or non-saturated and orientated binding surface. In a specific embodiment, the capture moiety comprises an immunoglobulin or fragment thereof, wherein the capture moiety is immobilized on the non-saturated or non-saturated and orientated binding surface by association with a ligand immobilized on a support coupler, wherein the support coupler is immobilized on a solid phase support. In a specific embodiment, the immunoglobulin or fragment thereof is biotinylated, the biotinylated immunoglobulin or fragment thereof is associated with a biotin-specific ligand binder (biotin-binding moiety), and the biotin-specific ligand binder is associated with biotin attached to the support directly or through a support coupler. In a specific embodiment, the support coupler comprises a protein, and the solid phase support comprises a microparticle.

In another aspect, the invention provides a method for performing an immunoassay using a support having a non-saturated or non-saturated and orientated binding surface, wherein the method comprises employing an immunoglobulin or fragment thereof to capture an analyte, wherein the immunoglobulin or fragment thereof is non-saturated or non-saturated and orientated on the binding surface. In various embodiments, the non-saturated or non-saturated and orientated nature of the immunoglobulin or fragment thereof is determined by the non-saturated or non-saturated and orientated nature of the binding surface with which the immunoglobulin or fragment thereof is bound.

In another aspect, the invention provides a support having a non-saturated or non-saturated and orientated binding surface, comprising ligands coupled with the support, either directly or indirectly via coupling to a support coupler which is coupled to the support, wherein the ligands are present at a density of about 1.0×10−3 to about 5.0×10−1 micromoles of ligand per square meter of support, or about 0.5×10−2 to about 2.0×10−1 micromoles of ligand per square meter of support, or about 1.0×10−2 to about 1.6×10−1 micromoles of ligand per square meter of support, or about 1.0×10−2 to about 2.0×10−1 micromoles of ligand per square meter of support, or alternatively the ligands are present at a density of about 0.5×10−4 to about 10×10−4 micromoles of ligand per milligram (mg) of microparticles, or about 1.0×10−4 to about 5.5×10−4 micromoles of ligand per mg of microparticles. In various embodiments, support couplers are 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 In various embodiments, the ligands are associated with ligand binders, wherein the ligand binders are present at a density of less than about 0.4×10−2 to less than about 8×10−2 micromoles of ligand binder per square meter of support, or at a density of about 1.0×10−2 to about 5.0×10−2 micromoles per square meter of the assay support.

Embodiments of the present invention are directed to having a capture moiety 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. In various embodiments, the binding surface further comprises a capture moiety, wherein the capture moiety is present at a density of less than about 2×10−3 to less than about 4×10−2 micromoles of capture moiety per square meter of support. In a specific embodiment, the ligand (e.g., biotin) is present at a density of about 1.9×10−2 to about 1.6×10−1 micromoles of ligand per square meter of support, the ligand binder (e.g., a biotin-binding moiety such as SA) is present at about 1.0×10−2 to about 5.0×10−2 micromoles per square meter of the assay support, or about 1.2×10−2 to less than about 4.6×10−2 micromoles of ligand binder per square meter of support, and the capture moiety (e.g., a biotinylated capture moiety such as biotinylated analyte-specific antibody) is present at a density of about 2.5×10−3 to about 1.4×10−2 micromoles of capture moiety per square meter of support. In at least one embodiment of the present invention, the support coupler is optional. In such an embodiment, the ligand could be biotin or a derivative thereof.

In a specific embodiment, the ligand comprises biotin or a derivative thereof. In another specific embodiment, the ligand binder comprises a biotin-binding moiety, or fragment thereof, such as, for example, avidin, SA, neutravidin, a fragment of SA, a fragment of avidin, a fragment of neutravidin, or mixtures thereof. In various embodiments, the capture moiety comprises one or more 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 single stranded polynucleotide, a double stranded oligonucleotide, a double stranded polynucleotide, an antigen, a peptide, or a protein. In various embodiments, the ligands are coupled with the support through support couplers, and the support couplers are in turn coupled with the support. In a specific embodiment, the support couplers comprise protein. In a specific embodiment, the protein is BSA, ovalbumin, a fragment of BSA, a fragment of ovalbumin, or mixtures thereof. In a specific embodiment, the support is a microparticle. In a specific embodiment, the binding surface has about 1×10−2 to about 5×10−1 micromoles of ligand per square meter of support, or alternatively, the binding surface has about 2×10−2 to about 2×10−1 micromoles of ligand per square meter of support. In another specific embodiment, the binding surface has about 1.9×10−2 to about 1.6×10−1 micromoles of ligand per square meter of support. In another specific embodiment, the binding surface has about 1.6×10−2 to about 6.6×10−2 micromoles of ligand per square meter of support. In another specific embodiment, the binding surface has about 1.6×10−2 to about 3.2×10−2 micromoles of ligand per square meter of support. In another specific embodiment, the binding surface has about 1.6×10−2 to about 2.0×10−1 micromoles of ligand per square meter of support.

In various embodiments, the binding surface comprises a ligand binder attached to the ligand and a capture moiety attached to the ligand binder, wherein the capture moiety is present at a density, in various embodiments, of about 75% of the density of the ligand, of about 50% of the density of the ligand, and of about 25% of the density of the ligand.

In a specific embodiment, the ligand comprises biotin, the biotin is coupled with the support through a support coupler protein selected from BSA, ovalbumin, a fragment of BSA, a fragment of ovalbumin, or mixtures thereof, the biotin is attached to a ligand binder comprising a biotin-binding moiety selected from avidin, SA, neutravidin, a fragment of SA, a fragment of avidin, a fragment of neutravidin, or mixtures thereof, and the biotin-binding moiety is attached to a biotinylated capture moiety, wherein the capture moiety is selected from the group consisting of one or more 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, or a protein.

In another aspect, the invention provides a support having a non-saturated and orientated binding surface, comprising: a plurality of support couplers disposed on the support; and ligands coupled with the support couplers; wherein the ligands are non-saturated and are orientated on the surface in a manner that provides sterically accessible ligands. In a specific embodiment, the ligands are associated with bi- or multivalent ligand binders capable of specifically associating with the ligands, and the binding surface is substantially free of unbound, free ligand.

In various embodiments, the support couplers are coupled with about 1.9×10−2 to about 1.6×10−1 micromoles of ligand per square meter of support.

The support couplers can be coupled with the support through covalent or non-covalent association. In various embodiments, the support couplers are covalently coupled with the support. In embodiments where the support couplers are covalently coupled with the support, any suitable binding chemistry known in the art can be used to attach the support coupler to the support. Suitable binding chemistries include, but are not limited to, attachment through one or more functional groups selected from the group consisting of carboxyl, hydroxyl, tosyl, epoxy, aldehyde, amine, amide, hydrazide, isothiocyanate, maleimide, and sulfhydryl. In a specific embodiment, the support coupler is covalently coupled with the support using tosyl chemistry for attachment.

The support couplers can comprise any suitable substance that can be coupled with a support and also with a ligand. The coupling with the support, and the coupling with the ligand, can be covalent. Suitable support couplers include, but are not limited to, macromolecules such as, for example, proteins or other polymers. In various embodiments, the support coupler comprises protein. The protein can be, for example, a monomer, a dimer, a multimer, or a fusion protein. In specific embodiments, the protein comprises at least one of an albumin such as, for example, BSA, ovalbumin, a fragment of BSA, a fragment of ovalbumin, or mixtures thereof.

In various embodiments, the ligands comprise biotin. Suitable biotin reagents for attaching biotin to a support surface or a support coupler include amine-reactive biotin labeling reagents such as, for example, sulfo-NHS-biotin, sulfo-NHS-LC-biotin, sulfo-NHS-LC-LC-biotin, sulfo-NHS-SS-biotin, NHS-PEO4-biotin, NHS-biotin, NHS-LC-biotin, NHS-LC-LC-biotin, PFP-biotin, TFP-PEO-biotin, or NHS-iminobiotin trifluoroacetamide, sulfhydryl-reactive biotin labeling reagents such as, for example, maleimide-PEO2-biotin, biotin-BMCC, PEO-lodoacetyl biotin, iodoacetyl-LC-biotin, or biotin-HPDP, carboxyl-reactive biotin labeling reagents such as, for example, biotin PEO-amine or biotin PEO-LC-amine, carbohydrate-reactive biotin labeling reagents such as, for example, biocytin hydrazide, biotin hydrazide, or biotin-LC-hydrazide, or photoreactive biotin labeling reagents such as, for example, psoralen-PEO-biotin. In a specific embodiment, the ligand comprises biotin and is attached to the support or support coupler using the amine reactive biotin labeling reagent sulfo-NHS-LC-biotin.

In embodiments where the ligands comprise biotin, the binding surface on the support can further comprise a ligand binder associated with the biotin. In certain embodiments, the ligand binder comprises a biotin-binding moiety. In various embodiments, the biotin-binding moiety comprises protein, for example, at least one of a biotin-binding protein such as avidin, SA, neutravidin, a fragment of SA, a fragment of avidin, a fragment of neutravidin, or mixtures thereof. The biotin-binding moiety could also comprise a fusion protein such as, for example, avidin fused to a different binding protein.

• Provisional Patent
• Utility Patent

• What Is a Patent?
• What Is a Trademark or Servicemark?
• What Is a Copyright?
• Patent Laws