Method and composition for removing biotin interference from assay via molecular trapping

Abstract

To provide a method and a composition for removing or reducing biotin interference from a specific assay, the method and the composition removing interfering hapten by adding a hapten trap to a reagent formulation, without involvement of assay components involved in the generation of an assay signal.SOLUTION: A hapten trap may be a soluble or solid cage. When the trap has small holes, the small holes need to be of sizes that allow free biotin to enter the small holes and prevent large molecules including biotin antibodies to enter the small holes. Alternatively or furthermore, the trap cannot be charged so that hapten is attracted to the cage. In the cage, interfering hapten molecules (biotin or fluorescein) can be captured through molecular imprinting involving hydrogen bonding, hydrophobic interactions, or specific binding partners.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] This application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Patent Application No. 62 / 735,911, filed on September 25, 2018. The entire content of the above patent application is hereby incorporated by reference into this specification explicitly.

[0002] The present invention relates to methods and compositions for assays and for removing interference, particularly biotin interference, from such assays.

Background Art

[0003] In modern in vitro clinical diagnostics, various methods are utilized for detecting analytes in samples. In one form of diagnostic method, immunoassays, one or more specifically binding species are used. Typical examples are sandwich immunoassays in which two specifically binding species (antibodies or antigens) bind to the analyte of interest, and competitive immunoassays in which the analyte of interest and an analog of this analyte compete for binding to a specifically binding species. In competitive immunoassays, the antigen is often also referred to as a hapten or ligand. One of the specifically binding species is generally attached to a so-called label or tag, which can be an atom (e.g., radioactive), a molecule (e.g., an enzyme, a fluorescent, or a luminescent compound), or a particle (magnetic or latex). This label enables the detection of the analyte of interest by various detection methods corresponding to the label utilized. In competitive assays, either the specifically binding species or the analog of the analyte can carry the label. The other specifically binding species often associates with a solid or suspendable substrate (the "solid phase") by covalent bonding or adsorption. Alternatively, the specifically binding species is linked to the first member of a second binding pair (e.g., biotin), while the second member of the second binding pair (e.g., streptavidin) is attached to the solid phase. This enables the specifically binding species to bind to the solid phase via the second binding pair interaction (e.g., biotin-streptavidin, etc.).

[0004] Haptens such as biotin and fluorescein are often used to conjugate with antibodies or other small drug molecules in assay reagents. Their tight binding to large protein molecules coated on a solid support (e.g., streptavidin to biotin and anti-FITC antibody to fluorescein) provides a convenient way to immobilize hapten-Ab or hapten-drug on the solid surface. Since the binding force between biotin and streptavidin or avidin exhibits one of the highest constants for biomolecules, biotin is very frequently used as a ligand and streptavidin is used as its specific binding partner.

[0005] For the stability and reproducibility of diagnostic assays, it is important that the binding ability of the solid-phase bound species to its binding partner is not impaired over time. This can lead to a decrease in reliability and a decrease in the sensitivity of the assay. One mechanism that can lead to such a drawback (apparent as instability) is that some of the solid-phase attached species leach into the surrounding medium. The free species compete with the solid-phase attached species for binding to the target and usually have a large kinetic advantage due to their fast diffusion. Therefore, it is advantageous to keep the amount of free species competing with the solid-phase attached species in the reagent constant, preferably very close to zero. This should allow the analyte to be detected with high sensitivity in a stable and reproducible manner. A preferred way to eliminate free species is to find a binding method that excludes dissociation. Covalent bonding, in contrast to association by adsorption, may be the optimal method because of its high binding strength. However, in many cases, this may be impossible or impractical for various reasons.

[0006] Biotin has found use as a supplement. Biotin as a dietary supplement is, for example, aimed at promoting the growth of healthy hair and nails and treating other medical conditions. Thus, the amount of biotin in serum can be very high. Since molecules such as biotin are used in many diagnostic assays to coat solid supports and bind to antibodies or hapten analogs, these high levels of biotin in blood can interfere with assay signals. Thus, when biotin is present in free form in a sample solution, biotin can occupy binding sites, resulting in false test results. This is extremely serious in the case of patients administered high doses of biotin. An amount of biotin exceeding 30 ng / ml in a sample is already thought to result in false results. In the case of patients treated with biotin, serum values of up to 180 ng / ml, or even up to 1500 ng / ml, and long-lasting values of about 70 ng / ml can occur.

[0007] One way to reduce such interference relates to the use of pre-formed reagents, i.e., pre-bound biotinylated assay components having a solid support coated with streptavidin during reagent manufacture. Due to the tight binding and slow off-rate between streptavidin and biotin, it is not the main way to replace the already bound biotin from streptavidin with biotin in the incoming patient sample.

[0008] A second way is to increase the streptavidin binding sites on the solid support, so that there are extra binding sites available for the biotin molecules in the sample in addition to the biotinylated assay components. A third way is to combine both of the above. However, none of the above truly solves the problem of biotin interference unless the use of biotin-streptavidin as an active assay component is completely avoided.

[0009] One major problem with all of the above solutions is that the assay components are involved in interference prevention, which can easily affect the magnitude of the assay signal itself. In such cases, the assay components used are taken out of the optimal conditions for detecting the intended analyte. Some examples are provided in Patent Document 1.

[0010] Another method is disclosed in Patent Document 2. It discloses using polymer particles having a biotin-binding core and a coating layer of protein, carbohydrate, or copolymer to filter free biotin but not biotin conjugated to large molecules.

[0011] This approach may be effective in some assay formats, but the introduction of particles may generate additional absorbance that can interfere with the assay signal.

Prior Art Documents

Patent Documents

[0012]

Patent Document 1

Patent Document 2

Summary of the Invention

Problems to be Solved by the Invention

[0013] Accordingly, an object of the present invention is to provide an improved method for reducing such substances in an assay so that the detection method is not hindered. Accordingly, a method for removing biotin is needed so that no interference in the test occurs.

Means for Solving the Problems

[0014] The use of the terms "a" or "an" when used in conjunction with the term "comprising" in the claims and / or the specification means "one", but is also not inconsistent with the meanings of "one or more", "at least one", and "more than one". Thus, the terms "a", "an", and "the" include plural referents unless the context clearly indicates otherwise. Thus, for example, a reference to "a compound" can refer to one or more compounds, two or more compounds, three or more compounds, four or more compounds, or a large number of compounds. The term "plural" refers to "two or more".

[0015] The use of the term "at least one" is understood to include one and any quantity greater than one including, but not limited to, two, three, four, five, ten, fifteen, twenty, thirty, forty, fifty, one hundred, etc. The term "at least one" can be extended up to a maximum of 100 or 1000 or more depending on the term to which it is attached; in addition, since larger upper limits can also yield satisfactory results, the quantities of 100 / 1000 are not considered limiting. Further, the use of the term "at least one of X, Y and Z" is understood to include X alone, Y alone and Z alone, as well as any combination of X, Y and Z. The use of ordinal terms (i.e., "first", "second", "third", "fourth", etc.) is for the sole purpose of distinguishing between two or more items and is not intended to imply, for example, an arrangement or order or importance or order of addition of one item as compared to another item.

[0016] The use of the term "or" in the claims is employed to mean inclusive "and / or" unless it is expressly stated that it refers only to alternatives or the alternatives are not mutually exclusive. For example, the condition "A or B" is satisfied by any one of the following: A and not B (where A is included and B is not), not A and B (where A is not included and B is included), and both A and B (where both A and B are included).

[0017] As used herein, any reference to "one embodiment", "an embodiment", "some embodiments", "one example", "for example", or "an example" means that a particular element, function, structure, or characteristic described in connection with that embodiment is included in at least one embodiment. For example, the phrases "in some embodiments" or "one example" may appear throughout this specification, but not necessarily all refer to the same embodiment. Further, any reference to one or more embodiments or examples should be construed as not limiting the claims in any way.

[0018] Throughout this application, the term "about" is used to indicate that a value includes the inherent error variability of the method used to determine that value for a composition / apparatus / device, or the variability that exists between the subjects being studied. For example, and not by way of limitation, when the term "about" is utilized, the indicated value can vary by ±20%, or ±15%, or ±12%, or ±11%, or ±10%, or ±9%, or ±8%, or ±7%, or ±6%, or ±5%, or ±4%, or ±3%, or ±2%, or ±1% from the specified value, as appropriate for the method disclosed such that such variability is understood by one of ordinary skill in the art.

[0019] As used in this specification and the claims, the words "comprising" (and any form of "comprising", such as "comprise" and "comprises"), "having" (and any form of "having", such as "have" and "has"), "including" (and any form of "including", such as "includes" and "include"), or "containing" (and any form of "containing", such as "contains" and "contain") are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0020] As used herein, the term "or combinations thereof" refers to all permutations and combinations of the items listed prior to this term. For example, "A, B, C, or combinations thereof" is intended to include at least one of A, B, C, AB, AC, BC, or ABC, and, where order is important in a particular context, also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB. Continuing with this example, combinations containing repetitions of one or more of the items or terms such as BB, AAA, AAB, BBC, AAABCCCC, CBBAAA, CABABB, etc. are also expressly included. Those skilled in the art will typically understand that there is no limit on the number of items or terms in any combination, unless it is apparent from the context that this is not the case.

[0021] As used herein, the term "substantially" means that the event or situation described subsequently occurs completely or occurs to a significant extent or degree. For example, when related to a particular event or situation, the term "substantially" means that the event or situation described subsequently occurs for at least 80% of the time, or at least 85% of the time, or at least 90% of the time, or at least 95% of the time. The term "substantially adjacent" can mean that two items are 100% adjacent to each other, or that two items are very close to each other but not 100% adjacent to each other, or that a part of one of the two items is not 100% adjacent to the other item but is very close to the other item.

[0022] As used herein, the phrases "associated with" and "coupled to" include that two parts are directly associated / coupled with each other and that two parts are indirectly associated / coupled with each other. Non-limiting examples of association / coupling include, for example, one part covalently bonding to another part either directly or via a spacer group, one part non-covalently bonding to another part directly or through a specific binding pair member that is bonded to that part, for example, incorporating one part into another part by dissolving or synthesizing one part in another part, and coating one part on another part.

[0023] As used herein, the terms "analog" and "derivative" are used interchangeably and refer to substances that contain the same basic carbon skeleton as a given compound and the carbon functionality in its structure, but may also contain one or more substitutions thereto. The term "substitution" as used herein is understood to refer to replacement with a residue R of at least one substituent on the compound. In certain non-limiting embodiments, R is selected from H, hydroxy, thiol, fluoride, chloride, bromide or iodide halides, C1-C4 compounds selected from one of the following: linear, branched, or cyclic, optionally substituted alkyl, and linear, branched, or cyclic alkenyl, where the optional substituents are one or more alkenylalkyl, alkynylalkyl, cycloalkyl, cycloalkenylalkyl, arylalkyl, heteroarylalkyl, heterocycloalkyl, optionally substituted heterocycloalkenylalkyl, arylcycloalkyl, and arylheterocycloalkyl, each of which is optionally substituted, where the optional substituents are one or more alkenylalkyl, alkynylalkyl, cycloalkyl, cycloalkenylalkyl, arylalkyl, alkylaryl, heteroarylalkyl, heterocycloalkyl, optionally substituted heterocycloalkenylalkyl, arylcycloalkyl, and arylheterocycloalkyl, phenyl, cyano, hydroxy, alkyl, aryl, cycloalkyl, cyano, alkoxy, alkylthio, amino, -NH(alkyl), -NH(cycloalkyl)2, carboxy and -C(O)-alkyl. -lalkyl, heteroarylalkyl, heterocycloalkyl, optionally substituted heterocycloalkenylalkyl, arylcycloalkyl, and arylheterocycloalkyl, each of which is optionally substituted, where the optional substituents are one or more alkenylalkyl, alkynylalkyl, cycloalkyl, cycloalkenylalkyl, arylalkyl, alkylaryl, heteroarylalkyl, heterocycloalkyl, optionally substituted heterocycloalkenylalkyl, arylcycloalkyl, and arylheterocycloalkyl, phenyl, cyano, hydroxy, alkyl, aryl, cycloalkyl, cyano, alkoxy, alkylthio, amino, -NH(alkyl), -NH(cycloalkyl)2, carboxy and -C(O)-alkyl.

[0024] As used herein, the term "sample" is understood to include any type of biological sample that can be utilized in accordance with the present disclosure. Examples of biological samples of fluids that can be utilized include, but are not limited to, whole blood or any portion thereof (i.e., plasma or serum), urine, saliva, sputum, cerebrospinal fluid (CSF), skin, intestinal fluid, intraperitoneal fluid, cyst fluid, sweat, interstitial fluid, extracellular fluid, tears, mucus, bladder washings, semen, feces, pleural fluid, nasopharyngeal fluid, combinations thereof, and the like.

[0025] As used herein, the term "specific binding partner" or "sbp", which is used particularly (but not by way of limitation) in the terms "biotin-specific binding partner" or "target analyte-specific binding partner", is understood to refer to any molecule that can specifically associate with biotin or a target analyte, respectively. For example, but not by way of limitation, the binding partner can be an antibody, receptor, ligand, aptamer, molecularly imprinted polymer (i.e., inorganic matrix), combinations or derivatives thereof, and any other molecule capable of specific binding to biotin or a target analyte, respectively.

[0026] The term "antibody" is used herein in its broadest sense and refers to, for example, intact monoclonal and polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), antibody fragments and conjugates thereof that exhibit the desired biological activity of analyte binding (including, but not limited to, Fab, Fab‘, F(ab‘)2, Fv, scFv, Fd, diabody, single-chain antibodies, and other antibody fragments and conjugates thereof that retain at least a portion of the variable region of an intact antibody), antibody surrogate proteins or peptides (i.e., engineered binding proteins / peptides), and combinations or derivatives thereof. Antibodies can be of any type or class (e.g., IgG, IgE, IgM, IgD, and IgA) or subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2).

[0027] As used herein, the term "hapten" refers to a small proteinaceous or non-protein antigenic determinant (or "epitope") that can be recognized by a target analyte-specific binding partner such as an antibody (but not limited to). As used herein, the term "polyhapten" is understood to refer to a synthetic molecule that contains multiple epitopes / antigenic determinants attached thereto.

[0028] An "analyte" is a macromolecule that can be recognized by an analyte-specific binding partner such as an antibody (but not limited to). Both the analyte and the hapten contain at least one antigenic determinant or "epitope", which is the region of the antigen or hapten that binds to the analyte-specific binding partner (i.e., the antibody). Typically, the epitope of a hapten is the entire molecule.

Brief Description of the Drawings

[0029]

Figure 1

Figure 2

Figure 3

Modes for Carrying Out the Invention

[0030] In one embodiment, a hapten trap is added to the reagent formulation to remove interfering haptens without the involvement of assay components that generate assay signals.

[0031] The hapten trap may be a soluble or solid cage. If the trap has pores, it needs to have a pore size such that only free biotin can enter the pores, but large molecules such as biotin antibodies cannot enter. Alternatively, or additionally, the trap can be charged so that the hapten is attracted to the cage. The interior of the cage needs to be able to capture interfering hapten molecules (biotin or fluorescein) by any of molecular imprinting such as hydrogen bonding, hydrophobic interaction, or specific binding partners.

[0032] Thus, the molecular cage captures the interfering hapten molecules but not the hapten-Ab conjugate, effectively reducing the accessibility of the hapten to the binding partner within the assay component of the hapten.

[0033] An example of a molecular cage is cyclodextrin. Cyclodextrin can be chemically modified to better exclude hapten conjugates. Other examples can include synthesizing an aptamer, avidin, streptavidin, or a solid (buoyant) support or soluble complex having an inner surface coated with an anti-FITC antibody. This solid support or complex needs to have a sufficiently small molecular size exclusion such that only the target hapten can enter the cage, but a large biotinylated antibody cannot enter. See Figure 1.

[0034] Accordingly, the present invention is a molecular trap for reducing free hapten interference in an assay, the molecular trap comprising: a molecular cage including a shell surrounding a cavity having the property of selectively capturing and retaining unconjugated or free hapten in an assay solution. The shell of the molecular cage can be selected from the group consisting of a cyclodextrin shell and a molecular imprint specific binding partner shell of a hapten. In certain embodiments, the property of the shell is selective permeability to free hapten, or a selective inhibitory force against relatively large assay components such as an assay conjugate of a hapten, or the shell may be a combination of both properties.

[0035] In some embodiments, the molecular cage further comprises a coating on the shell, the coating being selectively permeable to free hapten and impermeable to relatively large assay components present in the assay solution. Examples of relatively large assay components include an assay conjugate of a hapten, an assay specific binding partner (sbp) of a hapten, an assay conjugate of an sbp, and other assay molecules having a molecular weight greater than about 1000 daltons or more preferably greater than about 2000 daltons. In some embodiments, the coating comprises one or more of bovine serum albumin, dextran aldehyde, amino dextran, and ionically charged moieties.

[0036] In some embodiments, the cavity property of the molecular cage has a portion that selectively interacts with free hapten, selectively receives and retains free hapten, and has a molecular weight greater than about 1000 daltons or more preferably greater than about 2000 daltons present in the assay solution It includes one or both of the cavity size dimensions useful for preferentially excluding assay molecules of a certain amount. The cavity can include an internal specific binding moiety that selectively retains the received free hapten. The cavity properties include a cavity opening sized to selectively receive the free hapten and preferentially exclude assay molecules with a molecular weight greater than about 1000 Daltons present in the assay solution, and one or both of an internal cavity interaction with the free hapten including one or more of hydrogen bonding, van der Waals forces, polar bonds, hydrophilic interactions, hydrophobic interactions, ionic attractions, lock-and-key interactions.

[0037] Typically, the free hapten in the assay solution is about one-tenth the molecular weight of the hapten assay conjugate present in the assay solution, and the cavity properties of the molecular cage include a cavity opening with a molecular weight exclusion limit greater than the molecular weight of the free hapten and an internal cavity portion having a selective interaction with the free hapten.

[0038] The shell may be a cyclodextrin shell having a cyclodextrin selected from the group consisting of alpha-cyclodextrin, beta-cyclodextrin, and gamma-cyclodextrin.

[0039] The free hapten can be selected from the group consisting of free biotin and free fluorescein. The specific binding partner for biotin of the molecular imprint specific binding partner shell can include one or more of streptavidin, avidin, and traptavidin. When the free hapten is free fluorescein, the specific binding partner for fluorescein of the molecular imprint specific binding partner shell includes an anti-fluorescein antibody.

[0040] In another embodiment, a hapten trap is added to the reagent formulation to minimize interfering haptens from its binding partner without the involvement of assay components that generate an assay signal and without generating extra absorbance that could interfere with the true assay signal.

[0041] The trap has the following characteristics: a) It has a molecular structure soluble in aqueous solution, rather than being a particle or solid phase agent. b) It must bind only to free hapten and not to large molecules such as hapten-Ab conjugates. The functions described in a) and b) are sufficient to constitute a molecular hapten trap. Alternatively, the following functions are also considered sufficient for a molecular hapten trap: c) Due to the slow off-rate of binding, the bound hapten is effectively confined to a given position. The advantage of this function is that the captured (or confined) biotin does not readily dissociate and does not compete with the conjugate hapten for the assay signal that generates the hapten-binding partner. d) It is necessary to slow down the on-rate of binding so that the conjugate hapten preferentially binds to the hapten-binding partner that generates the assay signal. The molecular hapten trap functions adequately either alone or in combination with either function a) and b), or function c) and d).

[0042] Taking biotin-avidin (or streptavidin) as an example, avidin or streptavidin can be chemically modified with dexal or other spacer molecules via covalent bonds to form a surface layer that allows only free biotin to permeate, but not the biotin moiety of biotinylated antibodies. Traptavidin is a molecule with an on-rate of 1 / 2 and an off-rate of 1 / 10 for biotin binding, and pre-incubating with a sample containing biotin results in an excellent biotin trap. One proposed assay example for the LOCI PCT assay is as follows: 1) Incubate a sample containing biotin with a capture Ab-coated chemibead reagent (e.g., streptavidin or traptavidin modified with dextran or unmodified traptavidin) containing a soluble molecule biotin trap. Free biotin in the sample binds to the biotin trap. 2) Add a biotinylated antibody, followed by Sensibeads coated with streptavidin. If native (unmodified) traptavidin is used in step 1), Sensibeads coated with streptavidin need to be added immediately after the addition of the biotinylated antibody. This is to ensure that the biotinylated antibody preferentially binds to the streptavidin coated on the Sensibeads rather than traptavidin, which has a slower on-rate for biotin binding than streptavidin. When using a surface-modified traptavidin that does not bind to conjugated biotin, the bound free biotin molecules from the sample are trapped by the traptavidin molecules and thus do not compete with the biotinylated antibody for binding to the streptavidin Sensibeads.

[0043] In the field of medical diagnostics, many different forms of assay techniques are utilized. An example of an assay used commercially is the Luminescent Oxygen Channeling Assay (LOCI®) technology. For example, the advanced chemiluminescent assay of LOCI® is described in U.S. Patent No. 5,340,716 (Ullman et al.), the entire content of which is expressly incorporated herein by reference. The currently available LOCI® technology is highly sensitive and uses several reagents. In particular, in the LOCI® assay, two of these reagents (referred to as "Sensibeads" and "Chemibeads") need to be held by other specific binding partner assay reagents such that the Sensibeads and Chemibeads are held in very close proximity to each other to achieve a signal. When exposed to light of a particular wavelength, the Sensibeads release singlet oxygen, and if the two beads are in very close proximity, the singlet oxygen is transferred to the Chemibeads; this results in a chemical reaction and, as a result, the Chemibeads emit light that can be measured at a different wavelength.

[0044] Certain non-limiting examples of chemiluminescent compounds and photosensitizers that can be utilized in accordance with the present disclosure are described in U.S. Patent No. 5,340,716 (Ullman et al.), the entire content of which is expressly incorporated herein by reference.

[0045] Accordingly, the present invention includes a molecular trap for reducing free hapten interference in an assay, the molecular trap comprising: a molecular structure soluble in an assay solution for selectively binding to a free hapten, the molecular structure comprising a modified specific binding partner (sbp) for the free hapten. The modified sbp includes one or more of a dextran aldehyde component, a conjugated steric hindrance polymer, and a slower specific free hapten binding off-rate characteristic than other free hapten-specific binding partner (sbp) assay reagents.

[0046] Such a molecular structure can further include a coating, the coating including one or more of a selectively permeable material for the free hapten, an ionic charge for attracting the free hapten in the assay solution and repelling other assay molecules, and a polarity promoting one or both of the selective retention of the free hapten in the assay solution and the steric repulsion of other assay molecules.

[0047] The coating can include a protein or peptide such as bovine serum albumin, a polymer such as dextran aldehyde or amino dextran, a compound such as ethylenediamine, tetraethylenepentamine, an ionically charged moiety, a hydrophobic moiety (sulfo-N-hyd roxysuccinimide acetate), etc. The specific free hapten binding off-rate characteristic of the modified sbp is slower than the specific free hapten binding on-rate characteristic of the modified sbp.

[0048] In some embodiments, the free hapten is free biotin, the modified sbp is traptavidin having a slower specific free hapten binding off-rate than other free hapten sbps in the assay solution, and the other free hapten sbps in the assay solution are selected from streptavidin and avidin.

[0049] The modified sbp may include one or both of a dextran aldehyde component and a relatively large steric hindrance polymer with the assay components and assay conjugates in the assay solution, which selectively interfere with the binding of the modified sbp to the assay components and assay conjugates in the assay solution.

[0050] In some embodiments, the free hapten in the assay solution is selected from the group consisting of free biotin and free fluorescein, wherein the modified sbp for free biotin is one of modified streptavidin, modified avidin, and traptavidin, and wherein the modified sbp for free fluorescein is modified anti-fluorescein.

[0051] The modified sbp may be a genetically engineered sbp that includes a steric hindrance polymer conjugated to an amino acid of the modified sbp adjacent to the specific binding site of the free hapten, and the steric hindrance polymer interferes with the specific binding of the hapten-assay conjugate corresponding to the free hapten.

[0052] The steric hindrance polymer is selected from the group consisting of amino dextran and bovine serum albumin.

[0053] The hapten-assay conjugate is selected from the group consisting of one or more of a hapten-antibody conjugate, a hapten-antigen conjugate, a hapten-labeled enzyme conjugate, a hapten-analyte under test conjugate, a hapten-labeled conjugate, and a hapten-receptor conjugate.

[0054] Creating a hapten trap at the molecular level that is soluble in the aqueous reaction mixture should enable a much broader range of applications than hapten trap particles. The biotin lock-in mechanism provided by the traptavidin-like molecule greatly reduces the possibility that free biotin dissociates from the hapten trap and competes with the biotinylated antibody for the binding sensitive beads. Thirdly, due to the slower binding to traptavidin, when native (unmodified) traptavidin is used, the biotinylated antibody can preferentially bind to the sensitive beads.

[0055] In yet another embodiment, the hapten trap is a genetically engineered hapten-binding protein (free hapten trap). The genetically engineered hapten trap is added to the reagent formulation to sequester interfering free hapten from its binding partner without the involvement of assay components that generate an assay signal and without generating additional absorbance that could interfere with the assay signal. The production of the hapten trap includes the following: a) Site-directed mutagenesis changes amino acid residues near the biotin-binding site, enabling conjugation of another protein or polymer near the binding site, creating steric hindrance for large biotinylated antibodies rather than smaller free haptens to enter the binding site. b) Other genetic engineering techniques produce mutations similar to those in a). c) Genetically engineered streptavidin still binds free biotin with high affinity. d) Conjugate a protein (such as BSA) or polymer to the modified streptavidin to complete the creation of the free biotin trap. e) Genetically engineered by site-directed mutagenesis to change one amino acid residue to a unique amino acid not present in the current sequence. For example, change the amino acid residue at the junction between {37-{37, {35-{36, or {33-{34 (see Figure 2) to methionine (Met or M). Since the mutation is near the binding site, when the protein is conjugated using SMCC (maleimide chemistry) and / or other polymers, steric hindrance occurs with the biotinylated antibody reagent (see Figure 3).

[0056] Accordingly, the present invention includes a molecular trap for reducing free hapten interference in an assay, the molecular trap comprising a molecular complex soluble in an assay solution for selectively providing competitive specific binding of free hapten in the molecular complex. The molecular complex includes a conjugate comprising a hapten analog, a steric hindrance polymer relatively larger than the hapten analog, and a linking group for providing a flexible linker connection between the hapten analog and the steric hindrance polymer; and further, an anti-hapten specific binding partner (sbp) interconnected with the conjugate. The hapten analog has weaker specific binding properties to the anti-hapten sbp than the free hapten, the flexible linker connection provides freedom of movement between the hapten analog and the specific binding site on the anti-hapten sbp of the hapten, and the steric hindrance polymer prevents the hapten-assay conjugate in the assay solution from approaching the specific binding site.

[0057] In the above molecular trap, the hapten may be biotin, the hapten analog is selected from the group consisting of 4'-hydroxyazobenzene-2-carboxylic acid (HABA) and 2-iminobiotin, and the anti-hapten sbp is selected from streptavidin, avidin, and traptavidin.

[0058] Weaker specific binding properties of hapten analogs compared to free haptens may include pH-dependent specific binding. The steric hindrance polymer of the molecular trap can be selected from proteins or peptides such as bovine serum albumin, polymers such as dextran aldehyde or amino dextran, compounds such as ethylenediamine, tetraethylenepentamine, ionically charged moieties, or hydrophobic moieties such as sulfo-N-hydroxysuccinimide acetate.

[0059] The present invention is a method for reducing interference by free haptens in an assay, comprising: combining an assay component, and a sample from a patient having an analyte and excess free hapten under test to form an assay solution, the assay component comprising a specific binding pair comprising a hapten and a relatively large anti-hapten, respective assay conjugates of the hapten and the anti-hapten, and a molecular trap selective for the free hapten; selectively retaining the free hapten in the assay solution using the molecular trap; and wherein the molecular trap comprises one or a mixture of molecular structures including a molecular cage having a shell surrounding a cavity, a molecular complex, and a modified anti-hapten specific binding partner (sbp), and the modified anti-hapten sbp comprises one or more of a dextran aldehyde component, a bound steric hindrance polymer, and a slower specific free hapten binding off-rate characteristic than other anti-hapten specific binding partner (sbp) assay components. A method is included.

[0060] In such a method, the specific free hapten binding off rate characteristic of the modified anti-hapten sbp is slower than the specific free hapten binding on rate characteristic of the modified anti-hapten sbp.

[0061] Furthermore, in such a method, the molecular complex may be a hapten analog conjugated to a steric hindrance polymer using an anti-hapten sbp interconnected with a flexible linker and a hapten analog conjugate, the hapten analog having specific binding properties that are the same as or weaker than those of the free hapten to the anti-hapten sbp, the steric hindrance polymer preventing the hapten-assay conjugate in the assay solution from approaching the specific hapten binding site on the anti-hapten sbp, and the free hapten in the assay solution preferably binding to the anti-hapten sbp specific binding site.

[0062] In such a method, the assay conjugate of the hapten and the assay conjugate of the anti-hapten are each selected individually from the group of assay components consisting of antibodies, antigens, analytes under test, labels, labeled enzymes, receptors, and combinations of two or more of this group.

Claims

1. A molecular trap for reducing free hapten interference in an assay, comprising: a molecular structure soluble in an assay solution for selectively binding a free hapten, the molecular structure comprising a modified specific binding partner (sbp) for the free hapten, the modified sbp comprising a dextran aldehyde component, a bound steric hindrance polymer, and one or more of the specific free hapten binding off-rate characteristics slower than other free hapten specific binding partner (sbp) assay reagents, said molecular trap.

2. The molecular structure further comprises a coating, the coating comprising one or more of a selectively permeable material for the free hapten, an ionic charge for attracting the free hapten in the assay solution and repelling other assay molecules, and a polarity for promoting one or both of selective retention of the free hapten in the assay solution and steric repulsion of other assay molecules, the molecular trap according to claim 1.

3. The coating comprises one or more of a protein or peptide such as bovine serum albumin, a polymer such as dextran aldehyde or aminodextran, a compound such as ethylenediamine, tetraethylenepentamine, an ionically charged moiety, a hydrophobic moiety, the molecular trap according to claim 2.

4. The specific free hapten binding off-rate characteristic of the modified sbp is slower than the specific free hapten binding on-rate characteristic of the modified sbp, the molecular trap according to claim 1.

5. The free hapten is free biotin, the modified sbp comprises traptavidin having a specific free hapten binding off-rate slower than other free hapten sbps in the assay solution, and the other free hapten sbps in the assay solution are selected from streptavidin and avidin, the molecular trap according to claim 1.

6. The modified sbp comprising one or both of the dextran aldehyde component and the steric hindrance polymer is for selectively interfering with the binding of an assay component and an assay conjugate in the assay solution that are relatively larger than the free hapten, the molecular trap according to claim 1.

7. The free hapten in the assay solution is selected from the group consisting of free biotin and free fluorescein, the modified sbp for free biotin is one of modified streptavidin, modified avidin, and traptavidin, and the modified sbp for free fluorescein is modified anti-fluorescein. The molecular trap according to claim 1.

8. The modified sbp is a genetically engineered sbp comprising a steric hindrance polymer conjugated to an amino acid of the modified sbp adjacent to the specific binding site of the free hapten, and the steric hindrance polymer prevents specific binding of the hapten-assay conjugate corresponding to the free hapten. The molecular trap according to claim 1.

9. The steric hindrance polymer is selected from the group consisting of amino dextran and bovine serum albumin. The molecular trap according to claim 8.

10. The hapten-assay conjugate is selected from the group consisting of one or more of a hapten-antibody conjugate, a hapten-antigen conjugate, a hapten-labeled enzyme conjugate, a hapten-analyte under test conjugate, a hapten-label conjugate, and a hapten-receptor conjugate. The molecular trap according to claim 8.

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