Methods and compositions for removing biotin interference from assays using cyclodextrin traps

Abstract

Methods and compositions are provided that remove or reduce biotin interference from certain assays. [Solution] A hapten trap is added to the reagent formulation to remove interfering haptens without involving the assay components that generate the assay signal. The hapten trap can be a soluble or solid cage. If the trap has pores, it must have a pore size that allows only free biotin to enter the pores but not larger molecules such as biotin antibodies. Alternatively, or in addition, the trap can be charged so that the hapten is attracted to the cage. The interior of the cage must be able to capture the interfering hapten molecule (biotin or fluorescein) either by hydrogen bonding, hydrophobic interactions, or a molecular imprint such as a specific binding partner.

Description

[Technical Field]

[0001] This application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Patent Application No. 62 / 735,909, filed September 25, 2018, the entire contents of which are expressly incorporated herein by reference.

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

[0003] Modern in vitro clinical diagnostics utilize a variety of methods for detecting analytes in samples. One form of diagnostic method, immunoassays, utilize one or more specific binding species. Typical examples are sandwich immunoassays, in which two specific 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 the specific binding species. In competitive immunoassays, the antigen is often also called a hapten or ligand. One of the specific 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, fluorescent, or luminescent compound), or a particle (magnetic or latex). This label allows for detection of the analyte of interest by various detection methods corresponding to the label employed. In competitive assays, either the specific binding species or the analyte analog can carry the label. The other specific binding species is often associated with a solid or suspendable substrate ("solid phase") by covalent bonding or adsorption. Alternatively, the specifically binding species can be linked to a first member of a second binding pair (e.g., biotin), while a second member of the second binding pair (e.g., streptavidin) is attached to a solid phase, allowing the specifically binding species to bind to the solid phase via a 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 solid supports (e.g., streptavidin to biotin and anti-FITC antibody to fluorescein) provides a convenient method for immobilizing hapten-Ab or hapten-drug on solid surfaces. Because the binding strength between biotin and streptavidin or avidin is one of the highest constants for biomolecules, biotin is most often used as a ligand, with streptavidin as its specific binding partner.

[0005] It is important for the stability and reproducibility of diagnostic assays that the binding ability of solid-phase-bound species to their binding partners is not compromised over time. This can lead to reduced reliability and reduced assay sensitivity. One mechanism that can lead to such failure (manifested as instability) is leaching of some of the solid-phase-attached species into the surrounding medium. Free species compete with the solid-phase-attached species for binding to the target and typically have a significant kinetic advantage due to their rapid diffusion. Therefore, it is advantageous to maintain the amount of free species competing with the solid-phase-attached species in the reagent constant, preferably very close to zero. This should enable sensitive detection of analytes in a stable and reproducible manner. A preferred method for eliminating free species is to find a binding method that excludes dissociation. Covalent binding may be the method of choice due to its greater binding strength, as opposed to association via adsorption. However, in many cases, this is impossible or impractical for various reasons.

[0006] Biotin has found use as a dietary supplement. Biotin as a dietary supplement is intended, for example, to promote healthy hair and nail growth and treat other medical conditions. Therefore, the amount of biotin in serum can be very high. Because 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 the assay signal. Therefore, if biotin is present in free form in the sample solution, it can occupy binding sites, resulting in erroneous test results. This is particularly serious for patients receiving high doses of biotin. Amounts of biotin exceeding 30 ng / ml in a sample are considered to already produce false results. Patients treated with biotin may experience serum values ​​of up to 180 ng / ml, or even up to 1500 ng / ml, with long-lasting values ​​of approximately 70 ng / ml.

[0007] One method for mitigating such interference involves the use of preformed reagents, i.e., pre-bound biotinylated assay components with a streptavidin-coated solid support during reagent manufacture. Due to the tight binding and slow off-rate between streptavidin and biotin, displacement of already bound biotin from streptavidin with biotin in the incoming patient sample is not a primary method.

[0008] The second method is to increase the number of streptavidin binding sites on the solid support, so that there are extra binding sites available for biotin molecules in the sample in addition to the biotinylated assay components. The third method is to combine both of the above. However, neither of the above truly solves the problem of biotin interference unless the use of biotin-streptavidin as the active assay component is completely avoided.

[0009] One significant problem with all of the above solutions is that the assay components involved in interference prevention can easily affect the magnitude of the assay signal itself. In such cases, the assay components used are not optimal for detecting the intended analyte. Some examples are provided in U.S. Pat. No. 5,629,493.

[0010] Another method is disclosed in U.S. Patent No. 5,929,999, which discloses the use of polymer particles with a biotin-binding core and a coating layer of protein, carbohydrate, or copolymer to filter free biotin, but not biotin conjugated to larger molecules.

[0011] While this approach can be effective in some assay formats, the introduction of particles can generate extra absorbance that can interfere with the assay signal. [Prior art documents] [Patent documents]

[0012] [Patent Document 1] US8252605 [Patent Document 2] U.S. Patent No. 5,212,063 Summary of the Invention [Problem to be solved by the invention]

[0013] It is therefore an object of the present invention to provide an improved method for reducing such substances in an assay so that they do not interfere with the detection method. Thus, there is a need for a method for removing biotin so that it does not interfere with the test. [Means for solving the problem]

[0014] The use of the terms "a" or "an" in the claims and / or specification when used in conjunction with the term "comprising" means "one," but is also consistent with the meanings of "one or more," "at least one," and "one or more than one." Thus, the terms "a," "an," and "the" include plural referents unless the context clearly indicates otherwise. Thus, for example, 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 quantity of compounds. The term "plurality" refers to "two or more."

[0015] Use of the term "at least one" is understood to include 1 and any quantity greater than 1, including, but not limited to, 2, 3, 4, 5, 10, 15, 20, 30, 40, 50, 100, etc. The term "at least one" can extend up to 100 or 1000 or more, depending on the term with which it is attached; in addition, a quantity of 100 / 1000 is not considered limiting, as higher upper limits may also provide satisfactory results. Furthermore, 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. Use of ordinal terminology (i.e., "first," "second," "third," "fourth," etc.) is for the purpose of distinguishing two or more items only and is not intended to imply, for example, an order or sequence or importance or order of addition of one item compared to another.

[0016] The use of the term "or" in the claims is intended to mean an inclusive "and / or" unless expressly indicated to refer to only alternatives or unless the alternatives are mutually exclusive. For example, the condition "A or B" satisfies any one of the following: A and (including A) and not B and (excluding B), not A and (excluding A) and B and (including B), and both A and B and (including both A and B).

[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, feature, structure, or characteristic described in connection with that embodiment is included in at least one embodiment. For example, appearances of the phrases "in some embodiments" or "one example" in various places herein do not necessarily all refer to the same embodiment. Moreover, any reference to one or more embodiments or examples should not be construed as limiting the scope of the claims.

[0018] Throughout this application, the term "about" is used to indicate that a value includes the inherent variation of error for the composition / apparatus / device, the method being used to determine the value, or the variation that exists between study subjects. For example, and not as a limitation, when the term "about" is used, the indicated value may 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 such variations are appropriate for practicing the disclosed methods and would be understood by one of ordinary skill in the art.

[0019] As used in this specification and 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 preceding the 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 this example, combinations containing repeats of one or more items or terms, such as BB, AAA, AAB, BBC, AAABCCCC, CBBAAA, CABABB, etc., are also expressly included. Those of skill in the art will understand that there is typically no limit to the number of items or terms in any combination, unless otherwise clear from the context.

[0021] As used herein, the term "substantially" means that the subsequently described event or circumstance occurs perfectly or to a substantial extent or degree. For example, when referring to a particular event or circumstance, the term "substantially" means that the subsequently described event or circumstance occurs 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 portion of one of two items is very close to the other item but not 100% adjacent to the other item.

[0022] As used herein, the phrases "associated with" and "coupled to" include direct association / binding of two moieties to each other and indirect association / binding of two moieties to each other. Non-limiting examples of association / binding include, for example, covalent binding of one moiety to another moiety either by direct bonding or through a spacer group, non-covalent binding of one moiety to another moiety directly or through a specific binding pair member bonded to the moiety, incorporation of one moiety into another moiety, for example by dissolving one moiety in another moiety or by synthesis, and coating one moiety onto another moiety.

[0023] As used herein, the terms "analog" and "derivative" are used interchangeably and refer to a substance that contains the same basic carbon skeleton and carbon functionalities in that structure as a given compound, but that may contain one or more substitutions thereon. As used herein, the term "substituted" is understood to refer to the replacement of at least one substituent on the compound with a residue, R. In certain non-limiting embodiments, R can include H, hydroxy, thiol, a halide selected from fluoride, chloride, bromide, or iodide, a C1-C4 compound 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 of alkenylalkyl, alkynylalkyl, cycloalkyl, cycloalkenylalkyl, aryl, arylalkyl ... and arylheterocycloalkyl, each of which is optionally substituted, wherein the optional substituents are selected from 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), carboxy, and —C(O)-alkyl.

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

[0025] The term "specific binding partner" or "sbp," as used herein, 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 capable of specifically associating with biotin or a target analyte, respectively. For example, but not by way of limitation, a binding partner may be an antibody, a receptor, a ligand, an aptamer, a molecularly imprinted polymer (i.e., an 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 the broadest sense and refers to, for example, intact monoclonal and polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), antibody fragments and conjugates thereof (including, but not limited to, Fab, Fab', F(ab')2, Fv, scFv, Fd, diabodies, 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 that exhibit the desired biological activity of analyte binding. Antibodies may 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] The term "hapten," as used herein, refers to a small proteinaceous or non-proteinaceous antigenic determinant (or "epitope") capable of being recognized by a target analyte-specific binding partner, such as (but not limited to) an antibody. The term "polyhapten," as used herein, is understood to refer to a synthetic molecule that contains multiple epitopes / antigenic determinants attached thereto.

[0028] An "analyte" is a macromolecule capable of being recognized by an analyte-specific binding partner, such as (but not limited to) an antibody. Both analytes and haptens 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., an antibody). Typically, the epitope of a hapten is the entire molecule. [Brief explanation of the drawings]

[0029] [Figure 1] FIG. 1 is a schematic diagram of various cyclodextrin traps. [Figure 2] FIG. 1 is a schematic diagram of the amino acid structures of avidin and streptavidin. [Figure 3] FIG. 1 is a schematic representation of maleimide chemistry for possible mutations. DETAILED DESCRIPTION OF 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 an assay signal.

[0031] The hapten trap can be a soluble or solid cage. If the trap has a pore, it must have a pore size that allows only free biotin to enter the pore, but not larger molecules such as biotin antibodies. Alternatively, or in addition, the trap can be charged so that the hapten is attracted to the cage. The interior of the cage must be able to capture the interfering hapten molecule (biotin or fluorescein) either by hydrogen bonding, hydrophobic interactions, or molecular imprints such as specific binding partners.

[0032] Thus, the molecular cage traps the interfering hapten molecule but not the hapten-Ab conjugate, effectively reducing the accessibility of the hapten to its binding partner within the assay components.

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

[0034] Thus, the present invention provides a molecular trap for reducing free hapten interference in an assay, the molecular trap comprising: a molecular cage comprising a shell surrounding a cavity having properties for 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 the hapten. In certain embodiments, the shell's properties are selective permeability to free hapten or selective deterrence to larger assay components such as assay conjugates of the hapten, or the shell may have 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. The relatively large assay components can include assay conjugates of hapten, assay specific binding partners (sbp) of hapten, assay conjugates of 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, aminodextran, and an ionically charged moiety.

[0036] In some embodiments, the cavity characteristics of the molecular cage include a portion that has a selective interaction with a free hapten and a portion that selectively accepts and retains the free hapten and is compatible with molecules of molecular weight greater than about 1000 daltons, or more preferably greater than about 2000 daltons, present in the assay solution. The cavity may comprise one or both of a cavity size dimension that serves to preferentially exclude a quantity of assay molecules. The cavity may comprise an internal specific binding moiety that selectively retains the received free hapten. The cavity characteristics may include a cavity opening that is restricted in size to selectively receive the free hapten and preferentially exclude assay molecules with a molecular weight of more than about 1000 daltons present in the assay solution, and an internal cavity interaction with the free hapten that includes one or more of hydrogen bonding, van der Waals forces, polar bonding, hydrophilic interactions, hydrophobic interactions, ionic attraction, and lock-and-key interactions.

[0037] Typically, the free hapten in the assay solution is about one-tenth the molecular weight of the assay conjugate of the hapten present in the assay solution, and the cavity characteristics of the molecular cage include a cavity opening whose molecular weight exclusion limit is greater than the molecular weight of the free hapten and an internal cavity portion that has 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 their binding partners without the involvement of assay components that generate the assay signal and without generating excess absorbance that could interfere with the true assay signal.

[0041] Traps have the following characteristics: a) It is not a particle or solid phase agent, but a molecular structure that is soluble in aqueous solution. b) It should only bind to free haptens but not to larger molecules such as hapten-Ab conjugates. The functions described in a) and b) are sufficient to constitute a molecular hapten trap. Alternatively, the following features are also considered sufficient for molecular hapten trapping: c) The binding off-rate is slow, so the bound hapten is actually locked in place. The advantage of this feature is that the captured (or trapped biotin) does not readily dissociate and compete with the conjugated hapten for the assay signal that the hapten-binding partner generates. d) The on-rate of binding must be slow so that the conjugated hapten preferentially binds to the assay signal-generating hapten binding partner. The molecular hapten trap satisfactorily functions with either functions a) and b) or functions c) and d), either alone or in combination.

[0042] Taking biotin-avidin (or streptavidin) as an example, avidin or streptavidin can be chemically modified with dextran or other spacer molecules via covalent bonds to form a surface layer that is permeable to free biotin but not the biotin moiety of biotinylated antibodies. Traptavidin is a molecule with a 1 / 2-fold lower on-rate and a 1 / 10-fold lower off-rate for biotin binding, and is pre-incubated with a sample containing biotin. This makes it an excellent biotin trap. One proposed assay example for the LOCI PCT assay is as follows: 1) A sample containing biotin is incubated with a capture Ab-coated chemi-bead reagent containing a soluble molecular biotin trap (e.g., dextran-modified streptavidin or traptavidin, or unmodified traptavidin). Free biotin in the sample binds to the biotin trap. 2) Add the biotinylated antibody followed by streptavidin-coated SensiBeads. If native (unmodified) traptavidin is used in step 1), the streptavidin-coated SensiBeads must be added immediately after the biotinylated antibody. This ensures that the biotinylated antibody binds preferentially to the streptavidin coated on the SensiBeads rather than to the traptavidin, which has a slower on-rate for biotin binding than streptavidin. If surface-modified traptavidin that does not bind to conjugated biotin is used, the bound but free biotin molecules from the sample are trapped by the traptavidin molecules and therefore do not compete with the biotinylated antibody for binding to the streptavidin SensiBeads.

[0043] Many different forms of assay technology are utilized in the field of medical diagnostics. One example of a commercially used assay is the luminescent oxygen channeling assay (LOCI®) technology. For example, the LOCI® advanced chemiluminescent assay is described in U.S. Pat. No. 5,340,716 (Ullman et al.), the entire contents of which are expressly incorporated herein by reference. Currently available LOCI® technologies are highly sensitive and utilize several reagents. In particular, LOCI® assays require two of these reagents (called "SensiBeads" and "ChemiBeads") to be held by other specific binding partner assay reagents so that the SensiBeads and ChemiBeads are in close proximity to each other to achieve a signal. When exposed to light of a certain wavelength, the SensiBeads release singlet oxygen, which is transferred to the ChemiBeads when the two beads are in close proximity; this initiates a chemical reaction that causes the ChemiBeads to emit light at a different wavelength that can be measured.

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

[0045] Thus, the present invention includes a molecular trap for reducing free hapten interference in an assay, the molecular trap comprising: The assay reagents include a molecular structure soluble in an assay solution for selectively binding to a free hapten, the molecular structure including a modified specific binding partner (sbp) for the free hapten, the modified sbp including one or more of a dextran aldehyde moiety, an attached sterically hindered polymer, and a specific free hapten binding off-rate characteristic that is slower than other free hapten-specific binding partner (sbp) assay reagents.

[0046] Such molecular structures may further comprise a coating comprising one or more of a selectively permeable material for the free hapten, an ionic charge to attract the free hapten and repel other assay molecules in the assay solution, and a polarity to promote selective retention of the free hapten and steric repulsion of other assay molecules in the assay solution, or both.

[0047] The coating may be 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 (sulfo-N-hydroxybenzoate), or a hydroxybenzoate. The specific free hapten binding off-rate characteristics of the modified sbp are slower than the specific free hapten binding on-rate characteristics of the modified sbp.

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

[0049] The modified sbp may include one or both of a dextran aldehyde moiety and a sterically hindered polymer that selectively interferes with binding of assay components and assay conjugates in the assay solution and that is relatively larger than the free hapten.

[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 the free biotin is one of modified streptavidin, modified avidin, and traptavidin, and wherein the modified sbp for the 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, wherein the steric hindrance polymer prevents specific binding of a hapten-assay conjugate corresponding to the free hapten.

[0052] The steric hindrance polymer is selected from the group consisting of aminodextran 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 hapten traps at a molecular level that are soluble in aqueous reaction mixtures should enable a much broader range of applications than hapten trap particles. The biotin lock-in mechanism provided by traptabidine-like molecules significantly reduces the likelihood that free biotin will dissociate from the hapten trap and compete with biotinylated antibodies for Sensibiads. Third, slower binding to traptabidine allows biotinylated antibodies to preferentially bind to Sensibiads when native (unmodified) traptabidine is used.

[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 seize the interfering free hapten from its binding partner without the involvement of assay components that generate the assay signal and without generating excess absorbance that could interfere with the assay signal. Preparation of a hapten trap involves: a) Site-directed mutagenesis alters amino acid residues near the biotin binding site, allowing conjugation of another protein or polymer near the binding site, resulting in steric hindrance for the large biotinylated antibody but not the smaller free hapten to enter the binding site. b) Other genetic engineering techniques produce mutations similar to those in a). c) The engineered streptavidin still binds free biotin with high affinity. d) A protein (such as BSA) or polymer is conjugated to the modified streptavidin to complete the creation of a free biotin trap. e) Site-directed mutagenesis is used to engineer a single amino acid residue into a unique amino acid not present in the current sequence. For example, the amino acid residue at the junction between {37-{37, {35-{36, or {33-{34 (see Figure 2)} is changed to methionine (Met or M). Because the mutation is near the binding site, it creates steric hindrance to the biotinylated antibody reagent when the protein is conjugated using SMCC (maleimide chemistry) and / or other polymers (see Figure 3).

[0056] Thus, the present invention provides a molecular trap for reducing free hapten interference in an assay. The molecular trap comprises a molecular complex soluble in an assay solution that selectively provides competitive specific binding of the free hapten in the molecular complex. The molecular complex comprises a conjugate including a hapten analog, a steric hindrance polymer that is 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 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 a degree of freedom 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 accessing the specific binding site.

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

[0058] The weaker specific binding properties of the hapten analog compared to the free hapten 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 aminodextran, compounds such as ethylenediamine, tetraethylenepentamine, ionically charged moieties, or hydrophobic moieties such as sulfo-N-hydroxysuccinimide acetate.

[0059] The present invention provides a method for reducing interference from free hapten in an assay, comprising: combining assay components and a patient sample having the analyte under test and an excess of free hapten to form an assay solution, the assay components including a specific binding pair comprising a hapten and a relatively large anti-hapten, assay conjugates for each of the hapten and anti-hapten, and a molecular trap selective for the free hapten; selectively retaining free haptens in the assay solution using a molecular trap; Including, 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), wherein the modified anti-hapten sbp comprises one or more of a dextran aldehyde moiety, a conjugated steric hindrance polymer, and a specific free hapten binding off-rate characteristic that is slower than other anti-hapten specific binding partner (sbp) assay components; The method includes:

[0060] In such a method, the specific free hapten binding of the modified antihapten sbp is The kinetic profile is slower than the specific free hapten binding-on kinetic profile of the modified anti-hapten sbp.

[0061] Furthermore, in such methods, 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 the same or weaker specific binding properties as the free hapten for the anti-hapten sbp, the steric hindrance polymer preventing the hapten-assay conjugate in the assay solution from accessing 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 methods, the hapten assay conjugate and the anti-hapten assay conjugate are independently selected from the group of assay components consisting of antibodies, antigens, analytes under test, labels, labeling enzymes, receptors, and combinations of two or more of this group.

Claims

1. 1. A molecular trap suitable for reducing free hapten interference in an assay, said molecular trap being soluble in an assay solution, said molecular trap comprising: a molecular cage comprising a shell surrounding a cavity that has the property of selectively capturing and retaining unconjugated or free hapten in an assay solution; the cavity contains an internal specific binding moiety that selectively retains the received free hapten; the shell of the molecular cage is selected from the group consisting of a cyclodextrin shell and a shell comprising a hapten-specific binding partner formed by molecular imprinting; the free hapten is selected from the group consisting of free biotin and free fluorescein; and The molecular cage further comprises a coating on the shell, the coating being selectively permeable to free haptens and impermeable to assay components of greater than 1000 daltons molecular weight present in an assay solution, the assay components of greater than 1000 daltons molecular weight including a conjugate of a hapten used in the assay, a specific binding partner (sbp) of a hapten used in the assay, a conjugate of an sbp used in the assay, and other assay molecules of greater than 1000 daltons molecular weight, the coating comprising one or more of bovine serum albumin, dextran aldehyde, aminodextran, and an ionically charged moiety. The molecular trap.

2. The molecular trap of claim 1, wherein the shell properties include one or both of selective permeability to free haptens and selective deterrence to assay molecules with a molecular weight of more than 1000 daltons, including conjugates of the haptens used in the assay.

3. The molecular trap of claim 1, wherein the cavity characteristics of the molecular cage include one or both of a portion having a selective interaction with a free hapten and a cavity size dimension that serves to selectively receive and retain the free hapten and preferentially exclude assay molecules with a molecular weight of more than 1000 daltons present in the assay solution.

4. 2. The molecular trap of claim 1, wherein the cavity characteristics include one or both of a cavity opening restricted in size to selectively accept a free hapten and preferentially exclude assay molecules with a molecular weight of more than 1000 Daltons present in the assay solution, and internal cavity interactions with the free hapten including one or more of hydrogen bonds, van der Waals forces, polar bonds, hydrophilic interactions, hydrophobic interactions, ionic attractions, and lock-and-key interactions.

5. The molecular trap of claim 1, wherein the free hapten in the assay solution has a molecular weight that is one-tenth of the molecular weight of the assay conjugate of the hapten present in the assay solution, and the cavity characteristics of the molecular cage include a cavity opening whose molecular weight exclusion limit is greater than the molecular weight of the free hapten and an internal cavity portion that has a selective interaction with the free hapten.

6. 2. The molecular trap of claim 1, wherein the cyclodextrin shell is selected from the group consisting of alpha-cyclodextrin, beta-cyclodextrin, and gamma-cyclodextrin.

Images