Novel biotin-specific monoclonal antibodies and uses thereof

Abstract

This invention relates to novel biotin-specific monoclonal antibodies and their uses. The invention relates to monoclonal antibodies capable of binding to biotin. In one embodiment, the monoclonal antibody according to the invention also does not bind to the biotin portion of a biotinylated molecule, wherein the biotin portion is attached to the molecule via a carboxyl-functionalized carbon atom of the valerate portion of biotin. Methods for generating the antibodies disclosed herein are also disclosed. The monoclonal antibody according to the invention is particularly useful for methods of measuring analytes in samples, wherein (streptomycin) anti-biotin / biotin pairs are used to bind a biotinylated analyte-specific binder to a (streptomycin) anti-biotin-coated solid phase.

Description

[0001] This application is a divisional application of the international application PCT / EP2017 / 083572, filed on December 19, 2017, and filed in China with application number 201780080681.9, entitled "A novel biotin-specific monoclonal antibody and its use therein". Technical Field

[0002] This invention relates to monoclonal antibodies capable of binding to biotin. In one embodiment, the monoclonal antibody according to the invention also does not bind to the biotinylated portion of a molecule, wherein the biotinylated portion is attached to the molecule via a carboxyl-functionalized carbon atom of the valerate portion of biotin. Methods for generating the antibodies disclosed herein are also disclosed. The monoclonal antibody according to the invention is particularly useful for methods of measuring analytes in samples, wherein (streptomycin) anti-biotin / biotin pairs are used to bind a biotinylated analyte-specific binder to a (streptomycin) anti-biotin-coated solid phase. Background Technology

[0003] In living organisms and in in vitro biochemistry, biotin's primary function is as a co-substrate, required as a cofactor for enzymes with carboxyltransferase activity, such as pyruvate carboxylase and acetyl-CoA carboxylase. In bacteria, biotin attaches to biotin carboxyl carrier proteins via biotin-protein ligases. Furthermore, numerous chemical processes are known to allow biotin to covalently attach to suitable groups on virtually any molecule of interest. A common characteristic is that the carboxyl-functional carbon atom of the valerate side chain reacts with a suitable (acceptor) group on the molecule of interest, or with the reactive group of the linker, in a coupling reaction, where the linker itself is already attached to the molecule of interest, or the linker couples to the molecule once biotin has attached to it. This attachment of biotin to various chemical sites via the carboxyl-functional carbon atom of the valerate side chain is generally referred to as biotinylation.

[0004] Avidin and / or streptavidin (= (streptavidin) avidin) have exceptional affinity (K+) for biotin, respectively. a = 10 15 M -1 Avidin (STP) is one of the strongest known non-covalent interactions between proteins and ligands. It allows biotinylated molecules in complex mixtures to be specifically bound by streptomycin. For this reason, avidin and / or streptomycin are used in a wide range of immunological assays.

[0005] In addition to its strong affinity for (streptomycin) avidin, two further properties make biotin particularly suitable for labeling proteins and other macromolecules. First, biotin molecules are significantly smaller than proteins. This molecular size allows one or more biotin molecules to conjugate with the molecule of interest while minimizing the loss of its biological function. Second, the terminal carbon atom of the valerate side chain of biotin can be readily derivatized, thereby facilitating conjugation with reactive moieties on molecules of interest, particularly proteins. Notably, biotinylation does not alter the structure of the heterocyclic moiety of biotin.

[0006] After biotinylation via the terminal carbon atom of the valerate side chain, the biotin moiety retains its ability to specifically interact with (streptomycin) avidin because the portion of the biotin molecule responsible for specific interaction with the binding pocket of avidin-type proteins is unaffected by the heterocyclic structure represented by the ureidoyl ring fused with the tetrahydrothiophene ring.

[0007] The heterocyclic structure of biotin has also been targeted by existing monoclonal antibodies (mAbs) against biotin. Kohen F. et al. (Methods in Enzymology 279 (1997) 451-463) used biotinylated bovine serum albumin (BSA conjugated with N-hydroxysuccinimide biotin) as an immunogen to generate monoclonal antibodies. This literature reports the analysis of the amino acid sequence of the antigen-binding region of antibodies capable of binding the biotinylated portion of the protein. Sequence alignment was performed with homologous sequence segments of avidin and streptavidin polypeptide sequences, which have been reported to interact with the bicyclic system of biotin. Notably, similarity to the polypeptide sequences of avidin and streptavidin was identified in CDR2 and CDR3 of the biotin-specific antibody. The results are interpreted as a common pattern in the amino acid sequence of the biotin-binding pocket, which is essential for biotin binding.

[0008] Dakshinamurti, K et al. (Biochem. J. 237 (1986) 477-482) reported the generation and characterization of mouse mAbs using keyhole hemocyanin (KLH) conjugated with an activated form of biotin (N-hydroxysuccinimide biotin) as an immunogen. Several hybridoma clones were obtained, and their respective mAbs were effective in binding free biotin, hapten-conjugated biotin, protein-conjugated biotin, and biocytin. Notably, biocytin is a naturally occurring biotin derivative, an amide formed from the carboxyl functional group of valerate and the amino acid L-lysine. The fact that this derivatized biotin was bound by the mAbs of Dakshinamurti, K et al. (see above) indicates the binding specificity targeting the heterocyclic structure of biotin (i.e., the ureidoyl ring fused with a tetrahydrothiophene ring).

[0009] While the mAbs reported by Dakshinamurti, K et al. (see above) do bind both conjugated and free biotin, JP 2008-094721 discloses an mAb that specifically binds protein-conjugated biotin but not free biotin. Table 1 summarizes the properties of the prior art antibodies discussed.

[0010] Table 1 Binding properties of monoclonal antibodies against biotin Report Binding to the target of biotinylation Binding with free biotin Dakshinamurti, K et al. (Biochem. J. 237 (1986) 477-482) yes yes Kohen F. et al. (M. Enzymol. 279 (1997) 451-463) yes Undetermined JP 2008-094721 yes no No reports yet no yes Similar to the report by Dakshinamurti, K et al. (see above), WO 00 / 50088 A2 relates to antibodies for therapeutic interventions; specifically, it reports antibodies that have an affinity for conjugated biotin one to four orders of magnitude higher than the corresponding affinity for free biotin. This document discloses immunization with an antigen, in which biotin is conjugated to the antigen via a carboxyl-functionalized carbon atom of the valerate moiety. In a first screening step, antibodies binding to the conjugated biotin are identified; a subsequent second screening step is disclosed, aimed at identifying clones that secrete monoclonal antibodies capable of binding the conjugated biotin even in limited quantities of free biotin. Notably, this document does not mention monoclonal antibodies that, on the one hand, bind to free biotin (biotin that is not covalently bound to another molecule and is in a dissociated form in aqueous solution), but on the other hand, do not bind to the biotin moiety on the biotinylated molecule, i.e., the biotin moiety that can also be bound by (streptomycin) anti-biotinin.

[0011] Indyk HE et al. reported an optical biosensor assay for detecting free biotin in milk in the International Dairy Journal, 35 (2014), 25-31. A Biacore Q biosensor with a CM5 sensor chip was used; biotin was immobilized on an amine-modified sensor surface via covalent coupling of its NHS-activated valerate-terminal carboxyl group. Notably, the orientation of the biotin molecule coupled to the sensor chip was the same as that of the biotinylated molecule, i.e., corresponding to the biotin moiety that can also be bound by (streptomycin) anti-biotin. Therefore, the heterocyclic structure of the biotin, which is predominantly exposed, was used for antibody binding. The binding properties of three different biotin-specific polyclonal antibodies and two different monoclonal antibodies were characterized using the biotin sensor chip in the presence of different concentrations of free biotin as a competitor.

[0012] It is noteworthy that, to date, no publicly available technology describes monoclonal antibodies that specifically bind to biotin conjugated to target molecules, rather than biotinylated ones. Such antibodies would bind biotin primarily from their "tail," meaning they would significantly interact with the valeric acid moiety of biotin. Specifically, no antibody has yet been described that specifically binds to free biotin but not conjugated biotin, where the conjugated biotin attaches to the target molecule via the carboxyl-functionalized carbon atom of the valeric acid moiety, thus placing the heterocyclic "head" structure of biotin distantly from the target molecule. Furthermore, no such monoclonal antibody is known to date to exhibit an affinity K for free biotin in aqueous solution. s [Free] Affinity K higher than that of the same monoclonal antibody for conjugated biotin. s [Conjugation], wherein the conjugated biotin is conjugated via a carboxyl-functionalized carbon atom of the valeric acid moiety (see above), and wherein K s [Free] and K s [Aggregation] The difference is at least 50, 100, 500, 1,000, 5,000, 10,000, 50,000 or at least 100,000 times.

[0013] Johnson LC (“The synthesis of new biotin derivatives and their bioactivity,” a Master of Science thesis submitted to the Graduate Faculty of the Louisiana State University and Agricultural and Mechanical College (US) in December 2002, retrieved from the Internet on February 16, 2017; URL: http: / / etd.lsu.edu / docs / available / etd-0927102-135929 / unrestricted / Johnson_thesis.pdf) describes the chemical reaction for the derivatization of the heterocyclic “head” structure of biotin. An implementation scheme in which the 1'-N atom contained in the heterocyclic structure of biotin is targeted by derivatization is disclosed.

[0014] Wu F.-B. et al., Clinica Chimica Acta 308 (2001) 117-126, disclose a method for counteracting matrix interference in a competitive immunoassay. The initial assay configuration, consisting of a fixed second antibody that binds to a target-specific antibody (serum thyroxine), is replaced. This replacement is provided by first fixing biotinylated bovine serum albumin (BSA), followed by binding streptavidin to the biotinylated BSA, and then binding the target-specific antibody to the streptavidin, wherein the target-specific antibody is biotinylated prior to this step. This configuration was found to provide increased binding affinity to the assayed target (serum thyroxine) and thus increased resistance to matrix interference.

[0015] This invention provides a chemical structure of Formula I (see below), which specifically presents the valerate "tail" portion of the derivatized biotin as the distal (farthest) portion of the corresponding structure. By derivatizing biotin and attaching its "head" portion to the remainder of the Formula I structure, the valerate portion is primarily exposed and provided for physical interactions, including interactions with immune cell receptors and antibodies. It is assumed that antibodies interacting with the derivatized biotin according to Formula I will have binding properties capable of targeting the valerate "tail" portion. In this study, it was further tested whether monoclonal antibodies derived using the Formula I structure and capable of reacting with such a structure would also possess the antibody properties desired according to the invention.

[0016] Therefore, the desired antibody of the present invention is a monoclonal antibody that specifically binds biotin, wherein biotin is not covalently bound to another molecule. Similarly, consistent with the above reasoning, the desired antibody of the present invention is a monoclonal antibody that specifically binds biotin, wherein biotin is in a dissociated form in aqueous solution. Furthermore, the desired antibody of the present invention does not bind to biotin conjugated biotin on a biotinylated target molecule, wherein biotin is attached to the target molecule via the carboxyl group of the valeric acid moiety. In such a biotinylated target molecule, the conjugated form of the biotin “head” structure is present and can be bound, for example, by (streptomycin) anti-biotin protein.

[0017] Therefore, the inventors set out to determine whether antibodies could be prepared and isolated to specifically bind free biotin. This binding to biotin would have a completely different structural basis compared to antibodies that have been previously reported to bind to the "head" portion of biotin.

[0018] Therefore, the object of the present invention is to provide a monoclonal antibody that specifically binds to compound I. (Formula I) Its characteristic is that it is also bound to biotin, wherein Y is CH2 or C=O, and X is O or (CH2). n And n is an integer from 1 to 20, and R is H or ZL, where L is a linker, and Z is selected from haptens and peptides that do not contain the biotin moiety.

[0019] A further objective is to provide a monoclonal antibody that binds to a molecule of Formula I and biotin but not to conjugated biotin, said conjugated biotin being attached to the macromolecule via a carboxyl-functionalized carbon atom of the valeric acid moiety. As a further objective, the antibody of the present invention binds to biotin comprising a chemically unmodified valeric acid moiety. Therefore, the objective is to isolate a monoclonal antibody having an affinity for conjugated biotin on a biotinylated target molecule that is selected from an affinity for free biotin selected from at most 1 / 50, 1 / 100, 1 / 500, 1 / 1,000, 1 / 5,000, 1 / 10,000 or lower. In other words, the objective is to isolate a monoclonal antibody having an affinity for free biotin that is selected from an affinity for conjugated biotin on a biotinylated target molecule that is selected from at least 50, 100, 500, 1,000, 5,000 and at least 10,000 times.

[0020] It has now been surprisingly discovered that this monoclonal antibody can be produced and isolated.

[0021] High-dose biotin supplementation has recently become "popular." Biotin is considered a key contributor to keratin production, and therefore high-dose biotin can improve the quality and quantity of hair, nails, and skin. Biotin is water-soluble and rapidly excreted. However, with high-dose biotin supplementation, fairly high levels of biotin may be present in circulation, and this circulating biotin will also be present in samples used for in vitro analysis of analytes, i.e., in samples such as serum or plasma. Biotin contained in the sample, if present at high levels, may interfere with the determination of analytes using a solid phase coated with (streptomycin) anti-biotin protein and a biotinylated specific binding agent.

[0022] Therefore, with the increasing use of high-dose biotin supplements, there is a growing need to reduce the potential interference of abnormally high biotin levels in samples with analyte measurements from the same samples in assays based on (streptomycin) avidin-biotin binding pairs.

[0023] Further research is needed to determine whether the antibodies disclosed herein can be used to reduce potential interference from biotin. It has been surprisingly found that, in such assays, where the (streptomycin / biotin pair is used to bind a biotinylated analyte-specific binder to a (streptomycin)-coated solid phase, monoclonal antibodies capable of binding biotin but not the biotin moiety on the biotinylated target molecule are particularly useful for counteracting potential interference caused by abnormally high levels of biotin in the sample in methods used to measure analytes in such samples. Summary of the Invention

[0024] This article reports a monoclonal antibody that specifically binds to compound I. (Formula I) Its characteristic is that it is also bound to biotin, wherein Y is CH2 or C=O, and X is O or (CH2). n And n is an integer from 1 to 20, and R is H or ZL, where L is a linker, and Z is selected from haptens and peptides that do not contain the biotin moiety.

[0025] A method for measuring analytes in a sample is further reported, wherein a (streptomycin) avidin / biotin binding pair is used to bind a biotinylated analyte-specific binder to a (streptomycin) avidin-coated solid phase. The method includes adding (a) the antibody reported herein, (b) the biotinylated analyte-specific binder, and (c) the streptomycin-coated solid phase to the sample, followed by measuring the analyte bound to the solid by the (streptomycin) avidin and the biotinylated analyte-specific binder.

[0026] A method for measuring analytes in a sample is further reported, wherein a (streptomycin) avidin / biotin binding pair is used to bind a biotinylated analyte-specific binder to a label. The method includes adding (a) the antibody reported herein, (b) the biotinylated analyte-specific binder, and (c) the (streptomycin) avidin bound to the label to the sample, followed by separation of a complex containing the analyte, the biotinylated analyte-specific binder, and the labeled streptomycin, and determination of the amount of label bound to the analyte.

[0027] The use of the antibodies reported in this paper in methods for measuring analytes in samples is further reported, wherein (streptomycin) avidin / biotin pairs are used to bind biotinylated analyte-specific binders to a (streptomycin) avidin-coated solid phase or labeled (streptomycin).

[0028] Further reports describe immunoassay kits comprising at least (a) the antibodies disclosed herein, (b) a biotinylated analyte-specific binder, and (c) a solid phase coated with (streptomycin) antibiotin.

[0029] Further reports were made regarding the immunogens based on Formula I. (Formula I) Where Y is CH2 or C=O, and X is O or (CH2). n And n is an integer from 1 to 20, and R is ZL, where L is a linker, and Z is a polypeptide of at least 30 amino acids, preferably keyhole hemocyanin.

[0030] A method for generating the antibody disclosed herein is further reported, the method comprising the steps of: (a) immunizing experimental animals with the immunogen disclosed herein to induce B cells to produce antibodies that bind to the immunogen; (b) obtaining the monoclonal antibody that binds to the immunogen generated by the B cells in step (a) either by hybridoma technology or by B-cell PCR technology; and (c) further selecting the antibody from step (b) to bind with biotin to obtain the antibody disclosed herein.

[0031] A method for generating the antibodies disclosed herein, the method comprising the steps of: a) immunizing experimental animals with the immunogen disclosed herein to induce B cells to produce antibodies that bind to the immunogen; b) obtaining, either by hybridoma technology or by B-cell PCR technology, the monoclonal antibody that binds to the immunogen produced by the B cells in step (a); c) selecting the antibodies from step (b) for binding to biotin; and d) selecting those antibodies that do not bind to the compound of formula II. (Formula II) Thus, the antibody disclosed in this article was obtained. Invention Details In one embodiment, the present invention relates to monoclonal antibodies that specifically bind to compounds of formula I. (Formula I) Its distinguishing feature is that it also binds to biotin. in Y is CH2 or C=O. X is O or (CH2) n And n is an integer from 1 to 20, and R is H or ZL. in L is the connector, and Z is selected from haptens and polypeptides that do not contain the biotin moiety.

[0033] Surprisingly, it has been found that the method disclosed herein can reliably produce monoclonal antibodies that bind to both the structure of Formula I (described in more detail below) and biotin, which are also illustrated in more detail below.

[0034] For the purposes of this disclosure, the terms "(streptomycin) avidin" and avidin-type protein are used interchangeably in all aspects and embodiments mentioned herein. An avidin-type protein is generally understood to be a protein having at least one binding pocket capable of specifically binding to a heterocyclic structure of biotin, represented by a ureidoyl ring fused to a tetrahydrothiophene ring. By virtue of this property, an avidin-type protein is capable of binding to a biotinylated target molecule, wherein biotin is covalently bound to the molecule via a carboxyl-functionalized carbon atom of the valerate side chain of biotin. Several embodiments of avidin-type proteins are known in the art. More specifically, the avidin-type protein may be selected from avidin, neutral avidin, streptomycin, bradavidin, traptavidin, its biotin-binding variants, mixtures thereof, its monomers, dimers, trimers, tetramers, or polymers, its conjugated forms, and antibodies that bind to conventionally biotinylated molecules of interest. Many avidin-type proteins (especially those that are not antibodies), particularly avidin and streptavidin, are known to be homotetramers in their naturally occurring forms; that is, they consist of four identical subunits. In embodiments of monomeric avidin-type proteins, the naturally occurring form can be a di-, tri-, or tetra-oligomer, wherein each monomer has a biotin-binding pocket. In embodiments, the avidin-type protein is selected from monomers, homodimers, homotrimers, and homotetramers.

[0035] More specifically, the avidin-type protein can be an antibody having an antigen-binding pocket capable of specifically binding to the heterocyclic structure of biotin, represented by a ureidoyl ring fused to a tetrahydrothiophene ring. Examples of antibodies having this property are known in the prior art and cited above. In even more specific embodiments, if the avidin-type protein specifically binds... Figure 3 The biotin portion of structure A can be used to identify the avidin-type protein. In even more specific embodiments, the avidin-type protein binds non-specifically. Figure 3 The structure in B.

[0036] In one embodiment, the (streptomycin) avidin according to this disclosure is selected from avidin, neutral avidin, streptomycin, bradavidin, traptavidin, their biotin-binding variants, and mixtures thereof.

[0037] When referring to "(streptomycin) avidin" or avidin-type proteins in this disclosure, it should be understood that these terms equally include any variant thereof, provided that the variant is capable of non-covalently binding biotin in at least one binding pocket that is specifically capable of binding to a heterocyclic structure of biotin represented by a ureidyl ring fused to a tetrahydrothiophene ring. In this respect, a variant is a "functionally equivalent polypeptide" because the amino acids forming at least one binding pocket have electrostatic and stereochemical properties similar to the amino acid sequence of the original avidin-type protein under consideration, wherein the variant comprises one or more conserved amino acid substitutions, similar amino acid substitutions, and / or deletions and / or additions of amino acids that do not significantly affect or alter the function of the amino acids in the binding pocket. "Functionally equivalent" also includes homologous amino acid sequences relative to the respective referenced amino acid sequences.

[0038] The term "conservative substitution" applies to both amino acid and nucleic acid sequences. Regarding a specific nucleic acid sequence, "conservative substitution" refers to those nucleic acids encoding the same or substantially the same amino acid sequence, or, in cases where the nucleic acid does not encode an amino acid sequence, substantially the same sequence. Due to the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For example, codons GCA, GCC, GCG, and GCU all encode the amino acid alanine. Therefore, at each position where alanine is determined by a codon, the codon can be changed to any of the corresponding codons without altering the encoded polypeptide. This nucleic acid variation is a "silent variation," a type of conserved modification. Each nucleic acid sequence encoding a polypeptide described herein also describes every possible silent variation of that nucleic acid. Those skilled in the art will recognize that every codon in a nucleic acid (except for AUG, which is usually the only codon for methionine, and TGG, which is usually the only codon for tryptophan) can be modified to produce a functionally identical molecule. Therefore, every silent variation of a nucleic acid encoding a polypeptide is implicit in each described sequence.

[0039] Regarding amino acid sequences, those skilled in the art will recognize that in cases where changes result in the substitution of an amino acid with a chemically similar amino acid, individual substitutions of a single amino acid or a small percentage of amino acids in the amino acid sequence of a peptide, polypeptide, or protein are considered "conservative substitutions." Providing conservative representations of functionally similar amino acids is known to those skilled in the art. The following eight groups each contain amino acids that are conservatively substituted for each other: The term "conservative amino acid substitution" refers to all such substitutions in which the substituted amino acid has a similar structure or chemical properties to the corresponding amino acid in the reference sequence. For example, conservative amino acid substitution involves the substitution of one aliphatic or hydrophobic amino acid (e.g., alanine, valine, leucine, isoleucine, methionine, phenylalanine, or tryptophan) with another; the substitution of one hydroxyl-containing amino acid (e.g., serine and threonine) with another; the substitution of one acidic residue (e.g., glutamic acid or aspartic acid) with another; the substitution of one amide-containing residue (e.g., asparagine and glutamine) with another; the substitution of one aromatic residue (e.g., phenylalanine and tyrosine) with another; the substitution of one basic residue (e.g., lysine, arginine, and histidine) with another; and the substitution of one small amino acid (e.g., alanine, serine, threonine, and glycine) with another.

[0040] As used herein, “deletion” and “addition” in relation to an amino acid sequence refer to the deletion or addition of one or more amino acids at the amino terminus, carboxyl terminus, within the amino acid sequence, or in combination thereof. For example, an addition may be one of the antibody subjects of this application.

[0041] As used herein, a “homologous sequence” has an amino acid sequence that is at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% homologous to the corresponding reference sequence. A sequence that is at least 90% identical has no more than one alteration per 10 amino acids of the reference sequence, i.e., any combination of deletions, additions, or substitutions. This is achieved through methods such as DNA STAR. TM MEGALIGN in the program TM The project compares the amino acid sequences of variants with reference sequences to determine the percentage of homology.

[0042] In the context of two or more nucleic acid or polypeptide sequences, the term "identical" or percentage "identity" refers to two or more identical sequences or subsequences. When compared and aligned on a comparison window or designated region measured by manual alignment and visual inspection, with respect to maximum correspondence, the sequences are considered "substantially identical" (i.e., approximately 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity in the designated region) if they have a certain percentage of identical amino acid residues or nucleotides. This definition also relates to the complement of the test sequence. This identity may be present in a region of at least about 50 amino acids or nucleotides, or in a region of 75-100 amino acids or nucleotides, or, in unspecified cases, throughout the entire sequence of the polynucleotide or polypeptide. The polynucleotide encoding the polypeptide of the present invention, including homologs from species other than humans, can be obtained by a method comprising the steps of: screening a library under strict hybridization conditions with a probe or fragment thereof labeled with the polynucleotide sequence of the present invention, and isolating a full-length cDNA and a genomic clone containing said polynucleotide sequence. Such hybridization techniques are well known to those skilled in the art.

[0043] For sequence comparisons, one sequence typically serves as the reference sequence, which is then compared to the test sequence. When using a sequence comparison algorithm, the test and reference sequences are input into the computer, and the coordinates of the subsequences are specified if necessary, along with the sequence algorithm program parameters. Default program parameters can be used, or alternative parameters can be specified. The sequence comparison algorithm then calculates the percentage sequence identity of the test sequence relative to the reference sequence based on the program parameters.

[0044] For the purposes of this disclosure, the terms “biotin” or “free biotin” are used interchangeably and refer to naturally occurring compounds, namely, D(+)-biotin.

[0045] Biotin (D(+)-Biotin; C)10 H 16 N2O3S; MW = 244.31 g / mol; IUPAC name: 5-[(3aS,4S,6aR)-2-oxo-1,3,3a,4,6,6a-hexahydrothiophene[3,4-d]imidazol-4-yl]valeric acid), CAS Registry No. 58-85-5 contains a ureidoyl ring fused to the tetrahydrothiophene ring and a valeric acid substituent attached to one of the carbon atoms of the tetrahydrothiophene ring. The basic structure of biotin has long been known and reported, for example, by Melville DB et al. (J. Biol. Chem. 146(1942) 487-492). Biotin has three consecutive chiral carbon atoms, and therefore four diastereomeric racemic forms are possible. Of the diastereomeric racemic forms, only D(+)-biotin exists in nature, while the other isomers are of synthetic origin. The bioactive form is Figure 1 A and Figure 1 The configuration shown in B is (3aS,4S,6aR).

[0046] According to Marquet A. (Pure & Appl. Chem. 49 (1977) 183-196), in the crystal structure of D(+)-biotin, the ureidoyl ring is planar, while the tetrahydrothiophene ring has an envelope conformation, such as... Figure 1 As shown in B, the valeric acid side chain is not fully straightened but twisted, and there is an interaction between the C6 atom of the side chain and the N'3 atom of the urea ring; this interaction has been reported to affect the reactivity of biotin. An envelope conformation of the tetrahydrothiophene ring has also been reported in solution, as shown in NMR studies by Glasel JA (Biochemistry 5 (1966) 1851-1855) and Lett R. & Marquet A. / Tetrahedron 30 (1974) 3365-3377).

[0047] The term "biotin moiety" is used to refer to the biotin-related or biotin-derived portion of a molecule, such as, for example, obtained through any type of biotinylation or chemical coupling.

[0048] The appropriate chemical group that attaches biotin to the molecule of interest via a carboxyl-functionalized carbon atom in the valeric acid side chain is called "biotinylation" or "conventional biotinylation." Thus, the biotin residues in the "biotinylated" molecule of interest have an outward-facing ring structure (i.e., a ureidoyl ring fused to a tetrahydrothiophene ring), while the linear portion of the biotin residue faces inward, towards the surface of the biotinylated molecule. The outward-facing ring structure can be bound by avidin-type proteins. Therefore, it is important that the heterocyclic "head" structure of biotin be exposed for specific binding by avidin-type proteins.

[0049] The term "(streptomycin) avidin / biotin binding pair" is well known to those skilled in the art. It indicates the fact that biotin (including the biotinylated portion of the molecule) on one side and (streptomycin) avidin on the other side represent the two members of this binding pair. As mentioned above, this binding pair is noteworthy because it possesses one of the highest known binding affinities due to its non-covalent interactions.

[0050] The articles “a” and “an” used in this article refer to one or more (i.e., at least one) grammatical objects of the article. For example, “antibody” means one or more antibodies.

[0051] The term "antibody" includes various forms of antibody structures, including but not limited to complete antibodies and antibody fragments. Antibodies according to the invention are preferably goat, sheep, mouse, rabbit, or rat antibodies, chimeric antibodies, or further genetically engineered antibodies, provided they retain the characteristic properties according to the invention.

[0052] An "antibody fragment" comprises a portion of a full-length antibody, preferably its variable domains, or at least its antigen-binding site. Examples of antibody fragments include biantibodies, single-chain antibody molecules, and multispecific antibodies formed from antibody fragments. scFv antibodies are described, for example, in Huston, JS, Methods in Enzymol. 203 (1991) 46-88. Furthermore, antibody fragments include single-chain polypeptides having a V H The characteristic of a structural domain is that it can interact with V. L The domains are assembled together, or have a V that binds to IGF-1. L The characteristic of a structural domain is that it can interact with V. H The structural domains assemble together to form a functional antigen-binding site, thereby providing the properties of the antibody according to the invention.

[0053] As used herein, the terms “monoclonal antibody” or “monoclonal antibody composition” refer to formulations of antibody molecules consisting of a single amino acid.

[0054] The term "specific binder" is used to refer to a reagent that is capable of specifically binding to an analyte of interest or is specifically bound to an analyte of interest. Many different assay configurations for immunoassays are known in the art. Depending on the specific assay configuration, a variety of biotinylated specific binders may be used. In one embodiment, the biotinylated specific binder is selected from biotinylated analyte-specific binders, biotinylated analytes bound to a solid, and biotinylated antigens bound to a solid.

[0055] The term "analyte-specific binder" refers to a molecule that specifically binds to an analyte of interest. In the context of this disclosure, an analyte-specific binder generally includes a binding or trapping molecule capable of binding to an analyte (or, in other terms, the analyte of interest; the target molecule). In one embodiment, the analyte-specific binder has a binding affinity of at least 10⁻⁶ for its corresponding target molecule, i.e., the analyte. 7 An affinity of l / mol. In other embodiments, the analyte-specific binder has an affinity of 10 for its target molecule. 8 l / mol or even 10 9 Affinity of l / mol. As those skilled in the art will understand, the term specificity is used to indicate that other biomolecules present in the sample do not significantly bind to the analyte-specific binder. In some embodiments, the binding affinity with biomolecules other than the target molecule results in a binding affinity of only 10%, more preferably only 5% or less, of the affinity for the target molecule. In one embodiment, no binding affinity to any other molecule besides the analyte is measurable. In one embodiment, the analyte-specific binder will meet the minimum criteria for affinity and specificity described above.

[0056] In the context of antibodies, the term "analyte-specific binding" refers to the immunospecific interaction between an antibody and its target epitope on the analyte; that is, the binding of the antibody to the epitope on the analyte. The concept of analyte-specific binding of an antibody through its epitope on the analyte is fully apparent to those skilled in the art.

[0057] The terms “polypeptide,” “peptide,” and “protein” refer to polymers of amino acid residues. This terminology applies to naturally occurring amino acid polymers as well as amino acid polymers in which one or more amino acid residues are non-naturally encoded amino acids. As used herein, the term includes amino acid chains in which amino acid residues are linked by covalent peptide bonds. Polypeptides, peptides, and proteins are written using standard sequence notation, with the N-terminus on the left and the C-terminus on the right. Standard single-letter notations have been used as follows: A-alanine, C-cysteine, D-aspartic acid, E-glutamic acid, F-phenylalanine, G-glycine, H-histidine, S-isoleucine, K-lysine, L-leucine, M-methionine, N-asparagine, P-proline, Q-glutamine, R-arginine, S-serine, T-threonine, V-valine, W-tryptophan, Y-tyrosine. The term “peptide,” as used herein, refers to an amino acid polymer having a length of up to five amino acids. The term “polypeptide,” as used herein, refers to an amino acid polymer having a length of six or more amino acids. The term "protein" refers to a polypeptide chain or a polypeptide chain with further modifications, such as glycosylation, phosphorylation, acetylation, or other post-translational modifications.

[0058] A "hapten" is a small molecule (e.g., an insecticide, fungicide, drug, hormone, toxin, synthetic peptide, etc.) that does not directly induce an immune response, such as antibody formation. Techniques for generating antibodies against haptens by conjugating them to immunogenic carriers (e.g., antigenic macromolecules) have been established. For the purposes of this disclosure, a hapten is understood to be a low molecular weight molecule, particularly having a molecular weight of 10,000 Da or less, which does not elicit an immune response until and unless conjugated to an immunogenic carrier such as a protein. Once an antibody is formed, it can bind to the hapten. The resulting antibody can be used in many fields, particularly in the development of immunodiagnostic kits or biosensors. Therefore, the term "hapten" refers to a small molecule of 10,000 Da or less that can elicit an immune response only when attached to an immunogenic carrier (e.g., a polypeptide of at least 30 amino acids). In this sense, and in embodiments, a hapten is an incomplete antigen that cannot promote antibody formation on its own, but can when conjugated to a protein of at least 30 amino acids. Exemplary haptens are aniline, ortho-, meta-, and para-aminobenzoic acids, quinones, histamine-succinyl-glycine (HSG), hydralazine, haloalkanes, indium-DTPA, fluorescein, digitoxin, theophylline, bromodeoxyuridine, steroid compounds, and dinitrophenol. In specific embodiments, the hapten is not biotin and does not contain a biotinylated portion. In one specific embodiment, the hapten is digitoxin, theophylline, fluorescein, or bromodeoxyuridine. In the context of the disclosure of compounds according to Formula I, a hapten that is a part of the compound is understood to be covalently coupled to the remaining portion of the compound, wherein the hapten portion (i.e., a chemical structure of 10,000 Da or less) is capable of evoking an immune response only when attached to an immunogenic carrier such as a polypeptide of at least 30 amino acids.

[0059] "Isolated" antibodies are antibodies that have been identified, isolated, and / or recovered from components of their natural environment. These natural environmental contaminants are materials that would interfere with the research, diagnostic, or therapeutic use of the antibody and may include enzymes, hormones, and other protein or non-protein solutes. In some embodiments, the antibody is purified to greater than 95% by weight, and in some embodiments, to greater than 99%, as determined by SDS-PAGE under reducing or non-reducing conditions, such as using, for example, Coomassie blue or silver dye.

[0060] Immunoglobulin G antibodies are typically heterotetrameric glycoproteins of approximately 150,000 Daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to the heavy chain by a covalent disulfide bond, the number of which varies among different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has a variable domain (V) at one end. H Following this are many constant structural domains. Each light chain has a variable structural domain (V0) at one end. L The light chain has a constant domain at its other end; the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the variable domain of the light chain is aligned with the variable domain of the heavy chain. Specific amino acid residues are thought to form at the interface between the variable domains of the light and heavy chains.

[0061] The "variable region" or "variable domain" of an antibody refers to the amino-terminal domain of either the heavy or light chain. The variable domain of the heavy chain is called "VH," and the variable domain of the light chain is called "VL." These domains are typically the most variable parts of the antibody and contain the antigen-binding site.

[0062] The term "variable" refers to the fact that certain portions of the variable domain exhibit significant sequence differences within the antibody, contributing to the binding and specificity of each particular antibody to its specific antigen. However, variability is not uniformly distributed throughout the entire variable domain of the antibody. It is concentrated in three segments called hypervariable regions (HVRs) in both the light and heavy chain variable domains. The more highly conserved portions of the variable domain are called framework regions (FRs). The variable domains of the native heavy and light chains each contain four FR regions, predominantly adopting a β-sheet configuration, linked by three HVRs that form loops connecting the β-sheet structure and, in some cases, form part of the β-sheet structure. The HVRs in each chain are held together closely by the FR regions and, together with HVRs from the other chain, contribute to the formation of the antigen-binding site of the antibody (see Kabat et al.). Sequences of Proteins of Immunological Interest , 5th edition, National Institute of Health, Bethesda, MD (1991). The constant domain is not directly involved in the binding of antibodies to antigens, but exhibits various effector functions, such as antibody participation in antibody-dependent cytotoxicity.

[0063] The “light chains” of antibodies (immunoglobulins) from any vertebrate species can be assigned to one of two distinct types, called κ (κ) and λ (λ), based on the amino acid sequence of their constant structural domains.

[0064] The antibodies used in the method according to the invention can be derived from any animal source. In one embodiment, the antibody is a human, mouse (e.g., mouse and rat), donkey, monkey, rabbit, goat, guinea pig, camel, horse, or chicken antibody.

[0065] Antibodies (immunoglobulins) can be classified into different classes based on the amino acid sequence of their heavy chain constant domains. Human immunoglobulins have five main classes: IgA, IgD, IgE, IgG, and IgM, and several of these can be further subdivided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to different immunoglobulin classes are designated as α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional conformations of different immunoglobulin classes are well-known and are often described in works such as those by Abbas et al. Cellular and Mol. Immunology As described in the 4th edition (WBSaunders, Co., 2000). An antibody can be part of a larger fusion molecule formed by the covalent or non-covalent association of an antibody with one or more other proteins or peptides.

[0066] The terms “full-length antibody,” “intact antibody,” and “whole antibody” are used interchangeably herein to refer to an antibody in essentially its complete form, rather than an antibody fragment as defined below. The term specifically refers to antibodies having a heavy chain containing an Fc region.

[0067] An "antibody fragment" comprises a portion of a complete antibody, preferably including its antigen-binding region. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; single-chain antibody molecules; scFv, sc(Fv)2; biantibodies; and multispecific antibodies formed from antibody fragments.

[0068] Antibody digestion with papain produces two identical antigen-binding fragments, called "Fab" fragments, each with a single antigen-binding site, and a residual "Fc" fragment, the name reflecting its tendency to crystallize. Pepsin treatment produces the F(ab')2 fragment, which has two antigen-binding sites and remains capable of cross-linking antigens.

[0069] The Fab fragment contains variable domains for both the heavy and light chains, and also contains a constant domain for the light chain and a first constant domain (CH1) for the heavy chain. The Fab' fragment differs from the Fab fragment in that it has a few residues added to the carboxyl terminus of the heavy chain CH1 domain, including one or more cysteine ​​residues from the antibody-hinge region. Fab'-SH is the name for Fab' in this paper, where one or more cysteine ​​residues in the constant domain are accompanied by a free thiol group. The F(ab')2 antibody fragment was originally generated as a Fab' fragment pair with a hinge cysteine ​​residue between them. Other chemical conjugations of antibody fragments are also known.

[0070] "Fv" is the smallest antibody fragment containing a complete antigen-binding site. In one embodiment, the double-chain Fv species consists of a dimer of a heavy chain and a light chain variable domain in tight, non-covalent association. In the single-chain Fv (scFv) species, a heavy chain and a light chain variable domain are covalently linked by a flexible peptide linker, allowing the light and heavy chains to associate in a "dimeric" structure similar to that in the double-chain Fv species (sc(Fv)2). In this configuration, the three HVRs of each variable domain interact to define the antigen-binding site on the surface of the VH-VL dimer. In total, six HVRs confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv containing only three antigen-specific HVRs) can recognize and bind antigens, although its affinity is lower than that of the entire binding site.

[0071] This disclosure includes monovalent Fab fragments and single-chain Fvs derived from monoclonal antibodies capable of specifically binding free biotin, as disclosed herein. Compared to naturally occurring antibody forms, the monovalent type can diffuse more rapidly in aqueous solutions due to its smaller molecular weight. Furthermore, under suitable conditions, particularly scFv antibodies, recombinantly generated in prokaryotic expression systems is possible.

[0072] The term "biantibody" refers to an antibody fragment having two antigen-binding sites, comprising a heavy chain variable domain (VH) linked to a light chain variable domain (VL) on the same polypeptide chain (VH-VL). By using a linker that is too short to allow pairing between the two domains on the same chain, the domain is forced to pair with a complementary domain of the other chain, resulting in two antigen-binding sites. Biantibodies can be bivalent or bispecific. Biantibodies are more fully described, for example, in EP 404097; WO 1993 / 01161; Hudson et al. Nat. Med. 9:129-134 (2003); and Holliger et al., PNAS USA90:6444-6448 (1993). Triple-chain and quadruple-chain antibodies were also described by Hudson et al. Nat. Med. 9:129-134 (2003).

[0073] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibodies, meaning that the individual antibodies constituting the population are identical except for the possibility of small amounts of possible mutations (e.g., naturally occurring mutations). Therefore, the modifier "monoclonal" indicates that the antibody is not a mixture of discrete antibodies. In some embodiments, such a monoclonal antibody generally comprises an antibody containing a polypeptide sequence that binds to a target, wherein the target-binding polypeptide sequence is obtained by a method comprising selecting a single target-binding polypeptide sequence from a plurality of polypeptide sequences. For example, the selection method may be to select a unique clone from a plurality of clones, such as a library of hybridoma clones, phage clones, or recombinant DNA clones. It should be understood that the selected target-binding sequence can be further modified, for example, to increase affinity for the target, to humanize the target-binding sequence, to improve its production in cell cultures, to reduce its immunogenicity in vivo, to produce multispecific antibodies, etc., and antibodies containing modified target-binding sequences are also monoclonal antibodies of the present invention. In contrast to polyclonal antibody formulations, which generally comprise different antibodies targeting different determinants (epitopes), each monoclonal antibody in a monoclonal antibody formulation targets a single determinant on the antigen. In addition to their specificity, monoclonal antibody preparations are advantageous because they are generally not contaminated by other immunoglobulins.

[0074] The modifier "monoclonal" indicates that the antibody is obtained from a substantially homogeneous population of antibodies and should not be interpreted as requiring the antibody to be produced by any particular method. For example, the monoclonal antibody used according to the invention can be prepared by a variety of techniques, including, for example, hybridoma methods (e.g., Kohler and Milstein). Nature , 256:495-97 (1975); Hongo et al., Hybridoma, 14(3):253-260 (1995), Harlow et al., Antibodies: A Laboratory Manual , (Cold Spring Harbor Laboratory Press, 2 nd ed. 1988); Haemmerling et al., Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, NY, 1981), recombinant DNA methods (see, for example, U.S. Patent No. 4,816,567), phage display technology (see, for example, Clackson et al.), Nature, 352: 624-628 (1991); Marks et al., J. Mol. Biol. 222: 581-597 (1992); Sidhu et al., J. Mol. Biol. 338(2): 299-310 (2004); Lee et al., J. Mol. Biol. 340(5):1073-1093 (2004);Fellouse, PNAS USA 101(34): 12467-12472 (2004); and Lee et al., J. Immunol. Methods 284(1-2): 119-132 (2004), and techniques for producing human or human-like antibodies in animals possessing partial or complete human immunoglobulin loci or genes encoding human immunoglobulin sequences (see, e.g., WO 1998 / 24893; WO 1996 / 34096; WO 1996 / 33735; WO 1991 / 10741; Jakobovits et al., PNAS USA 90:2551 (1993); Jakobovits et al., Nature 362:255-258 (1993); Bruggemann et al., Year in Immunol. 7:33 (1993); U.S. Patent Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; and 5,661,016; Marks et al., Bio / Technology 10:779-783 (1992); Lonberg et al., Nature 368:856-859 (1994); Morrison, Nature 368:812-813 (1994); Fishwild et al., Nature Biotechnol. 14: 845-851 (1996); Neuberger, Nature Biotechnol. 14:826 (1996); and Lonberg and Huszar, Intern. Rev. Immunol. 13:65-93 (1995).

[0075] Monoclonal antibodies in this article specifically include “chimeric” antibodies, wherein a portion of the heavy and / or light chains is identical or homologous to the corresponding sequence in an antibody derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of one or more chains is identical or homologous to the corresponding sequence in an antibody derived from another species or belonging to another antibody class or subclass, and fragments of such antibodies, provided they exhibit the desired biological activity (e.g., U.S. Patent No. 4,816,567 and Morrison et al.). PNAS USA 81:6851-6855 (1984). Chimeric antibodies include PRIMATIZED® antibodies, wherein the antigen-binding region of the antibody is derived from an antibody produced, for example, by immunizing a rhesus monkey with an antigen of interest.

[0076] When used herein, the terms “hypervariant region,” “HVR,” or “HV” refer to regions of an antibody variable domain that are hypervariable in the sequence and / or form structurally defined loops. Typically, antibodies contain six HVRs: three of the VH (H1, H2, H3) and three of the VL (L1, L2, L3). In native antibodies, H3 and L3 exhibit the greatest diversity of the six HVRs, and H3, in particular, is considered to play a unique role in conferring fine specificity to antibodies. See, for example, Xu et al. Immunity 13:37-45 (2000); Johnson and Wu Methods in Molecular Biology 248:1-25 (Lo, ed., HumanPress, Totowa, NJ, 2003). In fact, naturally occurring camel antibodies consisting only of heavy chains are functional and stable even in the absence of light chains. See, for example, Hamers-Casterman et al. Nature 363:446-448 (1993) and Sheriff et al., Nature Struct. Biol. 3:733-736 (1996).

[0077] Many HVR descriptions are in use and are included in this paper. HVRs based on sequence variability, such as Kabat complementarity-determining regions (CDRs), are the most commonly used (Kabat et al.). Sequences of Proteins of Immunological Interest 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991)). Chothia is used instead to refer to the location of the structural ring (Chothia and Lesk). J. Mol. Biol.196:901-917 (1987)). AbM HVRs represent a compromise between Kabat CDRs and Chothia structural rings, and are used by the AbM antibody modeling software from Oxford Molecular. “Contact” HVRs are based on analysis of available complex crystal structures. The residues from each of these HVRs are recorded below.

[0078] HVRs can include the following “extended HVRs”: 24-36 or 24-34 (L1), 46-56 or 50-56 (L2), and 89-97 or 89-96 (L3) in VL, and 26-35 (H1), 50-65 or 49-65 (H2), and 93-102, 94-102, or 95-102 (H3) in VH. For each of these extended HVR definitions, the variable domain residues are numbered according to Kabat et al. above.

[0079] The expressions “as in Kabat variable domain residue numbering” or “as in Kabat amino acid position numbering” and their variations refer to the heavy chain or light chain variable domain numbering system used for antibody compilation, as described above by Kabat et al. Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to shortened or inserted FR or HVR of the variable domain. For example, the heavy chain variable domain may contain a single amino acid insertion after residue 52 of H2 (according to residue 52a in Kabat) and residues inserted after heavy chain FR residue 82 (e.g., residues 82a, 82b, and 82c, etc., according to Kabat). The Kabat number of residues for a given antibody can be determined by comparing the antibody sequence with homologous regions of sequences numbered in “standard” Kabat.

[0080] The term "laboratory animal" refers to a non-human animal. In one embodiment, the laboratory animal is selected from rats, mice, hamsters, rabbits, camels, llamas, non-human primates, sheep, dogs, cattle, chickens, amphibians, sharks, and reptiles. In one embodiment, the laboratory animal is a rabbit.

[0081] Due to the substituent at the N'1 atom, the molecule according to Formula I is incompatible with the binding pocket of (streptomycin) avidin because the essential tight interaction with the heterocyclic structure of biotin is sterically hindered. It is assumed that once the compound according to Formula I is used as an immunogen, this structure also prevents the formation of antibodies that bind to conventionally biotinylated molecules of interest. The structure of the derivatized biotin disclosed herein still retains the "tail" aspect of biotin, and therefore it is assumed that the derivatized diaminobiotin contained in the immunogen may be suitable for generating the desired monoclonal antibody against biotin.

[0082] In fact, in one embodiment, the monoclonal antibody according to the invention does not bind to conventionally biotinylated molecules, i.e., molecules conjugated with biotin, wherein the carboxyl-functional carbon atom of the valerate side chain of the biotin moiety is covalently coupled to said molecule. In a specific embodiment, the antibody according to the invention also does not bind to... Figure 3 The compound of formula II described in A.

[0083] Conversely, the monoclonal antibody according to the invention binds to a compound of formula I, wherein Y is CH2 or C=O, and X is O or (CH2). n And n is an integer from 1 to 20, and R is H or ZL, where L is a linker and Z is a hapten. In a specific embodiment, the monoclonal antibody according to the invention binds to... Figure 3 Compound of formula III as described in B.

[0084] In a more specific embodiment, the binding affinity of the monoclonal antibody to the compound of formula III is at least 50 times that to the compound of formula II. In yet another more specific embodiment, the binding affinity of the monoclonal antibody to the compound of formula III is at least 500, at least 1,000, at least 5,000, at least 10,000, at least 50,000, and at least 100,000 times that to the compound of formula II.

[0085] Therefore, in another specific embodiment of all aspects disclosed herein, the monoclonal antibody targets a biotinylated target molecule (including, but not limited to, exemplary compounds of formula II (see...)). Figure 3 The affinity of the biotin conjugated to biotin in A)) is selected from a maximum of 1 / 50, 1 / 100, 1 / 500, 1 / 1,000, 1 / 5,000, and at most 1 / 10,000, compared to the affinity for free biotin. In other words, the monoclonal antibody according to the invention has an affinity for unconjugated (free) biotin that is at least 50, 100, 500, 1,000, 5,000, and at least 10,000 times that for the affinity for biotin conjugated to the biotinylated target molecule.

[0086] Numerous methods and systems have been developed for the detection and quantification of analytes of interest in biochemical and biological samples. Methods and systems capable of measuring trace amounts of microorganisms, drugs, hormones, viruses, antibodies, nucleic acids, and other proteins are of great value to researchers and clinicians.

[0087] Many assays utilize analyte-specific binders to capture specific target molecules of interest from a sample, allowing for the assay of said target molecules. In other assays, the analyte of interest can be detected by the competitive binding of a solid-bound analyte and the analyte in the sample to a detectably labeled analyte-specific binder. In serological assays, antibodies against antigens such as infectious agents are detected directly or in so-called double-antigen sandwich assays.

[0088] Generally, the presence or absence of an observable “marker” attached to one or more analyte-specific binders indicates the presence of an analyte of interest.

[0089] The vast majority of immunoassays now employ solid-phase methods. Typically, at least one specific binding agent used in the assay binds directly or indirectly to the solid phase. The streptoavidin-biotin binding pair is characterized by its extremely high binding affinity. For this reason, the streptoavidin-biotin binding pair is widely used for the indirect binding of any suitable biotinylated specific binding agent to a solid phase coated with streptoavidin.

[0090] "Sandwich assay" is a type of assay that is one of the most useful and commonly used. Many variations of sandwich assay techniques exist, and all of these variations are intended to be included in this invention. In short, in a typical forward assay, an unlabeled antibody is immobilized on a "solid phase," and the analyte sample is brought into contact with the bound molecule. This immobilization of the capture antibody can be achieved by direct adsorption to the solid phase or indirectly, for example, by a specific binding pair, such as a streptavidin-biotin-binding pair. After an appropriate incubation period sufficient to allow for the formation of an antibody-antigen complex, a second antibody, labeled with a reporter molecule capable of producing a detectable signal and bound to the antigen, is then added, and incubated again to allow sufficient time for the formation of the antibody-antigen-labeled antibody sandwich complex. Any unreacted material is washed away, and the presence of the analyte is determined by observing the signal produced by the reporter molecule. The results can be qualitative, by simply observing a visible signal, or quantitative, by comparison with a control sample containing a known amount of the analyte.

[0091] For the purposes of this invention, in a typical sandwich assay, a first biotinylated analyte-specific binder, such as a biotinylated antibody, is non-covalently bound to a solid phase coated with (streptomycin)-avidin. The solid phase is typically glass or a polymer, most commonly cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride, or polypropylene. The solid phase can be in the form of tubes, beads, discs of microplates, or any other surface suitable for immunoassay. Coating methods are well known in the art and typically consist of cross-linking, covalent binding, or physical adsorption. The (streptomycin)-avidin-coated solid phase is typically treated to block non-specific binding and washed during preparation for use in the assay procedure. An aliquot of the test sample is contacted with a first or capture antibody and a labeled second antibody and incubated for a sufficient period of time (e.g., 2–40 minutes or overnight, if more convenient) and under suitable conditions (e.g., from room temperature to 40°C, or 25°C to 32°C, inclusive) to allow binding between the first or capture antibody and the corresponding antigen, as well as between the antigen and the second antibody binding to another epitope on the antigen, thereby forming a sandwich complex. Next, a solid phase coated with (streptolysin) avidin is added and incubated for a sufficient period of time (e.g., 2–40 minutes or overnight, if more convenient) and under suitable conditions (e.g., from room temperature to 40°C, or 25°C to 32°C, inclusive) to allow binding between the first or capture antibody and the solid phase. The second antibody is linked to a reporter molecule that indicates the binding of the second antibody to the complex of the first antibody and the antigen of interest.

[0092] The variations that can be measured include simultaneous assays, in which both the sample and the labeled antibody are added simultaneously to the antibody or the antibody that can bind to the solid via a (streptomycin) anti-biotin / biotin binding pair. These techniques are well known to those skilled in the art, including any minor variations that will be readily apparent.

[0093] In a further alternative setup, a sandwich complex is formed, wherein a first biotinylated antibody is provided, a second antibody labeled with a hapten is provided, and both antibodies are contacted with a sample containing the corresponding antigen. While incubating under conditions permissible for forming a sandwich comprising the first and second antibodies and the antigen, a labeled (streptomycin) avidin is added, and the complex is captured by a solid-phase trapping mechanism capable of specifically binding to the hapten to which the second antibody is attached. After a washing step, the amount of labeling bound to the solid indicates the presence and quantity of the antigen of interest.

[0094] Generally, this disclosure provides compounds of formula I for all aspects and embodiments thereof. (Formula I) Where Y is CH2 or C=O, and X is an oxygen atom or (CH2). n And n is an integer from 1 to 20, and R is H or ZL, where L is a linker, and Z is selected from haptens and peptides that do not contain the biotin moiety.

[0095] The first key feature of the Formula I structure is the biotin moiety with an unmodified, unconjugated valerate side chain. It is presumed that the conformation of the heterocyclic structure of the side chain relative to the biotin moiety is not entirely random. This view appears to be supported by NMR data and previous findings of interactions between the carbon atoms of the valerate side chain on one hand and the N'3 atom on the other. Therefore, the unanswered question until now is whether the "tail" portion of the biotin molecule is actually suitable for recognition by monoclonal antibodies, relative to (away from) the heterocyclic "head" portion that can be bound by (streptomycin) anti-biotin.

[0096] Another important feature is the N'3 atom in the heterocyclic portion of biotin in Formula I, because it is not directly modified. Furthermore, if this atom participates in the interaction with the carbon atom that is part of the valeric acid side chain, this interaction on the side chain conformation may be preserved.

[0097] A further key feature that forms the basis of this invention is the N'1 atom of the biotin heterocyclic moiety with substituent RXY, wherein Y is CH2 or C=O, and X is an oxygen atom or (CH2). n And n is an integer from 1 to 20. Therefore, substituted biotin is provided as a structural unit, which in particular allows for the formation of immunogens and molecules used for detecting and screening desired antibodies. Specifically, for this purpose, the terminal group R can be chosen as ZL, where L is a linker and Z is selected from haptens that do not contain the biotin moiety.

[0098] The term "joint" refers to a bifunctional or multifunctional portion that can be used to assemble (connect) a first portion with a second or more portions. Conjugates comprising first and second portions bonded together can be conveniently prepared using a joint having two reactive functionalities. In such a conjugate, the two portions are bonded "through" this joint. As will be apparent to those skilled in the art, in such a conjugate, the functional portion of the joint exists as part of a bond rather than as an unreacted functional portion.

[0099] As defined herein, the term “reactive group” or “reactive functional group” means any group that is suitable for reacting with an amine group, preferably an N-hydroxysuccinimide group or a maleimide group, to bind the linker to an amino group; or a group that is suitable for binding with a second functional group, for example for reacting with an SH- group, preferably a maleimide group, to bind the linker to an SH- group.

[0100] In specific embodiments, the heterobifunctional linker is selected from the NHS-maleimide linker, which is based on N-hydroxysuccinimide and maleimide reactive groups; succinimide-(PEG)n NHS-PEG-maleimide linker; NHS-haloacetyl linker; and NHS-pyridine dithio linker. In a particularly preferred embodiment, the heterobifunctional linker is the succinimide-(PEG)n NHS-PEG-maleimide linker.

[0101] In a specific implementation, L has a main chain length of 1 to 200 atoms. In other words, if the main chain length is between 1 and 200 atoms, the shortest connection between Z and R consists of 1 to 200 atoms.

[0102] In the presence of cyclic systems, the minimum number of atoms in the cyclic system is used when evaluating the joint length. For example, the length of the joint for a phenylene ring is calculated to be four atoms.

[0103] In one embodiment of Formula I, L is a linker having the following as the main chain: a straight or branched saturated, unsaturated, unsubstituted or substituted C1-C20 alkyl chain, or a chain of 1-200 atoms consisting of carbon atoms, substituted carbon atoms and / or one or more atoms selected from O, N, P and S or substituted N, P, S atoms, or a chain as described above, wherein the main chain contains one or more cyclic or heterocyclic aromatic or non-aromatic cyclic systems.

[0104] In one embodiment of Formula I, the connector L has the following as the main chain: a straight or branched saturated, unsaturated, unsubstituted or substituted C1-C100 alkyl chain, or a chain of 1-100 atoms consisting of carbon atoms, substituted carbon atoms and / or one or more atoms selected from O, N, P and S or substituted N, P or S atoms, or a chain as described above, wherein the main chain contains one or more cyclic or heterocyclic aromatic or non-aromatic cyclic systems.

[0105] In one embodiment of Formula I, the connector L has the following as the main chain: a straight or branched saturated, unsaturated, unsubstituted or substituted C1-C50 alkyl chain, or a chain of 1-50 atoms consisting of carbon atoms, substituted carbon atoms and / or one or more atoms selected from O, N, P and S or substituted N, P or S atoms, or a chain as described above, wherein the main chain contains one or more cyclic or heterocyclic aromatic or non-aromatic cyclic systems.

[0106] In a further embodiment of Formula I, the connector L has the following as the main chain: a straight or branched saturated, unsaturated, unsubstituted or substituted C1-C20 alkyl chain, or a chain of 1-20 atoms consisting of carbon atoms, substituted carbon atoms and / or one or more atoms selected from O, N, P and S or substituted N, P or S atoms, or a chain as described above, wherein the main chain contains one or more cyclic or heterocyclic aromatic or non-aromatic cyclic systems.

[0107] In another specific embodiment of Formula I, L is a connector, which is obtained by reacting a difunctional crosslinking agent with suitable chemical groups on each of the two portions connected by the connector, wherein the difunctional crosslinking agent is exemplified below. bis-(2,5-dioxo-pyrrolidone-1-yl) octanoate In another specific embodiment of Formula I, L is a connector, which is obtained by reacting a (hybrid)bifunctional crosslinking agent with suitable chemical groups on each of the two portions connected by the connector. The (hybrid)bifunctional crosslinking agent can be selected from... NHS-maleimide crosslinking agents, for example AMAS (N-α-maleimide acetoxysuccinimide ester) BMPS (N-β-maleimide propyl-oxosuccinimide ester) GMBS (N-γ-maleimide butyryloxysuccinimide ester) Sulfono-GMBS (N-γ-maleimide butyryloxysulfosuccinimide ester) MBS (m-maleimide benzoyl-N-hydroxysuccinimide ester) Sulfonyl-MBS (m-maleimide benzoyl-N-hydroxysulfosuccinimide ester) SMCC (4-(N-maleimidemethyl)cyclohexane-1-carboxylic acid succinimide ester) Sulfono-SMCC (4-(N-maleimidemethyl)cyclohexane-1-carboxylic acid sulfosuccinimide ester) EMCS (N-ε-maleimide hexanoyloxysuccinimide ester) Sulfono-EMCS (N-ε-maleimide hexanoyloxysulfosuccinimide ester) SMPB (4-(p-maleimide phenyl)butyrate succinimide ester) Sulfono-SMPB (4-(N-maleimidephenyl)butyrate sulfosuccinimide ester) SMPH (6-((β-maleimide propionamido)hexanoate succinimide ester) LC-SMCC (4-(N-maleimidemethyl)cyclohexane-1-carboxy-(6-aminohexanoic acid)succinimide ester) Sulfono-KMUS (N-κ-maleimide undecyloxysulfosuccinimide ester) Succinimide-(PEG)n-maleimide or NHS-PEG-maleimide crosslinking agents, for example, SM(PEG)2 (SMCC crosslinking agent with added polyethylene glycol) NHS-PEGn-maleimide Succinimide-([N-maleimidepropamido]-di-ethylene glycol) ester SM(PEG)4 (SMCC crosslinking agent with added polyethylene glycol) NHS-PEGn-maleimide Succinimide-([N-maleimidepropamido]-tetra-ethylenediol) ester SM(PEG)6 (a long-chain SMCC crosslinking agent with added polyethylene glycol) NHS-PEGn-maleimide Succinimide-([N-maleimidepropamido]-hexa-ethylenediol) ester SM(PEG)8 (a long-chain SMCC crosslinking agent with added polyethylene glycol) NHS-PEGn-maleimide Succinimide-([N-maleimidepropamido]-octa-ethylene glycol) ester SM(PEG)12 (a long-chain SMCC crosslinking agent with added polyethylene glycol) NHS-PEGn-maleimide Succinimide-([N-maleimidepropamido]-dodecylene glycol) ester SM(PEG)24 (a long-chain SMCC crosslinking agent with added polyethylene glycol) NHS-PEGn-maleimide Succinimide-([N-maleimidepropamido]-tetracosyl glycol) ester NHS-haloacetyl crosslinking agents, for example SIA (iodoacetic acid succinimide ester) SBAP (3-(bromoacetamido)propionate succinimide ester) SIAB ((4-iodoacetyl)aminobenzoic acid succinimide ester) Sulfon-SIAB ((4-iodoacetyl)aminobenzoic acid sulfosuccinimide ester) and NHS-pyridine dithiolated crosslinking agents, such as SPDP 3-(2-pyridinylthio)propionate succinimide ester LC-SPDP 6-[3(2-pyridinylthio)propamido]hexanoic acid succinimide ester Sulfono-LC-SPDP (6-(3'-(2-pyridinylthio)propamido)hexanoic acid sulfosuccinimide) SMPT (4-succinimideoxycarbonyl-α-methyl-α-(pyridinylthio)toluene) PEG4-SPDP (long-chain SPDP crosslinking agent with added polyethylene glycol) PEG12-SPDP (long-chain SPDP crosslinking agent with added polyethylene glycol) and α-[3-(o-pyridinyldithio)propamido]-ω-(succinimide propionate)hexadecyl(ethylene glycol) In embodiments of Formula I, R is ZL, and Z is a polypeptide. Therefore, the original disclosure herein and another general aspect of the invention (which also relates to all other aspects and embodiments disclosed herein) is an immunogen according to Formula I, where Y is CH2 or C=O, and X is O or (CH2). n And n is an integer from 1 to 20, and R is ZL, where L is a linker and Z is a polypeptide of at least 30 amino acids.

[0108] Importantly, the peptide is capable of evoking an immune response to the biotin moiety, while the biotin moiety alone has low immunogenicity. In embodiments, the peptide is selected from rat, rabbit, mouse, porcine, or bovine serum albumin, bovine or porcine thyroglobulin, ovalbumin, tetanus toxoid, gelatin, soybean trypsin inhibitor, keyhole cyanobacterium (KLH), and similar substances. In a specific embodiment, the peptide is keyhole cyanobacterium (KLH).

[0109] In one embodiment, this disclosure, relating to all other aspects and embodiments, also provides a method for preparing polyclonal antibodies (including antibodies, antibody fragments thereof, and antigen-binding fragments thereof) that specifically bind to an immunogen according to this disclosure. The method comprises: (a) providing an immunogen according to the invention; (b) immunizing an experimental animal with said immunogen under conditions that induce the immune system of an animal to produce antibodies; and (c) removing the antibody that specifically binds to biotin from said animal. The animal may be a sheep, goat, rabbit, rat, mouse, etc.

[0110] The original disclosure herein and another general aspect of the invention (which is also related to all other aspects and embodiments disclosed herein) is a method for producing antibodies according to the invention, the method comprising the steps of: (a) immunizing experimental animals with an immunogen according to the invention, thereby inducing B cells to produce antibodies that bind to the immunogen; (b) obtaining, either by hybridoma technology or by B-cell PCR technology, the monoclonal antibody that binds to the immunogen produced by the B cells in step (a); and (c) further selecting the antibody from step (b) to bind with biotin, thereby obtaining the antibody according to the invention.

[0111] In the embodiments, in Formula I, R is ZL, and Z is a hapten, wherein the hapten does not contain a biotinylate moiety. Therefore, the original disclosure herein and another general aspect of the invention (which also relates to all other aspects and embodiments disclosed herein) is a compound according to Formula I, wherein Y is CH2 or C=O, and X is O or (CH2). n And n is an integer from 1 to 20, and R is ZL, where L is a connector and Z is a hapten, wherein the hapten does not contain a biotinylate portion.

[0112] Compounds according to this aspect are particularly useful in capturing desired antibodies, for example, in methods for identifying polyclonal or monoclonal antibodies produced using the immunogen according to the invention. Such antibodies will specifically bind to the biotin moiety of the compound, meaning the compound is capable of capturing the desired antibody according to the invention. In this respect, the hapten must not and / or must not contain a further biotin moiety to maintain the capture specificity of the compound. Therefore, suitable haptens that are not biotinylated and / or do not contain biotin are selected from dinitrophenol, aniline, aminobenzoic acid, hydralazine, fluorescein, and digitoxin. In a specific embodiment, Z is digitoxin.

[0113] According to a specific embodiment of the compound of formula I, where Y is CH2 or C=O, and X is O or (CH2). n And n is an integer from 1 to 20, and R is ZL, where L is a connector and Z is a hapten, wherein the hapten does not contain a biotin moiety and is a compound of formula III.

[0114] (Formula III) The original disclosure herein and another general aspect of the invention (which is also related to all other aspects and embodiments disclosed herein) is a method for producing antibodies according to the invention, the method comprising the steps of: (a) immunizing experimental animals with an immunogen according to the invention, thereby inducing B cells to produce antibodies that bind to the immunogen; (b) obtaining, either by hybridoma technology or by B-cell PCR technology, the monoclonal antibody that binds to the immunogen produced by the B cells in step (a); and (c) further selecting the antibody from step (b) to bind with biotin, thereby obtaining the antibody according to the invention.

[0115] It is well known in the art that cell lines for producing monoclonal antibodies can be generated through hybridoma technology.

[0116] B-cell PCR techniques known to those skilled in the art utilize the fact that total mRNA can be isolated from B cells and transcribed into cDNA. Using specific primers, nucleic acids encoding homologous VH- and VL-regions can be amplified. Almost no identical sequences are obtained. This method provides highly diverse antibodies that bind to the same antigens.

[0117] Primers for amplifying nucleic acids encoding VH- can be used to obtain cDNA from cells of NMRI- mice, Armenian hamsters, Balb / c- mice, and Syrian hamsters and rabbits.

[0118] In one implementation of all the methods reported herein, the amino acid sequence is derived from the amplified VH-encoded nucleic acid, and the exact start and end points are identified by determining the amino acid sequences from EVQL / QVQL to VSS (VH-region) and from DIVM / DIQM to KLEIK (VL-region).

[0119] This article also reports a method for generating antibodies using B-cell PCR, comprising the following steps: (a) providing a (mature) population of B-cells (obtained from the blood of experimental non-human animals), (b) staining the cells of the B-cell population with at least one fluorescent dye (in one embodiment, 1-3 or 2-3 fluorescent dyes), (c) depositing individual cells of the stained B-cell population in individual containers (in one embodiment, the containers are wells of a multi-well plate), (d) culturing the deposited individual B-cells in the presence of feeder cells, (e) determining the binding specificity of antibodies secreted in the individual B-cell culture, (f) determining the amino acid sequences of the variable light chain and heavy chain domains of the specifically binding antibody by reverse transcriptase PCR and nucleotide sequencing, thereby obtaining the nucleic acid encoded by the variable light chain and heavy chain domains of the monoclonal antibody, (g) introducing the nucleic acid encoded by the variable light chain and heavy chain domains of the monoclonal antibody into an expression cassette for antibody expression, (h) introducing the nucleic acid into the cells, (i) culturing the cells and recovering the antibody from the cells or cell culture supernatant, thereby generating the antibody.

[0120] In one implementation, the non-human animal is selected from rats, mice, hamsters, rabbits, non-human primates, sheep, dogs, cattle, chickens, amphibians, and reptiles.

[0121] In a specific embodiment of the method for generating monoclonal antibodies disclosed herein, in step (c), selection is made in a competitive assay, wherein biotin is used as a competitive agent for the binding of the antibody to a compound according to formula I, wherein Y is CH2 or C=O, and X is O or (CH2). nAnd n is an integer from 1 to 20, and R is H or ZL, where L is a linker, and Z is selected from haptens and polypeptides without a biotin moiety. This compound has been discussed above. The compound is particularly useful in capturing desired antibodies. Such antibodies will specifically bind to the biotin moiety of the compound, that is, the compound is able to capture the desired antibody according to the invention. In this respect, the hapten must not and / or must not contain a further biotin moiety to maintain the capture specificity of the compound. Thus, the hapten is used to anchor the capture agent to a solid phase, as exemplified, for example, by digitoxin as a hapten in Example 5.

[0122] Another important step is to select and / or confirm that the antibody, particularly the monoclonal antibody produced using the immunogen disclosed herein and the method disclosed herein, is actually capable of binding biotin. Therefore, the competitive binding assay ensures that the antibody can bind biotin when contacted with free biotin in aqueous solution. Thus, in an embodiment of the method disclosed herein for producing monoclonal antibodies, in step (c), selection is made in a competitive assay where biotin is used as a competitor for the antibody to bind to the immunogen disclosed herein.

[0123] In another embodiment of the method for generating monoclonal antibodies disclosed herein, in step (c), selection is made in a competitive assay, wherein biotin is used as the antibody and also... Figure 3 The competing agent bound to the compound of formula III described in B.

[0124] More generally, in another embodiment of the method for generating monoclonal antibodies disclosed herein, in step (c), selection is made in a competitive assay, wherein biotin is used as a competitive agent for the binding of the antibody to a compound according to formula I, wherein Y is CH2 or C=O, and X is O or (CH2). n And n is an integer from 1 to 20, and R is ZL, where L is a connector and Z is a hapten, wherein the hapten does not contain the biotin portion.

[0125] As demonstrated in the embodiments of this disclosure, the present invention now provides, in its various aspects and embodiments, novel monoclonal antibodies that specifically bind to compounds of formula I. (Formula I) Its characteristic is that it is also bound to biotin, wherein Y is CH2 or C=O, and X is O or (CH2). n And n is an integer from 1 to 20, and R is H or ZL, where L is a linker, and Z is selected from haptens and peptides that do not contain the biotin moiety.

[0126] The monoclonal antibodies of the present invention can be obtained by means of the present invention and the methods disclosed herein.

[0127] As described above, the monoclonal antibody according to the invention is characterized by its non-binding of compounds of Formula II. In Formula II, the biotinylate moiety is coupled to the hapten via a linker through a carboxyl-functionalized carbon atom of the valerate side chain. Thus, this molecule exemplifies the conventionally biotinylated molecule that the antibody is desired to bind according to the invention.

[0128] Therefore, the original disclosure herein and another general aspect of the invention (which is also relevant to all other aspects and embodiments disclosed herein) is a method for generating antibodies that do not bind to biotinylated molecules, wherein the biotin moiety is coupled to a hapten via a linker through a carboxyl-functional carbon atom of the valerate side chain, the method comprising the steps of: (a) immunizing an experimental animal with an immunogen according to the invention, thereby inducing B cells to produce antibodies that bind to said immunogen; (b) obtaining a monoclonal antibody that binds to said immunogen, either by hybridoma technology or by B-cell PCR technology; (c) selecting the antibodies from step (b) for binding to biotin; and (d) selecting those antibodies that do not bind to biotinylated molecules, thereby obtaining antibodies according to the invention that do not bind to biotinylated molecules. In a specific embodiment, step (d) is performed using a compound according to Formula II.

[0129] Therefore, more specifically, a method for generating antibodies that do not bind to compounds of formula II is disclosed, the method comprising the steps of: (a) immunizing experimental animals with an immunogen according to the invention, thereby inducing B-cells to produce antibodies that bind to said immunogen; (b) obtaining monoclonal antibodies that bind said immunogen produced by B-cells in step (a) either by hybridoma technology or by B-cell PCR technology; (c) selecting the binding of the antibodies in step (b) to biotin; and (d) selecting those antibodies that do not bind to compounds of formula II, thereby obtaining antibodies according to the invention that do not bind to compounds according to formula II.

[0130] Especially in methods for measuring analytes in samples, where the (streptomycin) avidin / biotin binding pair is used to bind biotinylated analyte-specific binders to a (streptomycin) avidin-coated solid phase, the measurement of the analyte can become inaccurate if the sample contains particularly high amounts of biotin. For this reason, the removal of biotin is highly desirable. This technical problem can be addressed through sample pretreatment, i.e., by removing free biotin before the sample undergoes the method for measuring the analyte. However, this involves a large number of undesirable discrete working steps, such as, for example, mixing the sample with magnetic particles coated with (streptomycin), incubating the mixture to bind biotin to the particles, followed by magnetic removal of the particles. This sample pretreatment method not only consumes resources such as raw materials and time; moreover, it carries the risk of altering the sample (e.g., its volume and composition), which in turn can lead to undesirable effects in analyte detection and measurement.

[0131] Surprisingly, the monoclonal antibody according to the invention, capable of binding biotin without binding to conventionally biotinylated molecules of interest, provides a sophisticated solution for reducing biotin interference. When acting as a biotin scavenger, the antibody's biotin-binding properties are such that it does not interfere with, for example, the binding of biotinylated analyte-specific binders to (streptomycin) anti-biotin. Therefore, the monoclonal antibody according to the invention proves to be a powerful tool for counteracting interference caused by high levels of biotin that may be present in a sample.

[0132] The original disclosure herein and another general aspect of the invention (which is also related to all other aspects and embodiments disclosed herein) is the use of the antibody according to the invention in a method for measuring an analyte in a sample, wherein the (streptomycin) avidin / biotin pair is used to bind a biotinylated analyte-specific binder to a (streptomycin) avidin-coated solid phase.

[0133] The original disclosure herein and another general aspect of the invention (which is also relevant to all other aspects and embodiments disclosed herein) is a method for measuring an analyte in a sample, wherein a (streptomycin) avidin / biotin binding pair is used to bind a biotinylated analyte-specific binder to a (streptomycin) avidin-coated solid phase, the method comprising adding to the sample a) an antibody according to the invention, b) a biotinylated analyte-specific binder, c) a streptomycin-coated solid phase, followed by measuring the analyte bound to the solid by the (streptomycin) avidin and the biotinylated analyte-specific binder.

[0134] This disclosure provides a method for removing potentially interfering free biotin from a sample from which an analyte should be measured, and the measurement method utilizes a (streptomycin)-avidin / biotin binding pair. Preferably, the removal step is performed before the formation of the (streptomycin)-avidin / biotin pair. Thus, in an embodiment, a method for measuring an analyte in a sample is provided, wherein the (streptomycin)-avidin / biotin binding pair is used to bind a biotinylated analyte-specific binder to a (streptomycin)-avidin-coated solid phase, the method comprising adding to the sample a) an antibody according to the invention, b) a biotinylated analyte-specific binder, c) a (streptomycin)-avidin-coated solid phase, followed by measuring the analyte bound to the solid by the (streptomycin)-avidin and the biotinylated analyte-specific binder, wherein step (a) and optionally step (b) are performed before step (c). In yet another embodiment, steps (a) and (b) are performed simultaneously before step (c). In yet another embodiment, steps (b) and (c) are performed simultaneously after step (a) has been performed. In yet another embodiment, all three steps (a), (b), and (c) are performed simultaneously. Surprisingly, the monoclonal antibody of the present invention is even technically suitable as an effective scavenger of free biotin for use in methods for measuring analytes in samples containing free biotin, wherein (streptomycin) avidin / biotin pairs are used to bind a biotinylated analyte-specific binder to a (streptomycin) avidin-coated solid phase or labeled (streptomycin) avidin, wherein the sample, the monoclonal antibody of the present invention, the biotinylated binder, and any one of the (streptomycin) avidin-coated solid phase or labeled (streptomycin) avidin are simultaneously in contact with each other.

[0135] Regarding all aspects and embodiments of analyte detection and / or measurement as disclosed herein, aqueous liquid samples can be used in methods for the specific in vitro detection of analytes according to the methods of this disclosure. It may be known that the sample contains the analyte, or it may be suspected that the sample contains the analyte. In one embodiment, the sample used for in vitro diagnostics in the methods according to the disclosure is a bodily fluid selected from whole blood, serum, plasma, fluid, urine, or saliva. In one embodiment, the sample suspected of containing or containing the analyte is serum, plasma, or fluid. In one embodiment, the sample suspected of containing or containing the analyte is serum or plasma.

[0136] The original disclosure herein and another general aspect of the invention (which is also related to all other aspects and embodiments disclosed herein) is an immunoassay kit comprising, in a separate container, at least (a) an antibody according to any one of the claims of the invention, (b) a biotinylated analyte-specific binder, and (c) a solid phase coated with (streptomycin) antibiotin.

[0137] The term "single container unit" refers to the fact that, for many automated analyzers, such as the Elecsys® analyzer series from Rochediagnostics, the reagents required to measure an analyte are provided in the form of a "reagent pack," that is, a container unit that is installed on the analyzer and contains all the key reagents required to measure the analyte of interest in different compartments.

[0138] In one embodiment, the present invention relates to a kit in which the first partner of the binding pair is avidin or streptoavidin, and wherein the second partner of the binding pair is selected from biotin or biotin analogs, such as aminobiotin, iminobiotin, or desulfobiotin.

[0139] The following embodiments and accompanying drawings are provided to aid in understanding the invention, the true scope of which is set forth in the appended claims. It should be understood that modifications can be made to the stated procedures without departing from the spirit of the invention. Attached Figure Description

[0140] Figure 1 A: D(+)-Biotin; B: D(+)-Biotin Figure 2 A and B: Synthesis schemes of Examples 1 and 2 Figure 3 A: Compounds of Formula II; B: Compounds of Formula III Figure 4 Exemplary kinetic characteristics from antibody kinetic screening assays. Dashed line: SPR binding signal of Dig-biotin-conjugate-M-19.11-Fab' complex injection. Solid line: SPR binding signal of Dig-biotin-conjugate-M-19.11-Fab' complex supplemented with 300 nM d-biotin. Generally, three classes of free d-biotin blocking kinetics are observed: A: Complete d-biotin competition. B: Intermediate d-biotin blocking. C: No d-biotin signal interference. Suitable antibody candidates from classes A and B are selected for subsequent detailed studies.

[0141] Figure 5An exemplary SPR sensor overlay plot for guiding antibody candidate clone G SPR IC50 measurements. 270 nM Dig-biotin-conjugate was mixed with free d-biotin at concentrations of 270 nM (not shown), 90 nM, 30 nM (twice), 10 nM, 3.3 nM, and 1 nM. The mixture containing 90 nM of free d-biotin produced the lowest response signal, and 1 nM of free d-biotin produced the highest response signal (labeled). X indicates the location of the reporter point used in IC50 calculations.

[0142] Figure 6 A The kinetic screening assay was prepared using the SPR configuration. First, a pre-formed Dig-biotin-conjugate-M-19.11-Fab' was used as the analyte in solution to monitor the binding of the conjugate d-biotin with increasing molecular weight. Second, d-biotin was added to the analyte mixture to compete with the conjugate for binding.

[0143] B SPR assay setup. Dig-biotin-conjugate interactions were measured with and without d-biotin concentration series. In the absence of d-biotin, a concentration-dependent series of Dig-biotin-conjugate reactions was used to determine the relationship between rabbit antibody kinetics and the Dig-biotin-conjugate. IC50 was measured in the analyte sample mixture with constant concentrations of the Dig-biotin-conjugate and progressively increasing concentrations of free d-biotin.

[0144] C Alternative SPR assay configurations were used. Dig-biotin-conjugate interactions were measured with and without d-biotin concentration series. In the absence of d-biotin, concentration-dependent series of Dig-biotin-conjugates were used to determine the relationship between rabbit antibody kinetics and Dig-biotin-conjugates. In this case, the Dig-biotin-conjugates were captured by the surface-exposed M-19.11 antibody, while the rabbit antibody or a fragment thereof was used as a concentration-dependent analyte in solution. IC50 was measured in the analyte sample mixture using a constant concentration of rabbit antibody and progressively increasing concentrations of free d-biotin. Detailed Implementation

[0145] Example 1 Biotin derivatization at N'1 atom (1) FMOC-β-alanine-acyl chloride Slowly add approximately 20 mL of thionyl chloride to 4.7 g of FMOC-β-alanine (IRIS, FAA1300) in a flask under dry conditions. Stir the mixture at room temperature for 50 minutes, and then heat to reflux for 10 minutes. After cooling, evaporate the mixture, dissolve the residue in anhydrous toluene three times, evaporating each time.

[0146] The yield was 5.1 g.

[0147] (2) FMOC-β-alanyl-biotin The carboxylic acid of biotin was protected by reacting biotin (2.48 g) with tert-butylchlorodiphenylsilane (6.5 ml) in 15 mL of anhydrous pyridine overnight at room temperature under an inert argon atmosphere in the presence of DMAP (0.63 g), as described by Fang and Bergstrom in Nucleic Acids Research, 2003, Vol. 31, No. 2, 708.

[0148] Add 4.9 g of FMOC-β-alanine-acyl chloride dissolved in 10 mL of dichloromethane and stir at room temperature for 3.5 hours. Evaporate the mixture, add DMF, and evaporate again. Dissolve the residue in DMF-H2O (3:1), add 50 mmol of potassium carbonate, and stir the mixture for 30 minutes. Acidify to pH 4 with citric acid, extract the product with ethyl acetate, and purify by column chromatography (silica, eluent ethyl acetate / methanol).

[0149] HPLC-ESI-MS: M + = 538.3 Da. Yield is 1.5 g.

[0150] (3) β-Alanyl-Biotin = 5-[3-(3-amino-propionyl)-2-oxo-hexahydro-thieno[3,4-d]imidazol- 6-yl]-valeric acid The FMOC protecting group was cleaved by dissolving 1.67 g of FMOC-β-alanyl-biotin in a mixture of 20% piperidine in DMF (60 mL). The mixture was evaporated and dried under vacuum. The product was then separated by preparative HPLC using a reversed-phase silica and H2O-acetonitrile gradient.

[0151] HPLC-ESI-MS: M + = 316.3 Da. Yield is 0.55 g.

[0152] Example 2 Immunogen synthesis (4) Succinimide-cycloheptyl-β-alanyl-biotin = 7-{3-[6-(4-carboxy-butyl)-2-oxo- Hexahydro-thienro[3,4-d]imidazol-3-yl]-3-oxopropylcarbamoyl}-heptanoic acid 2,5-dioxo-pyrrolidine-1- ester 24 mg of disuccinimide octanoate and 10 μl of trimethylamine were added to a 10 mL flask, followed by the addition of 10 mg of β-alanyl-biotin dissolved in 1 mL of DMF. After stirring for 30 minutes, the solvent was removed under vacuum. The residue was purified by preparative HPLC.

[0153] HPLC-ESI-MS: M + = 569.3 Da. Yield is 12 mg.

[0154] (5) Antigen conjugate = KLH-SUB-β-ala-Biotin 10 mg of the NHS ester synthesized as described in Example 1 above was dissolved in 1000 μl of DMSO and added to a solution of 100 mg KLH (keyhole hemocyanin, Sigma H 8283). The pH was adjusted to pH = 8.3 and the solution was stirred overnight. The mixture was purified in an Amicon stirred assay cell.

[0155] Amino acid analysis: Antigen-KLH ratio - approximately 500:1.

[0156] Example 3 Reagents for synthesizing monoclonal antibodies (1) Dig-3-CME-AMCAP-DADOO-Biotin Digitalisin-3-CME-AMCAP-NHS ester (other names: digitalisin NHS-ester, cal-20(22)-enolactone, 3-[2-[[6-[(2,5-dioxo-1-pyrrolidinyl)oxy]-6-oxohexyl]amino]-2-oxoethoxy]-12,14-dihydroxy-,(3β,5β,12β)-) = CAS Nr. 129273-26-3 synthesized as described in DE3836656A1, or obtained as a commercially available product (Merck-Sigma).

[0157] 21 mg of digitoxin-3-CME-AMCAP-NHS ester was dissolved in 3 mL of DMF, and 6 μl of trimethylamine was added. Then, 14 mg of biotin-DADOO was added, and the mixture was stirred at room temperature for 2 hours. The solvent was removed by evaporation, and the product was separated by preparative HPLC.

[0158] HPLC-ESI-MS: M+ = 918.7 Da. Yield: 23 mg.

[0159] (2) Dig-3-CME-AMCAP-β-ala-Biotin β-alanyl-biotin=β-ala-biotin=5-[3-(3-amino-propionyl)-2-oxo-hexahydro-thieno[3,4-d]imidazol-6-yl]valerate was synthesized as described above (Example 1).

[0160] Digitalisin-3-CME-AMCAP-NHS ester (other names: digitalisin NHS-ester, cal-20(22)-enolactone, 3-[2-[[6-[(2,5-dioxo-1-pyrrolidinyl)oxy]-6-oxohexyl]amino]-2-oxoethoxy]-12,14-dihydroxy-,(3β,5β,12β)-) = CAS Nr. 129273-26-3 synthesized as described in DE3836656A1, or obtained as a commercially available product (Merck-Sigma).

[0161] 20 mg of digitoxin-3-CME-AMCAP-NHS ester was dissolved in 3 mL of DMF, and 6 μl of triethylamine was added. Then, 10.5 mg of β-ala-biotin was added, and the mixture was stirred at room temperature for 3.5 hours. The solvent was removed by evaporation, and the product was separated by preparative HPLC.

[0162] HPLC-ESI-MS: M+ = 859.6 Da. Yield: 16 mg.

[0163] Example 4 Immunizing rabbits to produce antibodies that bind to free biotin. This disclosure reports the development of antibodies capable of scavenging free biotin. To this end, antibodies that bind to biotin are generated only when the COOH- group of the valerate moiety of biotin is accessible and not used for conjugation. The antibodies according to the invention do not bind to biotinylated molecules, i.e., conventional biotin-conjugates, wherein biotin is covalently coupled via the carbon atom of the carboxyl group.

[0164] To generate this antibody, 12-16 week old NZW rabbits were immunized with KLH-SUB-β-ala-biotin (see above, Example 2). All rabbits underwent repeated immunizations. During the first month, animals were immunized weekly. From the second month onwards, animals were immunized monthly. For the first immunization, 500 μg of KLH-SUB-β-ala-biotin was dissolved in 1 mL of 140 mM NaCl and emulsified in 1 mL of CFA (complete Freund's adjuvant). For all subsequent immunizations, CFA was replaced with IFA (incomplete Freund's adjuvant). The titers were evaluated on day 45 after immunization began.

[0165] Example 5 Antibody titer analysis in immunized animals The experimental setup for serum titration was designed to determine the amount of polyclonal antibody that can distinguish between (i) conventional types of conjugated biotin, i.e., biotin conjugated to the carrier via the carboxyl functional carbon atom of the valeric acid moiety, and (ii) conjugated biotin covalently attached to the carrier via the N'1 atom of the urea ring. Example 3 and Figure 3 A and B provided compounds (1) for (i) the exemplary Dig-3-CME-AMCAP-DADOO-Biotin (compound of formula II), and (2) for (ii) the exemplary Dig-3-CME-AMCAP-β-ala-Biotin (compound of formula III).

[0166] Therefore, 96-well plates were first coated with 5 μg / mL polyclonal sheep anti-Dig antibody (Sigma). After washing, the plates were blocked with 5% BSA (Roche) to reduce background signal. To capture different biotin-Dig conjugates (CME-AMCAP-β-ala-biotin and Dig-3-CME-AMCAP-DADOO-biotin), the anti-Dig-coated plates were incubated with the conjugates at 250 ng / mL in separate wells. After another washing step, rabbit serum was diluted in PBS containing 1% BSA, and dilutions were added to the plates. Serum was tested at dilutions of 1:300, 1:900, 1:2,700, 1:8,100, 1:24,300, 1:72,900, 1:218,700, and 1:656,100. The binding of antibodies was detected using HRP-labeled F(ab')2 goat anti-rabbit Fcγ (Dianova) and ABTS (Roche) as substrates. The titers of the analyzed animals were set by diluting the curve to a 50% signal reduction.

[0167] As additional negative controls, the plates were also coated with different conventionally biotinylated molecules: a peptide-biotin conjugate (CD68-bi), a recombinant protein-biotin conjugate (CD4-bi), and a recombinant F(ab')2-biotin conjugate rK-F(ab')2. In all three negative controls, biotin was coupled to the linker via the carboxyl functional carbon atom of the valerate moiety and therefore should not be detected by polyclonal serum. Table 2 shows the results.

[0168] Table 2 Titration of serum from experimental animals immunized with biotin-KLH immunogen, wherein biotin is coupled to KLH via the N'1 atom of the urea ring.

[0169] Polyclonal sera from three immunized animals showed that only Dig-3-CME-AMCAP-β-ala-biotin bound, which is similar to the immunogen and accessible via the COOH- group of the valerate side chain. Of all the screening reagents in which biotin was coupled to the corresponding carrier via the carboxyl functional carbon atom of the valerate moiety, rK-F(ab')2, CD68-bi, CD4-bi, and Dig-3-CME-AMCAP-DADOO-biotin were weakly detected or not detected at all.

[0170] Example 6 Develop monoclonal antibodies that bind to free biotin To develop antibodies that bind to biotin in solution (i.e., free biotin) and are not cross-reactive with conventionally biotinylated targets, B-cell clones were used as described in Seeber et al. (2014), PLoS One. 4 Feb 2014; 9(2). To enrich antigen-reactive B-cells, biotinylated mouse anti-Dig antibody (Roche) was bound to streptoacidin-coated magnetic beads (Miltenyi). A library of PBMCs from immunized animals was then prepared and incubated with 250 ng / mL LDig-3-CME-AMCAP-β-ala-biotin. After 1 hour of incubation, the cells were washed with PBS and incubated with pre-coated anti-Dig magnetic beads. To enrich antigen-reactive B-cells, MACS columns (Miltenyi) were used. B-cell sorting and incubation were performed as described in Seeber et al. (2014), PLoS One. 4 Feb 2014; 9(2). 24 hours prior to ELISA, to identify antigen-reactive clones, 2 μg / mL streptavidin was added to the cell culture supernatant to neutralize free biotin. This was done as a precaution, as free biotin in the culture medium or cell-derived form can block the interaction between antibodies in the supernatant and the screening reagent Dig-3-CME-AMCAP-β-ala-biotin (Formula III). For ELISA, 96-well plates were coated with 5 μg / mL polyclonal goat anti-rabbit-IgG antibody. After a washing step, the plates were blocked with 5% BSA to reduce background signal. The plates were washed again, and 30 μl of rabbit B-cell culture was transferred to each 96-well plate and incubated at room temperature for 1 hour. After another washing step, 50 ng / mL of the positive screening reagent, Dig-3-CME-AMCAP-β-ala-biotin, or the negative screening reagent, Dig-3-CME-AMCAP-DADOO-biotin (Formula II), was added to the wells and incubated at room temperature for 1 hour. To detect antibodies binding to the screening reagent, 3 μg / mL of POD-labeled polyclonal sheep anti-Dig-antibody was added to the plate. After the final washing step, ABTS (Roche) was added as the POD substrate, and positive clones were identified by measuring OD at 405 nm. The results are shown in Table 3.

[0171] Table 3 The ELISA results of four identified clones that produced antibodies characterized by specific binding to the positive selection reagent according to Formula III but not to the negative selection reagent according to Formula II.

[0172] Example 7 Biotin-binding monoclonal antibody To demonstrate that the newly generated desired antibodies (including those represented by the clones shown in Table 3) additionally bind to free biotin, a competitive ELISA assay was designed and performed.

[0173] For this purpose, cloning antibodies were first performed as described by Seeber et al. (2014), PLoS One. February 4, 2014; 9(2). To produce a supernatant containing antibodies, HEK cells were transiently transfected with expression plasmids encoding the relevant heavy and light chains of antibiotin antibodies. After a 1-week culture period, the concentration of antibiotin antibodies in the transient transfection was determined, and the concentration of each selected clone was adjusted to 5 μg / ml.

[0174] For ELISA, 96-well plates were coated with 5 μg / mL polyclonal goat anti-rabbit IgG antibody. After washing, the plates were blocked with 5% BSA to reduce background signal. The plates were washed again, and 30 μl of antibody supernatant (5 μg / mL) from transient transfection was transferred to each well of the 96-well plate and incubated at room temperature for 1 hour.

[0175] Each selected clone was added in a row to eight different wells for biotin titration.

[0176] After incubation, the plate was washed three times to remove streptavidin / biotin complexes present in the cloning supernatant. For the positive control, 50 ng / mL of positive screening reagent, Dig-3-CME-AMCAP-β-ala-biotin (Formula III), was added to one well for each antibody and incubated at room temperature for 1 hour.

[0177] Titration was performed to test the binding of free biotin. For this purpose, free biotin was added to Dig-3-CME-AMCAP-β-ala-biotin at progressively increasing concentrations. Biotin concentrations of 10 ng / mL, 20 ng / mL, 40 ng / mL, 80 ng / mL, 160 ng / mL, 320 ng / mL, and 640 ng / mL were added to an amount of 50 ng / mL Dig-3-CME-AMCAP-β-ala-biotin. The plate was then washed again, and for the detection of antibodies binding to Dig-3-CME-AMCAP-β-ala-biotin, 3 μg / mL of POD-labeled polyclonal sheep anti-Dig-antibody was added to the plate. After the final washing step, ABTS (Roche) was added as a POD substrate, and the signal was detected by measuring the OD (optical density) at 405 nm. Notably, a decrease in signal was observed as a result of progressively increasing biotin concentrations in the presence of screening reagents. This indicates that free biotin in solution and Dig-3-CME-AMCAP-β-ala-biotin compete for binding to the selected antibody.

[0178] Example 8 Counteracting biotin interference in immunological assays For the control samples used in the Elecsys TSH assay, PreciControl Universal 2 was doped with gradually increasing concentrations of biotin (e.g., final biotin concentrations of 0, 100, and 200 ng / mL).

[0179] Reagent 2 of the Elecsys TSH commercial kit containing anti-TSH detection antibody is used in both unmodified (control) and modified forms, and also contains 300 μg / mL of monoclonal antibiotin antibody according to the present invention.

[0180] The sandwich assay was performed according to the standard procedure for Elecsys TSH assays: In short, 50 μL of sample (with or without biotin) was incubated with 60 μL of Reagent 1 from the Elecsys TSH kit containing biotinylated anti-TSH antibody and 50 μL of Reagent 2 containing, or without, ruthenylated anti-TSH antibody with or without the addition of 300 μg / mL monoclonal anti-biotin antibody. After incubation at 37°C for 9 minutes, 40 μL of a streptavidin-coated magnetic bead suspension from the Elecsys TSH kit was added, and the reaction was incubated for another 9 minutes. Finally, the reaction mixture was aspirated into the assay cell, where the particles were magnetically trapped onto the electrode surface. Unbound material was then removed using ProCell. A voltage was then applied to the electrode to induce electrochemiluminescence-based light emission, which was measured by a photomultiplier tube.

[0181] Table 4 shows the signals obtained for TSH-reagent variants with or without different antibiotin scavenger antibody clones: Using a control reagent, the presence of 100 or 200 ng / mL biotin in the sample resulted in a significant signal decrease to 53% or 16% of the reference signal (sample without biotin).

[0182] Conversely, in the modified form of the kit containing biotin-binding antibodies in reagent R2, the interference of biotin was significantly reduced.

[0183] Table 4 Signal and signal recovery from samples with different biotin concentrations using reagents that do not have or have different monoclonal anti-biotin antibody clones. Example 9 Kinetic antibody screening Kinetic screening was performed at 37°C on a GE Healthcare Biacore 4000 instrument. The Biacore CM5 Series S sensor was installed in the instrument and pre-conditioned according to the manufacturer's instructions. The system buffer was HBS-EP (10 mM HEPES (pH 7.4), 150 mM NaCl, 1 mM EDTA, 0.05% (w / v) P20). The sample buffer was a system buffer supplemented with 1 mg / ml CMD (carboxymethyl dextran, Fluka).

[0184] A rabbit antibody capture system was established on the biosensor. Polyclonal goat anti-rabbit IgG Fc capture antibody GARbFcγ (code number: 111-005-046, lot number #105332, Jackson Immuno Research) was chemically fixed using NHS / EDC according to the manufacturer's instructions. 30 μg / ml GARbFcγ in 10 mM sodium acetate buffer (pH 4.5) was pre-concentrated to spots 1, 2, 4, and 5 in flow assay cells 1, 2, 3, and 4, and fixed at 10,000 RU GARbFcγ. The sensor was then saturated with 1 M ethanolamine at pH 8.5. Spots 1 and 5 were used for interaction measurements, and spots 2 and 4 were used as references. Suspensions of each rabbit antibody clone were diluted 1:2 in sample buffer and injected at a flow rate of 30 μl / min for 1 minute. Rabbit antibody capture levels (CL), expressed in response units (RU), were monitored.

[0185] Because the molecular weight of the 0.9 kDa Dig-biotin-conjugate is too small for the sensitivity range of the SPR screening instrument, the 300 nM Dig-biotin-conjugate (Dig-3-cme-Amcap-β-Ala-biotin (BMO No. 15420318)) was incubated at room temperature with 900 nM... <dig>MD.G-Fab' pre-incubates for 2 hours. mAb <dig>MD.G-Fab binds to digitoxin with a KD 5 pM affinity. MD.G-Fab' also binds to the digitoxin moiety in the Dig-biotin-conjugate with a picomolar affinity and does not interfere with the GARbFcγ capture system. MD.G-Fab' does not interfere with the anti-biotin rabbit clone and free d-biotin. Using 50 kDa MD.G-Fab', an additional 0.9 kDa Dig-biotin-conjugate is loaded. This results in a highly stable immune complex with a molecular weight of 50.9 kDa, which is optimal for the sensitivity range of the SPR screening instrument. MD.G-Fab' is monomeric and neither produces nor detects analyte affinity. The pre-formed Dig-biotin-conjugate-MD.G-Fab' complex was injected separately at 30 μl / min for 5 min to monitor the association phase with the corresponding surface-exposed anti-biotin rabbit mAb. The dissociation of the conjugate from the rabbit clone was monitored for up to 5 min. After each kinetic rate determination cycle, the rabbit clone was completely washed from the biosensor capture system by injecting 10 mM glycine at pH 2.0 for 1 minute, followed by injecting 10 mM glycine at pH 2.25 at 20 μl / min for 2 minutes.

[0186] In the second screening configuration, rabbit anti-biotin antibodies sensitive to d-biotin binding interference were identified. An analyte mixture was prepared by overnight incubation at room temperature, consisting of 300 nM Dig-biotin-conjugate and 900 nM... <dig>The mixture consisted of MD.G-Fab' and 300 nM free d-biotin (d-biotin, CAS No.: 58-85-5, catalog number: 47868, Supelco). Kinetic screening was performed as described above, but with this analyte mixture in solution. In another embodiment, different Dig-biotin-conjugates and... <dig>MD.G-Fab' concentration.

[0187] Figure 6 The experimental setup used for screening is explained. This method was used to test antibodies on several thousand rabbits.

[0188] The kinetic traces of single-concentration kinetics for both analytes were monitored using Biacore 4000 evaluation software. Furthermore, the kinetic data were characterized by reporter points and interpreted by kinetic measurements. Two reporter points—the signal recorded shortly before the end of analyte injection, Binding Late (BL), and the signal recorded shortly before the end of dissociation time, Stability Late (SL)—were used to characterize analyte / antigen binding stability. Additionally, the dissociation rate constant was calculated according to the Langmuir model. k d (1 / s), and according to the formula ln(2) / (60* kd Calculate the half-life of the antibody / antigen complex in minutes. The molar ratio, combined with stoichiometry, is calculated using the following formula: MW(antibody) / MW(antigen) * BL(antigen) / CL(antibody).

[0189] Finally, the kinetic traces of each anti-biotin rabbit mAb were overlaid in an analytical plot. These overlays were primarily examined visually to select antibodies that showed an effective reduction in Dig-biotin-conjugate binding signal in the presence of 300 nM free d-biotin. Effective meant a reduction in Dig-biotin-conjugate binding signal of over 90%. In another embodiment, antibodies with 80%, 70%, 60%, and 50% signal reduction were selected. The selected antibodies were then transferred to a detailed IC50 analysis.

[0190] Figure 4 Three antibody blocking characteristics, as measured by SPR-based kinetic screening, are illustrated exemplarily. Class A exhibits complete d-biotin blockade upon binding to the Dig-biotin-conjugate. Class B is characterized by antibodies with only moderate sensitivity to d-biotin signaling interference, while Class C is insensitive to d-biotin.

[0191] Detailed functional analysis based on SPR Detailed kinetic studies were performed at 37°C on a GE Healthcare T200 instrument. The Biacore CM5 Series S sensor was installed in the instrument and pre-conditioned according to the manufacturer's instructions. The system buffer was HBS-EP (10 mM HEPES (pH 7.4), 150 mM NaCl, 1 mM EDTA, 0.05% (w / v) P20). The sample buffer was a system buffer supplemented with 1 mg / ml CMD (carboxymethyl dextran, Fluka).

[0192] In one embodiment, a rabbit antibody capture system was established on a CM5 biosensor. Polyclonal goat anti-rabbit IgG Fc capture antibody GARbFcγ (code number: 111-005-046, batch number #105332, Jackson ImmunoResearch) was chemically fixed using NHS / EDC according to the manufacturer's instructions. 30 μg / ml GARbFcγ was pre-concentrated in 10 mM sodium acetate buffer (pH 4.5) to flow assay cells 1, 2, 3, and 4, and fixed with 10,000 RU GARbFcγ. The sensor was then saturated with 1 M ethanolamine at pH 8.5.

[0193] Selected rabbit antibody clones from the initial kinetic screening step were diluted 500 nM each in sample buffer and captured on the biosensor at a flow rate of 5 μl / min for 2 min, followed by a 2 min wash at 60 μl / min with 10-fold concentrated HBS-EP system buffer. Rabbit antibody capture levels (CL), expressed in response units (RU), were monitored. The higher sensitivity of the T200 instrument avoids the limitations of traditional methods. <dig>Additional molecular weight loading of MD.G-Fab'. A series of analytes were injected at 60 μl / min for 5 min, with the association and dissociation phases monitored for 5 min. First, the Dig-biotin-conjugate analyte was injected at 300 nM, omitting d-biotin in solution. Then, progressively increasing d-biotin concentrations of 4 nM, 8 nM, 15 nM, 30 nM, 90 nM, and 270 nM were added to the Dig-biotin-conjugate mixture. Sensing overlay plots were generated to analyze the inhibition of Dig-biotin-conjugate binding signals by the presence of progressively increasing d-biotin concentrations. Late binding reporting points from the signal plateau of the analyte association phase were plotted within the progressively increasing d-biotin concentration range, and the d-biotin IC50 value was determined using point-to-point mode in Biaevaluation software. Furthermore, the competitive performance of d-biotin in the sensing overlay plots was visually investigated, and the % signal inhibition was estimated. In another implementation, the % signal blockade is calculated by comparing 0 nM d-biotin and 270 nM d-biotin sample injections.

[0194] Figure 6 The experimental setup for SPR measurement using IC50 is shown. Figure 6 B illustrates a preferred implementation scheme.

[0195] Figure 5 The results of this competitive assay are shown as an example. Antibody-guided candidate G is displayed. Higher concentrations of free d-biotin in the sample correspond to lower Dig-biotin-conjugate binding signals, and thus the sensitivity of the antibody to d-biotin binding can be estimated. The IC50 of clone G was determined to be IC50 = 30 nM d-biotin.

[0196] In another embodiment, the kinetic parameters ka [1 / Ms], kd [1 / s], t1 / 2 diss [min], KD [M], and binding stoichiometry (molar ratio) of the Dig-biotin-conjugate antibody are determined.

[0197] The kinetics of clone G at 37 °C were determined, with ka = 5.8 × 10⁵ 1 / Ms, kd = 2.1 × 10⁻² (1 / s), t¹ / ² diss = 1 min, and MR = 1.2. The affinity for the Dig-biotin-conjugate was KD = 36 nM.

[0198] The kinetics of clone A at 37 °C were determined, with ka = 1.0 × 10⁶ 1 / Ms, kd = 3.0 × 10⁻⁴ (1 / s), t¹ / ² diss = 39 min, and MR = 1.5. The affinity for the Dig-biotin-conjugate was KD = 0.3 nM.

[0199] In one embodiment, the T200 instrument was configured as described above. Dig-biotin-conjugates were injected serially at concentrations of 0 nM, 4 nM, 8 nM, 15 nM, 30 nM, 90 nM, and 270 nM. Kinetic parameters were determined using Biacore evaluation software.

[0200] In another embodiment, the kinetic parameters of Dig-biotin-conjugate antibody binding, ka [1 / Ms], kd [1 / s], t1 / 2 diss [min], KD [M], and binding stoichiometry (molar ratio), are determined by configuring an alternative sensor surface. In one embodiment, a mouse antibody capture system is established on a CM5 biosensor. Polyclonal rabbit anti-mouse IgG capture antibody (RbAMIgG (rabbit anti-mouse IgG), pAb) is chemically fixed using NHS / EDC according to the manufacturer's instructions. <m-igg>Rb-IgG (IS), BR-1008-38, 2017-08, GE Healthcare). 30 μg / ml GARbFcγ in 10 mM sodium acetate buffer (pH 5.0) was pre-concentrated into flow assay cells 1, 2, 3, and 4, and fixed with 10,000 RU. The sensor was then saturated with 1 M ethanolamine at pH 8.5.

[0201] Capture approximately 300 RU of 30 nM mAb at a rate of 10 μl / min. <dig>M-19 / 11-IgG (Q) (Roche, 28.1.1999, introduction 19.5.2017, Id. 2157861) was injected for 1 minute. Dig-biotin-conjugate was injected at 30 μl / min at 150 nM for 2 minutes to achieve stable capture by M-19 / 11-IgG.

[0202] Figure 6 The assay configuration is described. This assay is preferably used for the kinetic determination of recombinantly expressed rabbit antibiotin Fab' fragment.

[0203] Then, as described above, anti-biotin rabbit antibodies or fragments thereof were injected in a concentration-dependent serial manner to determine binding kinetics.

[0204] Example 10 ITC Experiment The purpose of the ITC experiment is to directly determine the affinity of the selected antibody for free biotin in solution, as well as the thermodynamic parameters and stoichiometry of the binding reaction.

[0205] All ITC experiments were performed on a VP-ITC microcalorimeter (Malvern Instruments). Experiments were conducted at 25°C in phosphate-buffered saline (25 mM potassium phosphate, pH 7.4, 150 mM potassium chloride). For monoclonal antibodies, the protein concentration was 750 μg / ml, resulting in a molar concentration of 10 μM for complementary sites. The biotin concentration was 100 μM. Titrations were performed between 10 μl injections with stirring at 310 rpm and at 200 s intervals. The first injection for each sample was excluded from the data fitting. Experimental data were fitted to theoretical curves using the NanoAnalyze Data Analysis software package (version 3.6.0) to provide K0. d The values ​​of dissociation constant (denoted by M), n (stoichiometry of binding), and ΔH (change of enthalpy in kcal / mol) are given. Thermodynamic parameters (ΔG and ΔS) are expressed using the following equation from K... d And ΔH calculation: Where R is the universal gas constant, T is the temperature, and ΔG, ΔH, and ΔS are the changes in Gibbs free energy, enthalpy, and entropy, respectively.

[0206] Table 5 Example 11 Purification of Fab fragments from prokaryotic cell fermentation The expressed polypeptides of this invention are secreted into and recovered from the periplasm of host cells. Cells can be harvested by centrifugation or filtration. After resuspending the biomass in a suitable buffer with a pH of 2.0-8.0 or using a complete fermentation culture, cells are lysed by physical, chemical, or enzymatic methods. Once the cells are lysed, cell debris or whole cells can be removed by centrifugation or filtration. Polypeptide purification may involve several precipitation steps and fractionation on cation exchange chromatography, anion exchange chromatography, hydrophobic interaction, mixing mode, affinity chromatography, or gel filtration chromatography. For the removal of trace amounts of bound biotin, treatment of the polypeptide solution with a binding agent such as streptoacidin may be necessary. For the final formulation of the polypeptide, dialysis, ultrafiltration, or ultradialysis steps can be performed. The polypeptide can be stored as a liquid, a cryopreservative, or as a spray-dried or freeze-dried solid.< / dig> < / dig> < / dig> < / dig> < / dig> < / dig>

Claims

1. Monoclonal antibodies that specifically bind to compound I. (Equation I) Its features It also binds to biotin. in Y is CH2 or C=O. X is O or (CH2) n And n is an integer from 1 to 20, and R is H or ZL. in L is the connector, and Z is selected from haptens and polypeptides that do not contain the biotin moiety.

2. The antibody of claim 1, further characterized in that it does not bind to the compound of formula II also described in Figure 3A. (Formula II).

3. The monoclonal antibody according to any one of claims 1 or 2, wherein the monoclonal antibody has a binding affinity for the compound of formula III, also described in Figure 3B, that is at least 50 times greater than its binding affinity for the compound of formula II. (Formula III).

4. The monoclonal antibody of claim 3, wherein the binding affinity of the monoclonal antibody to the compound of formula III is at least 500, at least 1,000, at least 5,000, at least 10,000, at least 50,000, and at least 100,000 times the binding affinity to the compound of formula II.

5. A method for measuring an analyte in a sample, wherein the (streptomycin) avidin / biotin binding pair is used to bind a biotinylated analyte-specific binder to a (streptomycin) avidin-coated solid phase. The method includes adding to the sample a) An antibody according to any one of claims 1-4, b) Biotinylated analyte-specific binders c) A solid phase coated with streptoacidin. The following is The analytes are measured by binding to the solid phase via (streptomycin) anti-biotin protein and biotinylated analyte-specific binders.

6. The method of claim 5, wherein step 5(a) and optionally step 5(b) are performed prior to step 5(c).

7. Use of the antibody according to any one of claims 1-4 in a method for measuring an analyte in a sample, wherein the (streptomycin) avidin / biotin pair is used to bind a biotinylated analyte-specific binder to the (streptomycin) avidin-coated solid phase.

8. An immunoassay kit, comprising at least the following components: a) An antibody according to any one of claims 1-4, b) Biotinylated analyte-specific binders, and c) Solid phase coated with streptomycin avidin.

9. Immunogens according to Formula I (Equation I) in Y is CH2 or C=O. X is O or (CH2) n And n is an integer from 1 to 20, and R is ZL, in L is the connector, and Z is a polypeptide of at least 30 amino acids, and preferably keyhole hemocyanin.

10. A method for producing the antibody according to claim 1, the method comprising the steps of: a) Immunizing experimental animals with the immunogen according to claim 9, thereby inducing B cells to produce antibodies that bind to said immunogen. b) Alternatively, by hybridoma technology or by B-cell PCR technology, obtain the monoclonal antibody that binds to the immunogen produced by B-cells in step (a). c) Further select the antibody from step (b) to bind with biotin. Thus, the antibody according to claim 1 is obtained.

11. The method of claim 10, wherein in step (c), selection is made in a competitive assay, wherein biotin is used as a competitor for the binding of the antibody to the compound according to formula I. (Equation I) in Y is CH2 or C=O. X is O or (CH2) n And n is an integer from 1 to 20, and R is H or ZL. in L is the connector, and Z is selected from haptens and polypeptides that do not contain the biotin moiety.

12. The method of claim 10, wherein in step (c), selection is made in a competitive assay, wherein biotin is used as a competitor for the binding of the antibody to the immunogen of claim 9.

13. The method of claim 10, wherein in step (c), selection is made in a competitive assay, wherein biotin is used as a competitor for the binding of the antibody to the compound of formula III, also described in Figure 3B. (Formula III).

14. A method for producing the antibody according to claim 2, the method comprising the following steps: a) Immunizing experimental animals with the immunogen according to claim 9, thereby inducing B cells to produce antibodies that bind to said immunogen. b) Alternatively, by hybridoma technology or by B-cell PCR technology, obtain the monoclonal antibody that binds to the immunogen produced by B-cells in step (a). c) Select the antibody from step (b) to bind with biotin, and d) Select antibodies that do not bind to compounds of formula II. (Formula II) Thus, the antibody according to claim 2 is obtained.

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