Novel anti-thymidine kinase antibody

Abstract

The present invention relates to novel monoclonal antibodies that specifically bind to conformation-dependent epitopes on human thymidine kinase 1 (hTK-1; SEQ ID NO: 1), methods for quantifying hTK-1 using the antibodies, and the use of anti-hTK-1 antibodies in quantifying hTK-1. [Selected Figure] Figure 1

Description

[Technical Field]

[0001] This invention relates to a novel monoclonal antibody that specifically binds to human thymidine kinase 1 (hTK-1; SEQ ID NO: 1), a method for quantifying hTK-1 using the antibody, and the use of an anti-hTK-1 antibody in the quantification of hTK-1. [Background technology]

[0002] Despite all the advances of the last few decades, cancer remains the second leading cause of death (Siegel et al., 2013). Biomarkers are crucial for a deeper understanding of cancer biology and for better evaluating important clinically relevant issues, such as, to point out just two major topics, diagnosing malignancy or detecting recurrence.

[0003] One class of biomarkers of particular interest in the field of oncology is the class of proliferation biomarkers, including Ki-67, proliferative nuclear antigen (PCNA), and thymidine kinase (TK-1). Since all tumors contain a relatively high proportion of proliferating cells, proliferation markers should be able to identify these proliferating malignant cells. Literature is available demonstrating that thymidine kinase is a fairly useful marker when measured at the tissue level, i.e., by immunohistochemistry.

[0004] In several different malignancies, primarily leukemia and lymphoma, measurement of serum TK1 activity has been used for monitoring and prognostic purposes. However, immunoassays for measuring circulating thymidine kinase from body fluids such as serum are rare, and immunoassays for serum TK-1 protein are not yet widely used in clinical diagnostic routines.

[0005] Thymidine kinase 1 (abbreviated as TK1 or TK-1), (ATP: thymidine 5'-phosphotransferase, EC2.7.1.21) is an enzyme involved in DNA precursor synthesis. The sequence of human thymidine kinase 1 (hTK-1 or hTK1) is publicly known and given by Sequence ID No. 1.

[0006] Serum TK1 activity can be measured using the radioactive substrate 1251-dUrd (PROLIFIGEN® TK-REA, DiaSorin Inc.). A non-radioactive TK1 activity assay (TK LIAISON® assay, DiaSorin Inc.) has recently become available. This is a highly sensitive and robust assay that provides clinically valuable information in humans and dogs with hematological malignancies, particularly for monitoring therapy and predicting recurrence.

[0007] This specification references several documents, including patent applications and manufacturer manuals. While the disclosure of these documents is not considered relevant to the patentability of the present invention, they are incorporated by reference in their entirety. More specifically, all referenced documents are incorporated by reference to the same extent that each individual document is specifically and individually indicated to be incorporated by reference.

[0008] Over the past several decades, numerous attempts have been made to generate useful and highly sensitive specific antibodies against hTK-1. However, the antibodies described and available to date have not met the requirements for the development of a reliable and highly sensitive immunoassay for the measurement of hTK-1 from bodily fluid samples. [Overview of the project] [Problems that the invention aims to solve]

[0009] Therefore, the problem that led to this disclosure was to develop a novel monoclonal antibody that would enable reliable detection and quantification of hTK-1 from body fluid samples when used in standard immunoassay methods.

[0010] This need is addressed by the present invention by providing embodiments as defined in the claims. Surprisingly, we were able to find and demonstrate that a monoclonal antibody against human thymidine kinase 1 (hTK-1; SEQ ID NO: 1) that binds to the higher-order structure-dependent epitope of hTK-1 but does not bind to the polypeptide consisting of amino acids 194-225 (SEQ ID NO: 2) of hTK-1, nor to any of the 15 consecutive amino acid polypeptides of hTK-1, solves the fundamental problem of this disclosure. [Means for solving the problem]

[0011] In one embodiment, the present disclosure relates to a monoclonal antibody that specifically binds to human thymidine kinase 1 (hTK-1; SEQ ID NO: 1), wherein this antibody a) Binds to a higher-order structure-dependent epitope of hTK-1, b) It does not bind to the polypeptide consisting of amino acids 194-225 (SEQ ID NO: 2) of hTK-1, and c) Does not bind to any of the 15-unit polypeptides of hTK-1. It is characterized by the following:

[0012] In one embodiment, the present disclosure is an in vitro method for quantifying hTK-1, a) A step of incubating a sample in which hTK-1 is to be quantified with an antibody that specifically binds to human thymidine kinase 1 (hTK-1; SEQ ID NO: 1), wherein the antibody (i) binds to a higher-order structure-dependent epitope of hTK-1, (ii) does not bind to the polypeptide consisting of amino acids 194-225 (SEQ ID NO: 2) of hTK-1, and (iii) does not bind to any polypeptide consisting of 15 consecutive amino acids of hTK-1 according to claim 1 or 2, thereby forming a complex between the antibody and hTK-1. b) A step to quantify the complex formed in step a), thereby quantifying hTK-1. Regarding methods including

[0013] Furthermore, the use of an antibody against hTK-1 as disclosed in the present disclosure in the quantification of hTK-1 is demonstrated.

BEST MODE FOR CARRYING OUT THE INVENTION

[0014] In a first embodiment, the present description relates to a monoclonal antibody that specifically binds to human thymidine kinase 1 (hTK-1; SEQ ID NO: 1), the antibody binding to a) a higher-order structure-dependent epitope of hTK-1, b) not binding to a polypeptide consisting of amino acids 194-225 (SEQ ID NO: 2) of hTK-1, and c) not binding to any polypeptide consisting of 15 consecutive amino acids of hTK-1.

[0015] Human thymidine kinase 1 is an enzyme consisting of 234 amino acids shown in SEQ ID NO: 1. In the human body, TK-1 exists in various forms such as dimers, tetramers, and high molecular weight polymers. These forms seem to depend on the presence or absence of a specific molecule, for example, the presence of adenosine triphosphate (ATP); the concentration of the hTK-1 protein itself, the type of protein, i.e., native or recombinant TK1; and the site / location of the protein, i.e., serum or cytoplasm.

[0016] Generally, cytosolic and recombinant human TK1 exist mainly as tetramers in the presence of ATP or at high concentrations, and as dimers in the absence of ATP or at low concentrations. The tetrameric forms of cytosolic and recombinant human TK1 have high TK1 activity, while the dimeric forms have lower TK1 activity.

[0017] Human serum TK1, in contrast, can be in the form of high molecular weight complexes, such as oligomers, or those containing such oligomers, having serum TK1 activity, and also in dimeric and tetrameric forms having very low or even lacking serum TK1 activity.

[0018] Numerous attempts have been made and described in the prior art to generate monoclonal antibodies against hTK-1. Some of these antibodies, such as 3B3.E11;EPR3194 and EPR3193 from Abcam and rabbit monoclonal antibodies from Abnova, are commercially available but do not react well with serum TK1. These anti-TK1 antibodies are generated based on human recombinant TK1. Therefore, it was assumed that the generation of monoclonal anti-TK1 antibodies based on human recombinant TK1 is generally inefficient and does not generally produce anti-TK1 antibodies capable of binding to the serum form of TK1 with sufficient binding strength (WO2015 / 094106).

[0019] Contrary to negative experiences and common hypotheses in the prior art, it has now been surprisingly found that recombinant hTK-1 can be used as an immunogen to obtain the antibody according to the present invention.

[0020] Since none of the antibodies disclosed in this invention bind to any of the 15-mer linear peptides shifted by one amino acid across the entire hTK-1 sequence, the antibodies according to the present invention bind to higher-order structure-dependent epitopes.

[0021] The overall structure of an antibody consists of two heavy chains and two light chains connected by disulfide bonds. Each of the heavy and light chains comprises one constant domain and one variable domain. Antigen binding specificity is provided by the variable domains of the light and heavy chains that form the antibody. More specifically, the antibody portion that determines their specificity and contacts the specific ligand is called the complementarity-determining region (CDR). The CDR is the most variable part of the molecule and contributes to the diversity of these molecules. Each variable domain has three CDR regions, CDR1, CDR2, and CDR3, embedded in four framework regions (FW). As used herein, CDR-HC (or CDR(HC)) represents the CDR region of the variable heavy chain, and CDR-LC (or CDR(LC)) refers to the CDR region of the variable light chain. Similarly, FW-HC (or FW(HC)) represents the framework region of the variable heavy chain, and FW-LC (or FW(LC)) relates to the framework region of the variable light chain.

[0022] As used in this invention, the term "including" means that in addition to the sequences and / or components specifically enumerated, further sequences / components may be included. However, this term also includes the fact that the claimed subject matter consists of the sequences and / or components precisely enumerated.

[0023] In embodiments of the present invention in which the antibody comprises more amino acid sequences than those listed, additional amino acids may be present at the N-terminus, C-terminus, or both. These additional sequences may include, for example, sequences introduced for purification or detection, as will be discussed in detail below. Furthermore, if individual sequences "contain" the listed sequences, they may also contain additional amino acids at the N-terminus, C-terminus, or both.

[0024] According to the present invention, the antibody specifically binds to human thymidine kinase 1 (hTK-1) of SEQ ID NO: 1. It is understood that even if the antibody of the present invention contains additional amino acids, as detailed above, the antibody must still specifically bind to hTK-1.

[0025] The term “specifically binding” (also referred to herein as “specifically interacting”) means, in this invention, that the antibody specifically binds only to hTK-1 but does not cross-react, or is virtually cross-reacting, with other proteins, particularly other proteins with similar structures, such as thymidine kinase 2 (SEQ ID NO: 5).

[0026] Corresponding methods for analyzing antibody specificity are described, for example, in Harlow & Lane (1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, and Harlow & Lane (1999) Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press. Non-limiting examples of suitable studies include, for example, binding studies, blocking and competition studies by structurally and / or functionally closely related molecules. These studies can be performed by methods such as FACS analysis, flow cytometry titration (FACS titration), surface plasmon resonance (SPR, e.g. by BIAcore®), isothermal titration calorimetry (ITC), fluorescence titration, or radiolabeled ligand binding assays. Further methods include, for example, Western blotting, ELISA (including competitive ELISA), RIA, ECL, and IRMA tests.

[0027] In connection with the present invention, the term “antibody” refers to the complete immunoglobulin molecule and its antigen-binding fragments such as Fab, Fab', F(ab')2, and Fv. Furthermore, the term refers to modified and / or altered antibody molecules, as well as antibodies produced / synthesized by recombinant or synthetic means. The term “antibody” also includes bifunctional antibodies, trifunctional antibodies, fully human antibodies, chimeric antibodies, and antibody constructs, such as single-chain Fv(scFv) or antibody fusion proteins.

[0028] As used herein, “Fab fragment” refers to a C12C fragment consisting of one light chain and one heavy chain.H 1 and a variable region. The heavy chain of the Fab molecule cannot form a disulfide bond with another heavy chain molecule. A "Fab' fragment" consists of one light chain and the V H domain and C H 1 domain, and also contains a part of one heavy chain between the C H 1 and C H 2 domains, so that interchain disulfide bonds can be formed between the two heavy chains of the two Fab' fragments to form an F(ab')2 molecule. An "F(ab')2 fragment" contains two light chains and the C H 1 and C H 2 domains and contains two heavy chains containing a part of the constant region between the C

[0029] The Fab / c fragment contains both Fc and Fab determinants. The "Fc" region contains two heavy chain fragments including the C H 2 and C H 3 domains. The two heavy chain fragments are joined by two or more disulfide bonds and the hydrophobic interaction of the C H 3 domains.

[0030] The "Fv region" includes variable regions from both the heavy and light chains but lacks the constant region. "Single-chain Fv" (also abbreviated as "scFv") is, in the context of the present invention, the V H and V L domains of an antibody fragment, and these domains are present in a single polypeptide chain. Generally, the scFv polypeptide allows the scFv to form the desired structure for antigen binding, the V H and V LIt further includes polypeptide linkers between domains. The techniques described for the production of single-chain antibodies are, for example, described in Pluckthun, The Pharmacology of Monoclonal Antibodies, edited by Rosenburg and Moore, Springer-Verlag, NY113 (1994), pp. 269–315.

[0031] The term "chimeric antibody" refers to an antibody containing a variable region of a human or non-human species that is fused to or chimerized with an antibody region (e.g., a constant region) from another human or non-human species (e.g., mouse, horse, rabbit, dog, cattle, chicken).

[0032] As noted above, the term “antibody” also includes antibody constructs such as antibody fusion proteins, and an antibody includes, in addition to the domains defined herein by specific amino acid sequences, additional domains (may include) for, for example, the isolation and / or preparation of recombinantly produced constructs.

[0033] The antibodies of the present invention can be produced such that they are recombinant antibodies, for example, recombinant rabbit antibodies or heterohybrid antibodies, that still contain the CDR disclosed and defined in the present invention.

[0034] The term “recombinant antibody” includes all antibodies prepared, expressed, produced, or isolated by recombinant means. Recombinant antibodies are, for example, antibodies obtained by B-cell PCR, or antibodies isolated from animals (e.g., mice) that are transgenic with respect to human immunoglobulin genes, antibodies expressed using recombinant expression vectors transfected into host cells, antibodies isolated from recombinant combinatorial human antibody libraries, or antibodies prepared, expressed, produced, or isolated by any other means, including splicing of human immunoglobulin gene sequences to other DNA sequences. Recombinant rabbit antibodies produced by B-cell PCR have variable and constant regions (if present) derived from rabbit germline immunoglobulin sequences. That is, the direct result of B-cell PCR is an antibody binding-associated fragment, and those skilled in the art have no problem interpreting this as, for example, a full-length antibody, a chimeric antibody, or any “antibody” desired / needed.

[0035] The term "heterohybrid antibody" refers to an antibody that possesses light and heavy chains originating from different organisms. For example, an antibody with a human heavy chain associated with a mouse light chain is a heterohybrid antibody. Examples of heterohybrid antibodies include chimeric and humanized antibodies.

[0036] The antibody or antigen-binding fragment according to the present invention a) binds to a higher-order structure-dependent epitope on human thymidine kinase 1 (hTK-1; SEQ ID NO: 1), b) does not bind to the polypeptide consisting of amino acids 194-225 (SEQ ID NO: 2) of hTK-1, and c) does not bind to any polypeptide consisting of 15 consecutive amino acids of hTK-1.

[0037] The binding and non-binding of the anti-hTK-1 antibody to the peptide of SEQ ID NO: 2 is determined by using the N-terminally biotinylated peptide of SEQ ID NO: 2. Such peptides are bound to a streptavidin-coated solid phase, and binding and non-binding are determined by standard procedures. In virtually the same manner, the binding and non-binding of the anti-hTK-1 antibody to any polypeptide consisting of 15 consecutive amino acids of hTK-1 is determined. For the latter purpose, each 15-mer polypeptide consisting of 15 consecutive amino acids of hTK-1 is synthesized, N-terminally biotinylated, and tested for antibody binding and non-binding, respectively. Each peptide is bound to a streptavidin-coated solid phase, and binding and non-binding are determined by standard procedures.

[0038] In one embodiment, the antibody according to the present invention binds to the same epitope as the antibody comprising the variable heavy chain (vHC) of SEQ ID NO: 3 and the variable light chain (vLC) of SEQ ID NO: 4. Such an antibody may be a variant of the given specific antibody and may, for example, include amino acid substitutions, or in alternative forms may have a different vHC, a different vLC, or both. The term "substitution" in this invention refers to the replacement of an amino acid with another amino acid. Therefore, the total number of amino acids remains the same. Deletion of an amino acid at a particular position, and introduction of one (or more) amino acids at different positions, are not expressly included in the term "substitution." Substitutions may be either conservative or non-conservative amino acid substitutions in this invention. The term "conservative amino acid substitution" is well known in the art and refers to the replacement of an amino acid with a different amino acid having similar structural and / or chemical properties. Such similarities include, for example, similarities in the polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphiphilicity of the residues involved. The amino acid substitution is a conservative amino acid substitution, in which one amino acid from one of the following groups is replaced by another amino acid from the same group: nonpolar (hydrophobic) amino acids include alanine, valine, leucine, isoleucine, proline, phenylalanine, tyrosine, tryptophan, and methionine; polar neutral amino acids include glycine, serine, threonine, cysteine, asparagine, and glutamine; positively charged (basic) amino acids include arginine, lysine, and histidine; and negatively charged (acidic) amino acids include aspartic acid and glutamic acid.

[0039] The "binding affinity" of an antibody measures the strength of the interaction between the epitope on the target antigen and the antibody's binding site using the following formula: KD = kd / ka During the ceremony: KD=dissociation equilibrium constant [M] kd=dissociation rate constant [s -1 ] ka = association rate constant [M -1 s -1 ] Further relevant parameters for antibody binding affinity are as follows: t / 2 = Dissociation complex half-life = ln2 / kd / 60 [minutes] R max = Maximum response value of the analyte [RU] MR: Molar ratio = Ratio of the maximum response values ​​(Rmax) of the analyte In one embodiment, the monoclonal antibody against hTK-1 disclosed herein binds to hTK-1 by t / 2-dissociation at 37°C for 10 minutes or more.

[0040] In one embodiment, the monoclonal antibody according to this disclosure is 10 against hTK-1. -9 In one embodiment, the monoclonal antibody according to this disclosure is characterized by having M or better binding affinity to hTK-1. -10 M or better, or 2×10 -10 It is characterized by having M or better binding affinity. Antibodies known from the prior art do not have such excellent affinity to the linear epitope on hTK-1, or do not bind to it, or both.

[0041] Several excellent monoclonal antibodies that specifically bind to human thymidine kinase 1 (hTK-1; SEQ ID NO: 1), characterized in that each such antibody a) binds to a higher-order structure-dependent epitope of hTK-1, b) does not bind to the polypeptide consisting of amino acids 194-225 (SEQ ID NO: 2) of hTK-1, and c) does not bind to any polypeptide consisting of 15 consecutive amino acids of hTK-1, were generated by the methods disclosed in the Examples section. Surprisingly, all three exemplary antibodies bind to very similar or the same epitope. Without wishing to be bound by theory, the conformational epitope to which the antibodies according to the present invention bind appears to be absolutely important in establishing highly sensitive immunoassays that are useful in clinical routines. Now that this definitive epitope has been identified, it will be fairly easy to find other monoclonal antibodies that bind to this epitope. Such antibodies can be readily produced by following the procedures disclosed herein or by modifying the sequences of the antibodies disclosed herein.

[0042] Surprisingly, all three antibodies were found to bind to the same epitope. Due to their production methods, the binding of these recombinant rabbit monoclonal antibodies is best defined by the sequences obtained in B-cell PCR, i.e., the heavy-chain and light-chain variable domain portions obtained by B-cell PCR. MAB 6C6 was selected as a prototype antibody for defining a common epitope. MAB 6C6 is characterized by the heavy-chain variable domain of SEQ ID NO: 3 and the light-chain variable domain of SEQ ID NO: 4.

[0043] In one embodiment, the present invention provides an antibody that binds to an epitope on human TK-1, which is identical to the monoclonal antibody or its binding fragment of the present invention having the heavy chain variable domain of SEQ ID NO: 3 and the light chain variable domain of SEQ ID NO: 4. The antibody that binds to the same epitope on hTK-1 is an antibody that competes with the antibody having the heavy chain variable domain of SEQ ID NO: 3 and the light chain variable domain of SEQ ID NO: 4 for binding to hTK-1.

[0044] Such competing antibodies can be identified based on their ability to compete with the monoclonal rabbit antibody MAB6C6, i.e., an antibody containing the heavy chain variable domain of SEQ ID NO: 3 and the light chain variable domain of SEQ ID NO: 4, in a standard hTK-1 binding assay. For example, BIAcore analysis, ELISA assay, or flow cytometry can be used to demonstrate competition with a monoclonal antibody or its binding fragment containing the heavy chain variable domain of SEQ ID NO: 3 and the light chain variable domain of SEQ ID NO: 4.

[0045] The ability of the test antibody to inhibit the binding of the monoclonal rabbit antibody MAB 6C6 to human TK-1 demonstrates that the test antibody can compete with the monoclonal rabbit antibody MAB 6C6 for binding to the same epitope on hTK-1 as the monoclonal rabbit antibody MAB 6C6, and therefore binds to it.

[0046] As noted, several different competitive assays can be used to identify antibodies that compete with the rabbit monoclonal antibody MAB 6C6 or its binding fragments, which have the heavy chain variable domain of SEQ ID NO: 3 and the light chain variable domain of SEQ ID NO: 4.

[0047] In an exemplary competition assay, immobilized hTK-1 is incubated in a solution containing a first labeled antibody that binds to hTK-1 (e.g., an anti-hTK-1 monoclonal antibody or its binding fragment having the heavy chain variable domain of SEQ ID NO: 3 and the light chain variable domain of SEQ ID NO: 4) and a second unlabeled antibody to be tested for its ability to compete with the first antibody for binding to hTK-1. The second antibody may be present in the hybridoma supernatant. As a control, immobilized hTK-1 is incubated in a solution containing the first labeled antibody but not the second unlabeled antibody. After incubation under conditions that allow binding of the first antibody to hTK-1, excess unbound antibody is removed and the amount of labeling associated with the immobilized hTK-1 is measured. If the amount of labeling associated with the immobilized hTK-1 is substantially reduced in the test sample compared to the control sample, it indicates that the second antibody is competing with the first antibody for binding to hTK-1. For example, see Harlow et al. Antibodies: A Laboratory Manual, Ch. 14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1988).

[0048] The binding characteristics of antibodies, such as anti-hTK-1 antibodies, are best determined by real-time biosensor-based molecular interaction measurements, such as surface plasmon resonance spectroscopy, for which Biacore technology has become synonymous. The Biacore system can also analyze antibodies in terms of competitive binding to the same epitope (i.e., binding to the same or overlapping epitopes). Experimental details are given in Example 3, and kinetic data are shown in Table 1. In one embodiment, competitive experiments characterizing antibodies for binding to higher-order structure-dependent epitopes identified herein are performed on the BIAcore instrument as described in the Examples section.

[0049] Several non-automated assays are available for the measurement of the hTK-1 protein. Perhaps the assay with the widest range is Arocell's hTK-1 assay, which is based on a microtiter plate format requiring several manual processing steps and a fairly long incubation time. However, as noted above, there are currently no available immunoassays for hTK-1 that can be performed on an automated immunoassay analyzer, likely due to the lack of a suitable antibody. However, the antibody according to the present invention solves this problem. It can be used with great advantage in the in vitro measurement of hTK-1.

[0050] In one embodiment, the present disclosure relates to an in vitro method for quantifying hTK-1, comprising the steps of: a) incubating a sample in which hTK-1 is to be quantified with an antibody that specifically binds to human thymidine kinase 1 (hTK-1; SEQ ID NO: 1), wherein the antibody (i) binds to a higher-order structure-dependent epitope of hTK-1, (ii) does not bind to the polypeptide consisting of amino acids 194-225 (SEQ ID NO: 2) of hTK-1, and (iii) does not bind to any polypeptide consisting of 15 consecutive amino acids of hTK-1 according to claim 1 or 2, thereby forming a complex between the antibody and hTK-1; and b) quantifying the complex formed in step a), thereby quantifying hTK-1.

[0051] Examples of immunoassays that can utilize the antibodies of the present invention are immunoassays in direct or indirect formats. Examples of such immunoassays include enzyme-linked immunosorbent assays (ELISA), enzyme-mediated immunoassays (EIA), radioimmunoassays (RIA), or immunoassays based on the detection of luminescence, fluorescence, chemiluminescence, or electrochemiluminescence.

[0052] In one embodiment, a sandwich immunoassay is used to measure hTK-1. This specification also discloses an in vitro method for quantifying hTK-1, comprising the steps of: a) incubating a sample in which hTK-1 is to be quantified with a first antibody, which is an antibody according to this disclosure, and a second antibody against hTK-1, thereby generating a sandwich complex between the first antibody, hTK-1, and the second antibody; and b) quantifying the sandwich complex formed in step a), thereby quantifying hTK-1.

[0053] Interestingly, the monoclonal antibody according to the present invention can be used for the detection of hTK-1 present in serum or plasma samples with or without pretreatment of such samples. This means that the antibody of the present invention also binds to oligomeric hTK-1 present in serum or plasma samples. In one embodiment, the present invention relates to the use of the antibody according to the present invention in the detection of hTK-1, wherein the sample is not pretreated with a reducing agent.

[0054] In conventional techniques, serum or plasma samples are typically pre-treated to generate a highly enzymatically active form of hTK-1 tetramer. Sample pre-treatment and measurement of hTK-1 from such pre-treated samples are very attractive methods because they can achieve a very good correlation with hTK-1 enzyme activity.

[0055] The selection of a suitable pretreatment reagent is entirely within the capabilities of those skilled in the art. To transform oligomeric hTK-1 into tetrameric hTK-1, such a pretreatment reagent must contain at least a reducing agent. As described, for example, by Sharif et al. (BMC Biochemistry (2012), 13:12), it is easy to evaluate by gel filtration whether the reducing agent was effective and whether hTK-1 exists primarily as a tetramer after treatment. Several different reducing agents, such as dithiothreitol (DTT), dithioerythritol (DTE), or dithiobutylamine (DTBA), are of interest to those skilled in the art. Furthermore, the concentration of the pretreatment reagent should be selected such that, after an appropriate incubation period or dilution step, it does not negatively affect any antibody used to measure the hTK-1 protein. When dithiobutylamine (DTBA) is used as the reducing agent, a suitable final concentration in the sample pretreatment step (mixture of sample and pretreatment reagent) is in the range of 5 mM. After pre-dilution and / or addition of a DTT blocking agent and / or dilution with a buffer containing the first antibody, the concentration of oxidized DTT is preferably 1.5 mM or less. In this method, the reducing agent does not affect any of the immunological reagents used.

[0056] As described above, ATP stabilizes the tetrameric form of hTK-1. The latter function of ATP makes it an attractive additive, for example, to pretreatment buffers. In one embodiment, the pretreatment buffer contains the reducing agent and ATP described above. The concentration of ATP in the sample pretreatment step should not be less than 1.25 mM. A good selection of ATP concentration in the pretreatment step is in the range of 2 mM to 20 mM.

[0057] In one embodiment, the present disclosure relates to an in vitro method for quantifying hTK-1, comprising the steps of: a) incubating a sample in which hTK-1 is to be quantified with a pretreatment solution containing a reducing agent and ATP; b) incubating the pretreatment sample obtained in step a) with an antibody according to the present invention to generate a complex between the antibody and hTK-1; and c) quantifying the complex formed in step b) to quantify hTK-1.

[0058] Sandwich immunoassays are widely used for the detection of analytes of interest. In such assays, the analyte is "sandwiched" between a first antibody and a second antibody. By appropriate means, such a sandwich complex is measured, and the analyte is quantified accordingly.

[0059] In one embodiment, the present disclosure relates to an in vitro method for quantifying hTK-1, comprising the steps of: a) incubating a sample in which hTK-1 is to be quantified with a pretreatment solution containing a reducing agent and ATP; b) incubating the pretreatment sample obtained in step a) with a first antibody, which is an antibody according to the present invention, and a second antibody against hTK-1, thereby generating a sandwich complex between the first antibody, hTK-1, and the second antibody; and c) quantifying the sandwich complex formed in step b), thereby quantifying hTK-1.

[0060] In one embodiment, the method of the present invention is carried out in a sandwich assay format. In a typical sandwich-type assay, a first antibody, bound to or capable of binding to a solid phase, and a second antibody, detectably labeled, each bind to the analyte at different non-overlapping epitopes. The first analyte-specific binding agent (e.g., antibody) is covalently or passively bound to the solid surface. The solid surface is typically glass or polymer, with the most commonly used polymers being cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride, or polypropylene. The solid support may be in the form of a tube, beads, a microplate disc, or any other surface suitable for performing the immunoassay. The binding process is well known in the art and generally consists of crosslinking covalent bonding or physical adsorption, and the polymer-antibody complex is washed for the preparation of the test sample. Aliquots of the sample to be tested are then added to the solid-phase complex and incubated for a sufficient time (e.g., 2–40 minutes, or overnight if more convenient) and under suitable conditions (e.g., room temperature to 40°C, e.g., between 25°C and 37°C including both ends) to allow binding between the first or capture antibody and the corresponding antigen. After the incubation period, the solid phase containing the first or capture antibody and the antigen bound to it can be washed and incubated with a secondary or labeled antibody that binds to another epitope on the antigen. The second antibody is linked to a reporter molecule used to indicate the binding of the second antibody to the complex of the first antibody and the antigen of interest.

[0061] An extremely versatile alternative sandwich assay format involves the use of a solid phase coated with a first partner of a binding pair, e.g., microparticles coated with paramagnetic streptavidin. Such microparticles are mixed and incubated with an analyte-specific binding agent conjugated to a second partner of the binding pair (e.g., a biotinylated antibody), which is a sample suspected of containing or containing the analyte, and the second partner of the binding pair is conjugated to the analyte-specific binding agent, which is detected by the analyte-specific binding agent and, for example, electrochemically luminescent labeling as used herein. As will be apparent to those skilled in the art, these components are incubated under appropriate conditions and for a sufficient time to conjugate the labeled antibody, the analyte-specific binding agent (conjugated to) the second partner of the binding pair, and the first partner of the binding pair to the solid phase microparticles via the analyte. Optionally, such assays may include one or more washing steps.

[0062] In one embodiment, the disclosure relates to a sandwich assay in which a first or second antibody is bound to or capable of binding to a solid phase, the second or first antibody is each detectably labeled, and at least one of these antibodies is an antibody disclosed in the present invention.

[0063] Typically, a sandwich assay requires that the capture and detection antibodies bind to different, non-overlapping epitopes on the analyte of interest. In the case of serum / plasma hTK-1, preferably the tetrameric form of the enzyme produced as described above is measured. In one embodiment of the sandwich assay method for the quantification of hTK-1, the antibody according to the present invention is used in combination with an antibody that binds to an epitope on the C-terminal portion of hTK-1 represented by amino acids in the range from position 195 to the C-terminus, i.e., position 234 (SEQ ID NO: 5), or an antibody that binds to an epitope contained in a polypeptide consisting of amino acids in the range from position 195 to 225 (SEQ ID NO: 2). In one embodiment, the antibody according to the present invention and an antibody that binds to an epitope contained in amino acids 211-230 (SEQ ID NO: 6) of hTK-1 are used in the sandwich assay for the measurement of hTK-1.

[0064] As the inventors have demonstrated through the present invention, the antibody according to the present invention can also be used for both purposes, namely as a capture antibody as part of a sandwich complex and as a detection antibody as the other part of a sandwich complex. In one embodiment, the quantification of hTK-1 is therefore performed by a sandwich assay using the antibody according to the present invention as both a capture antibody and a detection antibody.

[0065] In a sandwich immunoassay method for quantifying hTK-1, at least one antibody against hTK-1 contains a detectable label. The term "detectably labeled" encompasses labels that can be detected directly or indirectly.

[0066] Directly detectable labels either provide a detectable signal or interact with a second label to modify a detectable signal provided by a first or second label, for example, to give a FRET (fluorescence resonance energy transition). Labels such as fluorescent dyes and luminescent (including chemiluminescent and electrochemiluminescent) dyes (Briggs et al., "Synthesis of Functionalised Fluorescent Dyes and Their Coupling to Amines and Amino Acids," J. Chem. Soc., Perkin-Trans. 1 (1997), pp. 1051-1058) provide detectable signals and are generally applicable for labeling. In one embodiment, "detectably labeled" refers to a label capable of providing or inducing a detectable signal, i.e., a fluorescent label, a luminescent label (e.g., a chemiluminescent or electrochemiluminescent label), a radioactive label, or a metal chelate-based label, respectively.

[0067] Numerous labels (also called dyes) are available, which can generally be classified into the following categories, and all of them, and each of them, represent embodiments of this disclosure: (a) Fluorescent dyes Fluorescent dyes are described, for example, in Briggs et al., "Synthesis of Functionalized Fluorescent Dyes and Their Coupling to Amines and Amino Acids," J.Chem.Soc., Perkin-Trans.1 (1997), pp. 1051-1058.

[0068] Fluorescent labels or fluorophores include rare earth chelates (europium chelate), fluorescein-type labels such as FITC, 5-carboxyfluorescein, and 6-carboxyfluorescein; rhodamine-type labels such as TAMRA, dansyl, lysamine, cyanine, phycoerythrin, Texas Red, and their analogues. Fluorescent labels can be conjugated to aldehyde groups in target molecules using the techniques disclosed herein. Fluorescent dyes and fluorescent labeling reagents include those commercially available from Invitrogen / Molecular Probes (Eugene, Oregon, USA) and Pierce Biotechnology, Inc. (Rockford, Ill.).

[0069] (b) Luminescent dye Luminescent dyes or labels can be further subdivided into chemiluminescent and electrochemiluminescent dyes.

[0070] Different classes of chemiluminescent labels include luminols, acridinium compounds, coelenterazine and its analogues, dioxetanes, peroxyoxalic acid-based systems, and their derivatives. For immunodiagnostics, acridinium-based labels are primarily used (a detailed overview is given in Dodeigne C. et al., Talanta 51 (2000), pp. 415-439).

[0071] The most relevant labels used as electrochemiluminescence labels are ruthenium and iridium-based electrochemiluminescence complexes, respectively. Electrochemiluminescence (ECL) has proven to be highly beneficial as a sensitive and selective method in analytical applications. It combines the analytical advantages of chemiluminescence analysis (absence of background optical signals) with the ease of reaction control by applying electrode potential. Among common ruthenium complexes, [Ru(Bpy)3]2+ (emitting photons at approximately 620 nm), which is regenerated with TPA (tripropylamine) particularly in the liquid phase or at the liquid-solid interface, is used as an ECL label.

[0072] Electrochemiluminescence (ECL) assays provide highly sensitive and accurate measurements of the presence and concentration of an analyte of interest. Such techniques utilize labels or other reactants that can be induced to emit light when electrochemically oxidized or reduced in a suitable chemical environment. Such electrochemiluminescence is induced by a voltage applied to a working electrode for a specific time and in a specific manner. The light produced by the label is measured to indicate the presence or amount of the analyte. For a more complete description of such ECL techniques, see U.S. Patent Nos. 5,221,605, 5,591,581, 5,597,910, PCT Publication Nos. WO90 / 05296, WO92 / 14139, WO90 / 05301, WO96 / 24690, US95 / 03190, US97 / 16942, US96 / See also PCT publications 06763, WO95 / 08644, WO96 / 06946, WO96 / 33411, WO87 / 06706, WO96 / 39534, WO96 / 41175, WO96 / 40978, PCT / US97 / 03653, and U.S. Patent Application 08 / 437348 (U.S. Patent No. 5,679,519). See also the 1994 review of the analytical application of ECL by Knight et al. (Analyst, 1994, 119:879-890) and the references therein. In one embodiment, the method described herein is carried out using electrochemiluminescence labeling.

[0073] In recent years, iridium-based ECL markings have also been described (WO2012107419(A1)). In one embodiment, the directly detectable label is a chemiluminescent or electrochemiluminescent label. The light produced by the label is measured to directly or indirectly indicate the presence or quantity of the analyte.

[0074] (c) Radioactive labels may be radioactive isotopes (radionuclides), such as 3H, 11C, 14C, 18F, 32P, 35S, 64Cu, 68Gn, 86Y, 89Zr, 99TC, 111In, 123I, 124I, 125I, 131I, 133Xe, 177Lu, 211At, or 131Bi.

[0075] (d) Metal chelate complexes suitable as labels for imaging and therapeutic purposes are well known in the art (U.S. Patent Application Publication No. 2010 / 0111856; U.S. Patent Nos. 5,342,606; U.S. Patent Nos. 5,428,155; U.S. Patent Nos. 5,316,757; U.S. Patent Nos. 5,480,990; U.S. Patent Nos. 5,462,725; U.S. Patent Nos. 5,428,139; U.S. Patent Nos. 5,385,893; U.S. Patent Nos. 5,739,294; U.S. Patent Nos. 5,750,660; U.S. Patent Nos. 5,834,456; Hnatowich et al., J. Immunol. Methods 65 (1983), pp. 147-157; Meares et al., Anal. Biochem. 142 (1984), pp. 68-78; Mirzadeh et al., Bioconjugate Chem.1 (1990), pp. 59-65; Meares et al., J. Cancer (1990), Suppl.10: pp. 21-26; Izard et al., Bioconjugate Chem. 3 (1992) pp. 346-350; Nikula et al., Nucl. Med. Biol. 22 (1995) pp. 387-90; Camera et al., Nucl. Med. Biol. 20 (1993) pp. 955-62; Kukis et al., J. Nucl .Med.39 (1998) pp. 2105-2110; Verel et al., J. Nucl. Med. 44 (2003) pp. 1663-1670; Camera et al., J. Nucl. Med. 21 (1994) pp. 640-646; Ruegg et al., Cancer Res.50 (1990) pp. 4221-4226; Verel et al., J.Nucl.Med.44 (2003) pp. 1663-1670; Lee et al., Cancer Res.61 (2001) pp. 4474-4482; Mitchell et al., J.Nucl.Med.44 (2003) pp. 1105-1112; Kobayashi et al., Bioconjugate Chem.10 (1999) pp. 103-111; Miederer et al., J.Nucl.Med.45 (2004) pp. 129-137; DeNardo et al., Clinical Cancer Research 4 (1998) pp. 2483-2490; Blend et al., Cancer Biotherapy & Radiopharmaceuticals 18 (2003) pp. 355-363; Nikula et al., J.Nucl. Med. 40 (1999) pp. 166-76; Kobayashi et al., J. Nucl. Med. 39 (1998) pp. 829-36; Mardirossian et al., Nucl. Med. Biol. 20 (1993) pp. 65-74; Roselli et al., Cancer Biotherapy & Radiopharmaceuticals, 14 (1999) pp. 209-20). .

[0076] Despite the fact that hTK-1 has been known for decades, and despite numerous attempts yielding excellent immunoassays, it is puzzling that high-quality antibodies against hTK-1 that bind to the conformational epitope and are beneficial in highly sensitive detection of hTK-1 are still unavailable. While we do not wish to be bound by theory, one might imagine that this lack / failure is related to the fact that hTK-1 may be blocked to some extent by antibodies induced by immunization. It should not be forgotten that standard hybridoma techniques rely on HAT culture medium (hypoxanthine-aminopterin-thymidine) to select hybridomas, meaning that hybridomas must be able to use thymidine added to the selection medium in order to overcome the toxic effects of aminopterin. Now, successfully generated hTK-1 monoclonal antibodies have been obtained by B-cell PCR techniques. In B-cell PCR technology, HAT medium is not used, and the blocking of hTK-1 by antibodies produced by this technology may be less relevant or completely irrelevant.

[0077] In the embodiments shown in the corresponding sections of this disclosure, rabbits were used as experimental animals, immunized with hTK-1, and their B cells were used in rabbit B-cell PCR technology. As is evident, B-cell PCR technology can also be used for other experimental animals such as mice, or can be similarly established for other experimental animals if necessary. Thus, while B-cell PCR in rabbits is one embodiment, the use of B-cell PCR technology to produce anti-hTK-1 antibodies is not limited to this species.

[0078] In one embodiment, the antibody against hTK-1 according to the present invention is obtained by B-cell PCR technology. As described in much more detail herein, the antibodies against HTK-1 disclosed herein can be used with great advantage in the detection of hTK-1. Accordingly, in one embodiment, the present invention relates to the use of the antibodies disclosed herein in the quantification of hTK-1.

[0079] As will be apparent to those skilled in the art, it is advantageous to use the antibody according to the present invention in a method for detecting hTK-1. In one embodiment, the present disclosure relates to a method for detecting hTK-1 in a sample, comprising the steps of: a) contacting the sample with an anti-hTK-1 antibody according to the present disclosure under conditions and for a sufficient amount of time to form an anti-hTK-1 antibody / hTK-1 complex; and b) measuring the anti-hTK-1 antibody / hTK-1 complex, wherein the amount of the complex indicates the concentration of hTK-1 in the sample. For example, the term " / " in "anti-hTK-1 antibody / hTK-1 complex" is used to indicate that a non-covalent complex is formed between the anti-hTK-1 antibody on one hand and hTK-1 on the other.

[0080] In one embodiment, the present invention relates to a method for detecting hTK-1 in a sample, comprising the steps of: a) contacting a sample with a first antibody against hTK-1 and a second antibody against hTK-1 for a sufficient time and under conditions sufficient to form a first anti-hTK-1 antibody / hTK-1 / second anti-hTK-1 antibody complex, wherein the second antibody is detectably labeled; and b) measuring the complex formed in (a), wherein the amount of the complex indicates the concentration of hTK-1 in the sample, and the first or second antibody is an antibody according to the present invention.

[0081] As will be apparent to those skilled in the art, the sample can be brought into contact with the first and second antibodies in any desired order, i.e., first antibody first, second antibody; second antibody first, or simultaneously, for a sufficient amount of time and under conditions to form a first anti-hTK-1 antibody / hTK-1 / second anti-hTK-1 antibody complex.

[0082] As those skilled in the art will readily understand, establishing the appropriate time and conditions for the formation of a complex between a specific anti-hTK-1 antibody and the hTK-1 antigen / analyte (= anti-hTK-1 antibody / hTK-1 complex), or for the formation of a secondary or sandwich complex containing a first antibody against hTK-1, hTK-1 (analyte), and a second anti-hTK-1 antibody complex (= first anti-hTK-1 antibody / hTK-1 / second anti-hTK-1 antibody complex), is merely a routine experiment.

[0083] The detection of anti-hTK-1 antibody / hTK-1 complexes can be carried out by any suitable means. Those skilled in the art are well familiar with such means / methods. The terms “sample,” “target sample,” and “test sample” are used interchangeably herein. A sample is an in vitro sample, which is analyzed in vitro and not returned to the body. Examples of samples include, but are not limited to, liquid samples such as blood, serum, plasma, synovial fluid, urine, saliva, and lymph, or solid samples such as tissue extracts, cartilage, bone, synovial membrane, and connective tissue. In one embodiment, the sample is selected from blood, serum, plasma, synovial fluid, and urine. In one embodiment, the sample is selected from blood, serum, and plasma. In one embodiment, the sample is serum or plasma.

[0084] As used herein, the term “reference sample” refers to a sample that is analyzed in substantially the same manner as the sample of interest, and whose information is compared to that of the sample of interest. The reference sample thereby provides a standard that enables the evaluation of information obtained from the sample of interest. A reference sample can be derived from a healthy or normal tissue, organ, or individual, thereby providing a standard for the healthy state of the tissue, organ, or individual. Differences between the state of a normal reference sample and the state of the sample of interest may indicate a risk of developing a disease or the presence or further progression of such a disease or disorder. A reference sample can be derived from an abnormal or pathological tissue, organ, or individual, thereby providing a standard for the pathological state of the tissue, organ, or individual. Differences between the state of an abnormal reference sample and the state of the sample of interest may indicate a reduced risk of developing a disease or the absence or improvement of such a disease or disorder.

[0085] The terms “elevated” or “increased” levels of an indicator refer to a level of such indicator in a sample that is higher than the level of such indicator in a reference or reference sample. For example, a protein that is detectable in higher amounts in a liquid sample from an individual with a given disease has an elevated level.

[0086] In a particular embodiment, a sandwich is formed containing a first antibody against hTK-1, hTK-1 (analyte), and a second antibody against hTK-1, the second antibody being detectably labeled.

[0087] In one embodiment, a sandwich is formed containing a first antibody against hTK-1, hTK-1 (analyte), and a second antibody against hTK-1, wherein the second antibody is detectably labeled, and the first anti-hTK-1 antibody is either capable of binding to a solid phase or is bound to a solid phase.

[0088] In one embodiment, the anti-hTK-1 antibody disclosed herein is used in an immunoassay for measuring hTK-1. In one embodiment, the anti-hTK-1 antibody disclosed herein is used in a sandwich-type immunoassay. In one embodiment, the anti-hTK-1 antibody disclosed herein is used as a detection antibody. In one embodiment, the anti-hTK-1 antibody disclosed herein is detectably labeled with a luminescent dye, particularly a chemiluminescent dye or an electrochemiluminescent dye.

[0089] These and other embodiments are disclosed and incorporated by the description and examples of the present invention. Further literature relating to any one of the methods, uses, and compounds used by the present invention can be searched from public libraries and databases, for example, using electronic devices. For example, the public database "Medline," available on the Internet, can be accessed on the World Wide Web from, for example, ncbi.nlm.nih.gov / PubMed / medline.html. Further databases and addresses available on the World Wide Web, for example, ncbi.nlm.nih.gov / , fmi.ch / biology / research_tools.html.tigr.org / , or infobiogen.fr / are known to those skilled in the art and can also be obtained using the World Wide Web address from lycos.com.

[0090] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this invention pertains. In case of any conflict, the specification of this invention, including the definitions, shall prevail.

[0091] All amino acid sequences provided herein are presented starting from the N-terminal residue and ending at the C-terminal residue (N→C), as is customary in the art, and the one- or three-letter code abbreviations used to identify amino acids throughout the invention correspond to those commonly used for amino acids.

[0092] With respect to the embodiments characterized in this specification, and in particular in the claims, each embodiment pointed out in a dependent claim is intended to be combined with each embodiment of the claim (independent or dependent) to which the dependent claim depends. For example, in the case of independent claim 1 listing three alternatives A, B, and C, dependent claim 2 listing three alternatives D, E, and F, and claim 3 dependent on claims 1 and 2 and listing three alternatives G, H, and I, it should be understood that the specification expressly discloses embodiments corresponding to combinations A, D, G; A, D, H; A, D, I; A, E, G; A, E, H; A, E, I; A, F, G; A, F, H; A, F, I; B, D, G; B, D, H; B, D, I; B, E, G; B, E, H; B, E, I; B, F, G; B, F, H; B, F, I; C, D, G; C, D, H; C, D, I; C, E, G; C, E, H; C, E, I; C, F, G; C, F, H; C, F, I.

[0093] Similarly, if an independent and / or dependent claim does not enumerate alternatives, and if the dependent claim references multiple prior claims, it is understood that any combination of subject matter encompassed by it is explicitly disclosed. For example, in the case of independent claim 1, dependent claim 2 referencing claim 1, and dependent claim 3 referencing both claims 2 and 1, the combination of subject matter of claim 3 and 1 is explicitly and obviously disclosed, as is the combination of subject matter of claim 3, 2 and 1. If there is a further dependent claim 4 referencing any one of claims 1 through 3, the combinations of subject matter of claim 4 and 1, claim 4, 2 and 1, claim 4, 3 and 1, and claim 4, 3, 2 and 1 are explicitly and obviously disclosed.

[0094] The above considerations apply mutatis mutandis to all attached claims. To give a non-limiting example, in light of the structure of the claims, the combination of claims 8, 5, and 1 is clearly and obviously assumed. The same applies to combinations such as claims 8, 7, and 2.

[0095] Certain aspects of the present invention are illustrated in the accompanying drawings. [Brief explanation of the drawing]

[0096] [Figure 1] This is a schematic diagram of the Biacore assay configuration used to determine hTK-1 epitope accessibility. The sequence of various incubation steps is shown. [Figure 2] These are graphical representations of the immunoassay data obtained from prototype A) (=Figure 2a) and prototype B) (=Figure 2b), respectively. The left-hand portion of both Figure 2a and Figure 2b shows a box-whisker plot (box plot). The y-axis (log scale) shows the concentration in ng / ml. The right-hand portion of both figures shows the area under the curve (AUC). (Abbreviations: Ctr = control sample; DLBCL = sample from a patient with scattered large B-cell lymphoma). [Figure 3] This is a graphical representation of the LIAISON® thymidine kinase (activity) assay data. The left side of Figure 3 shows a box-whisker plot (box plot) with the y-axis (log scale) representing units per ml. The right side of this figure shows the area under the curve (AUC). (Abbreviations: Ctr = control sample; DLBCL = sample from a patient with scattered large B-cell lymphoma). [Figure 4a] Figure showing a comparison of methods. Deming Regression Fit is shown for the correlation between LIAISON® thymidine kinase (activity) assay data on the x-axis and immunoassay prototype A) on the y-axis in Figure 4a and prototype B) on the y-axis in Figure 4b, respectively. [Figure 4b] Figure showing a comparison of methods. Deming Regression Fit is shown for the correlation between LIAISON® thymidine kinase (activity) assay data on the x-axis and immunoassay prototype A) on the y-axis in Figure 4a and prototype B) on the y-axis in Figure 4b, respectively.

[0097] The following examples illustrate the present invention: [Examples]

[0098] Example 1 Materials and general methods Recombinant DNA technology Standard methods were used to artificially manipulate DNA as described in Sambrook, J. et al., Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989. Molecular biological reagents were used according to the manufacturer's instructions.

[0099] DNA sequencing The DNA sequence was determined by double-strand sequencing performed at Microsynth AG (Balgach, Switzerland).

[0100] DNA and protein sequence analysis and sequence data management Vector NT1 Advance suite version 11.5.0 was used for sequence generation, mapping, analysis, annotation, and visualization.

[0101] Protein chemistry and labeling technologies Standard protein chemistry and labeling techniques are provided, for example, in Hermanson, G., "Bioconjugate Techniques," 3rd edition (2013), Academic Press.

[0102] Bioinformatics Bioinformatics methods are provided, for example, in Keith JM (ed.), "Bioinformatics," Volumes I and II; Methods in Molecular Biology, Volumes 1525 and 1526 (2017), Springer; and Martin, ACR and Allen, J., "Bioinformatics Tools for Analysis of Antibodies"; and Dubel S. and Reichert JM (eds.), "Handbook of Therapeutic Antibodies," Wiley-VCH (2014).

[0103] Electrochemiluminescence immunoassay Immunoassays and related methods are provided, for example, in Wild D. (ed.), "The Immunoassay Handbook," 4th edition (2013), Elsevier. Ruthenium complexes as electrochemiluminescent labels are provided, for example, in Staffilani M. et al., Inorg. Chem. 42 (2003), pp. 7789-7798. Generally, for performing electrochemiluminescence (ECL)-based immunoassays, the Elecsys 2010 analyzer or successor systems, such as Roche analyzers (Roche Diagnostics GmbH, Mannheim, Germany), e.g., E170, cobas e 601 module, cobas e 602 module, cobas e 801 module, and cobas e 411, and Roche Elecsys assays designed for these analyzers were used, each under standard conditions unless otherwise indicated.

[0104] Example 2 Generation of anti-hTK-1 antibodies Numerous attempts were initially made to generate anti-hTK-1 monoclonal antibodies in mice using standard protocols. These experiments utilized various immunogens (recombinant hTK-1 produced in E. coli; recombinant hTK-1 produced in HEK cells; and several peptide immunogens). Only a very small number of monoclonal antibodies were obtained using this method, and none of the antibodies obtained in these early experiments showed good binding affinity to TK-1 in human serum samples. In the final successful attempt to produce anti-hTK1 antibodies, rabbits were used as experimental animals.

[0105] Immunization: Rabbits were immunized with rec hTK1 derived from E. coli or rec native hTK1 derived from HEK293 cells to produce antibodies against human thymidine kinase (hTK1). All rabbits underwent repeated immunization. During the first month, animals were immunized weekly. From the second month onward, animals were immunized monthly. For the initial immunization, 500 μg of rec hTK1 (E. coli or HEK293) was dissolved in 1 mL of 0.9 (w / v)% NaCl and emulsified in 3.5 mL of CFA. For all subsequent immunizations, 1.75 mL of IFA was used instead of CFA. Titer development was assessed at 45 and 105 days from the start of immunization. When the titer against the immunogen was detectable by ELISA, antibodies were obtained by B cell cloning as described below. For the production of full-length rabbit IgG, heavy and light chain coding plasmids derived from the recombinant IgG cloning process were used for transient transfection of HEK293 cells.

[0106] Development and expression of monoclonal antibodies against hTK1 using B-cell PCR technology: Antibodies against recombinant human thymidine kinase (hTK1) were obtained using the B-cell PCR method described by Seeber et al. (2014), PLoS One 4, 9(2). PBMCs and B cells for single-cell deposition were prepared from peripheral blood isolated from immunized rabbits at different time points. Individual hTK1 rabbit antibodies were ultimately expressed recombinantly in HEK293 cells. For the generation of full-length rabbit anti-hTK1 IgG, heavy and light chain coding plasmids derived from the recombinant IgG cloning process were used for transient transfection of HEK293 cells. HEK293 cells were grown in F17 medium (Gibco) at 37°C in air containing 8% CO2, using a 125 rpm shaker. Cells were divided the day before transfection into 0.7–0.8 × 10⁶ cells. 6 Cells were seeded at a density of cells / ml. On the day of transfection, 1–1.5 × 10⁶ cells were seeded in a 2 ml volume in a 48-well deep-well plate. 6 HEK293 cells were transfected with a 0.5 mg heavy chain plasmid plus a 0.5 mg light chain plasmid, suspended in 80 ml of OptiMEMH medium (Gibco) supplemented with 1 ml of PEIpro transfection reagent (Polyplus-transfection). The cultures were incubated at 37°C and 8% CO2 at 180 rpm for 7 days. After 7 days of incubation, the culture supernatant was collected and analyzed for antibody content and specificity.

[0107] Initial testing of recombinant antibodies for anti-hTK-1 binding: Binding to hTK-1 was first tested using an ELISA format. For this purpose, a biotinylated variant of recombinant natural hTK1 (derived from HEK293) was bound to streptavidin in the wells of a 96-well microtiter plate (MTP) pre-coated with streptavidin. The biotinylated protein was immobilized in 50 μl wells of MTP at a biotinylated hTK-1 concentration of 250 ng / mL. 30 μl of the transfection supernatant for each antibody was added to the MTP wells and incubated at room temperature for 30 minutes. After washing, the bound antibodies were used to test for HRP-labeled F(ab')2 goat anti-rabbit Fc γ The fragment (Dianova) and substrate were detected using ABTS (Roche).

[0108] Thus, four recombinant rabbit antibodies were obtained, named 4H4;4H11, 6C6, and 23C11, respectively. For all recombinant rabbit antibodies, the sequences of the variable light chain and the binding-related regions of the heavy chain (i.e., the variable heavy chain including the three CDRs, framework region, and constant region 1) were determined using a standard procedure.

[0109] hTK1, Klon 6C6, heavy chain: (SEQ ID NO: 3) METGLRWLLLVAVLKGVQCQEQLEESGGDLVKPEGSLTLTCTASRFSFSSSYWICWVRQAPGKGLEWIACIYAGDSGSSYYASWAKGRFTVSKTSSTTVTLQTTSLTAADTATYFCARASVGAAYDYFALWGPGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSG hTK1, Klon 6C6, light chain: (SEQ ID NO: 4) MDTRAPTQLLGLLLLWLPGARCALVMTQTPASVEAAMGGTVTIKCQASEDVSSHLAWYQQRPGQPPKLLIYGASDLASGVPSRFTGSGSGTQFTLAISDLECADAATYYCQGYYYISD SPYVFGGGTEVVVKGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC hTK1, Klon 4H4, heavy chain: (SEQ ID NO: 7) METGLRWLLLVAVLKGVQCQSLEESGGGLVQPEGSLTLTCTASGFSFSSGYDMCWVRQTPGKGLEWIACISVDSDGVTYYASWAKGRFTISKTSSTTVTLQMTSLTAADTATYFCARGYESSSGVYIPYFTLWGPGTLVTVSSGQPKAPSVFPLAPCCGDTPSTVTLGCLVKGYLPEPVTVTWNSG hTK1, Klon 4H4, light chain: (SEQ ID NO: 8) MDMRAPTQLLGLLLLWLPGARCADIVLTQTPASVEAAVGGTVTIKCQASQSIYSYLAWYQHKPGQPPKLLIYKASTLASGVPSRFKGSGSGTEYTLTISDLECADAATYYCQHYYYSST SGGGVFGGGTEVVVKGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC hTK1, Klon 23C11, heavy chain: (SEQ ID NO: 9) METGLRWLLLVAVLKGVQCQSLEESGGRLVTPGTPLTLTCTASGFSLSNYYMSWVRQAPGKGLEWIGIIYGDDNTYCANWTKGRFTISKTSTTVDLTITSPTTEDTATYFCARGPDYIAAKMDIWGPGTLVTVSLGQPKAPSVFPLAPCCGDTPSTVTLGCLVKGYLPEPVTVTWNSG hTK1, Klon 23C11, light chain: (Sequence ID 10) MDTRAPTQLLGLLLLWLPGARCDVVMTQTPASVEAAVGGTVTIKCQASQSISGYLSWYQQKPGQRPKLLIYRASTLESGVPSRFKGSGSGTEFTLTISDLECADAATYYCQCTYGSSTF SSYGNAFGGGTEVVVKGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC As is evident, full-length immunoglobulin or any binding fragment thereof can be readily interpreted by any person skilled in the art based on the sequences disclosed above, if desired / necessary.

[0110] Example 3: Epitope Characterization As described in Example 2, we were able to generate four different monoclonal antibodies that exhibited the required binding properties necessary for their usefulness in the development of immunoassays.

[0111] In the first attempt to characterize the epitopes that bind to the newly generated antibodies, PepScan analysis was performed. For this analysis, synthetic peptides were synthesized consisting of 15 amino acids each, shifted by one amino acid (e.g., 1-15:2-16), spanning the entire sequence of hTK-1 (SEQ ID NO: 1). These PepScan peptides were spotted onto microscope slides. After blocking for nonspecific binding, cell culture supernatants of various MABs were incubated on the microscope slides. Unbound MABs were washed away, and bound MABs were detected using HRP-labeled goat anti-rabbit IgG by a routine method.

[0112] Of the four MABs obtained by the method of Example 2, only one (antibody 4H11) reacted with the linear epitope. As can be shown, the epitope to which this antibody binds is contained in the sequence (SEQ ID NO: 6) spanning amino acid residues 211-230 of hTK-1.

[0113] Polyclonal and monoclonal antibodies that react with synthetic peptides corresponding to the polypeptide (SEQ ID NO: 2) consisting of amino acids 194-225 of hTK-1 are known in the prior art; see, for example, WO2015 / 094106. None of the three MABs, 4H4, 6C6, and 23C11, bound to the polypeptide (SEQ ID NO: 2) consisting of amino acids 194-225 of hTK-1, nor did they show significant binding to any of the PepScan peptides tested. This indicates that all three MABs bind to higher-order structure-dependent epitopes on hTK-1. Since these three MABs appeared quite promising from the outset for the development of further assays, further efforts were made to gain more knowledge about the epitopes to which these three MABs bound.

[0114] Epitope characterization by competitive experiments was performed at 25°C using a GE Healthcare Biacore 4000 instrument. The Biacore biotin capture kit, Series S sensor (catalog no. 28-9202-34) was mounted on the instrument, hydrodynamically addressed, and pre-adjusted according to the manufacturer's instructions. The system buffer was HBS-N (10 mM HEPES pH 7.4, 150 mM NaCl). The sample buffer was the system buffer. The biotin capture reagent provided by the manufacturer GE Healthcare was diluted 1:50 with the system buffer and injected at 10 μl / min for 60 seconds onto flow cells 1, 2, 3, and 4 to address spots 1, 2, 4, and 5. Spot 3 served as a reference. 10 nM biotinylated primary antibody was injected at 30 μl / min for a contact time of 120 seconds to address spots 1 and 5 on all four flow cells. Spots 2 and 4 served as controls. To address spots 1, 2, 4, and 5, 10 nM human recombinant thymidine kinase-1 (hTK-1, Roche, 114 kDa, tetramer) was injected into all flow cells at 30 μl / min for a contact time of 180 seconds. To block any remaining accessible epitopes of the primary antibody, 100 nM non-biotinized primary antibody was injected again at 30 μl / min for a contact time of 180 seconds to address spots 1, 2, 4, and 5 on all flow cells. 100 nM secondary antibody was injected into all flow cells at 30 μl / min for a contact time of 180 seconds to address spots 1, 2, 4, and 5. Finally, the complexes formed on the sensor surface were completely removed by a regeneration step of 120 seconds on all flow cells and all spots using a regeneration solution provided by the manufacturer, GE Healthcare.

[0115] Thus, four recombinant monoclonal rabbit IgG antibodies were investigated for their hTK-1 epitope access characteristics: antibody 4H11 (an antibody that binds to the linear epitope on sequence number 6), as well as rabbit MAB, 23C11, 6C6, and 4H4.

[0116] Reporting points were set before and after each sample injection. Reading of the reporting points in response units [RU] was performed using Biacore Evaluation V.1.1 software.

[0117] The initial biotinylated primary antibody capture signal (bi-Ab1, [RU]) was combined with a second binding response signal (block Ab1[RU]) for a non-biotinylated primary antibody. Molar ratio epitope accessibility MR EA The formula =Ab2[RU] / (bi-Ab1[RU]+block Ab1[RU]) was calculated and used as an estimate of the epitope accessibility of each antibody used in the assay.

[0118] To verify the tetrameric state of the hTK analyte, a second molar ratio was calculated from the hTK binding signal versus capture level of the biotinylated primary antibody using the formula MR = hTK[RU] / bi-Ab1[RU]. * Molecular weight bi-Ab1 (150kDa) / molecular weight hTK (114kDa).

[0119] For example, antibody 4H11 showed a 1:1 binding stoichiometric ratio (molar ratio) of antibody 4H11 / hTK-1 MR. In this biosensor assay, a single tetrameric hTK-1 molecule binds to a single antibody 4H11 molecule. Of the listed antibodies, only antibody 4H11 shows homologous hTK-1 complex formation when used as the blocker. Therefore, a sandwich assay would be possible using a sequential assay protocol with antibody 4H11 twice. For rabbit monoclonal antibodies MAB, 23C11, 6C6, and 4H4, homologous complex formation was not detected. This means that MAB, 23C11, 6C6, and 4H4 bind to the same epitope region. Antibodies 23C11, 6C6, and 4H4 form a sandwich with antibody 4H11 as secondary antibodies. In this assay, the highest-performing sandwich pair was 6C6 as the biotinylated primary antibody, which binds to antibody 4H11 in a molar ratio MR. EA It forms a complex exhibiting a value of =0.4, which means 40% epitope accessibility on the hTK analyte.

[0120] As is clear from the table below, antibody 4H11 (which binds to the C-terminal linear epitope included in SEQ ID NO: 6) can form immune complexes with 23C11, 6C6, and 4H4. On the other hand, it is evident that rabbit monoclonal antibodies MAB, 23C11, 6C6, and 4H4 share the same epitope.

[0121] [Table 1]

[0122] The table shows sandwich formation of four anti-hTK-1 antibodies using recombinant h-TK-1 as the analyte in solution. A value of 0.0 in the table indicates that the first and second antibodies used bind to the same epitope. Values ​​greater than 0.1 indicate sandwich formation despite an intermediate blocking step, i.e., the two antibodies investigated bind to different epitopes.

[0123] Example 4 Generation of MAB conjugates for use in Elecsys immunoassay experiments In short, the following steps were performed to obtain antibody conjugates for the capture and detection sides of an immunological assay.

[0124] Cell culture supernatant (containing recombinant antibodies) obtained from B-cell PCR-generating cells (see above) was used as the starting material. Recombinant antibodies contained in the tissue culture supernatant were purified to protein A by affinity chromatography.

[0125] The antibody used as a capture antibody was cleaved into F(ab')2 fragments by pepsin, and the F(ab')2 fragments were further purified by affinity chromatography and size exclusion chromatography. The F(ab')2 fragments were then reduced to Fab' by a thiol reaction and site-directed biotinylation, thereby obtaining monobiotylated Fab' fragments.

[0126] The antibody used as the detection antibody was sulfo-BPRuNHS ester (CAS registry number 482618-42-8, in this art, lutenate(2-), bis[[2,2'-bipyridine]-4,4'-dimethanesulfonate(2-)-κN 1 κN 1’ ][1-[4-(4'-methyl[2,2'-bipyridine]-4-yl-κN 1 κN 1’ The sulfo-ruthenium (WO2003 / 002974) was chemically conjugated using [-1-oxobutoxy]-2,5-pyrrolidinedione]-, sodium (1:2, also known as (OC-6-31)], and the unbound label was removed by size exclusion chromatography.

[0127] Example 5 Sample and hTK-1 measurement 5.1 Sample A "black and white" panel was investigated. On the one hand, serum samples from 50 healthy donors (only 49 were still available for some experiments) were used to quantify hTK-1 in various assays. On the other hand, hTK-1 was measured in 48 samples (only 47 were still available for some experiments) from patients with scattered large B-cell lymphoma (DLBCL).

[0128] 5.2 Prototype Electrochemiluminescence Immunoassay Several prototype immunoassays were established based on monoclonal rabbit antibodies obtained as described in Example 3, purified as described in Example 4, and conjugated, respectively.

[0129] A typical setup for a prototype electrochemiluminescence immunoassay utilizes a biotinylated capture antibody (or its antigen-binding fragment) and a detection antibody (or its antigen-binding fragment) labeled with a ruthenium complex.

[0130] Immunoassay data were generated in most cases using conventional Fab' fragments biotinylated by conventional procedures. Measurements were performed in a sandwich assay format using a cobas® E170 analyzer from Roche. Signal detection in the cobas® E170 analyzer is based on electrochemiluminescence. In this sandwich assay, the biotin conjugate (i.e., capture antibody) is immobilized on the surface of a streptavidin-coated magnetic bead. The detection antibody has a complexed ruthenium cation as the signaling moiety. In the presence of the analyte, the chromogenic ruthenium complex is crosslinked to the solid phase and emits light at 620 nm after excitation with a platinum electrode contained in the measurement cell of the cobas® E170 analyzer. The signal output is an arbitrary number of optic units.

[0131] Measurements were performed, for example, using recombinant hTK-1 from HEK cells and calibrators spiked with the human serum samples mentioned above. The experimental hTK-1 assay was performed as follows: 25 μl of human serum sample or spiked calibrator and 25 μl of pretreatment reagent (containing 10 mM DTBA) were mixed and incubated for 9 minutes; then 60 μl of capture antibody-biotin conjugate and 60 μl of detection antibody ruthenium-labeled conjugate were incubated together for a further 9 minutes, followed by the addition of 30 μl of streptavidin-coated paramagnetic microparticles. The final mixture was incubated for a further 9 minutes. hTK-1 was then detected as usual (i.e., by the electrochemiluminescence signal produced in these experiments).

[0132] Interestingly, each MAB to a higher-order structure-dependent epitope combined with MAB(4H11) to the C-terminus yielded fairly good sandwich combinations. That is, each of these combinations can be used to establish a high-quality immunoassay for the measurement of hTK-1.

[0133] Attempts were also made to use one and the same antibody as both the capture antibody and the detection antibody. The combination of 4H11 used as both the biotinylated capture antibody and the rutenylation detection antibody yielded a considerably low signal count, i.e., unsatisfactory results. Surprisingly, when used as both the biotinylated capture antibody and the rutenylation detection antibody, the antibody against the higher-order structure-dependent epitope, as a combination of one antibody against a linear epitope (4H11) and one of the antibodies against a higher-order structure-dependent epitope (4H4; 6C6; or 23C11), yielded a high but slightly lower signal count.

[0134] It was also possible to use 4H11 as a capture antibody and an antibody against a higher-order structural epitope as a detection antibody, or to change the orientation of the anti-hTK-1 antibody, that is, to use an antibody against a higher-order structural epitope as a capture antibody and a 4H11 antibody as a detection antibody.

[0135] Below are the results obtained for a) the combination of biotinylated 4H11 (used as Fab'-Bi) and ruthenylated 23C11 (used as IgG), and b) biotinylated 6C6 (used as Fab'-Bi) and ruthenylated 4H11 (used as IgG).

[0136] The calibration results for both prototypes a) and b) are shown in Table 2 below, respectively.

[0137] [Table 2]

[0138] Table 2 shows the signals obtained from two different immunoassay prototypes using various amounts of recombinant hTK-1 as the analyte. Duplicate measurements were performed. As can be seen, both assay prototypes yield excellent results in terms of agreement between duplicate determination / count numbers and concentrations (=conc), calculated / measured concentrations of hTK-1 (MWconc), and overall signal magnitude.

[0139] In both prototype assays, black and white panels were measured. Box-whisker plots were calculated, receiver operating characteristics (ROC) were analyzed, and the area under the curve (AUC) was determined. The AUC for assay prototype a) was 0.971, and for assay prototype b) it was 0.965, which were almost identical. Both box-whisker plots (box plots) for both prototype assays, with determined concentrations of hTK-1 and AUC, are shown in Figure 2.

[0140] 5.3 DiaSorin TK1 Activity Assay The LIAISON® thymidine kinase assay, manufactured by DiaSorin, is an indirectly modified two-step competitive chemiluminescence immunoassay (CLIA) for the quantitative measurement of TK in human serum and EDTA plasma. The LIAISON® thymidine kinase assay was performed using 50 control samples and 48 samples from DLBCL patients, according to the instructions provided by the manufacturer. It utilizes an initial enzymatic reaction in which TK in the sample converts AZT (3'-azido-3'-deoxythymidine) to AZTMP (3'-azido-3'-deoxythymidine monophosphate), followed by a competitive immunoassay for the quantitative measurement of AZTMP. The amount of AZT converted to AZTMP is a measure of the amount of TK present in the sample. In the assay, 50 μL of sample is incubated with 100 μL of assay buffer 1, 20 μL of assay buffer 2, and 20 μL of paramagnetic particles coated with anti-AZTMP polyclonal antibody. Rabbit anti-goat IgG, followed by anti-AZTMP goat polyclonal, is coated onto the solid phase. This is incubated for 40 minutes, then 100 μL of tracer, an AZTMP analog conjugated to an isoluminol derivative, is added. During the first incubation, AZTMP binds to the solid phase. In the second incubation, the tracer conjugate competes for binding with AZTMP in solution. After 20 minutes of incubation, unbound material is removed by a washing cycle. Starter reagents are then added to initiate the instantaneous chemiluminescence reaction. The light signal is measured as relative light units (RLU) by a photomultiplier tube and is proportional to the concentration of TK present in the calibrator, control, or sample.

[0141] Box-whisker plots were calculated, receiver operating characteristics (ROC) were analyzed, and the area under the curve (AUC) was determined. For the LIAISON® thymidine kinase assay, an AUC of 0.958 was found. Both the box-whisker plot (box plot) and AUC are shown in Figure 3.

[0142] Example 6 Comparison of TK-1 levels determined by activity assay / immunoassay On the one hand, values ​​obtained from the LIAISON® thymidine kinase assay were compared with those obtained from two prototype immunoassays. Considering that one assay measures thymidine kinase activity and the other two measure the amount of immunoreactive hTK-1, a surprisingly high correlation (within the range of 0.95 or even higher – depending on the statistical method used) was found between the two different assays. The excellent correlation between these different assays for hTK-1 is also clearly evident from Figure 4.

Claims

1. A monoclonal antibody that specifically binds to human thymidine kinase 1 (hTK-1; SEQ ID NO: 1), The heavy chain variable domain of SEQ ID NO: 3 and the light chain variable domain of SEQ ID NO: 4, The heavy chain variable domain of SEQ ID NO: 7 and the light chain variable domain of SEQ ID NO: 8, or The heavy chain variable domain of sequence number 9 and the light chain variable domain of sequence number 10, A monoclonal antibody having one of the following properties.

2. An in vitro method for quantifying hTK-1, a) A step of incubating a sample in which hTK-1 is to be quantified with the antibody of claim 1, thereby generating a complex between the antibody and hTK-1, b) A step to quantify the complex formed in step a), thereby quantifying hTK-1. A method that includes this.

3. An in vitro method for quantifying hTK-1, a) Incubating a sample in which hTK-1 is to be quantified with a first antibody, which is the antibody of claim 1, and a second antibody against hTK-1, thereby generating a sandwich complex between the first antibody, hTK-1, and the second antibody; b) A step to quantify the sandwich complex formed in step a), thereby quantifying hTK-1. A method that includes this.

4. An in vitro method for quantifying hTK-1, a) A step of incubating the sample to be quantified for hTK-1 with a pretreatment solution containing a reducing agent and ATP, b) Incubating the pre-treated sample obtained in step a) with the antibody according to claim 1, thereby generating a complex between the antibody and hTK-1, c) A step to quantify the complex formed in step b), thereby quantifying hTK-1. A method that includes this.

5. An in vitro method for quantifying hTK-1, a) A step of incubating the sample to be quantified for hTK-1 with a pretreatment solution containing a reducing agent and ATP, b) Incubating the pre-treated sample obtained in step a) with a first antibody, which is the antibody of claim 1, and a second antibody against hTK-1, thereby generating a sandwich complex between the first antibody, hTK-1, and the second antibody; c) A step to quantify the sandwich complex formed in step b), thereby quantifying hTK-1. A method that includes this.

6. The method according to claim 3 or 5, wherein the first or second antibody is bound to a solid phase or capable of binding to a solid phase, and the second or first antibody is detectably labeled.

7. The antibody according to claim 1, obtained by B-cell PCR technology.

8. Use of the antibody according to claim 1 or 7 in the quantification of hTK-1.

Images