umtck antibody or antigen-binding fragment thereof

Abstract

The present disclosure relates to the technical field of laboratory medicine, in particular to a uMtCK antibody or an antigen-binding fragment thereof. The uMtCK antibody or the antigen-binding fragment thereof can specifically bind to uMtCK, has little cross-reaction with CK-MM, CK-BB or CK-MB, has high affinity, and has good application prospect in the field of uMtCK detection.

Description

TECHNICAL FIELD

[0001] The present disclosure relates generally to the field of laboratory medicine, and in particular, to a uMtCK antibody or antigen-binding fragment thereof. BACKGROUND

[0002] Creatine kinase (CK, EC 2.7.3.2) belongs to the phosphagen kinase family and plays an important role in the energy metabolism of vertebrates by catalyzing reversible phosphorylation reactions. CK has four isoenzyme forms, including muscle type (CK-MM), brain type (CK-BB), hybrid type (CK-MB) and mitochondrial type (MtCK), which mainly exist in brain tissue, skeletal muscle and myocardium. Due to the important role of creatine kinase in cell energy cycle, it plays an important role in regulating glycolysis process, phagocytosis of phagocytes, release of neurotransmitters and ion transmembrane transport, etc. One of the important indicators for diagnosing myocardial infarction in clinic is the content of CK in serum, and CK is also considered as a potential clinical biomarker and therapeutic target for many diseases. Human mitochondrial ubiquitous isoform creatine kinase (uMtCK) plays an important role in mitochondrial oxidative phosphorylation and apoptosis, and recent studies have found that uMtCK has abnormal expression in many cancers.

[0003] At present, the method for determining uMtCK is mainly electrophoretic determination method, which needs enzyme activity determination to show the isoenzymes on different electrophoretic bands. However, uMtCK is sensitive to oxygen free radicals and is easily inactivated by free radicals, which may be the reason for the low detection rate of uMtCK in human serum. In addition, although immunological detection can avoid the disadvantages of electrophoretic determination, it is not easy to provide an antibody that can specifically recognize uMtCK due to the similar structure of the four isoenzymes of CK. SUMMARY

[0004] The present disclosure encompasses the following technical solutions:

[0005] (1) A uMtCK antibody or antigen-binding fragment thereof, comprising heavy chain complementarity determining regions with amino acid sequences shown in SEQ ID NO: 1-3 in order, and light chain complementarity determining regions with amino acid sequences shown in SEQ ID NO: 4-6 in order.

[0006] (2) The uMtCK antibody or antigen-binding fragment thereof according to (1), comprising a heavy chain variable region with an amino acid sequence shown in SEQ ID NO: 7, and a light chain variable region with an amino acid sequence shown in SEQ ID NO: 8.

[0007] (3) An isolated nucleic acid capable of encoding the uMtCK antibody or antigen-binding fragment thereof according to (1) or (2).

[0008] (4) a vector comprising the nucleic acid of (3).

[0009] (5) a host cell comprising the nucleic acid of (3), or transformed with the vector of (4).

[0010] (6) a method of producing the uMtCK antibody or antigen-binding fragment thereof of (1) or (2), comprising culturing the host cell of (5) under suitable conditions and recovering the desired product from the cell culture.

[0011] (7) an antibody combination product comprising the uMtCK antibody or antigen-binding fragment thereof of (1) or (2) and a second uMtCK antibody produced by Sino Biological, Cat. No. CSB-MA149995.

[0012] (8) a kit comprising the uMtCK antibody or antigen-binding fragment thereof of (1) or (2), or the antibody combination product of (7).

[0013] (9) use of the uMtCK antibody or antigen-binding fragment thereof of (1) or (2), or the antibody combination product of (7) in the preparation of a tumor diagnostic reagent or kit.

[0014] (10) the use of (9), wherein the tumor is selected from the group consisting of breast cancer, gastric cancer, prostate cancer, cervical cancer, liver cancer, ovarian cancer, lung cancer, lymphoma, colorectal cancer, nasopharyngeal carcinoma, and osteosarcoma.

[0015] Advantages:

[0016] The antibody or antigen-binding fragment thereof provided by the present disclosure can specifically bind to uMtCK, substantially not cross-react with CK-MM, CK-BB or CK-MB, and has a high affinity, and has a good application prospect in the field of uMtCK detection. DETAILED DESCRIPTION

[0017] Reference will now be made in detail to the embodiments of the present disclosure, one or more examples of which are set forth below. Each example is provided as an explanation and not as a limitation of the present disclosure. Indeed, it will be apparent to one of ordinary skill in the art that numerous modifications and variations of the present disclosure are possible in light of the above teachings. For example, features described or illustrated as part of one embodiment can be used with another embodiment to yield a still further embodiment.

[0018] Unless otherwise indicated, all terms, including technical and scientific terms, used herein in the disclosure have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. By further guidance, the following definitions are used for better understanding the teachings of the present disclosure. The terminology used in the specification of the present disclosure herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the present disclosure.

[0019] In the present disclosure, unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by a person of ordinary skill in the art. Also, the terms related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, immunology, and laboratory operation procedures used herein are terms and conventional procedures widely used in the corresponding fields. At the same time, for better understanding of the present disclosure, the definitions and explanations of related terms are provided as follows.

[0020] The selection range of the terms "and / or", "or / and", and "and / or" used herein includes any one of two or more related listed items, and also includes any and all combinations of the related listed items, including any two related listed items, any more related listed items, or all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or", "or / and", and "and / or", it should be understood that in the present disclosure, the technical solution undoubtedly includes the technical solution connected by "logical and", and also undoubtedly includes the technical solution connected by "logical or". For example, "A and / or B" includes three parallel solutions of A, B, and A+B. For another example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (i.e., the technical solution connected by "logical or"), and also includes any and all combinations of A, B, C, and D, i.e., includes the combination of any two or any three of A, B, C, and D, and also includes the four-item combination of A, B, C, and D (i.e., the technical solution connected by "logical and").

[0021] The terms "containing", "including", and "comprising" used in the present disclosure are synonymous terms, which are inclusive or open-ended, and do not exclude additional, unrecited members, elements, or method steps.

[0022] The numerical ranges in the present disclosure expressed in a range format should be interpreted as using the language "between X and Y" in the disclosure, and therefore include any and all values and sub-ranges between the upper and lower bounds of the range.

[0023] As used herein, reference to "about" a value or parameter herein includes (and describes) embodiments that are directed to that value or parameter per se. For example, description referring to "about X" includes description of "X".

[0024] In the present disclosure, the concentration values involved include fluctuations within a certain range. For example, they can fluctuate within a corresponding accuracy range. For example, 2% can fluctuate within ±0.1%. For values that are larger or do not need to be controlled too finely, the values involved can also include larger fluctuations. For example, 100 mM can fluctuate within ±1%, ±2%, ±5%, etc. For molecular weights, the values involved can include fluctuations of ±10%.

[0025] As used herein, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise.

[0026] In the present disclosure, "a plurality of", "a plurality of", and the like, unless otherwise specified, refer to greater than or equal to 2 in number.

[0027] In the present disclosure, in the technical features described in an open manner, both the closed technical solutions consisting of the listed features and the open technical solutions containing the listed features are included.

[0028] In the present disclosure, "preferably", "more preferably", "even more preferably", "suitably" only describe embodiments or examples with better effects, and it should be understood that they do not constitute a limitation on the protection scope of the present disclosure. In the present disclosure, "optionally", "optional", "optionally" means that it can or can not be present, i.e., it means to select either "yes" or "no" from the two parallel schemes. If there are multiple "options" in a technical solution, unless otherwise specified, and there is no contradictory or mutually restrictive relationship, each "option" is independent of each other.

[0029] In the present disclosure, the technical term "antibody" refers to a protein that binds to a specific antigen, and generally refers to all proteins and protein fragments containing complementarity determining regions (CDR regions), especially full-length antibodies or antibody functional fragments. The term "full-length antibody" includes polyclonal antibodies and monoclonal antibodies, and the term "antibody functional fragment" is a substance containing part or all of the CDR of the antibody, which lacks at least some of the amino acids present in the full-length chain but still can specifically bind to the antigen.

[0030] In the present disclosure, the term "complementarity determining region" or "CDR" refers to the highly variable regions of the heavy and light chains of immunoglobulins as defined by Kabat et al. (Kabat et al., Sequences of proteins of immunological interest, 5th Ed "US Department of Health and Human Services, NIH, 1991, and subsequent editions). There are three heavy chain CDRs and three light chain CDRs. Herein, the terms "CDR" and "CDRs" are used to refer to the region(s) comprising the amino acid residue(s) that play a major role in the binding affinity of an antibody to the antigen or epitope it recognizes, depending on the context.

[0031] As used herein, the term "signal material" refers to any ingredient, molecule, functional group, compound, fragment, or moiety that can provide a signal and be detected. In some embodiments, a detection entity is provided or used alone. In some embodiments, a detection entity is provided and / or used in conjunction with another agent. Examples of detection entities include, but are not limited to: various ligands, radionuclides (e.g. 3 H, 14 C, 18 F, 19 F, 32 P, 35 S, 135 I, 125 I, 123 I, 64 Cu, 187 Re, 111 In, 90 Y, 99 mTc, 177 Lu, 89 Zr, etc.), fluorescent dyes, chemiluminescent agents (e.g. acridinium esters, stabilized dioxetanes, etc.), bioluminescent agents, spectrally distinguishable inorganic fluorescent semiconductor nanocrystals (i.e. quantum dots), metallic nanoparticles (e.g. gold, silver, copper, platinum, etc.), nanoclusters, paramagnetic metal ions, enzymes, colorimetric labels (e.g. dyes, colloidal gold, etc.), biotin, digoxigenin, haptens, and proteins to which antisera or monoclonal antibodies are available.

[0032] All the documents mentioned in the present disclosure are cited as references in the present disclosure as if each document is individually cited as a reference. Unless and to the extent that the cited documents conflict with the purpose and / or technical solutions of the present disclosure, the cited documents are cited in the present disclosure in their entirety, in their entirety purpose. When the present disclosure refers to the cited documents, the definitions of the relevant technical features, terms, nouns, phrases, etc. in the cited documents are also cited. When the present disclosure refers to the cited documents, the examples and preferred modes of the cited relevant technical features can also be incorporated into the present disclosure as references, but are limited to the implementation of the present disclosure. It should be understood that when the cited content conflicts with the description in the present disclosure, the present disclosure is preferred or is modified according to the description in the present disclosure.

[0033] The first aspect of the present disclosure provides a uMtCK antibody or an antigen binding fragment thereof, which comprises a heavy chain complementarity determining region with the amino acid sequence shown in SEQ ID NO: 1-3 in turn, and a light chain complementarity determining region with the amino acid sequence shown in SEQ ID NO: 4-6 in turn.

[0034] In some embodiments, the uMtCK antibody or the antigen binding fragment thereof comprises a heavy chain variable region with the amino acid sequence shown in SEQ ID NO: 7, and a light chain variable region with the amino acid sequence shown in SEQ ID NO: 8.

[0035] According to the routine understanding of those skilled in the art, it should be considered that the variants of the above-mentioned amino acid sequences are also within the scope of the present disclosure. The variants of the uMtCK antibody or the antigen binding fragment thereof of the present disclosure, compared with any one of the polypeptides of SEQ ID NO: 1-SEQ ID NO: 6, respectively, comprise up to 1, 2 or 3 amino acid mutations in the CDR region (if any); or, the variants have a sequence with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with respect to the overall sequence of SEQ ID NO: 7-SEQ ID NO: 8. The mutation can be substitution, deletion or addition of amino acids or any combination thereof; preferably, the mutation is a conservative substitution.

[0036] "Conservative substitution" refers to the substitution of an amino acid in a protein with another amino acid having similar characteristics (e.g. charge, side chain size, hydrophobicity / hydrophilicity, backbone conformation and rigidity, etc.), so that changes can be frequently made without changing the biological activity of the protein.

[0037] Substitutions generally considered conserved include substitutions between aliphatic amino acids Ala, Val, Leu, and Ile; interchanges between hydroxyl residues Ser and Thr; exchange between acidic residues Asp and Glu; substitutions between amide residues Asn and Gln; exchange between basic residues Lys and Arg; and substitutions between aromatic residues Phe and Tyr. Those skilled in the art will recognize that, in general, single amino acid substitutions in non-essential regions of a polypeptide do not substantially alter biological activity (see, for example, Watson et al. (1987), Molecular Biology of the Gene, The Benjamin / Cummings Pub. Co., p. 224, (4th edition)). Furthermore, substitutions of structurally or functionally similar amino acids are unlikely to disrupt biological activity.

[0038] In some embodiments, the antigen-binding fragment is one of F(ab')2, Fab, scFv, and a bispecific antibody.

[0039] The term "F(ab')2" refers to the fragment obtained by digesting the entire full-length antibody with pepsin to remove most of the Fc region while retaining some hinge regions intact. The F(ab')2 fragment has two antigen-binding Fab moieties linked together by disulfide bonds, thus the F(ab')2 fragment is a bivalent antibody. Taking F(ab')2 prepared from IgG antibody as an example, its molecular weight is approximately 110 kDa.

[0040] The term "Fab" refers to an antibody structure that can still bind to the antigen; it is monovalent and does not contain an Fc portion. Papain digestion of a full-length antibody yields two Fab fragments and one Fc fragment, each Fab fragment being approximately 50 kDa.

[0041] The term "scFv" means a molecule comprising an antibody heavy chain variable domain (or region; VH) and an antibody light chain variable domain (or region; VL) connected by a linker. Such scFv molecules can have the general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. In the present disclosure, the term "linker" can be a flexible or rigid peptide, typically flexible, which can reduce steric hindrance between the fusion protein and the protein of interest, thus more favoring the correct folding of the protein. For example consisting of repeated GGGGS amino acid sequences or variants thereof, for example using 1-4 repeats of the variant (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90:6444-6448). Other linkers useful in the present disclosure are described by Alfthan et al. (1995), Protein Eng. 8:725-731, Choi et al. (2001), Eur. J. Immunol. 31:94-106, Hu et al. (1996), Cancer Res. 56:3055-3061, Kipriyanov et al. (1999), J. Mol. Biol. 293:41-56, and Roovers et al. (2001), Cancer Immunol. The linker is typically flexible, which can reduce steric hindrance between the fusion protein and the protein of interest, thus more favoring the correct folding of the protein.

[0042] In some embodiments, the uMtCK antibody or antigen-binding fragment thereof has a constant region.

[0043] In some embodiments, the species origin of the constant region is mouse, rat, guinea pig, hamster, rabbit, horse, sheep, goat, donkey, bovine, chicken, duck, and human.

[0044] In some embodiments, the heavy chain constant region sequence of the antibody or antigen-binding fragment thereof is selected from human IgGl.

[0045] In some embodiments, the heavy chain constant region sequence of the antibody or antigen-binding fragment thereof is set forth in SEQ ID NO: 9. In some embodiments, the light chain constant region sequence of the antibody or antigen-binding fragment thereof is set forth in SEQ ID NO: 10.

[0046] A second aspect of the present disclosure relates to an isolated nucleic acid capable of encoding a uMtCK antibody or antigen-binding fragment thereof as described above.

[0047] The term "isolated nucleic acid" refers herein to a deoxyribonucleic acid or ribonucleic acid polymer in either single- or double-stranded form. The isolated nucleic acid includes RNA genomic sequences, DNA (gDNA and cDNA) or RNA sequences transcribed from DNA, and, unless specifically indicated otherwise, the polypeptides also include natural polynucleotides, sugars, or base analogs. According to one aspect of the disclosure, the polynucleotide is a light chain polynucleotide.

[0048] The isolated nucleic acid includes nucleotide sequences encoding the amino acid sequences of the protein complex, as well as nucleotide sequences complementary thereto. The complementary sequences include sequences that are completely complementary and substantially complementary, which means sequences that hybridize to the nucleotide sequences encoding the amino acid sequences of the protein complex under stringent conditions known in the art.

[0049] Also, the nucleotide sequences encoding the amino acid sequences of the protein complex can be altered or mutated. The alterations include additions, deletions, or non-conservative substitutions or conservative substitutions. The polynucleotides encoding the amino acid sequences of the protein complex can be interpreted to include nucleotide sequences that are substantially identical to the isolated nucleic acid. The substantial identity aligns the nucleotide sequences in a manner that maximizes their correspondence, and when the aligned sequences are analyzed using algorithms common in the art, the sequences can show greater than 80% homology, such as greater than 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology.

[0050] In addition, the codons of the nucleic acid corresponding to the uMtCK antibody or antigen-binding fragment thereof can be optimized according to the species of the host cell.

[0051] A third aspect of the disclosure relates to a vector containing the nucleic acid as described above.

[0052] The term "vector" refers to a nucleic acid vehicle into which a polynucleotide can be inserted, for example, and which can be subsequently introduced into a host cell. When a vector is capable of mediating the expression of a polynucleotide inserted into it, the vector is said to be an expression vector. A vector can be introduced into a host cell by transformation, transduction or transfection, and the host cell into which the vector is introduced can be used to express the polynucleotide carried by the vector. Vectors are well known to those skilled in the art and include, but are not limited to, plasmids, phagemids, cosmids, artificial chromosomes such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1 -derived artificial chromosomes (PAC), bacteriophages such as lambda phage or M13 phage, and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papova viruses (such as SV40). In some embodiments, the vectors of the present disclosure comprise regulatory elements commonly used in genetic engineering, such as enhancers, promoters, internal ribosome entry sites (IRES) and other expression control elements (e.g., transcription termination signals, or polyadenylation signals and polyU sequences, etc.).

[0053] In the present disclosure, a vector can be a composition, for example, a mixture of multiple plasmids, different plasmids carrying a part of an antibody or an antigen-binding fragment thereof, for example, nucleic acids to be used to encode heavy and light chains of an antibody are inserted into different vectors independently.

[0054] A fourth aspect of the present disclosure relates to a host cell comprising a nucleic acid as described above, or transformed with a vector as described above.

[0055] Suitable host cells or cell lines for expressing the antigen-binding proteins of the present disclosure include mammalian cells such as NS0, Sp2 / 0, CHO, COS, HEK, fibroblast and myeloma cells. Human cells can be used, thus allowing the molecules to be modified with human glycosylation patterns. Alternatively, other eukaryotic cell lines can be employed. The selection of suitable mammalian host cells, and methods for transformation, culturing, amplification, screening and product production and purification, are known in the art.

[0056] It can be demonstrated that bacterial cells can be used as host cells that are suitable for expressing the proteins of the present disclosure or other embodiments. However, because proteins expressed in bacterial cells tend to be in an unfolded form or incorrectly folded form or non-glycosylated form, any protein produced in bacterial cells must be screened to retain antigen binding capacity. If the molecule expressed by the bacterial cell is produced in a properly folded form, the bacterial cell will be a desirable host, or, in an alternative embodiment, the molecule can be expressed in a bacterial host followed by refolding. For example, various strains of E. coli are well known host cells in the biotechnology field for expression. Various strains of B. subtilis, Streptomyces, other Bacillus species, and the like can also be used in the method.

[0057] If desired, strains of yeast cells and insect cells, e.g., Drosophila and lepidopteran insects and viral expression systems, known to those skilled in the art can also be used as host cells.

[0058] In some embodiments, the nucleic acid is inserted into the genome of the cell and is stably expressed. The nucleic acid can be inserted into the genome using a vector as described above, or the nucleic acid can be introduced directly into the cell without being inserted into a vector (e.g., by liposome-mediated transfection techniques).

[0059] A fifth aspect of the present disclosure relates to a method of making a uMtCK antibody or antigen binding fragment thereof as described above, comprising culturing a host cell as described above under suitable conditions and recovering the desired product from the cell culture.

[0060] The culturing methods of the present disclosure are typically serum-free culturing methods and the cells are typically cultured in serum-free suspension. Likewise, once the antibodies of the present disclosure are produced, they can be purified from the cell culture contents according to standard procedures in the art, including ammonium sulfate precipitation, affinity columns, column chromatography, gel electrophoresis, and the like. Such techniques are within the routine skill of the art and do not limit the present disclosure. The host cells can also be cultured to express the antibodies in animals, particularly transgenic animals or nude mice. This involves the use of an expression system that utilizes the animal casein promoter, which when transgenically incorporated into a mammal, allows the female animal to produce the desired recombinant protein in its milk. The culture fluid in which the antibodies are secreted can be purified using conventional techniques. For example, purification can be performed using an A or G Sepharose FF column with a modified buffer. Non-specifically bound components are washed away. The bound antibodies are eluted using a pH gradient and the antibody fragments are detected using SDS-PAGE and collected. The antibodies can be concentrated by filtration using conventional methods. Soluble aggregates and multimers can also be removed using conventional methods, such as molecular sieving, ion exchange. The resulting product is immediately frozen, e.g., at -70°C, or lyophilized.

[0061] The sixth aspect of the present disclosure relates to an antibody combination product comprising the uMtCK antibody or antigen-binding fragment thereof as described above and a second uMtCK antibody with the product number CSB-MA149995 from Ubiqiutic.

[0062] The above-mentioned antibody combination product is a paired antibody, and the two uMtCK antibodies can bind to different epitopes of uMtCK, thereby detecting uMtCK using a double antibody sandwich technique and obtaining higher detection specificity.

[0063] The seventh aspect of the present disclosure relates to a kit comprising the uMtCK antibody or antigen-binding fragment thereof as described above, or the antibody combination product as described above.

[0064] The term "kit" in the present disclosure can refer to any article (e.g., package or container) comprising at least one device, which comprises a detection agent as described in the present disclosure. The kit can further comprise instructions for use, supplementary reagents and / or components or components used in the methods described in the present disclosure or steps thereof.

[0065] In some embodiments, the kit comprises the antibody combination product as described above; wherein the uMtCK antibody or antigen-binding fragment thereof is labeled with a signal substance, and the second uMtCK antibody is conjugated with a solid support.

[0066] The signal substance comprises any moiety that can be detected using an assay, for example, due to its specific functional and / or chemical characteristics. Non-limiting examples of the agent include enzymes, radiolabels, haptens, fluorescent labels, phosphorescent molecules, chemiluminescent molecules, chromophores, luminescent molecules, photoaffinity molecules, colored particles or ligands (such as biotin). In some embodiments of the present disclosure, the signal substance is selected from the group consisting of luminol and its derivatives, isoluminol and its derivatives, alkaline phosphatase, and horseradish peroxidase.

[0067] According to the needs, any one or more of the following components can also be included in the kit: sample pretreatment reagents (such as sample purification enrichment reagents, lysates, etc.), washing solutions (such as PBS, etc.), buffers (such as phosphate buffer), color developing reagents for the signal substance (such as ECL for the signal substance horseradish peroxidase), preservatives (any one or a mixture of two or more of sodium azide, sodium nitrite, sodium benzoate, Proclin series, potassium sorbate). These components can be packaged separately, or two or more can be pre-mixed to facilitate use / preservation.

[0068] In the present disclosure, the solid support can be a substance for conjugating an antibody and facilitating detection, and in some embodiments, the solid support is a plastic, a microparticle, or a membrane support; the plastic can be polystyrene; the membrane support can be a nitrocellulose membrane, a glass cellulose membrane, or a nylon membrane. In some embodiments, the solid support is selected from a test tube, an EP tube, a multi-well plate, a chromatography column, a micro-reaction plate well.

[0069] In the present disclosure, the term "microparticle" can be a sphere, a near-sphere, a cube, a polyhedron, or an irregular shape. The diameter of the microsphere is preferably 10 nm to 1 mm, for example, 100 nm, 500 nm, 1 μm, 10 μm, 100 μm, 500 μm; preferably 400 nm to 10 μm. The microparticle is preferably a magnetic microparticle, which contains a magnetic substance in its composition. The magnetic substance can be a metal (a metal element or an alloy), a non-metal, or a composite of a metal and a non-metal. The metal is, for example, iron, aluminum-nickel-cobalt metal, etc.; the non-metal is, for example, a ferrite non-metal (preferably Fe2O3 or Fe3O4 magnetic nanoparticles); the composite of a metal and a non-metal is, for example, a neodymium-iron-boron rubber magnetic composite.

[0070] In some specific embodiments, the solid support is a membrane support and is used for lateral flow detection. Lateral flow detection is also known as lateral flow immunoassay assay (LFIA), laminar flow, immunochromatographic assay, or test strip test. LFIA test is a form of immunoassay in which the test sample flows along a solid substrate by capillary action. After the sample is applied to the test strip, it encounters a colored reagent (typically including an antibody specific for the test target antigen) bound to microparticles, which mixes with the sample and passes through a substrate (e.g., specific for a detectable label on the target nucleic acid or a complementary nucleic acid to the target nucleic acid) pre-treated with the antibody or pre-treated with a conjugated or unconjugated antibody as described herein. Depending on the level of target present in the sample, the signal substance (typically a colored substance) can be captured and bound at the test line or test zone. LFIA is essentially an immunoassay adapted for operation along a single axis to accommodate a test strip format or dipstick format. Test strip tests have many uses and can be easily modified by one skilled in the art to detect various antigens from fluid samples such as urine, blood, water, and / or homogenized tissue samples. Typically, a lateral flow test strip includes a sample pad, a conjugate pad, a detection membrane, and optionally an absorbent pad.

[0071] An eighth aspect of the present disclosure relates to the use of a uMtCK antibody or antigen-binding fragment thereof, or an antibody combination product as described above in the preparation of a tumor diagnostic reagent or kit.

[0072] In some embodiments, the tumor is selected from the group consisting of breast cancer, gastric cancer, prostate cancer, cervical cancer, liver cancer, ovarian cancer, lung cancer, lymphoma, colorectal cancer, nasopharyngeal cancer, and osteosarcoma.

[0073] The embodiments of the present disclosure will be described in detail below with reference to the examples. It should be understood that these examples are only used to illustrate the present disclosure and not to limit the scope of the present disclosure. The experimental methods in the following examples without specific conditions are preferred to refer to the guidance given in the present disclosure, and can also be carried out according to the experimental manuals or conventional conditions in the art, or can also refer to other experimental methods known in the art, or according to the conditions recommended by the manufacturer.

[0074] In the following specific examples, the measurement parameters of the raw material components may have slight deviations within the weighing accuracy range if not specifically stated. For temperature and time parameters, acceptable deviations caused by instrument testing accuracy or operation accuracy are allowed.

[0075] Example 1 Preparation and titer evaluation of antibody

[0076] 1. Antibody preparation

[0077] Synthesis of genes: According to the amino acid sequences of SEQ ID NO: 7-8, it is determined that the signal peptide sequence and the target protein sequence (the heavy chain signal peptide is shown in SEQ ID NO: 11, and the light chain signal peptide is shown in SEQ ID NO: 12) need to be included, and then the gene synthesis service (including codon optimization) is provided by Nanjing Kingsriver Biotechnology Co., Ltd. The upstream and downstream of the antibody light and heavy chains are HindIII and EcoRI restriction sites, and the downstream of the target fragment contains a stop codon. The light and heavy chain genes are synthesized into pUC57 vectors, respectively, and finally pUC57-AL and pUC57-AH are obtained.

[0078] Construction of recombinant plasmid: first, the light chain plasmid pUC57-AL and the vector pCDNA3.1 (from Invitrogen) are double-digested with HindIII and EcoRI, and the enzyme digestion system needs to be carried out according to the manufacturer's instructions of Invitrogen. After incubation at 37°C for 1h, the double-digested products are subjected to 1% agarose gel electrophoresis, and after identification on the gel imaging instrument, the gel block containing the target fragment is cut off and recovered with a gel recovery kit. The target fragment is ready for use. Add T4 DNA ligase, and after incubation at 22°C for 1h, transform into E. coli Top10 competent cells, and plate on ampicillin-resistant plates and incubate at 37°C for 12-16h.

[0079] Pick single colonies for PCR identification, and the reaction system needs to be carried out according to the manufacturer's instructions of Invitrogen. The identification primers are selected as the universal primers on the vector. The PCR reaction is carried out according to 95°C for 30s, 60°C for 30s, 72°C for 1min, 30 cycles, and then 4°C for 10min.

[0080] 60s, 30 cycles, 72°C 60s program for amplification. The PCR reaction system was subjected to 1% agarose gel electrophoresis, and positive clones were determined on a gel illuminator. The identified positive clones were inoculated into LB medium and cultured at 37°C for 12-16h, and the plasmid was extracted using the Omega general extraction kit. The plasmid was sent to Shanghai Shenguo Biological Engineering Co., Ltd. for sequencing. If the sequence is consistent with the synthesized gene sequence, it means that the light chain recombinant plasmid pCDNA3.1-AL is successfully constructed.

[0081] Similarly, the heavy chain recombinant plasmid pCDNA3.1-AH can be obtained.

[0082] Recombinant plasmid extraction: follow the instructions of Omega endotoxin plasmid extraction kit to extract recombinant plasmid and detect the concentration on ultraviolet spectrophotometer.

[0083] Transfection of mammalian cells: follow the instructions of Invitrogen liposome transfection to transfect recombinant plasmid into mammalian CHO cells, wherein the mass ratio of light chain and heavy chain recombinant plasmid is 2:1, the mass ratio of transfection reagent PEI and expression plasmid is 3:1, the passage density is 0.5E6, and the culture medium used is Mayban MaxY culture medium; after 72h culture at 37°C, 5% CO2 shaker, add the feed, the addition amount is 5% of the original volume, then add once every other day, and collect the sample on the 14th day.

[0084] Protein purification: follow the instructions of GE AKTA Pure protein separation and purification system to operate, and purify through A column to obtain the primary purified antibody. First, equilibrate the column with 20mM sodium phosphate, 20mM sodium citrate, pH 7.5, then load the supernatant of the centrifuged expression sample onto the Protein A column (HiTrap Protein a FF, 5mL, GE Healthcare). After washing with the same buffer, use 20mM sodium phosphate, 100mM sodium chloride, 100mM glycine pH 3.0 to elute gradually. The eluted antibody is neutralized with 0.5M sodium phosphate pH 8.0 (1:10), concentrated, and changed to obtain the purified antibody.

[0085] 2. Antibody titer evaluation

[0086] uMtck-1 antibody: purchased from Wuhan Huamei Biological

[0087] uMtck-2 antibody: the antibody prepared by the above method

[0088] uMtck-3 antibody: Wuhan Yunkong Biological

[0089] uMtck antigen: Wuhan Yunkong Biological

[0090] Antibodies from different manufacturers coated magnetic microspheres, and the luminescent marker labeled broad type mitochondria creatine kinase antigen.

[0091] Method for checking the affinity of antibodies from different manufacturers to uMtck antigen:

[0092] (1) Add magnetic spheres coated with antibodies from different manufacturers to different reaction cup holes, respectively.

[0093] (2) Add the luminescent marker labeled broad type mitochondria creatine kinase antigen to step (1) respectively, incubate, and make the luminescent marker labeled broad type mitochondria creatine kinase antigen bind to the antibody-coated magnetic microspheres; wherein the incubation temperature is 25-37°C, and the incubation time is 30 min.

[0094] (3) Magnetic separation to obtain the combination of the luminescent marker labeled broad type mitochondria creatine kinase antigen and the antibody-coated magnetic microspheres.

[0095] (4) Add a luminescent excitation substrate to the combination obtained in step (3) to detect the light signal intensity and compare the luminescent intensity; the luminescent excitation substrate includes but is not limited to sodium hydroxide and hydrogen peroxide.

[0096] The data are as follows:

[0097] Antibody uMtck-1 uMtck-2 uMtck-3 Mean luminescence intensity 542340 1346430 274367

[0098] As can be seen from the above table, the luminescent intensity of uMtck-2 antibody is the highest, and its affinity to uMtck antigen is the strongest.

[0099] Kit composition information of Example 2

[0100] 1. Component A: solution of magnetic microspheres coated with (Huawei; item number CSB-MA149995) diluted with phosphate buffer, wherein:

[0101] a) uMtck-1 antibody is a specific binding broad type mitochondria creatine kinase (obtained by monoclonal preparation).

[0102] b) Magnetic microspheres are any of the following: magnetic microspheres with epoxy, amino, aldehyde, sulfonyl, carboxyl, amide, sulfhydryl or hydroxyl groups on the surface.

[0103] The working concentration of the magnetic microspheres is 1-5 mg / ml, and the working concentration of the uMtck-1 antibody is 5-10 μg / ml.

[0104] 2. Component B: uMtck-2 antibody (antibody obtained in Example 1) marking the luminescent marker, wherein the working concentration of the luminescent marker is 1-1.50 μg / ml, and the working concentration of the uMtck-2 antibody is 300-500 ng / ml. The diluent is phosphate buffer.

[0105] The luminescent marker is any one of the following: luminol and its derivatives, isoluminol and its derivatives, alkaline phosphatase, horseradish peroxidase.

[0106] 3. Calibration solution: prepare a low-concentration calibration solution (concentration 39.06 ng / ml) and a high-concentration calibration solution (concentration 1250 ng / ml) using phosphate buffer.

[0107] 4. Reaction buffer: phosphate buffer.

[0108] Preparation of phosphate buffer: dissolve 0.2 g of potassium dihydrogen phosphate in 800 ml of purified water, add 2.9 g of disodium hydrogen phosphate dodecahydrate, add BSA and preservatives, and after complete dissolution, make up to 1000 ml, adjust the pH to 7.3, and use. The mass percentage concentration of BSA is 0.1%-0.5%, and the preservatives are any one or a mixture of two or more of sodium azide, sodium nitrite, sodium benzoate, Proclin series, and potassium sorbate.

[0109] Example 3 Kit Performance Verification

[0110] 1. Cross-reactivity verification

[0111] Prepare low-concentration calibration solution A1 (concentration 10.0 ng / ml), medium-concentration calibration solution A2 (concentration 30.0 ng / ml), and high-concentration calibration solution A3 (concentration 80.0 ng / ml) each 4 ml using phosphate buffer. Divide the three concentration gradient calibrations into 4 equal parts, and among them, the first part of the sample is not treated as the absolute control group sample; add potential cross-reactants to the second and third parts of the sample to prepare the test group samples; and add potential cross-reactants to the fourth part of the sample to prepare the relative control group sample. The potential cross-reactants are prepared as follows:

[0112] CK-MB cross interference mother liquor preparation: CK-MB antigen (purchased from Saituokang Biotechnology Co., Ltd.) is prepared into a 10 μg / ml concentration using phosphate buffer;

[0113] CK-BB cross interference mother liquor preparation: Take CK-BB antigen (purchased from Saituokang Biotechnology Co., Ltd.) and prepare it into a 200 μg / ml concentration using phosphate buffer;

[0114] CK-MM cross interference mother liquor preparation: CK-MM antigen (purchased from Siguokang Biotechnology Co., Ltd.) was prepared with phosphate buffer to a concentration of 700 μg / ml.

[0115] Phosphate buffer preparation: 0.2 g of potassium dihydrogen phosphate was dissolved in 800 ml of purified water, 2.9 g of 12 hydrated disodium hydrogen phosphate was added, BSA and preservatives were added, and after complete dissolution, the volume was adjusted to 1000 ml, the pH was adjusted to 7.4, and it was ready for use.

[0116] The cross interference control and test group samples C0, C1, C2 and C3 were prepared according to the following table, respectively, wherein C0 is the absolute control group sample, C1 is the relative control group sample, and C2 and C3 are the test group samples. Each sample was measured in triplicate, and the average of each sample test result was recorded.

[0117]

[0118] The test results are as follows:

[0119]

[0120]

[0121]

[0122] According to the above experimental results, when the cross interference CK-MB concentration is 500 ng / ml, the CK-BB concentration is 10 μg / ml, and the CK-MM concentration is 35 μg / ml, the detection result of 10.0 ng / ml / 30.0 ng / ml / 80.0 ng / ml uMtCK calibrator is within 10%, and the detection result is basically not interfered by CK-MB, CK-BB and CK-MM.

[0123] 2. Blank limit evaluation

[0124] The blank sample was repeatedly detected 20 times, and the average value (M) and standard deviation (SD) of the RLU value (relative luminescence value) of the 20 determination results were calculated, and the blank limit was determined by the concentration value of the MFI value of M+2SD. The test results of the blank limit are shown in the following table.

[0125]

[0126]

[0127] From the results in the table, it can be seen that the blank limit detection concentration is 1.0 ng / ml, and the maximum concentration that can be detected for the blank sample is only 1 ng / ml.

[0128] 3. Sensitivity evaluation

[0129] The uMtCK standard (purchased from Wuhan Yunclone Biotechnology) was used to prepare a ten-point curve with phosphate buffer, and the uMtCK kit in Example 2 was used for detection. The following table shows the ten-point curve RLU intensity detection data, where 0 is phosphate buffer, and RLU: relative light unit.

[0130] Formulation concentration ng / ml RLU 0 4449 9.75 11028 19.5 20133 39.06 45359 78.125 101610 156.25 236305 312.5 496942 625 821110 1250 1221613 2500 1584605

[0131] As can be seen from the above ten-point curve results, at a concentration of 0-2500 ng / ml, the difference in light intensity range is 1580156, and each stage of the concentration standard can be clearly distinguished.

[0132] 4. Reproducibility evaluation

[0133] Three different concentrations of calibrators were detected, and 10 parallel detections were performed to calculate the coefficient of variation CV, which can characterize the repeatability of the tumor-related index detection kit.

[0134]

[0135]

[0136] As can be seen from the above results, in the intra-batch experiment, the coefficients of variation CV of the low, medium and high calibrators are all within 6%.

[0137] 5. Positive detection rate evaluation:

[0138] Fifty clinical samples were detected using the uMtCK luminescence kit. The clinical samples were composed of serum collected from patients diagnosed with cancer and normal human serum.

[0139] Sensitivity = measured positive value / total number of cancer samples x 100%;

[0140] Specificity = number of negative samples / number of normal samples x 100%.

[0141]

[0142] As can be seen from the above table, the antibody of the present disclosure has good sensitivity and specificity for detecting clinical tumor samples.

[0143] The above examples only express several embodiments of the present disclosure, and the description is more specific and detailed, but it cannot be understood as limiting the scope of the patent. It should be noted that for ordinary skilled persons in the art, without departing from the concept of the present disclosure, several modifications and improvements can be made, which are within the scope of the present disclosure. Therefore, the protection scope of the present disclosure patent should be subject to the appended claims, and the description can be used to explain the content of the claims.

Claims

1. A uMtCK antibody or antigen-binding fragment thereof, characterized in that, a heavy chain complementarity determining region comprising the amino acid sequences of SEQ ID NOs: 1-3 in order, and a light chain complementarity determining region comprising the amino acid sequences of SEQ ID NOs: 4-6 in order.

2. The uMtCK antibody or antigen-binding fragment thereof of claim 1, wherein, a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 7, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:

8.

3. An isolated nucleic acid, characterized in that, a nucleic acid encoding the uMtCK antibody or antigen-binding fragment thereof of claim 1 or 2.

4. Vector, characterized in that, a nucleic acid of claim 3.

5. A host cell characterized in that, a host cell comprising the nucleic acid of claim 3, or transformed with the vector of claim 4.

6. A method of producing the uMtCK antibody or antigen binding fragment thereof according to claim 1 or 2, characterized in that, culturing the host cell of claim 5 under suitable conditions and recovering the desired product from the cell culture.

7. Antibody combination product, characterized in that a uMtCK antibody or antigen-binding fragment thereof of claim 1 or 2, and a second uMtCK antibody with product number CSB-MA149995 from Cusabio.

8. A kit, characterized in that, a uMtCK antibody or antigen-binding fragment thereof of claim 1 or 2, or the antibody combination product of claim 7.

9. Use of a uMtCK antibody or antigen-binding fragment thereof of claim 1 or 2, or the antibody combination product of claim 7, in the preparation of a tumor diagnostic reagent or kit. the tumor is selected from the group consisting of breast cancer, gastric cancer, prostate cancer, cervical cancer, liver cancer, ovarian cancer, lung cancer, and colorectal cancer.

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