Kit and method for detecting urine 11-dehydrothromboxane B2 / creatinine

Abstract

The present invention relates to the field of biological detection and analysis, and provides a kit and a detection method for detecting urine 11-dehydrothromboxane B2 / creatinine. Specifically, the present invention provides an antibody or antigen-binding fragment, which can specifically recognize 11-dehydrothromboxane B2, including at least one of the following CDR sequences: CDR sequences of heavy chain variable regions: SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3; CDR sequences of light chain variable regions: SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6. The detection kit prepared based on the antibody or its antigen-binding fragment can realize synchronous detection of 11-dehydrothromboxane B2 and creatinine in urine, has the advantages of good sensitivity, high accuracy, wide linear range, strong specificity, simple operation, and no interference from endogenous biotin, and provides a more accurate, convenient and quick method for clinical evaluation of platelet activity or aspirin antiplatelet effect.

Description

Technical Field

[0001] The present invention relates to the field of biological detection and analysis, and in particular to an 11-dehydrothromboxane B2 antibody or antigen-binding fragment and a kit for detecting urine 11-dehydrothromboxane B2 / creatinine using the antibody or antigen-binding fragment. Background Art

[0002] 11-dehydro-thromboxane B2 (11-dhTXB2) is the terminal metabolite of thromboxane A2 (TXA2). In normal blood circulation, platelets are in a static state. After vascular damage, the signal pathway in the platelets is activated, and the membrane phospholipids are cleaved to release arachidonic acid, which is converted into prostaglandin H under the action of platelet cyclooxygenase (COX-1 and COX-2). Prostaglandin H is chemically unstable and can be converted into a variety of biologically active prostaglandins including TXA2 under the action of isomerase. TXA2 is mainly synthesized in platelets through the COX-1 pathway, has a strong vasoconstrictive effect, and can also activate platelets by binding to thromboxane platelet receptor (TPR), promote their aggregation, and thus play a role in promoting thrombosis. Aspirin, as the most widely used antiplatelet aggregation drug, can reduce TXA2 synthesis by irreversibly inhibiting COX-1 activity. However, TXA2 is highly unstable, with an extremely short biological half-life of only about 30 seconds. It will be rapidly hydrolyzed into the more stable thromboxane B2 (TXB2), which is further metabolized into 11-dhTXB2 by the liver and excreted in the urine. Therefore, the urinary 11-dhTXB2 content can directly reflect the level of TXA2 production in the body and evaluate platelet activity and the antiplatelet effect of aspirin.

[0003] In order to make the determination of urine 11-dhTXB2 more standardized, it is necessary to use urine creatinine (CRE) concentration to correct the test value to reduce the difference in urine concentration or the influence of renal function caused by diet and drinking water, that is, to measure the values ​​of urine 11-dhTXB2 and urine creatinine respectively, and then calculate the relative creatinine value. Among them, the conventional detection methods of 11-dhTXB2 include high performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LC-MS), enzyme-linked immunosorbent assay (ELISA), chemiluminescence assay (CLIA), etc.; the detection methods of creatinine include picric acid method, dry chemical method, enzyme method, etc.

[0004] By analyzing the existing detection technologies and measurement methods, the following problems were found:

[0005] 1) Limited by the instrument: Since the measurement methods of 11-dehydrothromboxane B2 and urine creatinine are based on different methodologies, they must be measured separately using different instruments.

[0006] 2) The measurement steps are cumbersome: As mentioned in the previous point, different instruments are needed, which inevitably leads to the separate processing of the same sample to ensure the accuracy of the measurement of urine 11-dehydrothromboxane B2 and urine creatinine.

[0007] 3) Inaccurate measurement results: Since different instruments are required, there will inevitably be a certain time difference in the detection of urine 11-dehydrothromboxane B2 and urine creatinine. The difference between urine 11-dehydrothromboxane B2 and urine creatinine at different time points will amplify the deviation of 11-dehydrothromboxane B2 / creatinine results.

[0008] Therefore, there is an urgent need to develop a new method that can simultaneously detect the levels of 11-dehydrothromboxane B2 and urine creatinine in the same instrument to improve the accuracy of the test results. Summary of the invention

[0009] The purpose of the present invention is to overcome the deficiencies of the prior art. Therefore, the present invention develops an 11-dehydrothromboxane B2 antibody or antigen-binding fragment that can bind to 11-dehydrothromboxane B2. The antibody or antigen-binding fragment can specifically bind to 11-dehydrothromboxane B2. Based on this, the present invention uses the 11-dehydrothromboxane B2 antibody or antigen-binding fragment to prepare a urine 11-dehydrothromboxane B2 / creatinine detection kit. The kit is based on the immune competition method for the determination of urine 11-dehydrothromboxane B2 and urine creatinine. The kit uses the principle of combining chemiluminescence detection technology with magnetic particle immunoseparation technology to enable the two indicators to be synchronously detected on a luminometer. The kit has the advantages of good sensitivity, high accuracy, wide linear range, strong specificity, simple operation, and is not interfered by endogenous biotin. It provides a more accurate, convenient and quick method for clinical evaluation of platelet activity or the antiplatelet effect of aspirin.

[0010] In the first aspect of the present invention, the present invention provides an antibody or antigen-binding fragment. According to an embodiment of the present invention, the antibody or antigen-binding fragment comprises a CDR sequence selected from at least one of the following: a CDR sequence of a heavy chain variable region: SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3; a CDR sequence of a light chain variable region: SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6. The antibody or antigen-binding fragment according to an embodiment of the present invention has a binding affinity for 11-dehydrothromboxane B2 and can be used to detect 11-dehydrothromboxane B2 and detect cells containing 11-dehydrothromboxane B2.

[0011] In the second aspect of the present invention, the present invention provides a fusion protein. According to an embodiment of the present invention, the fusion protein comprises the antibody or antigen-binding fragment described in the first aspect; and a biologically active protein or a fragment thereof. The fusion protein of the present invention has a binding affinity for 11-dehydrothromboxane B2 and can be used to detect 11-dehydrothromboxane B2 and detect cells containing 11-dehydrothromboxane B2.

[0012] In the third aspect of the present invention, the present invention provides a nucleic acid molecule. According to an embodiment of the present invention, the nucleic acid molecule encodes the antibody or antigen-binding fragment described in the first aspect, and the fusion protein described in the second aspect. The nucleic acid molecule of the present invention can encode the antibody or antigen-binding fragment of the first aspect, and the fusion protein described in the second aspect.

[0013] In a fourth aspect of the present invention, the present invention provides an expression vector. According to an embodiment of the present invention, the expression vector carries the nucleic acid molecule described in the third aspect. Thus, the expression vector of the present invention can effectively achieve the expression of the antibody or antigen-binding fragment of the first aspect and the fusion protein of the second aspect, thereby achieving the in vitro acquisition of a large amount of the antibody or antigen-binding fragment or the fusion protein.

[0014] In the fifth aspect of the present invention, the present invention provides a recombinant cell. According to an embodiment of the present invention, the recombinant cell includes carrying the nucleic acid molecule described in the third aspect or the expression vector described in the fourth aspect; or expressing the antibody or antigen-binding fragment described in the first aspect or the fusion protein described in the second aspect. The recombinant cell can effectively express the aforementioned antibody or antigen-binding fragment, or fusion protein in the cell under suitable conditions.

[0015] In a sixth aspect of the present invention, the present invention provides a method for preparing an antibody or antigenic fragment. According to an embodiment of the present invention, the method comprises culturing the recombinant cell described in the fifth aspect under conditions suitable for protein expression and secretion, so as to obtain the antibody or antigen-binding fragment. Thus, the method of the present invention can be used to produce the aforementioned antibody or antigen-binding fragment.

[0016] In the seventh aspect of the present invention, the present invention proposes a conjugate. According to an embodiment of the present invention, the conjugate comprises the antibody or antigen-binding fragment described in the first aspect, the fusion protein described in the second aspect, or the antibody or antigen-binding fragment prepared according to the method described in the sixth aspect; and a coupling portion, wherein the coupling portion is connected to the antibody or antigen-binding fragment, or the fusion protein. The conjugate of the present invention has a binding affinity for 11-dehydrothromboxane B2 and can be used to detect 11-dehydrothromboxane B2 and detect cells containing 11-dehydrothromboxane B2.

[0017] In the eighth aspect of the present invention, the present invention provides the use of the antibody or antigen-binding fragment described in the first aspect in preparing a kit for detecting 11-dehydrothromboxane B2. According to an embodiment of the present invention, the antibody or antigen-binding fragment can bind to 11-dehydrothromboxane B2 with high specificity. Therefore, the kit containing the antibody or antigen-binding fragment can be used for detecting 11-dehydrothromboxane B2 with high specificity and high sensitivity.

[0018] In the ninth aspect of the present invention, the present invention proposes a kit. According to an embodiment of the present application, the kit comprises the antibody or antigen binding fragment described in the first aspect, the fusion protein described in the second aspect, the nucleic acid molecule described in the third aspect, the expression vector described in the fourth aspect, the recombinant cell described in the fifth aspect, the antibody or antigen binding fragment prepared according to the method described in the sixth aspect, or the conjugate described in the seventh aspect. According to an embodiment of the present invention, the antibody or its antigen binding fragment can bind to 11-dehydrothromboxane B2 with high specificity. In some scientific studies, the kit can be used for qualitative or quantitative detection of 11-dehydrothromboxane B2 in biological samples, and can also be used to judge the status of an individual, such as after obtaining the 11-dehydrothromboxane B2 level of the individual, judging whether the 11-dehydrothromboxane B2 level is higher or lower than the normal level.

[0019] In the tenth aspect of the present invention, the present invention proposes a method for detecting the content of 11-dehydrothromboxane B2. According to an embodiment of the present invention, the method comprises: using the kit described in the ninth aspect to detect and process the sample to be tested, so as to determine the content of the 11-dehydrothromboxane B2 in the sample to be tested. The method described in the present invention can detect 11-dehydrothromboxane B2 with high sensitivity, and is conducive to the detection of low-concentration 11-dehydrothromboxane B2. In addition, the method has the advantages of strong specificity, wide linear range, simple operation, and no need for sample pretreatment. In practical clinical applications, a large number of samples can be quickly and high-throughput detected with the help of a chemiluminescence instrument.

[0020] In the eleventh aspect of the present invention, the present invention proposes a method for detecting the relative content of 11-dehydrothromboxane B2. According to an embodiment of the present invention, the method comprises: using the kit described in the ninth aspect to detect and process the sample to be tested, so as to determine the relative content of the 11-dehydrothromboxane B2 in the sample to be tested. Since 11-dehydrothromboxane B2 and creatinine are both small molecules, the small molecules have a large degree of freedom, which is not conducive to the exposure of the antibody binding site, and is also not conducive to the binding of the antigen and the antibody. Before determining the content of 11-dehydrothromboxane B2 and creatinine in the sample, the method of the present invention allows the small molecule antigen (11-dehydrothromboxane B2 and creatinine) in the sample to react with the enzyme-labeled antibody first, and then adds the small molecule derivative coated with magnetic beads to compete, so it is conducive to the detection of low-concentration antigens and improves the sensitivity of the reagent. In addition, the method has the advantages of strong specificity, wide linear range, simple operation, and no need for pre-treatment of the sample. In practical clinical applications, a large number of samples can be quickly and high-throughput detected with the help of a chemiluminometer.

[0021] Additional aspects and advantages of the present invention will be given in part in the following description and in part will be obvious from the following description, or will be learned through practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The above and / or additional aspects and advantages of the present invention will become apparent and easily understood from the description of the embodiments in conjunction with the following drawings, in which:

[0023] Figure 1 is a calibration curve diagram of the detection system of 11-dehydrothromboxane B2 in the kit according to an embodiment of the present invention;

[0024] Figure 2 is a calibration curve diagram of a creatinine detection system of a kit according to an embodiment of the present invention;

[0025] Figure 3 is a linear graph of the detection system of 11-dehydrothromboxane B2 in the kit according to an embodiment of the present invention;

[0026] Figure 4 is a linear graph of the creatinine detection system of the kit 1 according to an embodiment of the present invention;

[0027] Figure 5 is a correlation diagram between the 11-dehydrothromboxane B2 detection system in the kit according to an embodiment of the present invention and a commercially available ELISA kit;

[0028] Figure 6 is a correlation diagram between the creatinine detection system in the kit according to an embodiment of the present invention and a commercially available enzymatic kit;

[0029] Figure 7is the correlation between the results of the kit 11-dehydrothromboxane B2 / creatinine according to an embodiment of the present invention and the results calculated with commercially available reagents;

[0030] Figure 8 It is a gel map of subtype identification of antibodies expressed by hybridoma cell line 11DH025 and cloning of antibody variable regions according to an embodiment of the present invention. The lane sequence is shown in Table 5. DETAILED DESCRIPTION

[0031] The present invention will be further described below by specific examples. It should be noted that the examples described below are only used to explain the present invention, but not to limit the present invention.

[0032] In order to make the present invention more easily understood, certain technical and scientific terms are specifically defined below. Unless otherwise clearly defined elsewhere in this document, all other technical and scientific terms used herein have the meanings commonly understood by those skilled in the art to which the present invention belongs.

[0033] In the present application, unless otherwise specified, the terms "comprise" or "include" are open expressions, that is, including the contents specified in the present application but not excluding other contents.

[0034] In this application, unless otherwise stated, the terms "optionally", "optional" or "optionally" generally mean that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not occur.

[0035] In this application, unless otherwise specified, the terms "first" and "second" are used for descriptive purposes only and are not to be understood as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one of the features. In the description of this application, "plurality" means at least two, such as two, three, etc., unless otherwise clearly and specifically defined.

[0036] The endpoints and any values ​​of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0037] Herein, the term "fragment" refers to a target protein or polypeptide, and a target protein or polypeptide having an N-terminal (N-terminus) or C-terminal (C-terminus) truncation, and / or internal deletion.

[0038] In order to make the present invention more easily understood, certain technical and scientific terms are specifically defined below. Unless otherwise clearly defined elsewhere in this document, all other technical and scientific terms used herein have the meanings commonly understood by those of ordinary skill in the art to which the present invention belongs. The abbreviations for amino acid residues are standard 3-letter and / or 1-letter codes used in the art to refer to one of the 20 commonly used L-amino acids.

[0039] In this article, the term "antibody" is used in the broadest sense, which can include full-length monoclonal antibodies, multispecific antibodies, and chimeric antibodies, and the specific structure is not limited as long as they show the desired biological activity. It usually includes a light chain with a lighter molecular weight and a heavy chain with a heavier molecular weight, and the heavy chain (H chain) and the light chain (L chain) are connected by a disulfide bond to form an antibody molecule. Among them, the amino acid sequence of the amino terminal (N terminal) of the peptide chain varies greatly, which is called the variable region (V region); the carboxyl terminal (C terminal) is relatively stable and varies little, which is called the constant region (C region). The V regions of the L chain and the H chain are called VL and VH, respectively.

[0040] In this article, the heavy chain complementary determining region (heavy chain variable region CDR) is represented by "HCDRs" or "HCDR", which includes HCDR1 (also known as CDR-H1), HCDR2 (also known as CDR-H2) and HCDR3 (also known as CDR-H3); the light chain complementary determining region (light chain variable region CDR) is represented by "LCDRs" or "LCDR", which includes LCDR1 (also known as CDR-L1), LCDR2 (also known as CDR-L2) and LCDR3 (also known as CDR-L3). Commonly used CDR definition schemes in the art include: Kabat definition, Chothia definition, IMGT definition, Contact definition and AbM definition.

[0041] In this article, the term "antibody variable region" refers to a domain in the heavy and light chains of antibodies. In nature, antibody variable regions are encoded by V, D (only heavy chain is applicable), and J fragments in immunoglobulin (heavy and light chain) genes through gene recombination splicing. The amino acid sequence of the variable region has a high degree of diversity between different antibodies (the amino acid sequences in other regions of the antibody are relatively highly identical), and is responsible for identifying and combining with specific antigenic determinants. In the antibody variable region, the framework region and CDR region (comlementarity determining region) can be further subdivided. A typical antibody variable region has 3 framework regions and 3 CDR regions (they are interspersed with each other). The framework region mainly plays the role of protein domain framework formation, while the CDR region mainly plays the role of antigen-antibody specific recognition and combination.

[0042] As used herein, the term "antigen-binding fragment" or "antibody fragment" is a fragment comprising part or all of an antibody, which lacks at least some of the amino acids present in the full-length chain but is still capable of specifically binding to an antigen, for example, the fragment may comprise part or all of an antibody CDR. Such fragments are biologically active because they bind to an antigen and can compete with other antigen-binding molecules (including intact antibodies) for binding to a given epitope. Such fragments are selected from Fab, F(ab)2, Fab', F(ab')2, Fv, scFv or single domain antibodies. Such fragments may be produced by recombinant nucleic acid technology, or may be produced by enzymatic or chemical cleavage of antigen-binding molecules (including intact antibodies).

[0043] It should be noted that, for the antibodies or antigen-binding fragments, CDRs, light chain variable regions or heavy chain variable regions, etc. described in the present invention, without substantially affecting the antibody activity (retaining at least 80%, 90%, or 95% activity), those skilled in the art may replace, add and / or delete one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more) amino acids in the sequence of the present invention to obtain variants of the sequence of the antibody or its functional fragment. They are all considered to be included in the scope of protection of the present invention. For example, amino acids with conservative modifications are replaced in the variable region. The variant sequence of the present invention may have at least 80% identity (or homology) with the reference sequence. The sequence identity of the present invention can be measured using sequence analysis software. For example, the computer program BLAST, especially BLASTP or TBLASTN, using default parameters is used.

[0044] In this document, the amino acid sequences described in the present invention are all shown from N-terminus to C-terminus.

[0045] The antibody or antigen-binding fragment described in the present invention is usually prepared by a biosynthetic method. According to the nucleotide sequence described in the present invention, a person skilled in the art can easily prepare the encoding nucleic acid of the present invention by various known methods. These methods are, for example, but not limited to: PCR, DNA artificial synthesis, etc., and specific methods can be found in J. Sambrook, "Molecular Cloning Laboratory Guide". As an embodiment of the present invention, the encoding nucleic acid sequence of the present invention can be constructed by synthesizing the nucleotide sequence in segments and then performing overlap extension PCR. Among them, the antibody or antigen fragment in the present invention is numbered and defined using the Kabat numbering system. However, a person of ordinary skill in the art is fully capable of converting the sequence Kabat numbering of the sequence table into "HCDRs" and / or "LCDRs" under other numbering systems (including but not limited to AbM, IMGT, Chothia), and the CDR determined based on the heavy chain variable region and light chain variable region disclosed in the present application using other rules disclosed in the art also belongs to the protection scope of the present disclosure.

[0046] As used herein, the "Kabat definition" refers to the definition system described by Kabat et al., U. S. Patent. of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983). The "Chothia definition" is found in Chothia et al., J Mol Biol 196:901-917 (1987).

[0047] As used herein, the term "conservatively modified forms of an amino acid sequence" refers to amino acid modifications that do not significantly affect or alter the binding properties of an antibody comprising the amino acid sequence, including amino acid substitutions, additions, and deletions. Modifications can be introduced into the antibodies of the invention by standard techniques such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are substitutions in which an amino acid residue is replaced by an amino acid residue with a similar side chain. Families of amino acid residues with similar side chains have been identified in the art. These families include amino acids with basic side chains (such as lysine, arginine, histidine), amino acids with acidic side chains (such as aspartic acid, glutamic acid), amino acids with uncharged polar side chains (such as glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), amino acids with non-polar side chains (such as alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), amino acids with beta-branched side chains (such as threonine, valine, isoleucine), and amino acids with aromatic side chains (such as tyrosine, phenylalanine, tryptophan, histidine).

[0048] As used herein, the term "identity" is used to describe an amino acid sequence or a nucleic acid sequence relative to a reference sequence, and the percentage of identical amino acids or nucleotides between two amino acid sequences or nucleic acid sequences is determined by conventional methods, for example, see Ausubel et al., eds. (1995), Current Protocols in Molecular Biology, Chapter 19 (Greene Publishing and Wiley-Interscience, New York); and the ALIGN program (Dayhoff (1978), Atlas of Protein Sequence and Structure 5: Suppl. 3 (National Biomedical Research Foundation, Washington, DC). There are many algorithms for aligning sequences and determining sequence identity, including the homology alignment algorithm of Needleman et al. (1970) J. Mol. Biol. 48:443; the local homology algorithm of Smith et al. (1981) Adv. Appl. Math. 2:482; the similarity search method of Pearson et al. (1988) Proc. Natl. Acad. Sci. 85:2444; the Smith-Waterman algorithm (Meth. Mol. Biol. 70:173-187 (1997); and BLASTP, BLASTN, and BLASTX algorithms (see Altschul et al. (1990) J. Mol. Biol. 215:403-410). Computer programs utilizing these algorithms are also available and include, but are not limited to, ALIGN or Megalign (DNASTAR) software, or WU-BLAST-2 (Altschul et al., Meth. Enzym., 266:460-480 (1996)); or GAP, BESTFIT, BLASTAltschul et al., supra, FASTA, and TFASTA, available in the Genetics Computing Group (GCG) package, Version 8, Madison, Wisconsin, USA; and CLUSTAL in the PC / Gene program provided by Intelligenetics, Mountain View, California.

[0049] As used herein, the term "at least 80% identity" refers to at least 80%, and may be 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% identity to each reference sequence.

[0050] In this article, the term "fusion protein" refers to a new protein formed by the fusion of at least two proteins or polypeptides. The above fusion operation can usually be achieved through genetic engineering and other technologies, for example, the expression product of two genes recombined by DNA recombination technology.

[0051] In this article, the term "expression vector" generally refers to a nucleic acid molecule that can be inserted into a suitable host and replicates itself, the nucleic acid molecule comprises a nucleotide sequence that can express a target protein, and the nucleic acid molecule can be transferred into a host cell and / or between host cells. The expression vector may include a vector that is mainly used to insert DNA or RNA into a cell, a vector that is mainly used to replicate DNA or RNA, and a vector that is mainly used for the expression of transcription and / or translation of DNA or RNA. The expression vector also includes vectors with a variety of the above functions. The expression vector can be a polynucleotide that can be transcribed and translated into a polypeptide when introduced into a suitable host cell. Typically, the expression vector can produce a desired expression product by culturing a suitable host cell containing the expression vector.

[0052] In this article, the term "recombinant cell" generally refers to the use of genetic engineering technology or cell fusion technology to modify or reorganize the genetic material of the recipient cell (or host cell) to obtain a cell with a unique trait of stable inheritance. Among them, the term "host cell" refers to a prokaryotic cell or eukaryotic cell that can be introduced into a recombinant expression vector. The terms "transformed" or "transfected" used herein refer to the introduction of nucleic acids (such as expression vectors) into cells by various techniques known in the art. Suitable host cells can be transformed or transfected with the DNA sequence of the present invention and can be used for the expression and / or secretion of target proteins. Examples of suitable host cells that can be used in the present invention include immortalized hybridoma cells, NS / 0 myeloma cells, 293 cells, Chinese hamster ovary (CHO) cells, HeLa cells, Cap cells (cells derived from human amniotic fluid) and CoS cells.

[0053] In this article, the terms "magnetic particles", "magnetic particles", "magnetic microspheres" and "magnetic beads" have the same meaning, which refers to colloidal composite materials that can be uniformly dispersed in a certain base liquid. Due to their superparamagnetic properties, high specific surface area, and modifiable functional groups, antigens / antibodies, enzymes, nucleic acids / oligonucleotides, small molecule drugs, etc. can be fixed on their surfaces and enriched in a magnetic field.

[0054] In this article, for ease of expression, 11-dehydrothromboxane B2 is sometimes abbreviated as "11-dhTXB2" and creatinine is abbreviated as "CRE". Unless otherwise specified, the abbreviations are no different from the original expressions.

[0055] Detailed description of the kit for detecting urine 11-dehydrothromboxane B2 and creatinine of the present invention

[0056] The present invention provides an antibody or antigen-binding fragment, a fusion protein, a nucleic acid molecule, an expression vector, a recombinant cell, a conjugate, a kit, a method for preparing the antibody or antigen-binding fragment, a method for detecting the content of 11-dehydrothromboxane B2 or a method for detecting the relative content of 11-dehydrothromboxane B2, which will be described in detail below.

[0057] Antibodies or antigen-binding fragments

[0058] In the first aspect of the present invention, the present invention provides an antibody or antigen-binding fragment. According to an embodiment of the present invention, the antibody or antigen-binding fragment comprises a CDR sequence selected from at least one of the following: a CDR sequence of a heavy chain variable region: SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3; a CDR sequence of a light chain variable region: SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6. The antibody or antigen-binding fragment according to an embodiment of the present invention has a binding affinity for 11-dehydrothromboxane B2 and can be used to detect 11-dehydrothromboxane B2 and detect cells containing 11-dehydrothromboxane B2.

[0059] According to some embodiments of the present invention, the antibody or antigen-binding fragment comprises HCDR1-3 and LCDR1-3; wherein the HCDR1 has an amino acid sequence as shown in SEQ ID NO:1; the HCDR2 has an amino acid sequence as shown in SEQ ID NO:2; the HCDR3 has an amino acid sequence as shown in SEQ ID NO:3; the LCDR1 has an amino acid sequence as shown in SEQ ID NO:4; the LCDR2 has an amino acid sequence as shown in SEQ ID NO:5; and the LCDR3 has an amino acid sequence as shown in SEQ ID NO:6.

[0060] In an optional embodiment of the present invention, "the amino acid sequence as shown in SEQ ID NO:1" refers to an amino acid sequence that is completely identical to the amino acid sequence shown in SEQ ID NO:1, and an amino acid sequence that has one or more conservative amino acid substitutions compared to the amino acid sequence shown in SEQ ID NO:1. "Conservative amino acid substitution" refers to the replacement of an amino acid with another amino acid residue that is biologically, chemically or structurally similar. Biological similarity refers to the biological activity of the 11-dehydrothromboxane B2 antigen. Structurally similar refers to amino acids having side chains of similar length, such as alanine, glycine or serine, or having side chains of similar size. Chemical similarity refers to amino acids having the same charge or being hydrophilic or hydrophobic. For example, hydrophobic residues isoleucine, valine, leucine or methionine are substituted for each other. Or polar amino acids are used, such as arginine replacing lysine, glutamic acid replacing aspartic acid, glutamine replacing asparagine, serine replacing threonine, and the like. The same as “the amino acid sequence shown in SEQ ID NO:2”, “the amino acid sequence shown in SEQ ID NO:3”, “the amino acid sequence shown in SEQ ID NO:4”, “the amino acid sequence shown in SEQ ID NO:5”, “the amino acid sequence shown in SEQ ID NO:6”, “the amino acid sequence shown in SEQ ID NO:7”, “the amino acid sequence shown in SEQ ID NO:8”, “the amino acid sequence shown in SEQ ID NO:9”, “the amino acid sequence shown in SEQ ID NO:10”, “the amino acid sequence shown in SEQ ID NO:11”, or “the amino acid sequence shown in SEQ ID NO:12”.

[0061] According to an embodiment of the present invention, the HCDR1 has an amino acid sequence as shown in SEQ ID NO: 1 or an amino acid sequence having 1 to 2 amino acid differences therefrom.

[0062] According to an embodiment of the present invention, the HCDR2 has an amino acid sequence as shown in SEQ ID NO: 2 or an amino acid sequence having 1 to 2 amino acid differences therefrom.

[0063] According to an embodiment of the present invention, the HCDR3 has an amino acid sequence as shown in SEQ ID NO: 3 or an amino acid sequence having 1 to 2 amino acid differences therefrom.

[0064] According to an embodiment of the present invention, the LCDR1 has an amino acid sequence as shown in SEQ ID NO: 4 or an amino acid sequence having 1 to 2 amino acid differences therefrom.

[0065] According to an embodiment of the present invention, the LCDR2 has an amino acid sequence as shown in SEQ ID NO: 5 or an amino acid sequence having 1 to 2 amino acid differences therefrom.

[0066] According to an embodiment of the present invention, the LCDR3 has an amino acid sequence as shown in SEQ ID NO: 6 or an amino acid sequence having 1 to 2 amino acid differences therefrom.

[0067] As used herein, the term "or an amino acid sequence having 1 to 2 amino acid differences therefrom" refers to an amino acid sequence having 1 or 2 amino acid differences compared to the aforementioned amino acid sequence, and the difference does not significantly affect or change the binding properties of the antibody comprising the amino acid sequence, and the modification includes amino acid substitution, addition and deletion.

[0068] According to an embodiment of the present invention, the antibody or antigen-binding fragment thereof specifically recognizes 11-dehydrothromboxane B2, and in particular specifically recognizes an antigen having a sequence as shown in SEQ ID NO:14.

[0069] According to some embodiments of the invention, the antibody or antigen-binding fragment comprises a heavy chain framework region and / or a light chain framework region.

[0070] According to some embodiments of the present invention, at least a portion of the heavy chain framework region and / or light chain framework region is derived from at least one of a mouse antibody, a primate antibody, a bovine antibody, a horse antibody, a dairy cow antibody, a porcine antibody, a sheep antibody, a goat antibody, a dog antibody, a cat antibody, a rabbit antibody, a camel antibody, a donkey antibody, a deer antibody, a mink antibody, a chicken antibody, a duck antibody, a goose antibody, a turkey antibody, a fighting cock antibody, or a mutant thereof.

[0071] According to some embodiments of the present invention, at least a portion of the heavy chain framework region and / or the light chain framework region is derived from at least one of a murine antibody or a mutant thereof and a human antibody or a mutant thereof.

[0072] According to some embodiments of the present invention, the antibody or antigen-binding fragment comprises: a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO:7; and a light chain variable region having an amino acid sequence as shown in SEQ ID NO:8.

[0073] According to some embodiments of the present invention, it further comprises a heavy chain constant region and / or a light chain constant region.

[0074] According to some embodiments of the present invention, at least a portion of at least one of the heavy chain constant region and the light chain constant region is from at least one of a mouse antibody, a primate antibody, a bovine antibody, a horse antibody, a dairy cow antibody, a porcine antibody, a sheep antibody, a goat antibody, a dog antibody, a cat antibody, a rabbit antibody, a camel antibody, a donkey antibody, a deer antibody, a mink antibody, a chicken antibody, a duck antibody, a goose antibody, a turkey antibody, a fighting cock antibody or a mutant thereof.

[0075] According to some embodiments of the invention, the heavy chain constant region comprises a heavy chain constant region selected from IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD.

[0076] According to some embodiments of the invention, the light chain constant region comprises a light chain constant region selected from a kappa-type or a lambda-type.

[0077] According to some embodiments of the present invention, the light chain constant region and the heavy chain constant region are both derived from at least one of a murine antibody or a mutant thereof and a human antibody or a mutant thereof.

[0078] According to some embodiments of the present invention, the N-terminus of the heavy chain constant region is connected to the C-terminus of the heavy chain variable region; and / or the N-terminus of the light chain constant region is connected to the C-terminus of the light chain variable region.

[0079] According to some embodiments of the present invention, the heavy chain constant region has the amino acid sequence shown in SEQ ID NO:9; and / or the light chain constant region has the amino acid sequence shown in SEQ ID NO:10.

[0080] According to some embodiments of the present invention, the antibodies include polyclonal antibodies, full-length monoclonal antibodies, F(ab') 2 Antibody, Fab' antibody, Fab antibody, F(ab) 2 At least one of an antibody, an Fv antibody, a single-chain antibody and a minimum recognition unit.

[0081] According to some embodiments of the present invention, the antigen-binding fragment comprises F(ab') 2 fragment, Fab' fragment, Fab fragment, F(ab) 2 At least one of a fragment, an Fv fragment, a scFv fragment, a scFv-Fc fusion protein, a scFv-Fv fusion protein and a minimum recognition unit.

[0082] As used herein, the terms "full-length antibody", "full-length monoclonal antibody" or "full-length monoclonal antibody" are composed of at least two identical light chains and at least two identical heavy chains connected by interchain disulfide bonds, such as immunoglobulin G (IgG), immunoglobulin A (IgA), immunoglobulin M (IgM), immunoglobulin D (IgD) or immunoglobulin E (IgE).

[0083] In this article, the terms "polyantibody" and "multispecific antibody" are synonymous and both refer to antibodies that can recognize multiple antigenic epitopes, such as antibodies that can recognize two antigenic epitopes (bispecific antibodies, abbreviated as bispecific antibodies), antibodies that can recognize three antigenic epitopes, or antibodies that can recognize four antigenic epitopes. This is understood in a broad sense and the specific structure is not limited as long as it can recognize multiple antigenic epitopes.

[0084] In this article, the terms "single domain antibody", "nanoantibody" and "VHH antibody" are used interchangeably, which were originally described as antigen-binding immunoglobulin (variable) domains of "heavy chain antibodies" (i.e., "antibodies lacking light chains") (Hamers-Casterman C, Atarhouch T, Muyldermans S, Robinson G, Hamers C, Songa EB, Bendahman N, Hamers R.: "Naturally occurring antibodies devoid of light chains"; Nature 363, 446-448 (1993)), comprising a heavy chain variable region (VH) and conventional CH2 and CH3 regions, which specifically bind to antigen proteins through the heavy chain variable region.

[0085] As used herein, the term "Fab antibody" or "Fab fragment" generally refers to an antibody or fragment containing only the Fab molecule, which is composed of the VH and CH1 of the heavy chain and a complete light chain, with the light chain and the heavy chain connected by a disulfide bond.

[0086] As used herein, the term "F(ab')2 antibody" or "F(ab')2 fragment" has two antigen-binding F(ab') portions linked together by a disulfide bond.

[0087] As used herein, the term "Fv antibody" or "Fv fragment" generally refers to an antibody or fragment consisting only of a light chain variable region (VL) and a heavy chain variable region (VH) connected by non-covalent bonds, and is the smallest functional fragment of an antibody molecule that retains a complete antigen binding site.

[0088] As used herein, the terms "single-chain antibody" and "scFv fragment" refer to antibodies or fragments formed by connecting the heavy chain variable region and the light chain variable region of an antibody via a short peptide.

[0089] In this article, the terms "minimum recognition unit" and "MRU" both refer to antibodies or fragments consisting of only one CDR, and their molecular weight is very small, accounting for only about 1% of the complete antibody.

[0090] According to some embodiments of the present invention, the antibody or antigen-binding fragment comprises: a heavy chain having an amino acid sequence as shown in SEQ ID NO:11; and a light chain having an amino acid sequence as shown in SEQ ID NO:12.

[0091] Fusion Protein

[0092] In the second aspect of the present invention, the present invention provides a fusion protein. According to an embodiment of the present invention, the fusion protein comprises the antibody or antigen-binding fragment described in the first aspect; and a biologically active protein or a fragment thereof. The fusion protein of the present invention has a binding affinity for 11-dehydrothromboxane B2 and can be used to detect 11-dehydrothromboxane B2 and detect cells containing 11-dehydrothromboxane B2.

[0093] According to some embodiments of the present invention, the biologically active protein or fragment thereof comprises at least one selected from a protein tag, albumin or a fragment thereof, and an Fc fragment.

[0094] In this article, "protein tag" generally refers to a polypeptide or protein fused with a target protein (antibody or antigen-binding fragment), which can be used for the expression, detection, disappearance or purification of the target protein, etc., including but not limited to His tag (also known as His-Tag, sequence HHHHHH), Flag tag (also known as Flag-Tag, sequence DYKDDDDK), GST tag (also known as GST-Tag, glutathione sulfhydryl transferase tag), MBP tag (also known as MBP-Tag, maltose binding protein tag), SUMO tag and C-Myc tag, etc.

[0095] Herein, "Fc fragment" generally refers to the Fc region from IgG (e.g., IgG1, IgG2, IgG3 or IgG4 subtype), IgA1, IgA2, IgD, IgE or IgM, including CH2, CH3 region and optionally hinge region. Preferably, IgG, IgA1, IgA2, IgD, IgE or IgM is derived from mouse, human, primate or alpaca.

[0096] According to some embodiments of the invention, the albumin or a fragment thereof is serum albumin or a fragment thereof.

[0097] Nucleic acid molecule, expression vector, recombinant cell and method for preparing antibody or antigen fragment

[0098] In the process of preparing or obtaining these antibodies or antigen-binding fragments thereof, nucleic acid molecules expressing these antibodies or antigen-binding fragments can be connected to different vectors and then expressed in different cells to obtain the corresponding antibodies or antigen-binding fragments thereof.

[0099] To this end, in the third aspect of the present invention, the present invention provides a nucleic acid molecule. According to an embodiment of the present invention, the nucleic acid molecule encodes the antibody or antigen-binding fragment described in the first aspect, and the fusion protein described in the second aspect. The provided nucleic acid molecule can be obtained by techniques known to those skilled in the art. Moreover, it can be species-optimized and more easily expressed in mammalian cells.

[0100] According to some embodiments of the invention, the nucleic acid molecule is DNA.

[0101] It should be noted that, for the nucleic acid molecules mentioned herein, those skilled in the art will understand that they actually include any one or both of the complementary double strands. For convenience, in this specification and claims, although only one strand is provided in most cases, the other strand complementary thereto is actually disclosed. In addition, the nucleic acid sequence in the present invention includes a DNA form or an RNA form, and disclosing one of them means that the other is also disclosed.

[0102] In the fourth aspect of the present invention, the present invention proposes an expression vector. According to an embodiment of the present invention, the expression vector carries the nucleic acid molecule described in the third aspect. When the above-mentioned nucleic acid molecule is connected to the expression vector, the nucleic acid molecule can be directly or indirectly connected to the control element on the expression vector, as long as these control elements can control the translation and expression of the nucleic acid molecule. Of course, these control elements can come directly from the expression vector itself, or they can be exogenous, that is, not from the expression vector itself. Of course, the nucleic acid molecule can be operably connected to the control element.

[0103] Herein, "operably linked" means connecting the foreign gene to the expression vector so that the control elements in the expression vector, such as transcription control sequences and translation control sequences, can play their intended functions of regulating the transcription and translation of the foreign gene. Commonly used expression vectors can be, for example, plasmids, bacteriophages, and the like.

[0104] According to some specific embodiments of the present invention, after the expression vector is introduced into a suitable recipient cell, the expression of the aforementioned antibody or antigen-binding fragment, or the aforementioned fusion protein can be effectively achieved under the mediation of a regulatory system, thereby achieving large-scale in vitro acquisition of the antibody or antigen-binding fragment, or the fusion protein.

[0105] According to an embodiment of the present invention, the expression vector comprises a vector selected from a eukaryotic expression vector or a prokaryotic expression vector.

[0106] In an optional embodiment of the present invention, the expression vector is a plasmid expression vector, a viral expression vector, such as a lentiviral expression vector.

[0107] In the fifth aspect of the present invention, the present invention provides a recombinant cell. According to an embodiment of the present invention, the recombinant cell includes carrying the nucleic acid molecule described in the third aspect or the expression vector described in the fourth aspect; or expressing the antibody or antigen-binding fragment described in the first aspect, or the fusion protein described in the second aspect. The recombinant cell can effectively express the aforementioned antibody or antigen-binding fragment, or fusion protein in the cell under suitable conditions.

[0108] It should be noted that "suitable conditions" refer to conditions suitable for the expression of the antibody or antigen-binding fragment or fusion protein of the present invention. It is easy for those skilled in the art to understand that conditions suitable for the expression of the antibody or antigen-binding fragment or fusion protein include but are not limited to suitable transformation or transfection methods, suitable transformation or transfection conditions, healthy host cell state, suitable host cell density, suitable cell culture environment, and suitable cell culture time. "Suitable conditions" are not particularly limited, and those skilled in the art can optimize the most suitable conditions for the expression of the above-mentioned antibody or antigen-binding fragment or fusion protein according to the specific environment of the laboratory.

[0109] According to some embodiments of the present invention, the recombinant cell is obtained by introducing the expression vector described in the fourth aspect into a host cell.

[0110] It should be noted that the recombinant cells of the present invention are not particularly limited and may be prokaryotic cells, eukaryotic cells or bacteriophages. The prokaryotic cells may be Escherichia coli, Bacillus subtilis, Streptomyces or Proteus mirabilis, etc. The eukaryotic cells include fungi such as Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Trichoderma, insect cells such as armyworms, plant cells such as tobacco, mammalian cells such as BHK cells, CHO cells, COS cells, myeloma cells, etc. In some embodiments, the recombinant cells of the present invention are preferably mammalian cells, including BHK cells, CHO cells, NSO cells or COS cells, and do not include animal germ cells, fertilized eggs or embryonic stem cells.

[0111] According to an embodiment of the present invention, the recombinant cell is a eukaryotic cell, preferably a mammalian cell.

[0112] In a sixth aspect of the present invention, the present invention provides a method for preparing an antibody or antigenic fragment. According to an embodiment of the present invention, the method comprises culturing the recombinant cell described in the fifth aspect under conditions suitable for protein expression and secretion, so as to obtain the antibody or antigen-binding fragment. Thus, the method of the present invention can be used to produce the aforementioned antibody or antigen-binding fragment.

[0113] Conjugates and kits

[0114] In the seventh aspect of the present invention, the present invention proposes a conjugate. According to an embodiment of the present invention, the conjugate comprises the antibody or antigen-binding fragment described in the first aspect, the fusion protein described in the second aspect, or the antibody or antigen-binding fragment prepared according to the method described in the sixth aspect; and a coupling portion, wherein the coupling portion is connected to the antibody or antigen-binding fragment, or the fusion protein. The conjugate of the present invention has a binding affinity for 11-dehydrothromboxane B2 and can be used to detect 11-dehydrothromboxane B2 and detect cells containing 11-dehydrothromboxane B2.

[0115] According to some embodiments of the present invention, the coupling moiety comprises at least one selected from a carrier, a drug, a toxin, a cytokine, a protein tag, a modifier and a chemotherapeutic agent.

[0116] In this article, the carrier can be a substance that can be suspended or dispersed in a liquid phase (for example, solid phase carriers such as particles and magnetic beads), or a solid phase that can contain or carry a liquid phase (for example, a support such as a plate, a membrane, a test tube, and a container such as a well plate, a microfluidic channel, a glass capillary, a nanocolumn, a monolithic column, etc.); it can also be a labeling carrier for labeling an antibody or antigen-binding fragment, or a fusion protein, such as an enzyme (for example, peroxidase, alkaline phosphatase, luciferin, β-galactosidase ), affinity substances (for example, one of streptavidin and biotin, one of the complementary positive and antisense nucleic acids), fluorescent dyes (for example, fluorescein, fluorescein isothiocyanate, rhodamine, green fluorescent protein, red fluorescent protein), luminescent substances (for example, luciferin, aequorin, acridinium ester, tris(2,2'bipyridine)ruthenium, luminol), radioactive isotopes (for example, 3H, 14C, 32P, 35S, 125I) and gold colloids, etc.

[0117] According to some embodiments of the invention, the carrier is selected from an enzyme.

[0118] According to some embodiments of the present invention, the enzyme is selected from any one or more of peroxidase, alkaline phosphatase, luciferin, and β-galactosidase.

[0119] According to an embodiment of the present invention, the drug is a small molecule drug that can be combined with an antibody or an antigen-binding fragment, or a fusion protein.

[0120] According to an embodiment of the present invention, the protein tag includes but is not limited to a His tag, a Flag tag, a GST tag, an MBP tag, a SUMO tag, a C-Myc tag, and the like.

[0121] According to the embodiments of the present invention, the modifier should be understood in a broad sense, and may refer to a substance used to modify a protein. For example, it may be polyethylene glycol or a derivative thereof.

[0122] It should be noted that the binding method of the coupling part and the antibody or antigen binding fragment or fusion protein can be a method known in the art. For example, physical adsorption, covalent binding, methods using affinity substances (e.g., biotin, streptavidin) and ion binding methods can be cited.

[0123] In the ninth aspect of the present invention, the present invention proposes a kit. According to an embodiment of the present application, the kit comprises the antibody or antigen binding fragment described in the first aspect, the fusion protein described in the second aspect, the nucleic acid molecule described in the third aspect, the expression vector described in the fourth aspect, the recombinant cell described in the fifth aspect, the antibody or antigen binding fragment prepared according to the method described in the sixth aspect, or the conjugate described in the seventh aspect. According to an embodiment of the present invention, the antibody or its antigen binding fragment can bind to 11-dehydrothromboxane B2 with high specificity. In some scientific studies, the kit can be used for qualitative or quantitative detection of 11-dehydrothromboxane B2 in biological samples, and can also be used to judge the status of an individual, such as after obtaining the 11-dehydrothromboxane B2 level of the individual, judging whether the 11-dehydrothromboxane B2 level is higher or lower than the normal level.

[0124] The kit of the present invention is prepared according to the principle of the competition method. The antigen derivative coated with magnetic particles competes with the antigen in the sample to be tested for the labeled antibody in the liquid phase. The magnetic particles are evenly dispersed in the entire reaction system. Due to the influence of the contact surface, the specific binding rate between the antigen in the sample to be tested and the antibody is faster than the binding between the solid phase antigen and the liquid phase antibody. The more obvious the rate difference is, the greater the inhibition rate is, that is, the higher the sensitivity is, and the more conducive it is to detect low-concentration antigens to be tested.

[0125] As mentioned above, the antibodies or antigen-binding fragments of some specific embodiments of the present invention can effectively bind to 11-dehydrothromboxane B2, and therefore, the reagents or kits containing the antibodies or antigen-binding fragments can effectively detect 11-dehydrothromboxane B2 qualitatively or quantitatively. The reagents or kits provided by the present invention can be used, for example, for immunoblotting, immunoprecipitation, and other reagents or kits that involve detection using the specific binding of 11-dehydrothromboxane B2 and antibodies.

[0126] According to some embodiments of the present invention, the kit further comprises at least one of the following additional technical features:

[0127] According to some embodiments of the invention, the kit further comprises a first magnetic particle.

[0128] According to some embodiments of the present invention, the kit further comprises first magnetic particles, creatinine antibodies, second magnetic particles, 11-dehydrothromboxane B2 calibrators, creatinine calibrators and luminescent substrate solution.

[0129] According to some embodiments of the invention, the antibody or antigen-binding fragment and the creatinine antibody are respectively labeled with a biomarker.

[0130] According to some embodiments of the present invention, the biomarker is selected from at least one of a biological enzyme, a fluorescein and a chemiluminescent marker.

[0131] According to some embodiments of the present invention, the biological enzyme is selected from at least one of horseradish peroxidase and alkaline phosphatase.

[0132] According to some embodiments of the present invention, the first magnetic particles are coated with 11-dehydrothromboxane B2 derivatives, and the second magnetic particles are coated with creatinine derivatives.

[0133] According to some embodiments of the present invention, the first magnetic particles and the second magnetic particles are respectively selected from magnetic microspheres.

[0134] The magnetic microparticles described in the present invention are magnetic beads with superparamagnetism and corresponding magnetic field responsiveness. According to the type of surface groups of the magnetic beads, the magnetic beads include carboxyl magnetic beads, amino magnetic beads, silica-based magnetic beads, tosyl magnetic beads, aldehyde magnetic beads, thiol magnetic beads and streptavidin magnetic beads, etc., preferably carboxyl magnetic beads.

[0135] According to some embodiments of the present invention, the surface of the magnetic microspheres is modified with active groups.

[0136] According to some embodiments of the present invention, the active group is selected from at least one of a hydroxyl group, a thiol group, a carboxyl group, an amino group, a silicon group, a tosyl group, a streptavidin group, and a derivative group thereof.

[0137] According to some embodiments of the present invention, the particle size of the magnetic microspheres is 0.5 to 5 μm.

[0138] According to some embodiments of the present invention, the particle size of the magnetic microspheres is 1-3 μm.

[0139] According to some embodiments of the present invention, the first magnetic particle and the second magnetic particle are the same or different. The same refers to the same magnetic particle size and surface groups, while different magnetic particles have different particle sizes or active groups.

[0140] According to some embodiments of the present invention, the substrate solution is selected from at least one of AMPPD and APS-5.

[0141] Application and detection method

[0142] In the eighth aspect of the present invention, the present invention provides the use of the antibody or antigen-binding fragment described in the first aspect in preparing a kit for detecting 11-dehydrothromboxane B2. According to an embodiment of the present invention, the antibody or antigen-binding fragment can bind to 11-dehydrothromboxane B2 with high specificity. Therefore, the kit containing the antibody or antigen-binding fragment can be used for detecting 11-dehydrothromboxane B2 with high specificity and high sensitivity.

[0143] In the tenth aspect of the present invention, the present invention proposes a method for detecting the content of 11-dehydrothromboxane B2. According to an embodiment of the present invention, the method comprises: using the kit described in the ninth aspect to detect and process the sample to be tested, so as to determine the content of the 11-dehydrothromboxane B2 in the sample to be tested. The method described in the present invention can detect 11-dehydrothromboxane B2 with high sensitivity, and is conducive to the detection of low-concentration 11-dehydrothromboxane B2. In addition, the method has the advantages of strong specificity, wide linear range, simple operation, and no need for sample pretreatment. In practical clinical applications, a large number of samples can be quickly and high-throughput detected with the help of a chemiluminescence instrument.

[0144] According to some embodiments of the present invention, the detection process includes: performing a first mixing treatment on the sample to be tested and the antibody or antigen-binding fragment, wherein the antibody or antigen-binding fragment is labeled with a biomarker; performing a second mixing treatment on the first mixing treatment product and the first magnetic particles; performing a sedimentation treatment on the second mixing treatment product, removing the supernatant, and obtaining a first precipitate; performing a first contact treatment on the first precipitate and the luminescent substrate solution, and detecting and obtaining a first luminescence value RLU; based on the first luminescence value RLU, obtaining the content of 11-dehydrothromboxane B2 in the sample to be tested.

[0145] According to some embodiments of the present invention, the biomarker is selected from at least one of a biological enzyme, a fluorescein and a chemiluminescent marker.

[0146] According to some embodiments of the present invention, the biological enzyme is selected from at least one of horseradish peroxidase and alkaline phosphatase.

[0147] According to some embodiments of the present invention, the method used for labeling the antibody or antigen-binding fragment with a biomarker can be obtained based on any method known in the art. For example, a commonly used activator 2-Iminothiolane hydrochloride (2-IT, 2-iminothiolane hydrochloride) in the art is added to the antibody or antigen-binding fragment, and a commonly used activator Succinimidyl4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC, 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid succinimide ester) is added to the biomarker. The mixed mass ratio of the activated biomarker to the antibody or antigen-binding fragment is (0.5-1.5):1.

[0148] According to some embodiments of the present invention, the detection process further comprises: using the 11-dehydrothromboxane B2 calibrator to draw a 11-dehydrothromboxane B2 concentration-luminescence value standard curve; wherein the 11-dehydrothromboxane B2 calibrator comprises 11-dehydrothromboxane B2 with a predetermined concentration gradient.

[0149] According to some embodiments of the present invention, the step of obtaining the content of 11-dehydrothromboxane B2 in the sample to be tested based on the first luminescence value RLU includes: calculating the content of 11-dehydrothromboxane B2 in the sample to be tested based on the 11-dehydrothromboxane B2 concentration-luminescence value standard curve and the first luminescence value RLU.

[0150] According to some embodiments of the present invention, the sample to be tested is urine.

[0151] In the eleventh aspect of the present invention, the present invention proposes a method for detecting the relative content of 11-dehydrothromboxane B2. According to an embodiment of the present invention, the method comprises: using the kit described in the ninth aspect to detect and process the sample to be tested, so as to determine the relative content of the 11-dehydrothromboxane B2 in the sample to be tested. Since both 11-dehydrothromboxane B2 and creatinine are small molecules, the small molecules have a large degree of freedom, which is not conducive to the exposure of the site for binding with the antibody, and is also not conducive to the binding of the antigen with the antibody. Before determining the content of 11-dehydrothromboxane B2 and creatinine in the sample, the method of the present invention allows the small molecule antigen (11-dehydrothromboxane B2 and creatinine) in the sample to react with the enzyme-labeled antibody first, and then adds the small molecule derivative coated with magnetic beads to compete, so it is conducive to the detection of low-concentration antigens and improves the sensitivity of the reagent. In addition, the method has the advantages of strong specificity, wide linear range, simple operation, and no need for pre-treatment of the sample. In practical clinical applications, a large number of samples can be quickly and high-throughput detected with the help of a chemiluminometer.

[0152] According to some embodiments of the present invention, the detection process includes: performing a first mixing process and a second mixing process on the sample to be tested, the antibody or antigen binding fragment, and the creatinine antibody, respectively, wherein the antibody or antigen binding fragment and the creatinine antibody are respectively labeled with a biomarker; performing a third mixing process on the first mixing process product and the first magnetic particle, wherein the first mixing process product contains the antibody or antigen binding fragment; performing a fourth mixing process on the second mixing process product and the second magnetic particle, wherein the second mixing process product contains the creatinine antibody; performing a sedimentation process on the third mixing process product and the fourth mixing process product, respectively, removing the supernatant to obtain a third precipitate and a fourth precipitate; performing a first contact process and a second contact process on the third precipitate and the fourth precipitate, respectively, with the luminescent substrate solution, and detecting and obtaining a first luminescence value RLU and a second luminescence value RLU; obtaining the content of 11-dehydrothromboxane B2 in the sample to be tested based on the first luminescence value RLU; obtaining the content of creatinine in the sample to be tested based on the second luminescence value RLU; determining the relative content of 11-dehydrothromboxane B2 in the sample to be tested based on the following formula

[0153]

[0154] According to some embodiments of the present invention, the biomarker is selected from at least one of a biological enzyme, a fluorescein and a chemiluminescent marker.

[0155] According to some embodiments of the present invention, the biological enzyme is selected from at least one of horseradish peroxidase and alkaline phosphatase.

[0156] According to some embodiments of the present invention, the method by which the antibody or antigen-binding fragment and the creatinine antibody are respectively labeled with a biomarker can be obtained based on any method known in the art, for example, 2-Iminothiolane hydrochloride (2-IT, 2-iminothiolane hydrochloride), an activator commonly used in the art, is added to the antibody or antigen-binding fragment and the creatinine antibody, respectively, and Succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC, 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid succinimide ester), an activator commonly used in the art, is added to the biomarker, and the mixed mass ratio of the activated biomarker to the antibody or antigen-binding fragment or the creatinine antibody is (0.5-1.5):1.

[0157] According to some embodiments of the present invention, the detection process further includes: using 11-dehydrothromboxane B2 calibrator and creatinine calibrator to draw a 11-dehydrothromboxane B2 concentration-luminescence value standard curve and a creatinine concentration-luminescence value standard curve, respectively; wherein the 11-dehydrothromboxane B2 calibrator contains 11-dehydrothromboxane B2 with a predetermined concentration gradient; and the creatinine calibrator contains creatinine with a predetermined concentration gradient.

[0158] According to some embodiments of the present invention, the step of obtaining the content of 11-dehydrothromboxane B2 in the sample to be tested based on the first luminescence value RLU includes: calculating the content of 11-dehydrothromboxane B2 in the sample to be tested based on the 11-dehydrothromboxane B2 concentration-luminescence value standard curve and the first luminescence value RLU.

[0159] According to some embodiments of the present invention, the step of obtaining the creatinine content in the sample to be tested based on the second luminescence value RLU includes: calculating the creatinine content in the sample to be tested based on the creatinine concentration-luminescence value standard curve and the second luminescence value RLU.

[0160] According to some embodiments of the present invention, the sample to be tested is urine.

[0161] In one example of the present application, the 11-dehydrothromboxane B2 concentration-luminescence value standard curve is obtained by the following method, including:

[0162] (1) Immune reaction: 50 μL of 11-dehydrothromboxane B2 calibrator (11-dehydrothromboxane B2 concentrations are 0 pg / mL, 100 pg / mL, 500 pg / mL, 2000 pg / mL, 5000 pg / mL, and 10000 pg / mL) and 50 μL of the antibody or antigen-binding fragment labeled with a biomarker are sequentially added to a reaction tube, incubated at 37°C for 10 min, and then the first magnetic particles coated with the 11-dehydrothromboxane B2 derivative are added, and the incubation continues at 37°C for 10 min;

[0163] (2) Magnetic separation: Place the reaction tube in a magnetic field, allow the magnetic particles to settle in the magnetic field, remove the supernatant, remove the magnetic field, add 200-500 μL of cleaning solution, and then allow the magnetic particles to settle in the magnetic field again, and remove the supernatant; repeat this 2-4 times to remove other unreacted substances;

[0164] (3) Reading: Add 200 μL of luminescent substrate solution and read the luminescence value (RLU) after 2 minutes;

[0165] (4) Based on the detected luminescence values, a logistic four-parameter fitting was used to obtain a 11-dehydrothromboxane B2 concentration-luminescence value standard curve.

[0166] In the twelfth aspect of the present invention, the present invention proposes a method for detecting creatinine content. According to an embodiment of the present invention, the method comprises: using the kit described in the ninth aspect to detect and process the sample to be tested, so as to determine the content of creatinine in the sample to be tested. The method described in the present invention can detect creatinine with high sensitivity, and is conducive to the detection of low concentration creatinine. In addition, the method has the advantages of strong specificity, wide linear range, simple operation, and no need for pre-treatment of samples. In practical clinical applications, large quantities of samples can be quickly and high-throughput detected with the help of a chemiluminometer.

[0167] According to some embodiments of the present invention, the detection process includes: performing a first mixing treatment on the sample to be tested and the creatinine antibody, wherein the creatinine antibody is labeled with a biomarker; performing a second mixing treatment on the first mixing treatment product and the first magnetic particles; performing a sedimentation treatment on the second mixing treatment product, removing the supernatant, and obtaining a first precipitate; performing a first contact treatment on the first precipitate and the luminescent substrate solution, and detecting and obtaining a first luminescence value RLU; based on the first luminescence value RLU, obtaining the creatinine content in the sample to be tested.

[0168] According to some embodiments of the present invention, the biomarker is selected from at least one of a biological enzyme, a fluorescein and a chemiluminescent marker.

[0169] According to some embodiments of the present invention, the biological enzyme is selected from at least one of horseradish peroxidase and alkaline phosphatase.

[0170] According to some embodiments of the present invention, the method used for labeling the creatinine antibody with a biomarker can be obtained based on any method known in the art. For example, a commonly used activator in the art, 2-Iminothiolanehydrochloride (2-IT, 2-iminothiolane hydrochloride) is added to the creatinine antibody, and a commonly used activator in the art, Succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC, 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid succinimide ester) is added to the biomarker. The mixed mass ratio of the activated biomarker to the antibody or antigen-binding fragment is (0.5-1.5):1.

[0171] According to some embodiments of the present invention, the detection process further comprises: using the creatinine calibrator to draw a creatinine concentration-luminescence value standard curve; wherein the creatinine calibrator comprises creatinine with a predetermined concentration gradient.

[0172] According to some embodiments of the present invention, the step of obtaining the creatinine content in the sample to be tested based on the first luminescence value RLU includes: calculating the creatinine content in the sample to be tested based on the creatinine concentration-luminescence value standard curve and the first luminescence value RLU.

[0173] According to some embodiments of the present invention, the sample to be tested is urine.

[0174] The above detection kit and detection method have the following beneficial effects:

[0175] The present invention is based on the immunocompetitive method for the determination of urine 11-dehydrothromboxane B2 and urine creatinine, and utilizes the principle of combining chemiluminescence detection technology with magnetic particle immunoseparation technology, so that the two indicators can be detected synchronously on a luminometer, and has the advantages of good sensitivity, high accuracy, wide linear range, strong specificity, simple operation, and no interference from endogenous biotin, and provides a more accurate, convenient and quick method for clinical evaluation of platelet activity or antiplatelet effect of aspirin.

[0176] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention. If no specific techniques or conditions are specified in the embodiments, the techniques or conditions described in the literature in this area or the product specifications are used. The reagents or instruments used that do not specify the manufacturer are all conventional products that can be obtained commercially.

[0177] in,

[0178] Fully automatic chemiluminescence analyzer: Cosma SMART 500H;

[0179] 11-Dehydrothromboxane B2 derivative: coupled with BSA, purchased from CD Company, USA;

[0180] 11-Dehydrothromboxane B2: purchased from Cyman Company, USA;

[0181] 2,3-dinor thromboxane B2: purchased from Cyman Company, USA;

[0182] Creatinine antibody: sheep polyclonal antibody, purchased from CD Company, USA;

[0183] Creatinine derivatives: coupled with BSA, purchased from CD Company, USA;

[0184] Creatinine: purchased from Sigma;

[0185] Magnetic beads: with carboxyl groups, particle size 1.0-3.0 μm, purchased from JSR Corporation of Japan;

[0186] Alkaline phosphatase (ALP): purchased from BBI, UK;

[0187] EDC: purchased from Sigma;

[0188] Luminescent substrate solution: from our company's self-developed reagent, the main component is APS-5;

[0189] Coating buffer: 0.05 mmol / L MES (biological buffer), pH 6.0;

[0190] Blocking solution: 100 mmol / L glycine, 10% BSA (bovine serum albumin), pH 7.4;

[0191] Cleaning solution: 10mmo / L PB (phosphate buffer), 0.05% Tw-20, pH 7.4;

[0192] Coating storage solution: 50mmo / L PB (phosphate buffer), 1.0% BSA (bovine serum albumin), pH 7.4;

[0193] Enzyme label storage solution: 50mmo / L PB (phosphate buffer), 1.0% BSA (bovine serum albumin), pH 7.4;

[0194] Calibrator diluent: 50mmol / L PBST (phosphate buffered saline).

[0195] The amino acid sequences involved in this article are shown in Table 1:

[0196] Table 1: Amino acid sequences

[0197]

[0198] Practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry, and immunology, which are within the capabilities of a skilled artisan. This technique is fully explained in the literature, such as Molecular Cloning: A Laboratory Manual, 2nd Edition (Sambrook et al., 1989); Oligonucleotide Synthesis (MJ Gait, ed., 1984); Animal Cell Culture (RI Freshney, ed., 1987); Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (DM Weir and CC Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (JM Miller and MP Calos, eds., 1987); Current Protocols in Molecular Biology (FM Ausubel et al., eds., 1987); PCR: The Polymerase Chain Reaction (PCR: The Polymerase Chain Reaction) (Academic Press, Inc., 1987). Reaction" (Mullis et al., eds., 1994); and Current Protocols in Immunology (JE Coligan et al., eds., 2011), each of which is expressly incorporated herein by reference.

[0199] In the embodiments of the present invention, the nucleotide sequence used to prepare the expression vector can be obtained according to its amino acid sequence using conventional methods or conventional software (such as the online program Vectorbuilder (website: https: / / www.vectorbuilder.cn / tool / codon-optimization.html), GeneOptimizer online program, etc.).

[0200] Example 1: Large-scale preparation of monoclonal antibodies

[0201] 1. 11-dehydrothromboxane B2 was coupled to KLH (hemocyanin) using the N-hydroxysuccinimide ester method to serve as the 11-dehydrothromboxane B2 immunogen.

[0202] 2. BALB / c mice aged 6 to 8 weeks were selected for the following immunization procedure: for the first immunization, 25 μg of 11-dehydrothromboxane B2 immunogen was mixed with an equal amount of Freund's complete adjuvant, emulsified and injected subcutaneously at multiple points; 14 days after the first immunization, 12.5 μg of 11-dehydrothromboxane B2 immunogen was mixed with Freund's incomplete adjuvant, emulsified and boosted. 14 days after the second immunization, 12.5 μg of 11-dehydrothromboxane B2 immunogen was mixed with Freund's incomplete adjuvant, emulsified and boosted. 14 days after the third immunization, blood was collected and serum was separated, and 1 μg / mL 11-dehydrothromboxane B2 derivative was used to coat ELISA plates for indirect ELISA tests to determine serum titers.

[0203] The results showed that the titer of the prepared mouse antiserum was greater than 72,000. The typical immune mouse serum test results are shown in Table 2.

[0204] Table 2: Test results of serum from immunized mice

[0205] Serum dilution multiple <![CDATA[OD 450 ]]> 100 1.754 300 1.707 900 1.646 2700 1.548 8100 1.388 24300 0.991 72900 0.596 blank 0.137

[0206] 3. Carry out cell fusion in the above-mentioned immunized mice. The specific process is as follows: after removing the mouse eyeball to collect blood, the mouse is killed by dislocation, placed in a 70% alcohol bottle for 2 minutes, and then fixed on a foam board in a biosafety cabinet. Open the abdominal skin to find the spleen, remove it with tweezers, put it in a 200-mesh stainless steel filter and gently grind it, and gently rinse the cells with DMEM culture medium (Thermo, 11965092), then centrifuge at 200×g for 10 minutes at room temperature, discard the supernatant and set aside; when preparing feeder cells, the mouse is killed by dislocation, placed in a 70% alcohol bottle for 2 minutes, and then fixed on a foam board in a biosafety cabinet, open the abdominal skin, use a syringe to draw PBS and gently inject it into the peritoneum, and then wash out the liquid containing feeder cells from the other side, and then centrifuge at 200×g for 10 minutes at room temperature, and discard the supernatant and set aside. 2.0×10 7 FO myeloma cells and 2.0×10 8The spleen cells were mixed, centrifuged at 200×g for 10 minutes, the supernatant was discarded, and the mixture was gently shaken and mixed. In a 37°C water bath, 1 ml of a 50% PEG-1450 (Merk, P1458) aqueous solution was added dropwise within 90 seconds, and then 20 mL of DMEM medium was added dropwise. The mixture was centrifuged at 200×g for 10 minutes, the supernatant was discarded, and the mixture was washed again. The mixture was centrifuged at 200×g for 10 minutes, the supernatant was discarded, and the hybridoma cells were plated in 10 96-well culture plates, 150 μL per well. 10,000 feeder layer cells / well were added to the above 10 96-well cell culture plates, 100 μL per well. The culture plates were marked and placed in a 37°C incubator containing 5% CO 2 Culture in a cell culture incubator, add HAT screening medium (Merk, H0262) on the second day, and then use HAT selection culture for 1-2 days. A large number of tumor cells will die, and the tumor cells will disappear after 3-4 days. The hybrid cells will form small colonies. After 7-10 days of HAT screening culture medium, it should be replaced with HT culture medium (Merk, H0137), and then maintained for another 2 weeks. Then, use 20% FBS (ExCell, FSP500) DMEM culture medium to continue culture. During the above selection culture period, when the hybridoma cells cover 1 / 10 of the bottom of the well, you can start to detect specific antibodies and screen out the required hybridoma cell lines. During the selection culture period, half of the culture medium is generally replaced every 2-3 days.

[0207] 4. Determine the optimal coating amount of 11-dehydrothromboxane B2 derivative as antigen by orthogonal experiment. 0.5, 1.0, 2.0, 4.0 μg of 11-dehydrothromboxane B2 derivative was coated on 96-well plate, and 6 wells of each concentration were set as 3 positive and 3 negative. Square titration was performed with positive serum of mice immunized with 11-dehydrothromboxane B2 at different dilution multiples, and negative serum of unimmunized mice was used as negative control. 96-well ELISA plate was coated with 0.5 μg purified 11-dehydrothromboxane B2 derivative per well, incubated at 4°C overnight; washed twice with PBST; added 200 μL of 1% BSA in PBS to each well, blocked at room temperature for 2 hours, and then placed on trifold paper and patted dry; sample addition: add 0.1 mL of the sample to be tested to the reaction well, incubate at 37°C for 1 hour, then wash, and make blank wells (without sample), negative control wells and positive control wells at the same time. Add 0.1 mL of newly diluted antibody to each reaction well, incubate at 37°C for 1 hour, and then wash 3 times. Add enzyme-labeled secondary antibody: Add 0.1 mL of newly diluted enzyme-labeled antibody to each reaction well. Incubate at 37°C for 1 hour, and wash 3 times. Add substrate solution for color development: Add 0.1 mL of TMB substrate solution to each reaction well, and let stand at room temperature for 10 minutes. Add 1M H 2 SO 40.1 mL. Measure the OD value to determine the result, and use a microplate reader to measure the absorbance value (A450) at 450nm. A value 2.1 times or more of the negative control OD value is considered positive (calculated after adjusting the blank control well to zero). Select the hybridoma cell monoclone that is resistant to 11-dehydrothromboxane B2.

[0208] According to the above method, the positive hybridoma cells screened were subcloned, and the original wells were diluted with HAT selection medium by limiting dilution method and redistributed into 96-well culture plates, and then the morphology and number of cells were observed. The cells were adjusted to 3-10 cells / mL. Take the cell culture plate with the feeder cell layer prepared the day before, and add 100 μL of diluted cells to each well. Incubate at 37°C and 5% CO 2 Culture in a static incubator. Change the medium on the 7th day, and every 2-3 days thereafter. Cell clones can be seen on the 8th to 9th day, and the antibody activity can be detected in time. Move the cells in the positive wells to a 24-well plate for expansion and culture. Each clone should be frozen as soon as possible, and finally the clone number 11DH025 hybridoma cell line was selected for antibody production.

[0209] 5. 8-week-old BALB / C mice were intraperitoneally injected with 0.5 mL of Freund's incomplete adjuvant. Two weeks later, 1×10 6Hybridoma cells with clone number 11DH025 can produce ascites 7 to 10 days after inoculation. Closely observe the health status and ascites signs of the animals. When ascites is as much as possible and before the mice die, kill the mice and use a dropper to suck the ascites into a test tube. 5 to 10 ml of ascites can be obtained from one mouse. Ascites can also be extracted with a syringe and can be collected repeatedly several times. The ascites obtained was centrifuged at 3000×g for 10 minutes, the upper layer of fat and the bottom sediment were discarded, the supernatant was collected and distributed at -20℃, and after the ascites supernatant was thawed and equilibrated to room temperature, 1 / 10 volume of 1M Tris-HCl PH8.0 was added to adjust the sample pH to 8.0. The protein G affinity column was balanced with 20 column volumes of 100 mM Tris-HCl at pH 8.0, the ascites supernatant after adjusting the pH to 8.0 was loaded onto the column, and then washed with 20 column volumes of 100 mM Tris-HCl at pH 8.0, and finally eluted with 100 mM Glycine-HCL pH 2.5 (i.e., 11-dehydrothromboxane B2 antibody 11DH025, which may be referred to as antibody 11DH025 or 11DH025). The antibody eluate was added to a concentrator tube (Millipore, UFC801008, 10K) and centrifuged at room temperature at 3000 × g for 20 minutes in a centrifuge (Xiangyi, L550). The solution was centrifuged in batches until the volume of the solution reached 1 ml / concentrator tube (2 tubes), 4 mL of 10 mM PBS pH 7.4 buffer was added, and the solution was centrifuged at room temperature at 3000 × g for 20 minutes. Repeat centrifugation 3 times to make the antibody buffer 10mM PBS PH = 7.4, add 10mM PBS PH = 7.4 to a total volume of 10mL. Finally, 2mL / tube of concentrated antibody solution was dispensed into centrifuge tubes and stored at -80°C. The antibody concentration was determined using a BCA kit (Solabo, PC0020), and the concentration of the purified monoclonal antibody was determined to be 1.832mg / mL.

[0210] Example 2: Antibody titer determination

[0211] The titer of 11-dehydrothromboxane B2 antibody 11DH025 was detected by indirect ELISA method. The 11-dehydrothromboxane B2 derivative was diluted with PBS and coated in a 96-well ELISA plate at 0.2μg / mL and 100μL / well. After overnight at 4℃, the ELISA plate was washed twice with PBST solution, 300μL / well each time, and patted dry after washing. The ELISA plate was blocked with 1% BSA PBS solution, 200μL / well, at room temperature for 2 hours, and patted dry after blocking; 11-dehydrothromboxane B2 antibody 11DH025 diluted to 20ng / mL with PTB was added, and reacted in a 37℃ incubator for 1 hour. The ELISA plate was washed twice with PBST solution, 300μL / well each time. Patted dry after washing. Add HRP-labeled goat anti-mouse antibody diluted 5000 times with PTB, react in a 37°C incubator for 1 hour, wash the plate twice with PBST solution, 300 μL / well each time. Pat dry after washing. Add TMB substrate solution, 100 μL / well, react at room temperature for 10 minutes, add 100 μL / well 1M H 2 SO 4 The reaction was terminated. The absorbance (A450) was measured at 450 nm using an ELISA reader. The results are shown in Table 3, and it can be seen that the titer of the purified antibody is greater than 1.6 million.

[0212] Table 3: Antibody titer determination results

[0213]

[0214]

[0215] Example 3: Sequence analysis of monoclonal antibodies

[0216] (1) Monoclonal antibody subtype identification

[0217] Hybridoma cell line 11DH025 was cultured in a 10 cm diameter cell culture dish (37°C, 5% CO) using DMEM medium (GIBCO, #C11995500BT) supplemented with 10% serum. 2 After 7 days of culture, the cells were transferred to a 15 ml centrifuge tube and 4 × 10 6 After centrifugation at 200×g for 5 minutes, the supernatant was discarded, and the centrifuge tube was inverted to drain the liquid in the tube. The cells in the tube were synthesized into cDNA using the reverse transcription kit (Qiagen, 74134) of QIAGEN.

[0218] The antibody subtype was determined by PCR using antibody subtype-specific primers. The above-synthesized cDNA was used as a PCR reaction template. PCR reaction solution system: TAKARA Ex Taq (5U / μL, TAKARA, RR001B), 0.25μL; 10×Ex Taq Buffer, 5μL; dNTP mixture (2.5mM each), 4μL; template cDNA, 1μL; upstream primer (100μM), 1μL; downstream primer (100μM), 1μL; add double distilled water to a total volume of 50μL. PCR reaction temperature program: 94℃5 minutes pre-denaturation, temperature cycle 30 times (94℃1 minute, 57℃1 minute, 72℃1 minute), 72℃10 minutes extension. After the reaction, 10μL of each PCR product was loaded on a 1% agarose gel for electrophoresis. The order of samples added to each lane is shown in Table 5. The antibody subtype can be inferred based on the PCR product results (Table 4). For specific results, see Figure 8 The monoclonal antibody 11DH025 obtained in the present invention has a heavy chain of IgG1 and a light chain of kappa.

[0219] Table 4: PCR primer information

[0220]

[0221] Where S=C or G, M=A or C, R=A or G, and W=A or T.

[0222] Table 5: Antibody information

[0223]

[0224]

[0225] (2) Sequencing of the variable region (V region) of the hybridoma cell line 11DH025 antibody

[0226] The fragment obtained after PCR amplification of the V region of the antibody of the cell line 11DH025 in step (1) of this embodiment was cut from the agarose gel and extracted using a DNA extraction kit (Qiagen, 74134). The extracted DNA fragment was linked to the pEASY-T1 cloning vector and transformed into Trans1-T1 competent cells (Transgen, CT101-1). The transformed bacterial colonies were picked into LB culture medium and DNA sequencing was performed after overnight culture. The light chain V region nucleic acid sequence of the antibody (11DH025) for recognizing 11-dehydrothromboxane B2 provided by the present invention is shown in SEQ ID NO:8, and the nucleic acid sequence of the heavy chain V region is shown in SEQ ID NO:7.

[0227] Example 4: Kit prepared using 11-dehydrothromboxane B2 antibody

[0228] 1. Preparation of magnetic bead-antigen complex (particle size 1.0-3.0 μm)

[0229] The magnetic bead-antigen complex refers to magnetic particles A coated with 11-dehydrothromboxane B2 derivatives, or magnetic particles B coated with creatinine derivatives, which are used for 11-dehydrothromboxane B2 detection and creatinine detection, respectively. The preparation processes are as follows:

[0230] Preparation of magnetic microparticles A: Add a magnetic field to the uniformly mixed magnetic beads (the concentration of magnetic beads in the system is 0.01-5 mg / mL) to remove the supernatant, wash once with coating buffer, then add the activator EDC to 0.01-2 mg / mL, shake and react at room temperature for 0.5-2h, then add a magnetic field to remove the supernatant, add coating buffer and an appropriate amount of 11-dehydrothromboxane B2 derivative (the concentration of 11-dehydrothromboxane B2 derivative in the system is 0.1-5μg / mL), shake and react at room temperature for 1-3h, add a magnetic field to remove the supernatant after the reaction, add blocking solution, and continue to shake and react for 1-3h. After the blocking is completed, wash the magnetic beads 3 times with cleaning solution, and finally dilute the magnetic microparticles A to the use concentration with coating storage solution and store at 4°C.

[0231] Preparation of magnetic microparticles B: Add a magnetic field to the uniformly mixed magnetic beads (the concentration of magnetic beads in the system is 0.01-5 mg / mL) to remove the supernatant, wash once with coating buffer, then add the activator EDC to 0.01-2 mg / mL, shake and react at room temperature for 0.5-2h, then add a magnetic field to remove the supernatant, add coating buffer and creatinine derivatives (the concentration of creatinine derivatives in the system is 0.1-5μg / mL), shake and react at room temperature for 1-3h, add a magnetic field to remove the supernatant after the reaction, add blocking solution, continue to shake and react for 1-3h, wash the magnetic beads 3 times with cleaning solution after blocking, finally dilute magnetic microparticles A to the use concentration with coating storage solution, and store at 4°C.

[0232] 2. Preparation of Alkaline Phosphatase-labeled Antibodies

[0233] The alkaline phosphatase labeled antibody refers to the alkaline phosphatase labeled 11-dehydrothromboxane B2 antibody prepared in the previous embodiment, or the alkaline phosphatase labeled creatinine antibody, which is respectively combined with the 11-dehydrothromboxane B2 derivative connected to the magnetic particle A and the creatinine derivative connected to the magnetic particle B during the detection process, and is used for 11-dehydrothromboxane B2 detection and urine creatinine detection. The preparation process is as follows:

[0234] (1) Preparation of alkaline phosphatase-labeled 11-dehydrothromboxane B2 antibody:

[0235] 1.0 mg of the 11-dehydrothromboxane B2 antibody prepared in the previous example was concentrated to 2.5 mg / mL, and 5 μL of 13.76 mg / mL activator 2-iminothiolane hydrochloride (2-IT) solution was added, and the mixture was reacted at room temperature for 15 minutes. The activated 11-dehydrothromboxane B2 antibody was collected after desalting using a Sephadex G25 gel column.

[0236] 1.2 mg of alkaline phosphatase was concentrated to 2.5 mg / mL, and 12 μL of activator 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid succinimidyl ester (SMCC) solution with a concentration of 6.69 mg / mL was added, and the mixture was reacted at room temperature for 15 minutes. The activated alkaline phosphatase was collected after desalting using a Sephadex G25 gel column.

[0237] The activated 11-dehydrothromboxane B2 antibody and alkaline phosphatase were mixed at a ratio of 1.0 mg of antibody to 1.0 mg of alkaline phosphatase (mass ratio of 1:1), and allowed to react at 4°C for 18 hours.

[0238] Use Supperdex200 gel chromatography column to separate and purify, remove unconnected 11-dehydrothromboxane B2 antibody and alkaline phosphatase, and store the connection at 4°C. When using, dilute it with enzyme labeling storage solution to the working concentration.

[0239] (2) Preparation of creatinine antibody labeled with alkaline phosphatase:

[0240] 1.0 mg of creatinine antibody was concentrated to 2.5 mg / mL, and 5 μL of activator 2-iminothiolane hydrochloride (2-IT) solution with a concentration of 13.76 mg / mL was added, and the mixture was reacted at room temperature for 15 minutes. The activated creatinine antibody was collected after desalting using a Sephadex G25 gel column.

[0241] 1.2 mg of alkaline phosphatase was concentrated to 2.5 mg / mL, and 12 μL of activator 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid succinimidyl ester (SMCC) solution with a concentration of 6.69 mg / mL was added, and the mixture was reacted at room temperature for 15 minutes. The activated alkaline phosphatase was collected after desalting using a Sephadex G25 gel column.

[0242] The activated creatinine antibody and alkaline phosphatase were mixed at a ratio of 1.0 mg of antibody to 1.0 mg of alkaline phosphatase (mass ratio of 1:1), and allowed to react at 4°C for 18 hours.

[0243] Use Supperdex200 gel chromatography column to separate and purify, remove unconnected creatinine antibody and alkaline phosphatase, and store the connection at 4°C. When using, dilute it with enzyme labeling storage solution to the working concentration.

[0244] 3. In addition to the magnetic bead-antigen complex and alkaline phosphatase-labeled antibody prepared in step 1 and step 2, the kit also contains a calibrator and a luminescent substrate solution. The calibrator is composed of a calibrator diluent and a mixed solution of different levels of 11-dehydrothromboxane B2 and different levels of creatinine. The luminescent substrate solution comes from the company's self-developed kit, and its main component is APS-5.

[0245] Example 5: Magnetic microparticle separation chemiluminescent immunoassay method of the kit

[0246] The steps of using the kit of Example 4 to detect the relative content of 11-dehydrothromboxane B2 in urine are as follows:

[0247] 1.11-Dehydrothromboxane B2 one-and-a-half-step detection procedure:

[0248] (a1) Immunoreaction: 50 μL of calibrator and 50 μL of alkaline phosphatase-labeled 11-dehydrothromboxane B2 antibody were added to a reaction tube in sequence, incubated at 37°C for 10 min, and then 50 μL of magnetic microparticles A coated with 11-dehydrothromboxane B2 derivatives were added, and incubated at 37°C for another 10 min, wherein the calibrator was a mixed solution consisting of a calibrator diluent and different levels of 11-dehydrothromboxane B2 (11-dehydrothromboxane B2 concentrations were 0 pg / mL, 100 pg / mL, 500 pg / mL, 2000 pg / mL, 5000 pg / mL, and 10000 pg / mL, respectively);

[0249] (a2) Magnetic separation: Place the incubated reaction tube in a magnetic field to allow the magnetic particles to settle in the magnetic field, remove the supernatant, remove the magnetic field, add 200-500 μL of cleaning solution, and then allow the magnetic particles to settle in the magnetic field again, and remove the supernatant; repeat this 2-4 times to remove other unreacted substances;

[0250] (a3) Reading: Add 200 μL of luminescent substrate solution and read the luminescence value (RLU) after 2 minutes;

[0251] (a4) A logistic four-parameter fit was used based on the detected values ​​to obtain a standard curve of 11-dehydrothromboxane B2 concentration-luminescence value.

[0252] (a5) Repeat the above steps (a1) to (a3) ​​to detect the urine sample to be tested and read the corresponding luminescence value (RLU);

[0253] (a6) Substituting the luminescence value of the urine sample to be tested into the Logistic four-parameter equation fitted in step (a4), the content of 11-dehydrothromboxane B2 in the sample to be tested can be calculated.

[0254] 2. Creatinine one-and-a-half-step test procedure:

[0255] (b1) Immune reaction: 50 μL of calibrator and 50 μL of alkaline phosphatase-labeled creatinine antibody were added to a reaction tube in sequence, incubated at 37°C for 10 min, and then magnetic particles B coated with creatinine derivatives were added, and incubated at 37°C for another 10 min, wherein the calibrator was a mixed solution consisting of a calibrator diluent (50 mmol / L PBST buffer) and different levels of creatinine (creatinine concentrations were 0 mg / dL, 0.5 mg / dL, 2.5 mg / dL, 5 mg / dL, 10 mg / dL, and 20 mg / dL, respectively);

[0256] (b2) Magnetic separation: Place the reaction tube in a magnetic field, allow the magnetic particles to settle in the magnetic field, remove the supernatant, remove the magnetic field, add 200-500 μL of cleaning solution, and then allow the magnetic particles to settle in the magnetic field again, and remove the supernatant; repeat this 2-4 times to remove other unreacted substances;

[0257] (b3) Reading: Add 200 μL of luminescent substrate solution and read the luminescence value (RLU) after 2 minutes;

[0258] (b4) A Logistic four-parameter fit was used according to the detected values ​​to obtain a creatinine concentration-luminescence value standard curve.

[0259] (b5) After the urine sample to be tested is diluted 10 times, 50 μL is taken for testing, and the above steps (b1) to (b3) are repeated to read the corresponding luminescence value (RLU);

[0260] (b6) Substitute the luminescence value of the diluted sample into the Logistic four-parameter equation fitted in step (b4) to calculate the creatinine content in the diluted sample. Multiply the result by 10 to obtain the creatinine concentration of the urine sample to be tested.

[0261] 3.11-dehydrothromboxane B2 / creatinine (11-dehydrothromboxane B2 relative content) result calculation formula:

[0262]

[0263] Example 6: Kit performance test

[0264] 1. Kit calibration curve

[0265] The kit of the present invention was used to measure calibrators containing different levels of 11-dehydrothromboxane B2 and different levels of creatinine according to the method described in Example 5, and the 11-dehydrothromboxane B2 calibration curves (such as Figure 1 ) and the creatinine calibration curve (as shown Figure 2 shown).

[0266] 2. Kit reproducibility

[0267] The kit of the present invention was used to repeat the determination of high and low quality control samples 10 times according to the method described in Example 5. The experimental results are shown in Table 6, indicating that the repeatability CV of the kit of the present invention was controlled within 5%, and the repeatability was good.

[0268] Table 6: Repeatability test data and analysis

[0269]

[0270] 3. Kit linearity

[0271] The high concentration calibrator of the kit is diluted proportionally with the calibrator diluent of the present invention, and the linearity of the kit is analyzed, such as Figure 3 , 4 The linearity of the 11-dehydrothromboxane B2 detection system (magnetic particles A and 11-dehydrothromboxane B2 antibody labeled with alkaline phosphatase) in the kit of the present invention can reach 10000 pg / mL, and the linearity of the creatinine detection system (magnetic particles B and creatinine antibody labeled with alkaline phosphatase) can reach 20 mg / dL.

[0272] 4.11-Dehydrothromboxane B2 Detection System Specificity

[0273] In order to verify that the 11-dehydrothromboxane B2 detection system (magnetic particles A and alkaline phosphatase-labeled 11-dehydrothromboxane B2 antibody) in the kit of the present invention has specificity for 11-dehydrothromboxane B2, the 11-dehydrothromboxane B2 detection system in the kit of the present invention was used to perform specific analysis on 2,3-dinor thromboxane B2, another metabolite of thromboxane A2 in urine. 2,3-dinor thromboxane B2 was diluted to a gradient concentration of 0.02 ng / mL, 0.2 ng / mL, 2 ng / mL, and 20 ng / mL using the calibrator diluent of the present invention, and tested using the 11-dehydrothromboxane B2 detection system of the present invention. Each gradient concentration was measured three times, and no concentration value was detected (data not shown), indicating that there was no cross reaction and the 11-dehydrothromboxane B2 detection system of the present invention had good specificity.

[0274] 5. Kit relevance

[0275] 29 urine samples to be tested were collected, and the 11-dhTXB2 concentration was measured using a commercial kit (ELISA method, from Corgenix) and the 11-dehydrothromboxane B2 detection system (magnetic particles A and alkaline phosphatase-labeled 11-dehydrothromboxane B2 antibody) in the kit of the present invention, and the creatinine concentration was measured using a commercially available enzymatic kit and the creatinine detection system (magnetic particles B and alkaline phosphatase-labeled creatinine antibody) in the kit of the present invention. The test data are shown in Table 7, and the correlation analysis is shown in Table 7. Figure 5 , Figure 6 As shown, the test results show that the kit of the present invention has high accuracy in detecting 11-dhTXB2 / CRE samples and can meet clinical detection needs.

[0276] Table 7: Correlation test data

[0277]

[0278] In summary, the present invention combines chemiluminescence detection technology with magnetic microparticle immunoseparation technology, so that the two indicators of urine 11-dehydrothromboxane B2 and urine creatinine can be detected synchronously on a luminometer based on the principle of immune competition method, which is easy to operate, eliminates the possibility of deviation of different measurement results due to detection time differences, has good repeatability and accuracy, and a wide linear range. The linearity of urine 11-dehydrothromboxane B2 detection can reach 10ng / mL. In addition, each detection system adopts the method of directly coating magnetic beads with corresponding small molecule derivatives, and the reagent has high sensitivity, is not interfered by endogenous biotin, and has strong specificity. It provides a more accurate, convenient and quick method for clinical evaluation of aspirin metabolism.

[0279] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims

1. An antibody or antigen-binding fragment, characterized in that The antibody or antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3; wherein, The HCDR1 is the amino acid sequence shown in SEQ ID NO: 1; The HCDR2 is the amino acid sequence shown in SEQ ID NO: 2; The HCDR3 is the amino acid sequence shown in SEQ ID NO: 3; The LCDR1 is the amino acid sequence shown in SEQ ID NO: 4; The LCDR2 is an amino acid sequence as shown in SEQ ID NO: 5; and The LCDR3 is the amino acid sequence shown in SEQ ID NO: 6; The antibody or antigen-binding fragment specifically binds to 11-dehydrogenated isocyanate B2.

2. The antibody or antigen-binding fragment according to claim 1, characterized in that The antibody or antigen-binding fragment comprises a heavy chain framework region and / or a light chain framework region.

3. The antibody or antigen-binding fragment according to claim 2, characterized in that: At least a portion of the heavy chain framework region and / or the light chain framework region is derived from at least one of a murine antibody or a mutant thereof and a human antibody or a mutant thereof.

4. The antibody or antigen-binding fragment according to claim 3, characterized in that The antibody or antigen-binding fragment comprises: A heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 7; and The amino acid sequence of the light chain variable region is shown in SEQ ID NO:

8.

5. The antibody or antigen-binding fragment according to claim 1, characterized in that: Further comprising a heavy chain constant region and / or a light chain constant region.

6. The antibody or antigen-binding fragment according to claim 5, characterized in that The heavy chain constant region comprises a heavy chain constant region selected from IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD; or The light chain constant region comprises a light chain constant region selected from a kappa-type or a lambda-type.

7. The antibody or antigen-binding fragment according to claim 6, characterized in that The light chain constant region and the heavy chain constant region are both derived from at least one of a murine antibody or a mutant thereof and a human antibody or a mutant thereof.

8. The antibody or antigen-binding fragment according to claim 7, characterized in that The N-terminus of the heavy chain constant region is connected to the C-terminus of the heavy chain variable region; and / or The N-terminus of the light chain constant region is connected to the C-terminus of the light chain variable region.

9. The antibody or antigen-binding fragment according to claim 8, characterized in that The amino acid sequence of the heavy chain constant region is shown in SEQ ID NO: 9; and / or The amino acid sequence of the light chain constant region is shown in SEQ ID NO:

10.

10. The antibody or antigen-binding fragment according to claim 9, characterized in that The antibody or antigen-binding fragment comprises: A heavy chain having an amino acid sequence as shown in SEQ ID NO: 11; and The light chain has an amino acid sequence as shown in SEQ ID NO:

12.

11. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the antibody or antigen-binding fragment according to any one of claims 1 to 10.

12. The nucleic acid molecule according to claim 11, characterized in that The nucleic acid molecule is DNA.

13. An expression vector, characterized in that: Carrying the nucleic acid molecule according to any one of claims 11 to 12.

14. The expression vector according to claim 13, characterized in that The expression vector includes one selected from a eukaryotic expression vector or a prokaryotic expression vector.

15. A recombinant cell, characterized in that The recombinant cell carries the nucleic acid molecule according to any one of claims 11 to 12 or the expression vector according to any one of claims 13 to 14; or Expressing the antibody or antigen-binding fragment according to any one of claims 1 to 10.

16. The recombinant cell according to claim 15, characterized in that The recombinant cell is obtained by introducing the expression vector according to any one of claims 13 to 14 into a host cell.

17. A method for preparing the antibody or antigen-binding fragment according to any one of claims 1 to 10, characterized in that: include: The recombinant cell according to any one of claims 15 to 16 is cultured under conditions suitable for protein expression and secretion to obtain the antibody or antigen-binding fragment.

18. Use of the antibody or antigen-binding fragment according to any one of claims 1 to 10 in the preparation of a kit for detecting 11-dehydrothromboxane B2.

19. A kit, characterized in that: include: At least one of the antibody or antigen-binding fragment of any one of claims 1 to 10, the nucleic acid molecule of any one of claims 11 to 12, the expression vector of any one of claims 13 to 14, the recombinant cell of any one of claims 15 to 16, and the antibody or antigen-binding fragment prepared according to the method of claim 17.

20. The kit according to claim 19, characterized in that The kit comprises the antibody or antigen-binding fragment according to any one of claims 1 to 10, first magnetic particles, creatinine antibodies, second magnetic particles, 11-dehydrothromboxane B2 calibrators, creatinine calibrators and luminescent substrate solution; The first magnetic particles are coated with 11-dehydrothromboxane B2 derivatives; the second magnetic particles are coated with creatinine derivatives.

21. The kit according to claim 20, characterized in that The antibody or antigen-binding fragment and the creatinine antibody are respectively labeled with a biomarker.

22. The kit according to claim 21, characterized in that The biomarker is selected from at least one of a biological enzyme, a fluorescein and a chemiluminescent marker.

23. The kit according to claim 22, characterized in that The biological enzyme is selected from at least one of horseradish peroxidase and alkaline phosphatase.

24. The kit according to claim 20, characterized in that The first magnetic particles and the second magnetic particles are respectively selected from magnetic microspheres.

25. The kit according to claim 24, characterized in that The surface of the magnetic microspheres is modified with active groups.

26. The kit according to claim 25, characterized in that The active group is selected from at least one of a hydroxyl group, a thiol group, a carboxyl group, an amino group, a silicon group, a tosyl group and streptavidin.

27. The kit according to claim 26, characterized in that The particle size of the magnetic microspheres is 0.5-5 μm.

28. The kit according to claim 20, characterized in that The luminescent substrate liquid is selected from at least one of AMPPD and APS-5.

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