CN118255891A

CN118255891A - Kit for detecting urine 11-dehydrothromboxane B2/creatinine and detection method

Info

Publication number: CN118255891A
Application number: CN202410294367.2A
Authority: CN
Inventors: 张静茹; 郭健夫; 任蔷; 何宋兵
Original assignee: Beijing Sisweide Biotechnology Co ltd
Current assignee: Beijing Sisweide Biotechnology Co ltd
Filing date: 2024-03-14
Publication date: 2024-06-28

Abstract

The invention relates to the field of biological detection and analysis, and provides a kit for detecting urine 11-dehydrothromboxane B2/creatinine and a detection method. In particular, the present invention provides an antibody or antigen binding fragment capable of specifically recognizing 11-dehydrothromboxane B2 comprising a CDR sequence from at least one of the following: CDR sequences of heavy chain variable region: SEQ ID NO. 1, SEQ ID NO. 2 and SEQ ID NO. 3; CDR sequences of the light chain variable region: SEQ ID NO. 4, SEQ ID NO. 5 and SEQ ID NO. 6. The detection kit prepared based on the antibody or the antigen binding fragment thereof 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 and simple operation, is not interfered by endogenous biotin, and provides a more accurate, convenient and quick method for clinically evaluating platelet activity or aspirin anti-platelet effect.

Description

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

Technical Field

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

Background

11-Dehydrothromboxane B2 (11-dehydro-thrombexane B2, 11-dhTXB 2) is the final metabolite of thromboxane A2 (thrombexane A2, TXA 2). In normal blood circulation, platelets are in a quiescent state, and after vascular injury, the platelet signaling pathway is activated, its membrane phospholipids are cleaved, arachidonic acid is released, and the arachidonic acid generates prostaglandin H by the action of platelet cyclooxygenase (COX-1 and COX-2). Prostaglandin H is chemically unstable and can be converted to a variety of biologically active prostanoids including TXA2 by the action of an isomerase. TXA2 is synthesized in platelets mainly through COX-1 pathway, has strong vasoconstriction effect, and can activate platelets through binding with thromboxane platelet receptor (thromboxane platelet receptor, TPR) to promote aggregation of the platelets, so that the thrombopoiesis effect is exerted. Aspirin, as the most widely used anti-platelet aggregation drug, can reduce TXA2 synthesis by irreversibly inhibiting COX-1 activity. However, TXA2 is highly unstable, has a very short biological half-life of only about 30 seconds, and is rapidly hydrolyzed to the more stable thromboxane B2 (TXB 2), and TXB2 is further metabolized to 11-dhTXB2 by the liver and excreted with urine. Thus, the urine 11-dhTXB2 content can directly reflect the generation level of TXA2 in the body, and evaluate the platelet activity and the anti-platelet effect of aspirin.

To make the measurement of urine 11-dhTXB2 more standard, it is necessary to correct the measured value with urinary creatinine (creatinine, CRE) concentration to reduce the influence of urine concentration differences or renal function due to diet and drinking water, i.e., to measure the urinary 11-dhTXB2 and urinary creatinine respectively, and calculate the relative creatinine. The conventional detection method of 11-dhTXB is High Performance Liquid Chromatography (HPLC), liquid chromatography-mass spectrometry (LC-MS), enzyme-linked immunoassay (ELISA), chemiluminescence method (CLIA), etc.; creatinine detection methods include picric acid method, dry chemical method, and enzyme method.

Analyzing the existing detection technology and measurement method, the following problems are found:

1) Limited by the instrument: since the measurement methods of 11-dehydrothromboxane B2 and urinary creatinine are established based on different methodologies, separate measurements are performed with different instruments.

2) The measuring steps are complicated: the need to use different instruments, as mentioned above, necessarily results in separate treatments of the same sample, in order to ensure the accuracy of the separate measurement of urinary 11-dehydrothromboxane B2 and urinary creatinine.

3) Measurement results are inaccurate: due to the need to use different instruments, the detection of urinary 11-dehydrothromboxane B2 and urinary creatinine must have a certain time difference, and the difference between urinary 11-dehydrothromboxane B2 and urinary creatinine at different time points can amplify the deviation of the 11-dehydrothromboxane B2/creatinine results.

Therefore, there is an urgent need to develop a novel method capable of simultaneously detecting the contents of 11-dehydrothromboxane B2 and urinary creatinine in the same instrument to improve the accuracy of the detection result.

Disclosure of Invention

The invention aims to overcome the defects of the prior art, and therefore, the invention develops an 11-dehydrothromboxane B2 antibody or antigen binding fragment capable of being combined with 11-dehydrothromboxane B2, the antibody or antigen binding fragment can be specifically combined with 11-dehydrothromboxane B2, and based on the invention, the 11-dehydrothromboxane B2 antibody or antigen binding fragment is utilized to prepare a urine 11-dehydrothromboxane B2/creatinine detection kit, and the kit is based on an immune competition method for the measurement of urine 11-dehydrothromboxane B2 and urine creatinine, and utilizes the principle of combining a chemiluminescence detection technology and a magnetic particle immune separation technology, so that the two indexes can be synchronously detected on a luminometer, and the kit has the advantages of good sensitivity, high accuracy, wide linear range, strong specificity, simplicity in operation and no interference by endogenous biotin, and provides a more accurate, convenient and quick method for clinical evaluation of platelet activity or aspirin anti-platelet effect.

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

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

In a third aspect of the invention, the invention provides a nucleic acid molecule. According to an embodiment of the invention, the nucleic acid molecule encodes an antibody or antigen binding fragment according to the first aspect, a fusion protein according to the second aspect. The nucleic acid molecule of the invention may encode an antibody or antigen binding fragment of the first aspect, a fusion protein of the second aspect.

In a fourth aspect of the invention, the invention provides an expression vector. According to an embodiment of the invention, the expression vector carries a nucleic acid molecule according to the third aspect. Thus, the expression vector of the invention can effectively realize the expression of the antibody or antigen binding fragment of the first aspect and the fusion protein of the second aspect, and further realize the in vitro mass acquisition of the antibody or antigen binding fragment or the fusion protein.

In a fifth aspect of the invention, the invention provides a recombinant cell. According to an embodiment of the invention, the recombinant cell comprises an expression vector carrying the nucleic acid molecule of the third aspect or the fourth aspect; or expressing the antibody or antigen-binding fragment of the first aspect or the fusion protein of the second aspect. The recombinant cells can be used to efficiently express the aforementioned antibodies or antigen-binding fragments, or fusion proteins in cells under appropriate conditions.

In a sixth aspect of the invention, the invention provides a method of making an antibody or antigen fragment. According to an embodiment of the invention, the method comprises culturing the recombinant cell of the fifth aspect under conditions suitable for protein expression and secretion in order to obtain the antibody or antigen-binding fragment. Thus, the methods of the invention can be used to produce the aforementioned antibodies or antigen-binding fragments.

In a seventh aspect of the invention, the invention provides a conjugate. According to an embodiment of the invention, the conjugate comprises an antibody or antigen-binding fragment according to the first aspect, a fusion protein according to the second aspect or an antibody or antigen-binding fragment prepared according to the method of the sixth aspect; and a coupling moiety attached to the antibody or antigen binding fragment, or the fusion protein. The conjugate of the invention has 11-dehydrothromboxane B2 binding affinity and can be used for detecting 11-dehydrothromboxane B2 and detecting cells containing 11-dehydrothromboxane B2.

In an eighth aspect of the invention, the invention provides the use of an antibody or antigen binding fragment according to the first aspect for the preparation of a kit for the detection of 11-dehydrothromboxane B2. According to embodiments of the invention, the antibody or antigen binding fragment is capable of binding 11-dehydrothromboxane B2 with high specificity. Thus, kits comprising the antibodies or antigen binding fragments can be used for high specificity, high sensitivity detection of 11-dehydrothromboxane B2.

In a ninth aspect of the application, the application provides a kit. According to an embodiment of the application, the kit comprises an antibody or antigen binding fragment according to the first aspect, a fusion protein according to the second aspect, a nucleic acid molecule according to the third aspect, an expression vector according to the fourth aspect, a recombinant cell according to the fifth aspect, an antibody or antigen binding fragment prepared according to the method according to the sixth aspect, or a conjugate according to the seventh aspect. According to an embodiment of the application, the antibody or antigen binding fragment thereof is capable of binding 11-dehydrothromboxane B2 with high specificity. In some scientific researches, the kit can be used for qualitatively or quantitatively detecting 11-dehydrothromboxane B2 in a biological sample, and can also be used for judging the state of an individual, such as judging whether the 11-dehydrothromboxane B2 level of the individual is higher or lower than the normal level after the 11-dehydrothromboxane B2 level of the individual is obtained.

In a tenth aspect of the invention, the invention provides a method for detecting the B2 content of 11-dehydrothromboxane. According to an embodiment of the invention, the method comprises: performing detection treatment on a sample to be detected by using the kit according to the ninth aspect so as to determine the content of the 11-dehydrothromboxane B2 in the sample to be detected. The method disclosed by the invention can be used for detecting the 11-dehydrothromboxane B2 with high sensitivity, is favorable for detecting the low-concentration 11-dehydrothromboxane B2, and has the advantages of strong specificity, wide linear range, simplicity in operation, no need of pretreatment on a sample and the like. In clinical practice, large batches of samples can be rapidly detected with high throughput by means of a chemiluminescent instrument.

In an eleventh aspect of the invention, the invention provides a method of detecting the relative content of 11-dehydrothromboxane B2. According to an embodiment of the invention, the method comprises: performing detection treatment on a sample to be detected by using the kit according to the ninth aspect so as to determine the relative content of the 11-dehydrothromboxane B2 in the sample to be detected. Because the 11-dehydrothromboxane B2 and the creatinine are small molecules, the degree of freedom of the small molecules is large, the exposure to the binding site of the antibody is not facilitated, and the binding of the antigen and the antibody is not facilitated, but the method disclosed by the invention can enable the small molecule antigen (11-dehydrothromboxane B2 and creatinine) in the sample to react with the enzyme-labeled antibody before the content of the 11-dehydrothromboxane B2 and the creatinine in the sample is measured, and then the small molecule derivative coated by the magnetic beads is added for competition, so that the detection of the low-concentration antigen is facilitated, and the reagent sensitivity is improved. In addition, the method has the advantages of strong specificity, wide linear range, simple operation, no need of pretreatment on the sample and the like. In clinical practice, large batches of samples can be rapidly detected with high throughput by means of a chemiluminescent instrument.

Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention.

Drawings

The foregoing and/or additional aspects and advantages of the invention will become apparent and may be better understood from the following description of embodiments taken in conjunction with the accompanying drawings in which:

FIG. 1 is a calibration graph of a kit 11-dehydrothromboxane B2 detection system according to an embodiment of the present invention;

FIG. 2 is a calibration graph of a kit creatinine detection system according to an embodiment of the present invention;

FIG. 3 is a linear diagram of a kit 11-dehydrothromboxane B2 detection system according to an embodiment of the present invention;

FIG. 4 is a linear diagram of a creatinine detection system of kit 1 according to an embodiment of the present invention;

FIG. 5 is a graph showing the correlation of the 11-dehydrothromboxane B2 detection system in a kit according to an embodiment of the present invention with a commercially available ELISA kit;

FIG. 6 is a graph showing the correlation of a creatinine detection system in a kit according to an embodiment of the present invention with a commercially available enzymatic kit;

FIG. 7 is a correlation of the 11-dehydrothromboxane B2/creatinine results of a kit according to an embodiment of the present invention with calculated results of a commercially available reagent;

FIG. 8 is a clone gel chart of subtype identification and antibody variable region of an antibody expressed by hybridoma cell line 11DH025 according to an embodiment of the present invention, the lane sequences being shown in Table 5.

Detailed Description

The invention will be further illustrated by means of specific examples. It should be noted that the examples described below are only for explaining the present invention, and are not limiting of the present invention.

In order that the invention may be more readily understood, certain technical and scientific terms are defined below. Unless clearly defined otherwise herein in this document, all other technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

In the present application, unless otherwise indicated, the terms "comprise" and "comprising" are open-ended terms that include the teachings of the present application, but do not exclude other aspects.

In the present application, unless otherwise indicated, the terms "optionally," "optional," or "optionally" generally refer to the occurrence of a subsequently described event or condition, which may but need not occur, and this description includes instances where the event or condition occurs, as well as instances where the event or condition does not occur.

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

The endpoints and any values of the ranges disclosed herein are not limited to the precise range or value, and are understood to encompass values approaching those ranges or values. For numerical ranges, one or more new numerical ranges may be found between the endpoints of each range, between the endpoint of each range and the individual point value, and between the individual point value, in combination with each other, and are to be considered as specifically disclosed herein.

As used herein, the term "fragment" refers to a protein or polypeptide of interest, as well as a protein or polypeptide of interest having an N-terminal (N-terminal) or C-terminal (C-terminal) truncation, and/or internal deletion.

In order that the invention may be more readily understood, certain technical and scientific terms are defined below. Unless clearly defined otherwise herein in this document, all other technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this 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.

As used herein, the term "antibody" is used in its broadest sense and may include full length monoclonal antibodies, multispecific antibodies, and chimeric antibodies, and the specific structure is not limited so long as they exhibit the desired biological activity. It generally comprises a light chain of relatively light molecular weight and a heavy chain of relatively heavy molecular weight, the heavy chain (H chain) and the light chain (L chain) being linked by disulfide bonds to form an antibody molecule. Wherein the amino-terminal (N-terminal) amino acid sequence of the peptide chain varies greatly and is called variable region (V region); the carboxy terminus (C-terminus) is relatively stable with little variation and is referred to as the constant region (C-region). The V chains of the L chain and H chain are referred to as VL and VH, respectively.

Herein, the heavy chain complementarity determining region (heavy chain variable region CDR) is denoted 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); light chain complementarity determining regions (light chain variable region CDRs) are denoted by "LCDRs" or "LCDRs," and include LCDR1 (also known as CDR-L1), LCDR2 (also known as CDR-L2) and LCDR3 (also known as CDR-L3). CDR definition schemes commonly used in the art include: kabat definition, chothia definition, IMGT definition, contact definition, and AbM definition.

Herein, the term "antibody variable region" refers to one domain in the heavy and light chains of an antibody. In nature, antibody variable regions are encoded by V, D (heavy chain only, applicable) and J fragments in immunoglobulin (heavy and light chain) genes by gene recombination splicing. The amino acid sequences of the variable regions between different antibodies are highly variable (the amino acid sequences of other regions of the antibodies are relatively highly identical) and are responsible for recognition and binding to specific epitopes. In the antibody variable region, framework regions and CDR regions can be subdivided (comlementarity determining region). A typical antibody variable region has 3 framework regions and 3 CDR regions (which are arranged in an alternating fashion). The framework regions serve primarily for protein domain framing, while the CDR regions serve primarily for antigen-antibody specific recognition binding.

As used herein, the term "antigen binding fragment" i.e., an "antibody fragment" is a fragment comprising a portion or all of an antibody that lacks at least some of the amino acids present in the full-length chain but is still capable of specifically binding an antigen, e.g., the fragment may comprise a portion or all of the CDRs of an antibody. Such fragments are biologically active in that 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 techniques, or may be produced by enzymatic or chemical cleavage of antigen binding molecules, including intact antibodies.

The antibody or antigen binding fragment, CDRs, light chain variable region or heavy chain variable region, etc. of the invention may be substituted, added and/or deleted for one or more (e.g., 1,2, 3, 4, 5, 6, 7, 8, 9 or 10 or more) amino acids by a person of skill in the art to obtain variants of the sequences of the antibody or functional fragment thereof without substantially affecting the activity of the antibody (retaining at least 80%, 90%, or 95% of the activity). They are all considered to be included within the scope of the present invention. Amino acids with conservatively modified forms are substituted, such as in the variable region. The variant sequences of the invention may have at least 80% identity (or homology) to a reference sequence. Sequence identity as described herein can be measured using sequence analysis software. Such as computer programs BLAST, in particular BLASTP or TBLASTN, using default parameters.

In this context, the amino acid sequences mentioned in the present invention are all shown in N-terminal to C-terminal fashion.

Antibodies or antigen binding fragments of the application are typically prepared by biosynthetic methods. The coding nucleic acids according to the application can be prepared by various known methods, conveniently by the person skilled in the art, based on the nucleotide sequences according to the application. Such as, but not limited to: PCR, DNA synthesis, etc., and specific methods can be found in J.Sam Brookfield, guidelines for molecular cloning experiments. As one embodiment of the present application, the coding nucleic acid sequence of the present application can be constructed by a method of synthesizing nucleotide sequences in segments followed by overlap extension PCR. Wherein the antibody or antigen fragment of the application is numbered and defined using the Kabat numbering system. However, CDRs determined based on the heavy and light chain variable regions of the present disclosure using other rules disclosed in the art are also within the scope of the present disclosure, as one of ordinary skill in the art is fully capable of converting the sequence Kabat numbering of the sequence listing to "HCDRs" and/or "LCDRs" under other numbering systems, including but not limited to AbM, IMGT, chothia.

As used herein, "Kabat definition" refers to the definition system described by Kabat et al, U.S. Dept. Of HEALTH AND Human Services, "Sequence of Proteins of Immunological Interest" (1983). "Chothia definition" see Chothia et al, J Mol Biol 196:901-917 (1987).

In this context, the term "conservatively modified form of an amino acid sequence" refers to an amino acid modification which does not significantly affect or alter the binding properties of an antibody comprising the amino acid sequence, including amino acid substitutions, additions and deletions. Modifications may 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 with an amino acid residue having 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 (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

In this context, the term "identity" is used to describe the percentage of identical amino acids or nucleotides between two amino acid sequences or nucleic acid sequences when compared to the amino acid sequence or nucleic acid sequence of a reference sequence, using conventional methods, e.g., see, ausubel et al, eds. (1995), current Protocols in Molecular Biology, chapter 19 (Greene Publishing and Wiley-Interscience, new York); and ALIGN program (Dayhoff(1978),Atlas of Protein Sequence and Structure 5：Suppl.3(National Biomedical Research Foundation,Washington,D.C.). there are many algorithms for aligning sequences and determining sequence identity, including, needleman et al (1970) J.mol. Biol.48:443 homology comparison algorithm; smith et al (1981) adv.appl.Math.2:482, a local homology algorithm; pearson et al (1988) Proc.Natl. Acad.Sci.85:2444 similarity search method; computer programs utilizing 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)), are also available and include, but are not limited to, ALIGN or Megalign (DNASTAR) software, or WU-BLAST-2 (Altschul et al, meth. Enzyme, 266:460-480 (1996)); or GAP, BESTFIT, BLAST Altschul et al, supra, FASTA, and TFASTA, available in the Genetics Computing Group (GCG) package, 8 th edition, madison, wisconsin, USA, and CLUSTAL in the PC/Gene programs provided by Intelligenetics, mountain View, california.

The term "at least 80% identity" as used herein refers to at least 80% identity to each reference sequence, which 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.

In this context, the term "fusion protein" refers to a novel protein in which at least two proteins or polypeptides are fused, and such fusion operations are usually achieved by techniques such as genetic engineering, for example, by recombinant expression products of two genes obtained by DNA recombination techniques.

In this context, the term "expression vector" generally refers to a nucleic acid molecule capable of insertion into a suitable host for self-replication, which nucleic acid molecule comprises a nucleotide sequence capable of expressing a protein of interest, and which nucleic acid molecule is capable of being transferred into and/or between host cells. The expression vector may include a vector mainly used for inserting DNA or RNA into cells, a vector mainly used for replicating DNA or RNA, and a vector mainly used for expression of transcription and/or translation of DNA or RNA. The expression vector also includes vectors having a plurality of the above functions. The expression vector may be a polynucleotide capable of transcription and translation into a polypeptide when introduced into a suitable host cell. Typically, the expression vector will produce the desired expression product by culturing a suitable host cell containing the expression vector.

As used herein, the term "recombinant cell" generally refers to a cell that has been modified or recombined with genetic material of a recipient cell (or host cell) using genetic engineering techniques or cell fusion techniques to obtain a unique trait of stable inheritance. Wherein the term "host cell" refers to a prokaryotic or eukaryotic cell into which a recombinant expression vector may be introduced. The term "transformed" or "transfected" as used herein refers to the introduction of a nucleic acid (e.g., an expression vector) into a cell by various techniques known in the art. Suitable host cells can be transformed or transfected with the DNA sequences of the invention and can be used for expression and/or secretion of a target protein. 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 (human amniotic fluid derived cells) and CoS cells.

As used herein, the terms "magnetic particles", "magnetic microspheres" and "magnetic beads" are used in the same sense and refer to colloidal composites that are uniformly dispersed in a matrix fluid. Because of the characteristics of superparamagnetism, higher specific surface area, modifiable functional groups and the like, antigens/antibodies, enzymes, nucleic acid/oligonucleotides, small molecule drugs and the like can be immobilized on the surfaces of the antibodies and enriched in a magnetic field.

For ease of expression herein, 11-dehydrothromboxane B2 is sometimes abbreviated as "11-dhTXB2" and creatinine as "CRE", the abbreviated form being indistinguishable from the original expressed form, unless otherwise indicated.

Detailed description of the kit for detecting urine 11-dehydrothromboxane B2 and creatinine according to the present invention

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

Antibodies or antigen binding fragments

According to some embodiments of the invention, the antibodies or antigen binding fragments comprise HCDR 1-3 and LCDR 1-3; wherein, the HCDR1 has an amino acid sequence shown as SEQ ID NO. 1; the HCDR2 has an amino acid sequence shown as SEQ ID NO. 2; the HCDR3 has an amino acid sequence shown as SEQ ID NO. 3; the LCDR1 has an amino acid sequence shown as SEQ ID NO. 4; the LCDR2 has an amino acid sequence shown as SEQ ID NO. 5; and said LCDR3 has an amino acid sequence as set forth in SEQ ID NO. 6.

In an alternative embodiment of the invention, the "amino acid sequence as shown in SEQ ID NO. 1" refers to an amino acid sequence that is identical to the amino acid sequence shown in SEQ ID NO. 1, and an amino acid sequence that has more than one conservative amino acid substitution compared to the amino acid sequence shown in SEQ ID NO. 1. "conservative amino acid substitution" refers to the substitution of an amino acid with another amino acid that is biologically, chemically, or structurally similar. Biologically similar means biologically active with 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 that have the same charge or are both hydrophilic or hydrophobic. For example, the hydrophobic residues isoleucine, valine, leucine or methionine are substituted for each other. Or with polar amino acids such as arginine for lysine, glutamic for aspartic acid, glutamine for asparagine, serine for threonine, and the like. 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.

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 1-2 amino acid differences therefrom.

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 1-2 amino acid differences therefrom.

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 1-2 amino acid differences therefrom.

According to an embodiment of the invention, the LCDR1 has an amino acid sequence as shown in SEQ ID NO. 4 or an amino acid sequence differing from it by 1-2 amino acids.

According to an embodiment of the invention, the LCDR2 has an amino acid sequence as shown in SEQ ID NO.5 or an amino acid sequence differing from it by 1-2 amino acids.

According to an embodiment of the invention, the LCDR3 has an amino acid sequence as shown in SEQ ID NO. 6 or an amino acid sequence differing from it by 1-2 amino acids.

In this context, the term "or an amino acid sequence having a1-2 amino acid difference thereto" means that there is a1 or 2 amino acid difference compared to the amino acid sequence described previously, which difference does not significantly affect or alter the binding properties of an antibody comprising the amino acid sequence, and such modifications include amino acid substitutions, additions and deletions.

According to an embodiment of the invention, the antibody or antigen binding fragment thereof specifically recognizes 11-dehydrothromboxane B2. In particular specifically recognizing an antigen having the sequence shown in SEQ ID NO. 14.

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.

According to some embodiments of the invention, at least a portion of the heavy and/or light chain framework regions is derived from at least one of a murine, primates, bovine, equine, dairy, porcine, ovine, caprine, canine, feline, rabbit, camel, donkey, deer, mink, chicken, duck, goose, turkey, or mutants thereof.

According to some embodiments of the 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 mutant thereof and a human antibody or mutant thereof.

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

According to some embodiments of the invention, further comprising a heavy chain constant region and/or a light chain constant region.

According to some embodiments of the invention, at least a portion of at least one of the heavy and light chain constant regions is derived from at least one of a murine, primates, bovine, equine, dairy, porcine, ovine, caprine, canine, feline, rabbit, camel, donkey, deer, mink, chicken, duck, goose, turkey, or a mutant thereof.

According to some embodiments of the invention, the heavy chain constant region comprises a heavy chain constant region selected from the group consisting of IgG1, igG2, igG3, igG4, igA, igM, igE, or IgD.

According to some embodiments of the invention, the light chain constant region comprises a light chain constant region selected from the group consisting of kappa-type and lambda-type.

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

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

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

According to some embodiments of the invention, the antibody comprises at least one of a polyclonal antibody, a full length mab, a F (ab ') ₂ antibody, a Fab' antibody, a Fab antibody, a F (ab) ₂ antibody, a Fv antibody, a single chain antibody, and a minimal recognition unit.

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

As used herein, the term "full length antibody", "full length monoclonal antibody" or "full length monoclonal antibody" is made up of at least two identical light chains and at least two identical heavy chains joined by interchain disulfide bonds, such as immunoglobulin G (IgG), immunoglobulin A (IgA), immunoglobulin M (IgM), immunoglobulin D (IgD) or immunoglobulin E (IgE).

The terms "polyclonal antibody" and "multispecific antibody" are synonymous herein, and refer to an antibody that recognizes multiple epitopes, for example, an antibody that recognizes two epitopes (bispecific antibody, abbreviated as "diabody"), an antibody that recognizes three epitopes, or an antibody that recognizes four epitopes, which is understood in a broad sense, and the specific structure is not limited, and may recognize multiple epitopes.

The terms "single domain antibody", "nanobody" and "VHH antibody" are used interchangeably herein, and are initially described as an antigen-binding immunoglobulin (variable) domain (Hamers-Casterman C,AtarhouchT,Muyldermans S,Robinson G,Hamers C,Songa EB,Bendahman N,Hamers R.:"Naturallyoccurring antibodies devoid of light chains";Nature 363,446-448(1993)), of a "heavy chain antibody" (i.e., an "antibody lacking a light chain") comprising a heavy chain variable region (VH) and conventional CH2 and CH3 regions, which specifically bind to an antigen protein via the heavy chain variable region.

In this context, the term "Fab antibody" or "Fab fragment" generally refers to an antibody or fragment comprising only Fab molecules, consisting of VH and CH1 of the heavy chain and the complete light chain, linked by a disulfide bond between the light and heavy chains.

As used herein, the term "F (ab ') 2 antibody" or "F (ab ') 2 fragment" has two antigen binding F (ab ') moieties linked together by disulfide bonds.

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

As used herein, the terms "single chain antibody", "scFv fragment" are antibodies or fragments that are made up of antibody heavy and light chain variable regions linked by short peptides.

As used herein, the terms "minimal recognition unit" and "MRU" refer to antibodies or fragments consisting of only one CDR, which have a molecular weight that is sufficiently small to account for only about 1% of the total antibody.

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

Fusion proteins

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

In this context, a "protein tag" generally refers to a polypeptide or protein that is expressed in fusion with a protein of interest (antibody or antigen binding fragment), which may be used for expression, detection, missing or purification of the protein of interest, etc. Including but not limited to His tags (also known as His-Tag, sequence HHHHHH), flag tags (also known as Flag-Tag, sequence DYKDDDDK), GST tags (also known as GST-Tag, glutathione-sulfhydryl transferase tags), MBP tags (also known as MBP-Tag, maltose binding protein tags), SUMO tags, C-Myc tags, and the like.

In this context, "Fc fragment" generally refers to an Fc region from IgG (e.g., igG1, igG2, igG3, or IgG4 subtype), igA1, igA2, igD, igE, or IgM, including the CH2, CH3 region, and optionally the hinge region. Preferably, the IgG, igA1, igA2, igD, igE or IgM is of murine, human, primate or alpaca origin.

According to some embodiments of the invention, the albumin or fragment thereof is serum albumin or fragment thereof.

Nucleic acid molecules, expression vectors, recombinant cells and methods for producing antibodies or antigen fragments

In preparing or obtaining these antibodies or antigen-binding fragments thereof, nucleic acid molecules expressing these antibodies or antigen-binding fragments may be used, linked to different vectors, and then expressed in different cells to obtain the corresponding antibodies or antigen-binding fragments thereof.

For this purpose, in a third aspect of the invention, the invention proposes a nucleic acid molecule. According to an embodiment of the invention, the nucleic acid molecule encodes an antibody or antigen binding fragment according to the first aspect, a fusion protein according to the second aspect. The nucleic acid molecules provided may be obtained by techniques known to those skilled in the art. But also can be optimized by species and is easier to express in mammalian cells.

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

It is noted that, for the nucleic acid molecules mentioned herein, one skilled in the art will understand that either one or both of the complementary double strands are actually included. For convenience, in the present description and claims, although only one strand is shown in most cases, the other strand complementary thereto is actually disclosed. In addition, the nucleic acid sequences of the present invention include DNA forms or RNA forms, one of which is disclosed, meaning the other is also disclosed.

In a fourth aspect of the invention, the invention provides an expression vector. According to an embodiment of the invention, the expression vector carries a nucleic acid molecule according to the third aspect. In the case of ligating the above-mentioned nucleic acid molecule to an expression vector, the nucleic acid molecule may be directly or indirectly linked to control elements on the expression vector, as long as these control elements are capable of controlling translation, expression, etc. of the nucleic acid molecule. These control elements may of course be derived directly from the expression vector itself or may be exogenous, i.e. not derived from the expression vector itself. Of course, the nucleic acid molecule may be operably linked to a control element.

"Operably linked" herein refers to linking the exogenous gene to the expression vector such that control elements within the expression vector, such as transcription control sequences and translation control sequences, and the like, are capable of performing their intended functions of regulating transcription and translation of the exogenous gene. The expression vector used in common can be, for example, a plasmid, phage, or the like.

According to some embodiments of the present invention, after the expression vector is introduced into a suitable receptor cell, the expression of the antibody or antigen binding fragment and the fusion protein can be effectively achieved under the mediation of a regulatory system, so that the in vitro mass acquisition of the antibody or antigen binding fragment and the fusion protein can be further achieved.

According to an embodiment of the invention, the expression vector comprises a vector selected from eukaryotic expression vectors or prokaryotic expression vectors.

In an alternative embodiment of the invention, the expression vector is a plasmid expression vector, a viral expression vector, such as a lentiviral expression vector.

In a fifth aspect of the invention, the invention provides a recombinant cell. According to an embodiment of the invention, the recombinant cell comprises an expression vector carrying the nucleic acid molecule of the third aspect or the fourth aspect; or expressing the antibody or antigen-binding fragment of the first aspect, the fusion protein of the second aspect. The recombinant cells can be used to efficiently express the aforementioned antibodies or antigen-binding fragments, or fusion proteins in cells under appropriate conditions.

"Suitable conditions" means conditions suitable for expression of the antibody or antigen-binding fragment, or fusion protein of the present invention. Those skilled in the art will readily appreciate that conditions suitable for expression of the antibody or antigen binding fragment, or fusion protein, include, but are not limited to, suitable transformation or transfection means, suitable transformation or transfection conditions, healthy host cell status, suitable host cell density, suitable cell culture environment, suitable cell culture time. The "suitable conditions" are not particularly limited, and those skilled in the art can optimize the conditions for expression of the above-described antibody or antigen-binding fragment, or fusion protein, optimally according to the specific environment of the laboratory.

According to some embodiments of the invention, the recombinant cell is obtained by introducing the expression vector of the fourth aspect into a host cell.

It should be noted that the recombinant cells of the present invention are not particularly limited, and may be prokaryotic cells, eukaryotic cells, or phage. The prokaryotic cell can be escherichia coli, bacillus subtilis, streptomycete or proteus mirabilis and the like. The eukaryotic cells comprise fungi such as pichia pastoris, saccharomyces cerevisiae, schizosaccharomyces, trichoderma and the like, insect cells such as armyworm and the like, plant cells such as tobacco and the like, and mammalian cells such as BHK cells, CHO cells, COS cells, myeloma cells and the like. In some embodiments, the recombinant cells of the 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.

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

Conjugates and kits

According to some embodiments of the invention, the conjugate moiety comprises at least one member selected from the group consisting of a carrier, a drug, a toxin, a cytokine, a protein tag, a modification, and a chemotherapeutic agent.

Herein, the carrier may be a substance capable of being suspended or dispersed in a liquid phase (for example, a solid-phase carrier such as particles or magnetic beads), or a solid phase capable of accommodating or carrying a liquid phase (for example, a support such as a plate, a membrane, a test tube, or a container such as an orifice plate, a microchannel, a glass capillary, a nanopillar, a monolith, or the like); also useful are labeling vectors for labeling antibodies or antigen binding fragments, or fusion proteins, such as enzymes (e.g., peroxidase, alkaline phosphatase, luciferase (luciferin), beta-galactosidase), affinity substances (e.g., one of streptavidin and biotin, one of the nucleic acids of the sense and antisense strands that are complementary to each other), fluorescent dyes (e.g., fluorescein isothiocyanate, rhodamine, green fluorescent protein, red fluorescent protein), luminescent substances (e.g., luciferin, aequorin (Aequorin), acridine, tris (2, 2' bipyridine) ruthenium, luminol), radioisotopes (e.g., 3H, 14C, 32P, 35S, 125I), gold colloids, and the like.

According to some embodiments of the invention, the carrier is selected from enzymes.

According to some embodiments of the invention, the enzyme is selected from any one or more of peroxidase, alkaline phosphatase, luciferase (luciferin), beta-galactosidase.

According to an embodiment of the invention, the drug is a small molecule drug that can bind to an antibody or antigen binding fragment, or fusion protein.

According to an embodiment of the present invention, the protein tag includes, but is not limited to, his tag, flag tag, GST tag, MBP tag, SUMO tag, C-Myc tag, and the like.

According to embodiments of the present invention, the modification is to be understood in a broad sense and may refer to a substance for modifying a protein. Illustratively, polyethylene glycol or derivatives thereof may be used.

The method for binding the conjugate moiety to the antibody or antigen-binding fragment, or the fusion protein may be any method known in the art. Examples thereof include physical adsorption, covalent bonding, and methods using affinity substances (e.g., biotin and streptavidin), and ion bonding.

The kit is prepared according to the principle of competition method, the antigen derivative coated with the magnetic particles competes with the antigen in the sample to be detected for the labeled antibody in the liquid phase, the magnetic particles are uniformly dispersed in the whole reaction system, and the specific binding rate of the antigen in the sample to be detected and the antibody is faster than the binding rate of the solid-phase antigen and the liquid-phase antibody due to the influence of the contact surface, so that the higher the rate difference is, the higher the inhibition rate is, namely the higher the sensitivity is, and the lower concentration of the antigen to be detected is more favorable.

As previously mentioned, the antibodies or antigen-binding fragments of some embodiments of the invention are effective in binding 11-dehydrothromboxane B2, and thus, reagents or kits comprising the antibodies or antigen-binding fragments are effective in the qualitative or quantitative detection of 11-dehydrothromboxane B2. The reagent or the kit provided by the invention can be used for example for immunoblotting, immunoprecipitation and the like, and relates to a reagent or a kit for detection by utilizing the specific binding property of 11-dehydrothromboxane B2 and an antibody.

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

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

According to some embodiments of the invention, the kit further comprises a first magnetic particle, a creatinine antibody, a second magnetic particle, an 11-dehydrothromboxane B2 calibrator, a creatinine calibrator, and a luminescent substrate solution.

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

According to some embodiments of the invention, the biomarker is selected from at least one of a biological enzyme, a fluorescein, and a chemiluminescent label.

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

According to some embodiments of the invention, the first magnetic particle is coated with a 11-dehydrothromboxane B2 derivative and the second magnetic particle is coated with a creatinine derivative.

According to some embodiments of the invention, the first magnetic particle and the second magnetic particle are each selected from magnetic microspheres.

The magnetic particles are magnetic beads with superparamagnetism and corresponding magnetic field responsiveness, and according to the types of groups on the surfaces of the magnetic beads, the magnetic beads comprise carboxyl magnetic beads, amino magnetic beads, silicon-based magnetic beads, tosyl magnetic beads, aldehyde magnetic beads, mercapto magnetic beads, streptavidin magnetic beads and the like, and carboxyl magnetic beads are preferred.

According to some embodiments of the invention, the magnetic microsphere surface is modified with reactive groups.

According to some embodiments of the invention, the reactive group is selected from at least one of hydroxyl, thiol, carboxyl, amino, silicon, tosyl, streptavidin, and derivatives thereof.

According to some embodiments of the invention, the magnetic microspheres have a particle size of 0.5 to 5 μm.

According to some embodiments of the invention, the magnetic microspheres have a particle size of 1-3 μm.

According to some embodiments of the invention, the first magnetic particle is the same as or different from the second magnetic particle. Wherein, the same means that the particle size and the surface groups of the magnetic beads are the same, and the particle sizes or active groups of different magnetic beads are different.

According to some embodiments of the invention, the substrate liquid is selected from at least one of AMPPD and APS-5.

Use and detection method

According to some embodiments of the invention, the detection process comprises: performing first mixing treatment on the sample to be tested and the antibody or antigen binding fragment, wherein the antibody or antigen binding fragment is marked by a biomarker; performing second mixing treatment on the first mixed treatment product and the first magnetic particles; settling the second mixed treatment product, and removing the supernatant to obtain a first precipitate; carrying out first contact treatment on the first precipitate and the luminescent substrate liquid, and detecting to obtain a first luminescence value RLU; and obtaining the content of 11-dehydrothromboxane B2 in the sample to be detected based on the first luminescence value RLU.

According to some embodiments of the invention, the method by which the antibody or antigen-binding fragment is labeled with a biomarker may be obtained based on any method known in the art, for example, adding the commonly used activator 2-Iminothiolane hydrochloride (2-IT, 2-iminothiolane hydrochloride) in the art to the antibody or antigen-binding fragment, adding the commonly used activator Succinimidyl- (N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC, 4- (N-maleimidomethyl) cyclohexane-1-carboxylic acid succinimidyl ester) in the biomarker, the mixing mass ratio of the activated biomarker to the antibody or antigen-binding fragment being (0.5-1.5): 1.

According to some embodiments of the invention, the detection process further comprises: drawing a standard curve of the concentration-luminous value of the 11-dehydrothromboxane B2 by using the 11-dehydrothromboxane B2 calibrator; wherein the 11-dehydrothromboxane B2 calibrator comprises a predetermined concentration gradient of 11-dehydrothromboxane B2.

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

According to some embodiments of the invention, the sample to be tested is urine.

In an eleventh aspect of the invention, the invention provides a method of detecting the relative content of 11-dehydrothromboxane B2. According to an embodiment of the invention, the method comprises: performing detection treatment on a sample to be detected by using the kit according to the ninth aspect so as to determine the relative content of the 11-dehydrothromboxane B2 in the sample to be detected. Because the 11-dehydrothromboxane B2 and the creatinine are small molecules, the degree of freedom of the small molecules is large, the exposure of the binding sites of the small molecules and the antibodies is not facilitated, and the binding of the antigen and the antibodies is also not facilitated, but the method disclosed by the invention can enable the small molecule antigen (11-dehydrothromboxane B2 and creatinine) in the sample to react with the enzyme-labeled antibody before the content of the 11-dehydrothromboxane B2 and the creatinine in the sample is measured, and then the small molecule derivative coated by the magnetic beads is added for competition, so that the detection of the antigen with low concentration is facilitated, and the reagent sensitivity is improved. In addition, the method has the advantages of strong specificity, wide linear range, simple operation, no need of pretreatment on the sample and the like. In clinical practice, large batches of samples can be rapidly detected with high throughput by means of a chemiluminescent instrument.

According to some embodiments of the invention, the detection process comprises: respectively carrying out first mixing treatment and second mixing treatment on the sample to be tested, the antibody or antigen binding fragment and the creatinine antibody, wherein the antibody or antigen binding fragment and the creatinine antibody are respectively marked by biological markers; performing third mixing treatment on the first mixed treatment product and the first magnetic particles, wherein the first mixed treatment product contains the antibody or antigen binding fragment; performing fourth mixing treatment on the second mixed treatment product and the second magnetic particles, wherein the second mixed treatment product contains creatinine antibodies; respectively carrying out sedimentation treatment on the third mixed treatment product and the fourth mixed treatment product, and removing supernatant to obtain a third precipitate and a fourth precipitate; carrying out first contact treatment and second contact treatment on the third precipitate and the fourth precipitate and the luminescent substrate liquid respectively, and detecting to obtain a first luminescence value RLU and a second luminescence value RLU; based on a first luminescence value RLU, obtaining the content of 11-dehydrothromboxane B2 in the sample to be detected; based on a second luminescence value RLU, obtaining the creatinine content in the sample to be detected; determining the relative content of 11-dehydrothromboxane B2 in the sample to be tested based on the following formula

According to some embodiments of the invention, the method employed for labeling the antibody or antigen-binding fragment and the creatinine antibody, respectively, with a biomarker may be based on any method known in the art, for example, adding the commonly used in the art activator 2-Iminothiolane hydrochloride (2-IT, 2-iminothiolane hydrochloride) to the antibody or antigen-binding fragment and the creatinine antibody, respectively, adding the commonly used in the art activator Succinimidyl- (N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC, 4- (N-maleimidomethyl) cyclohexane-1-carboxylic acid succinimidyl ester) to the biomarker, the ratio of the mass of the activated biomarker to the combined mass of the antibody or antigen-binding or fragment or the creatinine antibody, respectively, is (0.5-1.5): 1.

According to some embodiments of the invention, the detection process further comprises: respectively drawing a standard curve of 11-dehydrothromboxane B2 concentration-luminous value and a standard curve of creatinine concentration-luminous value by using the 11-dehydrothromboxane B2 calibrator and the creatinine calibrator; wherein the 11-dehydrothromboxane B2 calibrator comprises a predetermined concentration gradient of 11-dehydrothromboxane B2; the creatinine calibrator comprises a predetermined concentration gradient of creatinine.

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

In one example of the application, the 11-dehydrothromboxane B2 concentration-luminescence value standard curve is obtained by a method comprising:

(1) Immune response: sequentially adding 50. Mu.L of the 11-dehydrothromboxane B2 calibrator (the concentration of 11-dehydrothromboxane B2 is 0pg/mL,100pg/mL,500pg/mL,2000pg/mL,5000pg/mL,10000 pg/mL) and 50. Mu.L of the antibody or antigen binding fragment labeled with the biomarker into a reaction tube, incubating for 10min at 37 ℃, adding the first magnetic particles coated with the 11-dehydrothromboxane B2 derivative, and continuing to incubate for 10min at 37 ℃;

(2) Magnetic separation: placing the reaction tube in a magnetic field, settling the magnetic particles in the magnetic field, removing the supernatant, removing the magnetic field, adding 200-500 mu L of cleaning liquid, settling the magnetic particles in the magnetic field again, and removing the supernatant; repeating the steps for 2-4 times to remove unreacted other substances;

(3) Reading: 200. Mu.L of a luminescence substrate solution was added thereto, and after 2 minutes, a luminescence value (RLU) was read;

(4) A standard curve of the concentration-luminescence value of 11-dehydrothromboxane B2 is obtained by using Logistic four-parameter fitting according to the detected luminescence value.

In a twelfth aspect of the invention, the invention provides a method for detecting creatinine content. According to an embodiment of the invention, the method comprises: and carrying out detection treatment on the sample to be detected by adopting the kit in the ninth aspect so as to determine the creatinine content in the sample to be detected. The method provided by the invention can be used for detecting creatinine with high sensitivity, is favorable for detecting low-concentration creatinine, and has the advantages of strong specificity, wide linear range, simplicity in operation, no need of pretreatment on samples and the like. In clinical practice, large batches of samples can be rapidly detected with high throughput by means of a chemiluminescent instrument.

According to some embodiments of the invention, the detection process comprises: performing first mixing treatment on the sample to be tested and the creatinine antibody, wherein the creatinine antibody is marked by a biomarker; performing second mixing treatment on the first mixed treatment product and the first magnetic particles; settling the second mixed treatment product, and removing the supernatant to obtain a first precipitate; carrying out first contact treatment on the first precipitate and the luminescent substrate liquid, and detecting to obtain a first luminescence value RLU; and obtaining the creatinine content in the sample to be detected based on the first luminescence value RLU.

According to some embodiments of the invention, the method employed for labeling the creatinine antibody with a biomarker may be based on any method known in the art, for example, adding the activator 2-Iminothiolane hydrochloride (2-IT, 2-iminothiolane hydrochloride) commonly used in the art to the creatinine antibody, adding the activator Succinimidyl 4- (N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC, 4- (N-maleimidomethyl) cyclohexane-1-carboxylic succinimidyl ester) commonly used in the art to the biomarker, the mixing mass ratio of the activated biomarker to the antibody or antigen binding fragment being (0.5-1.5): 1.

According to some embodiments of the invention, the detection process further comprises: drawing a creatinine concentration-luminescence value standard curve by using the creatinine calibrator; wherein the creatinine calibrator comprises a predetermined concentration gradient of creatinine.

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

The detection kit and the detection method thereof have the following beneficial effects:

The invention is based on immune competition method for the measurement of urine 11-dehydrothromboxane B2 and urine creatinine, and utilizes the principle of combining the chemiluminescence detection technology and the magnetic particle immune separation technology to ensure that two indexes can be synchronously detected on a luminometer, has the advantages of good sensitivity, high accuracy, wide linear range, strong specificity and simple operation, is not interfered by endogenous biotin, and provides a more accurate, convenient and quick method for clinically evaluating platelet activity or aspirin anti-platelet effect.

Embodiments of the present invention are described in detail below. The following examples are illustrative only and are not to be construed as limiting the invention. The examples are not to be construed as limiting the specific techniques or conditions described in the literature in this field or as per the specifications of the product. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.

Wherein,

Full-automatic chemiluminescence analyzer: kesmal SMART 500H;

11-dehydrothromboxane B2 derivative: coupled with BSA, available from CD company, usa;

11-dehydrothromboxane B2: purchased from Cyman company, usa;

2,3-dinor thromboxane B2: purchased from Cyman company, usa;

creatinine antibodies: sheep polyclonal antibody, purchased from american CD company;

creatinine derivatives: coupled with BSA, available from CD company, usa;

Creatinine: purchased from Sigma;

Magnetic beads: with carboxyl groups, particle size 1.0-3.0 μm, available from JSR corporation of Japan;

alkaline phosphatase (ALP): purchased from BBI company, uk;

EDC: purchased from Sigma;

Luminescent substrate solution: the main component of the self-grinding reagent from the company is APS-5;

coating buffer solution: 0.05mmol/L MES (biological buffer), pH 6.0;

sealing liquid: 100mmol/L glycine, 10% BSA (bovine serum albumin), pH 7.4;

cleaning liquid: 10mmo/L PB (phosphate buffer), 0.05% Tw-20, pH 7.4;

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

Enzyme-labeled preservation solution: 50mmo/L PB (phosphate buffer), 1.0% BSA (bovine serum albumin), pH 7.4;

calibrator dilution: 50mmol/L PBST (phosphate buffer).

The amino acid sequences referred to herein are shown in table 1:

Table 1: amino acid sequence

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 ability of a person skilled in the art. This technique is well explained in the literature, as is the case for molecular cloning: laboratory Manual (Molecular Cloning: A Laboratory Manual), second edition (Sambrook et al, 1989); oligonucleotide Synthesis (Oligonucleotide Synthesis) (M.J.Gait, eds., 1984); animal cell Culture (ANIMAL CELL Culture) (r.i. freshney, 1987); the methods include methods of enzymology (Methods in Enzymology) (academic Press, inc. (ACADEMIC PRESS, inc.), manual of experimental immunology (Handbook of Experimental Immunology) (D.M.Weir and C.C. Blackwell, inc.), gene transfer Vectors for mammalian cells (GENE TRANSFER vector for MAMMALIAN CELLS) (J.M.Miller and M.P.Calos, inc., 1987), methods of contemporary molecular biology (Current Protocols in Molecular Biology) (F.M.Ausubel, et al, 1987), polymerase chain reaction (PCR: the Polymerase Chain Reaction) (Mullis, et al, 1994), and methods of contemporary immunology (Current Protocols in Immunology) (J.E.Coligan, et al, 2011), each of which are expressly incorporated herein by reference.

In the present embodiment, the nucleotide sequence used for preparing the expression vector may be obtained by a conventional method or a conventional software (e.g., on-line program Vectorbuilder (website: https:// www.vectorbuilder.cn/tool/code-optimization. Html), geneOptimizer on-line program, etc.) according to the amino acid sequence thereof.

Example 1: large-scale preparation of monoclonal antibodies

1. 11-Dehydrothromboxane B2 was coupled to KLH (hemocyanin) by the N-hydroxysuccinimide ester method as 11-dehydrothromboxane B2 immunogen.

2. BALB/c mice 6-8 weeks old were selected for the following immunization procedure: in the primary immunization, 25 mug 11-dehydrothromboxane B2 immunogen is mixed with equivalent Freund's complete adjuvant, and emulsified and injected subcutaneously in multiple points; 14 days after the first immunization, the immunization was boosted after emulsification by mixing 12.5. Mu.g of 11-dehydrothromboxane B2 immunogen with Freund's incomplete adjuvant. 14 days after the second immunization, the immunization was boosted after emulsification by mixing 12.5. Mu.g of 11-dehydrothromboxane B2 immunogen with Freund's incomplete adjuvant. The serum was collected and isolated 14 days after three immunizations, and the ELISA plate was coated with 1. Mu.g/mL of 11-dehydrothromboxane B2 derivative, and an indirect ELISA assay was performed to determine serum titers.

The results show that the prepared mouse antiserum has a titer of more than 7.2 ten thousand, and the typical serum detection results of immunized mice are shown in Table 2.

Table 2: serum test results of immunized mice

Serum dilution factor	OD₄₅₀
		100	1.754
300	1.707
		900	1.646
2700	1.548
		8100	1.388
24300	0.991
		72900	0.596
Blank space	0.137

3. Cell fusion was performed in the immunized mice as described above, and the specific procedure was as follows: after taking out the eyeballs of the mice to obtain blood, killing the mice through dislocation, placing the mice in a 70% alcohol bottle for 2 minutes, fixing the mice on a foam plate in a biosafety cabinet, unlocking the abdominal skin to find the spleen, taking the spleen out by forceps, putting the spleen into a 200-mesh stainless steel filter membrane to lightly grind the spleen, lightly flushing down cells by using a DMEM (Thermo, 11965092), centrifuging the cells at 200 Xg at room temperature for 10 minutes, and discarding the supernatant for later use; when preparing feeder cells, the mice are killed after dislocation, the mice are fixed on a foam plate in a biosafety cabinet after being placed in a 70% alcohol bottle for 2 minutes, the abdominal skin is untied, PBS is sucked by a syringe and gently injected under the peritoneum, liquid containing feeder cells is washed out from the other side, and then 200 Xg is centrifuged at room temperature for 10 minutes, and the supernatant is discarded for later use. 2.0X10 ⁷ FO myeloma cells were mixed with 2.0X10 ⁸ spleen cells, centrifuged at 200 Xg for 10 minutes in a centrifuge, the supernatant was discarded, mixed with gentle shaking, 1mL of 50% by volume PEG-1450 (Merk, P1458) aqueous solution was added dropwise over 90 seconds, then 20mL of DMEM medium was added dropwise, centrifuged at 200 Xg for 10 minutes in the centrifuge, the supernatant was discarded, washing was repeated once, centrifuged at 200 Xg for 10 minutes in the centrifuge, the supernatant was discarded to obtain hybridoma cells, and the cells were plated in 10 96-well plates at 150. Mu.L per well. 10000 cells/hole of feeder cells are added into the 10 96-hole cell culture plates, 100 mu L of each hole is marked, the culture plates are placed into a cell culture box containing 5% CO ₂ at 37 ℃ for culture, HAT screening culture medium (Merk, H0262) is added the next day, then HAT is used for selection culture for 1-2 days, a large number of tumor cells die after 3-4 days, tumor cells disappear, hybrid cells form a small colony, HT culture solution (Merk, H0137) is replaced after the HAT screening culture solution is maintained for 7-10 days, and further maintenance is performed for 2 weeks, and DMEM culture medium containing 20% FBS (Excell, FSP 500) is used for continuous culture. During the selection culture period, when the hybridoma cells are distributed to be 1/10 area of the hole bottom, specific antibodies can be detected, and the needed hybridoma cell lines can be screened out. During the selection culture, half of the culture medium is generally changed every 2 to 3 days.

4. The optimal coating amount of the 11-dehydrothromboxane B2 derivative as antigen was determined by orthogonal experiments. 0.5, 1.0, 2.0, 4.0. Mu.g of 11-dehydrothromboxane B2 derivative was coated in 96-well plates, and 6 wells at each concentration were set to 3 positives and 3 negatives, respectively. Orthotopic titration was performed with 11-dehydrothromboxane B2 immunized murine positive serum at different dilution fold, while non-immunized murine negative serum was used as negative control. Coating 96-well ELISA plates with 0.5 μg of purified 11-dehydrothromboxane B2 derivative per well, overnight at 4 ℃; PBST wash 2 times; 200 mu L of PBS containing 1% BSA is added into each hole, and the mixture is placed on a piece of folded paper for beating after being blocked for 2 hours at room temperature; sample adding: 0.1mL of the sample to be detected is added into the reaction well, the reaction well is incubated for 1 hour at 37 ℃, then the reaction well is washed, and blank wells (without adding the sample), negative control wells and positive control wells are simultaneously made. Each reaction well was added with 0.1mL of the newly diluted antibody, incubated at 37℃for 1 hour, and then washed 3 times. Adding enzyme-labeled secondary antibodies: each reaction well was added with 0.1mL of the newly diluted enzyme-labeled antibody. Incubate at 37℃for 1 hour and wash 3 times. Adding a substrate solution for color development: to each reaction well was added 0.1mL of TMB substrate solution, and the mixture was allowed to stand at room temperature for 10 minutes. 1M H ₂SO₄ 0.1: 0.1mL was added to each well. The OD value was measured and the absorbance was determined at 450nm using an ELISA reader (A450). Positive was found to be 2.1 times greater than the OD of the negative control (calculated after zeroing the blank control wells). Hybridoma cells were selected for monoclonal against 11-dehydrothromboxane B2.

The positive hybridoma cells obtained by screening were subcloned according to the above method, and the original wells were diluted with HAT selective medium by limiting dilution, and then re-divided into 96-well plates, followed by observation of the morphology and number of cells. The cells were adjusted to 3-10 cells/mL. The cell culture plates of the feeder cell layer prepared the day before were taken and 100. Mu.L of diluted cells were added to each well. The culture was allowed to stand at 37℃in a 5% CO ₂ incubator. The liquid was changed at day 7, and then 1 time every 2-3 days. The cell clone formation is seen in 8-9 days, and the antibody activity is detected in time. Cells from the positive wells were transferred to 24-well plates for expansion. Each clone should be frozen as soon as possible and finally clone number 11DH025 hybridoma cell line was selected for antibody production.

5. The method comprises the steps of injecting 0.5mL of Freund incomplete adjuvant into 8-week-old BALB/C mice in an intraperitoneal mode, injecting 1X 10 ⁶ hybridoma cells with clone number 11DH025 in the intraperitoneal mode after 2 weeks, inoculating the cells for 7-10 days, generating ascites, closely observing the health condition and ascites symptoms of the animals, killing the mice before the mice die as much as possible, sucking the ascites into a test tube by using a dropper, and obtaining 5-10 mL of ascites by one mouse. The ascites can also be extracted by a syringe, and can be repeatedly collected for a plurality of times. Centrifuging the obtained ascites at 3000 Xg for 10min, discarding upper layer oil and bottom precipitate, collecting supernatant, sub-packaging at-20deg.C, thawing the supernatant, balancing to room temperature, and adding 1/10 volume of 1M Tris-HCl pH8.0 to make pH of sample reach 8.0. The ascites supernatant after pH adjustment to 8.0 was loaded onto 20 Tris-HCl affinity columns with a column volume of 100mM pH=8.0, followed by washing with 20 Tris-HCl with a column volume of 100mM pH=8.0 and finally eluting the antibody with 100mM Glycine-HCl pH 2.5 (i.e.11-dehydrothromboxane B2 antibody 11DH025, abbreviated as antibody 11DH025 or 11DH 025). The antibody eluate was added to a centrifuge (Hunan instrument, L550) in a concentration tube (Millipore, UFC801008, 10K) and centrifuged at 3000 Xg for 20 minutes at room temperature. The solution was centrifuged in portions to a volume of 1 mL/concentrated tube (2 tubes), and 4mL of 10mM PBS pH=7.4 buffer was added, followed by centrifugation at 3000 Xg at room temperature for 20 minutes. Centrifugation was repeated 3 times to make the buffer of the antibody 10mM PBS PH=7.4, and 10mM PBS PH=7.4 was added to a total volume of 10mL. Finally, the concentrated antibody solution is split into 2 mL/tube and packed into a centrifuge tube for preservation at-80 ℃. The antibody concentration was measured using BCA kit (solebao, PC 0020) and the concentration of purified monoclonal antibody was 1.832mg/mL.

Example 2: antibody titer determination

The potency of 11-dehydrothromboxane B2 antibody 11DH025 was detected by ELISA indirect method. The 11-dehydrothromboxane B2 derivative was diluted with PBS and washed with PBST solution 2 times at 4℃overnight in 96-well ELISA plates at 0.2. Mu.g/mL and 100. Mu.L/Kong Baobei, each time at 300. Mu.L/well, and the plates were dried by pipetting. Blocking the ELISA plate with 1% BSA PBS solution at 200 μl/well, standing at room temperature for 2 hr, and drying; the 11-dehydrothromboxane B2 antibody 11DH025 diluted to 20ng/mL with PTB was added and reacted in an incubator at 37℃for 1 hour, and the ELISA plate was washed with PBST solution 2 times, 300. Mu.L/well each time. Cleaning and then drying. HRP-labeled goat anti-mouse antibody diluted 5000-fold with PTB was added, and the reaction was carried out in an incubator at 37 ℃ for 1 hour, and the elisa plate was washed 2 times with PBST solution, 300 μl/well each time. Cleaning and then drying. The reaction was terminated by adding 100. Mu.L/well of TMB substrate solution, 100. Mu.L/well of 1M H ₂SO₄, and allowing the reaction to proceed at room temperature for 10 minutes. Absorbance was measured at 450nm using a microplate reader (a 450). The results are shown in Table 3, and it can be seen that the antibody titer after purification is more than 160 ten thousand.

Table 3: antibody titer determination results

Example 3: sequence analysis of monoclonal antibodies

(1) Monoclonal antibody subtype identification

Hybridoma cell line 11DH025 was cultured in 10cm diameter cell culture dishes (37 ℃ C., 5% CO ₂) with DMEM medium (GIBCO, #C11995500 BT) supplemented with 10% serum. After 7 days of incubation, the cells were transferred to a 15ml centrifuge tube, 4X 10 ⁶ cells were removed after counting by a hemocytometer, centrifuged at 200 Xg for 5 minutes, the supernatant was discarded, and the tube was inverted and the tube was drained. The in-tube cells were synthesized with cDNA using the QIAGEN reverse transcription kit (Qiagen, 74134).

Antibody subtypes are determined by PCR with primers specific for the antibody subtype. The cDNA synthesized as described above was used as a template for PCR reaction. PCR reaction solution system: TAKARA Ex Taq (5U/. Mu.L, TAKARA, RR 001B), 0.25. Mu.L; 10 xEx Taq Buffer, 5. Mu.L; dNTP mix (2.5 mM each), 4. Mu.L; template cDNA, 1. Mu.L; upstream primer (100. Mu.M), 1. Mu.L; downstream primer (100. Mu.M), 1. Mu.L; double distilled water was added to a total volume of 50. Mu.L. PCR reaction temperature procedure: pre-denaturation at 94℃for 5min, temperature cycling 30 times (94℃for 1 min, 57℃for 1 min, 72℃for 1 min) and extension at 72℃for 10 min. After the reaction, 10. Mu.L of each PCR product was subjected to electrophoresis on a 1% agarose gel, and the order of the samples applied to each lane was as shown in Table 5. From the PCR product results, the subtype of the antibody can be deduced (table 4), see fig. 8 for specific results. The monoclonal antibody 11DH025 obtained by the invention has a heavy chain of IgG1 and a light chain of kappa.

Table 4: PCR primer information

Where s=c or G, m=a or C, r=a or G, and w=a or T.

Table 5: antibody information

(2) Hybridoma cell line 11DH025 antibody variable region (V region) sequencing

The fragment obtained by PCR amplification of the V region of the antibody of cell line 11DH025 in step (1) of this example was excised from the agarose gel and extracted with a DNA extraction kit (Qiagen, 74134). The extracted DNA fragment was ligated with pEASY-T1 cloning vector and transformed into Trans1-T1 competent cells (Transgen, CT 101-1). Transformed bacterial colonies were picked into LB medium and subjected to DNA sequencing after overnight culture. The nucleic acid sequence of the light chain V region of the antibody (11 DH 025) for recognizing 11-dehydrothromboxane B2 is shown as SEQ ID NO. 8, and the nucleic acid sequence of the heavy chain V region is shown as SEQ ID NO. 7.

Example 4: kit prepared by 11-dehydrothromboxane B2 antibody

1. Preparation of magnetic bead-antigen Complex (particle size 1.0-3.0 μm)

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 respectively used for detecting 11-dehydrothromboxane B2 and creatinine, and the preparation process is as follows:

Preparation of magnetic particles a: removing the supernatant by applying a magnetic field to the uniformly mixed magnetic beads (the concentration of the magnetic beads in the system is 0.01-5 mg/mL), washing the magnetic beads once by using a coating buffer solution, adding an activating agent EDC to 0.01-2mg/mL, performing a shaking reaction at normal temperature for 0.5-2h, adding the magnetic field to remove the supernatant, adding the coating buffer solution and a proper amount of 11-dehydrothromboxane B2 derivative (the concentration of the 11-dehydrothromboxane B2 derivative in the system is 0.1-5 mu g/mL), performing a shaking reaction at normal temperature for 1-3h, adding a magnetic field to remove the supernatant after the reaction is finished, adding a sealing solution, continuing the shaking reaction for 1-3h, washing the magnetic beads for 3 times by using a washing liquid after the sealing is finished, and finally diluting the magnetic particles A to the use concentration by using a coating preservation solution, and preserving the magnetic particles at 4 ℃.

Preparation of magnetic particles B: removing supernatant by applying a magnetic field to uniformly mixed magnetic beads (the concentration of the magnetic beads in a system is 0.01-5 mg/mL), washing the magnetic beads once by using a coating buffer solution, adding an activating agent EDC to 0.01-2mg/mL, performing a normal-temperature shaking reaction for 0.5-2h, adding the magnetic field to remove supernatant, adding the coating buffer solution and a creatinine derivative (the concentration of the creatinine derivative in the system is 0.1-5 mug/mL), performing a normal-temperature shaking reaction for 1-3h, adding a magnetic field to remove supernatant after the reaction is finished, adding a sealing solution, continuing the shaking reaction for 1-3h, washing the magnetic beads for 3 times by using a washing liquid after the sealing is finished, and finally diluting the magnetic particles A to the use concentration by using a coating preservation solution, and preserving the magnetic particles at 4 ℃.

2. Preparation of alkaline phosphatase-labeled antibodies

The alkaline phosphatase-labeled antibody refers to an alkaline phosphatase-labeled 11-dehydrothromboxane B2 antibody prepared in the previous example, or an alkaline phosphatase-labeled creatinine antibody, which is respectively combined with the 11-dehydrothromboxane B2 derivative connected to the magnetic particles A and the creatinine derivative connected to the magnetic particles B in the detection process, and is used for the detection of 11-dehydrothromboxane B2 and the detection of urinary creatinine, and the preparation processes thereof are as follows:

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

1.0mg of the 11-dehydrothromboxane B2 antibody obtained in the previous example was concentrated to 2.5mg/mL, 5. Mu.L of an activator 2-iminothiolane hydrochloride (2-IT) solution having a concentration of 13.76mg/mL was added thereto, and the mixture was reacted at room temperature for 15 minutes. The activated 11-dehydrothromboxane B2 antibody was collected by desalting using a Sephadex G25 gel column.

1.2Mg of alkaline phosphatase was concentrated to 2.5mg/mL, and 12. Mu.L of an activator 4- (N-maleimidomethyl) cyclohexane-1-carboxylic acid succinimidyl ester (SMCC) solution having a concentration of 6.69mg/mL was added thereto and reacted at room temperature for 15 minutes. The alkaline phosphatase was collected after desalting using a Sephadex G25 gel column.

The activated 11-dehydrothromboxane B2 antibody was mixed with alkaline phosphatase in a ratio of 1.0mg of the antibody to 1.0mg of alkaline phosphatase (mass ratio: 1:1), and reacted at 4℃for 18 hours.

The unbound 11-dehydrothromboxane B2 antibody and alkaline phosphatase were removed by separation and purification using Supperdex.sup.200 gel chromatography columns and the ligation was stored at 4 ℃. When in use, the enzyme-labeled preservative solution is diluted to the use concentration.

(2) Preparation of alkaline phosphatase-labeled creatinine antibodies:

1.0mg of creatinine antibody was concentrated to 2.5mg/mL, and 5. Mu.L of an activator 2-iminothiolane hydrochloride (2-IT) solution having a concentration of 13.76mg/mL was added thereto and reacted at room temperature for 15 minutes. The salt was removed using Sephadex G25 gel column and the activated creatinine antibodies were collected.

The activated creatinine antibody and alkaline phosphatase were mixed in a ratio of 1.0mg of the antibody to 1.0mg of alkaline phosphatase (mass ratio: 1:1), and reacted at 4℃for 18 hours.

The unbound creatinine antibody and alkaline phosphatase were removed by separation and purification using Supperdex200,200 gel chromatography columns and the ligation was stored at 4 ℃. When in use, the enzyme-labeled preservative solution is diluted to the use concentration.

3. Besides the magnetic bead-antigen complex and alkaline phosphatase labeled antibody prepared in the step 1 and the step 2, a calibrator and a luminescent substrate solution are also arranged in the kit, wherein the calibrator consists of a calibrator diluent and mixed solutions of 11-dehydrothromboxane B2 with different levels and creatinine with different levels, and the luminescent substrate solution is from a company self-research kit and mainly comprises APS-5.

Example 5: magnetic particle separation chemiluminescence immune detection method of kit

The procedure for detecting the relative content of 11-dehydrothromboxane B2 in urine using the kit of example 4 is as follows:

1.11-one-step half-method detection procedure for dehydrothromboxane B2:

(a1) Immune response: sequentially adding 50 mu L of calibrator and 50 mu L of alkaline phosphatase-labeled 11-dehydrothromboxane B2 antibody into a reaction tube, incubating for 10min at 37 ℃, adding 50 mu L of magnetic particles A coated with 11-dehydrothromboxane B2 derivatives, and continuously incubating for 10min at 37 ℃, wherein the calibrator consists of a mixed solution of calibrator diluent and different levels of 11-dehydrothromboxane B2 (the concentration of 11-dehydrothromboxane B2 is 0pg/mL,100pg/mL,500pg/mL,2000pg/mL,5000pg/mL and 10000pg/mL respectively);

(a2) Magnetic separation: placing the reaction tube after incubation in a magnetic field, settling the magnetic particles in the magnetic field, removing the supernatant, removing the magnetic field, adding 200-500 mu L of cleaning liquid, settling the magnetic particles in the magnetic field again, and removing the supernatant; repeating the steps for 2-4 times to remove unreacted other substances;

(a3) Reading: 200. Mu.L of a luminescence substrate solution was added thereto, and after 2 minutes, a luminescence value (RLU) was read;

(a4) A Logistic four-parameter fitting was used to obtain a standard curve of 11-dehydrothromboxane B2 concentration-luminescence value from the values detected.

(A5) Repeating the steps (a 1) - (a 3), detecting a urine sample to be detected, and reading a corresponding luminescence value (RLU);

(a6) And (3) bringing the luminescence value of the urine sample to be detected into the Logistic four-parameter equation fitted in the step (a 4), and then calculating the content of the 11-dehydrothromboxane B2 in the sample to be detected.

2. Creatinine one-step-half assay procedure:

(b1) Immune response: sequentially adding 50 mu L of calibrator and 50 mu L of alkaline phosphatase-labeled creatinine antibody into a reaction tube, incubating for 10min at 37 ℃, adding magnetic particles B coated with creatinine derivatives, and continuously incubating for 10min at 37 ℃, wherein the calibrator consists of a mixed solution of a calibrator diluent (50 mmol/L of PBST buffer) and creatinine with different levels (creatinine concentrations are 0mg/dL,0.5mg/dL,2.5mg/dL,5mg/dL,10mg/dL and 20mg/dL respectively);

(b2) Magnetic separation: placing the reaction tube in a magnetic field, settling the magnetic particles in the magnetic field, removing the supernatant, removing the magnetic field, adding 200-500 mu L of cleaning liquid, settling the magnetic particles in the magnetic field again, and removing the supernatant; repeating the steps for 2-4 times to remove unreacted other substances;

(b3) Reading: 200. Mu.L of a luminescence substrate solution was added thereto, and after 2 minutes, a luminescence value (RLU) was read;

(b4) A standard creatinine concentration-luminescence curve was obtained using Logistic four-parameter fitting from the detected values.

(B5) Diluting a urine sample to be tested by 10 times, taking 50 mu L of the urine sample to be tested, detecting, repeating the steps (b 1) - (b 3), and reading a corresponding luminescence value (RLU);

(b6) And (3) bringing the luminescence value of the diluted sample into the Logistic four-parameter equation fitted in the step (b 4), calculating the creatinine content in the diluted sample, and multiplying the result by 10 to obtain the creatinine concentration of the urine sample to be measured.

3.11-Dehydrothromboxane B2/creatinine (11-dehydrothromboxane B2 relative content) results calculation formula:

example 6: kit performance detection

1. Calibration curve of kit

Calibration samples containing different levels of 11-dehydrothromboxane B2 and different levels of creatinine were assayed using the kit of the present invention as described in example 5, yielding an 11-dehydrothromboxane B2 calibration curve (shown in FIG. 1) and a creatinine calibration curve (shown in FIG. 2), respectively.

2. Kit reproducibility

The quality control was measured at two levels, high and low, respectively, 10 times in duplicate using the kit of the present invention as described in example 5. As shown in Table 6, the results of the experiment show that the reproducibility CV of the kit of the present invention was controlled to be within 5% and the reproducibility was good.

Table 6: repeatability test data and analysis

3. Kit linearity

The high-concentration calibrator of the kit is diluted by the calibrator diluent according to the invention in proportion, and the kit is linear, as shown in figures 3 and 4. The linearity of the 11-dehydrothromboxane B2 detection system (magnetic particle A and alkaline phosphatase marked 11-dehydrothromboxane B2 antibody) in the kit disclosed by the invention can reach 10000pg/mL, and the linearity of the creatinine detection system (magnetic particle B and alkaline phosphatase marked creatinine antibody) can reach 20mg/dL.

4.11-Dehydrothromboxane B2 detection System specificity

To verify that the 11-dehydrothromboxane B2 detection system (magnetic particle A and alkaline phosphatase labeled 11-dehydrothromboxane B2 antibody) in the kit of the invention was specific for 11-dehydrothromboxane B2, the 11-dehydrothromboxane B2 detection system in the kit of the invention was used to conduct a specific analysis on another metabolite 2,3-dinor thromboxane B2 of thromboxane A2 in urine, 2,3-dinor thromboxane B2 was diluted to a gradient concentration of 0.02ng/mL, 0.2ng/mL, 2ng/mL and 20ng/mL with the calibrator dilution of the invention, and the 11-dehydrothromboxane B2 detection system of the invention was used to conduct a test, and the concentration of each gradient was repeatedly measured 3 times, and as a result, no concentration value was detected (data not shown), indicating that no cross reaction was present, the 11-dehydrothromboxane B2 detection system of the invention was better in specificity.

5. Kit relativity

The concentration of 11-dhTXB2 in the collected 29 urine samples to be tested was measured by using a commercial kit (ELISA method, from Corgenix) and the 11-dehydrothromboxane B2 detection system (magnetic particle A and alkaline phosphatase-labeled 11-dehydrothromboxane B2 antibody) in the kit of the present invention, respectively, and the concentration of creatinine was measured by using a commercial enzyme method kit and the creatinine detection system (magnetic particle B and alkaline phosphatase-labeled creatinine antibody) in the kit of the present invention, respectively. The detection data are shown in Table 7, the correlation analysis is shown in FIG. 5 and FIG. 6, and the detection result shows that the kit provided by the invention has higher accuracy in detecting 11-dhTXB/CRE samples, and can meet the clinical detection requirement.

Table 7: correlation detection data

In summary, the invention combines the chemiluminescence detection technology and the magnetic particle immune separation technology, so that two indexes of urine 11-dehydrothromboxane B2 and urinary creatinine can be synchronously detected on a luminometer based on the immune competition principle, the operation is simple and convenient, the possibility of deviation of different detection results due to detection time difference is eliminated, the repeatability and the accuracy are good, the linearity range is wide, wherein the urine 11-dehydrothromboxane B2 detection linearity can reach 10ng/mL, in addition, each detection system adopts a mode of directly coating magnetic beads by corresponding small molecule derivatives, the reagent sensitivity is high, the interference of endogenous biotin is avoided, and the specificity is strong. Provides a more accurate, convenient and quick method for clinically evaluating the metabolic capacity of aspirin.

While embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and not to be construed as limiting the invention, and that variations, modifications, alternatives and variations may be made to the above embodiments by one of ordinary skill in the art within the scope of the invention.

Claims

1. An antibody or antigen-binding fragment comprising a CDR sequence selected from at least one of the following:

CDR sequences of heavy chain variable region: SEQ ID NO.1, SEQ ID NO. 2 and SEQ ID NO. 3;

CDR sequences of the light chain variable region: SEQ ID NO. 4, SEQ ID NO. 5 and SEQ ID NO. 6.

2. The antibody or antigen-binding fragment of claim 1, wherein the antibody or antigen-binding fragment comprises HCDR 1-3 and LCDR 1-3; wherein,

The HCDR1 has an amino acid sequence shown as SEQ ID NO. 1;

the HCDR2 has an amino acid sequence shown as SEQ ID NO. 2;

the HCDR3 has an amino acid sequence shown as SEQ ID NO. 3;

the LCDR1 has an amino acid sequence shown as SEQ ID NO. 4;

the LCDR2 has an amino acid sequence shown as SEQ ID NO. 5; and

The LCDR3 has an amino acid sequence as shown in SEQ ID NO. 6.

3. The antibody or antigen-binding fragment of claim 2, wherein the antibody or antigen-binding fragment comprises a heavy chain framework region and/or a light chain framework region;

Optionally, at least a portion of the heavy chain framework region and/or light chain framework region is derived from at least one of a murine antibody, a primatized antibody, a bovine antibody, a equine antibody, a dairy-bovine antibody, a porcine antibody, a ovine antibody, a caprine antibody, a canine antibody, a feline 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 bucket chicken antibody, or a mutant thereof;

Optionally, 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 mutant thereof and a human antibody or mutant thereof;

Optionally, the antibody or antigen binding fragment comprises:

a heavy chain variable region with an amino acid sequence shown as SEQ ID NO. 7; and

Has a light chain variable region with an amino acid sequence shown as SEQ ID NO. 8.

4. The antibody or antigen-binding fragment of claim 1, further comprising a heavy chain constant region and/or a light chain constant region;

Optionally, at least a portion of at least one of the heavy and light chain constant regions is derived from at least one of a murine, primates, bovine, equine, dairy, porcine, ovine, caprine, canine, feline, rabbit, camel, donkey, deer, mink, chicken, duck, goose, turkey, bucket, or mutant thereof;

Optionally, the heavy chain constant region comprises a heavy chain constant region selected from the group consisting of IgG1, igG2, igG3, igG4, igA, igM, igE, or IgD; or alternatively

The light chain constant region comprises a light chain constant region selected from the group consisting of kappa-type or lambda-type;

optionally, the light chain constant region and the heavy chain constant region are both derived from at least one of a murine antibody or mutant thereof and a human antibody or mutant thereof;

optionally, the N-terminus of the heavy chain constant region is linked to the C-terminus of the heavy chain variable region; and/or

The N-terminal of the light chain constant region is connected with the C-terminal of the light chain variable region;

Optionally, the heavy chain constant region has an amino acid sequence as set forth in SEQ ID NO. 9; and/or

The light chain constant region has an amino acid sequence shown as SEQ ID NO. 10;

Optionally, the antibody comprises at least one of a polyclonal antibody, a full length monoclonal antibody, a F (ab ') ₂ antibody, a Fab' antibody, a Fab antibody, a F (ab) ₂ antibody, an Fv antibody, a single chain antibody, and a minimal recognition unit; or alternatively

The antigen binding fragments include at least one of a F (ab ') ₂ fragment, a Fab' fragment, a Fab fragment, a F (ab) ₂ fragment, an Fv fragment, an scFv-Fc fusion protein, an scFv-Fv fusion protein, and a minimal recognition unit;

Optionally, the antibody or antigen binding fragment comprises:

Has a heavy chain with an amino acid sequence shown as SEQ ID NO. 11; and

Has a light chain with an amino acid sequence shown as SEQ ID NO. 12.

5. A fusion protein comprising:

the antibody or antigen-binding fragment of any one of claims 1-4; and

A biologically active protein or fragment thereof;

optionally, the biologically active protein or fragment thereof comprises at least one selected from the group consisting of a protein tag, albumin or fragment thereof, and an Fc fragment.

6. A nucleic acid molecule encoding the antibody or antigen-binding fragment of any one of claims 1 to 4, the fusion protein of claim 5;

optionally, the nucleic acid molecule is DNA.

7. An expression vector carrying the nucleic acid molecule of claim 6;

Optionally, the expression vector comprises a vector selected from eukaryotic expression vectors or prokaryotic expression vectors.

8. A recombinant cell comprising:

Carrying the nucleic acid molecule of claim 6 or the expression vector of claim 7; or (b)

Expressing the antibody or antigen-binding fragment of any one of claims 1 to 4 or the fusion protein of claim 5;

Optionally, the recombinant cell is obtained by introducing the expression vector of claim 7 into a host cell.

9. A method of making the antibody or antigen-binding fragment of any one of claims 1-4, comprising:

Culturing the recombinant cell of claim 8 under conditions suitable for protein expression and secretion to obtain the antibody or antigen-binding fragment.

10. A conjugate, comprising:

the antibody or antigen-binding fragment of any one of claims 1-4, the fusion protein of claim 5, or the antibody or antigen-binding fragment prepared according to the method of claim 9; and

A coupling moiety attached to the antibody or antigen binding fragment, or the fusion protein;

optionally, the conjugate moiety comprises at least one selected from the group consisting of a carrier, a drug, a toxin, a cytokine, a protein tag, a modification, and a chemotherapeutic agent.

11. Use of the antibody or antigen binding fragment of any one of claims 1 to 4 in the preparation of a kit for the detection of 11-dehydrothromboxane B2.

12. A kit, comprising:

The antibody or antigen-binding fragment of any one of claims 1-4, the fusion protein of claim 5, the nucleic acid molecule of claim 6, the expression vector of claim 7, the recombinant cell of claim 8, the antibody or antigen-binding fragment prepared according to the method of claim 9, or the conjugate of claim 10.

13. The kit of claim 12, further comprising a first magnetic particle;

optionally, further comprising a creatinine antibody, a second magnetic particle, an 11-dehydrothromboxane B2 calibrator, a creatinine calibrator, and a luminescent substrate solution;

Optionally, the antibody or antigen-binding fragment and the creatinine antibody, respectively, are labeled with a biomarker;

optionally, the biomarker is selected from at least one of a biological enzyme, a fluorescein, and a chemiluminescent label;

Optionally, the biological enzyme is selected from at least one of horseradish peroxidase and alkaline phosphatase;

optionally, the first magnetic particles are coated with an 11-dehydrothromboxane B2 derivative;

optionally, the second magnetic particles are coated with a creatinine derivative;

Optionally, the first magnetic particle and the second magnetic particle are each selected from magnetic microspheres;

optionally, the surface of the magnetic microsphere is modified with active groups;

Optionally, the reactive group is selected from at least one of hydroxyl, thiol, carboxyl, amino, silicon-based, tosyl, streptavidin, and derivatives thereof;

optionally, the magnetic microspheres have a particle size of 0.5-5 μm;

Optionally, the luminescent substrate liquid is selected from at least one of AMPPD and APS-5.

14. A method for detecting the B2 content of 11-dehydrothromboxane, comprising;

Performing detection treatment on a sample to be detected by using the kit according to claim 12 or 13 so as to determine the content of the 11-dehydrothromboxane B2 in the sample to be detected.

15. The method of claim 14, wherein the detecting process comprises:

performing first mixing treatment on the sample to be tested and the antibody or antigen binding fragment, wherein the antibody or antigen binding fragment is marked by a biomarker;

Performing second mixing treatment on the first mixed treatment product and the first magnetic particles;

settling the second mixed treatment product, and removing the supernatant to obtain a first precipitate;

carrying out first contact treatment on the first precipitate and the luminescent substrate liquid, and detecting to obtain a first luminescence value RLU;

Based on a first luminescence value RLU, obtaining the content of 11-dehydrothromboxane B2 in the sample to be detected;

Optionally, the biological enzyme is selected from at least one of horseradish peroxidase and alkaline phosphatase.

16. The method of claim 15, wherein the detecting process further comprises:

Drawing a standard curve of the concentration-luminous value of the 11-dehydrothromboxane B2 by using the 11-dehydrothromboxane B2 calibrator; wherein,

The 11-dehydrothromboxane B2 calibrator comprises a predetermined concentration gradient of 11-dehydrothromboxane B2;

optionally, the step of obtaining the content of 11-dehydrothromboxane B2 in the sample to be tested based on the first luminescence value RLU comprises:

Calculating the content of 11-dehydrothromboxane B2 in the sample to be detected based on the standard curve of the concentration-luminous value of 11-dehydrothromboxane B2 and the first luminous value RLU;

optionally, the sample to be tested is urine.

17. A method for detecting the relative content of 11-dehydrothromboxane B2, comprising;

Performing detection treatment on a sample to be detected by using the kit according to claim 12 or 13 so as to determine the relative content of the 11-dehydrothromboxane B2 in the sample to be detected.

18. The method of claim 17, wherein the detecting process comprises:

Respectively carrying out first mixing treatment and second mixing treatment on the sample to be tested, the antibody or antigen binding fragment and the creatinine antibody, wherein the antibody or antigen binding fragment and the creatinine antibody are respectively marked by biological markers;

Performing third mixing treatment on the first mixed treatment product and the first magnetic particles, wherein the first mixed treatment product contains the antibody or antigen binding fragment;

performing fourth mixing treatment on the second mixed treatment product and the second magnetic particles, wherein the second mixed treatment product contains creatinine antibodies;

respectively carrying out sedimentation treatment on the third mixed treatment product and the fourth mixed treatment product, and removing supernatant to obtain a third precipitate and a fourth precipitate;

Carrying out first contact treatment and second contact treatment on the third precipitate and the fourth precipitate and the luminescent substrate liquid respectively, and detecting to obtain a first luminescence value RLU and a second luminescence value RLU;

Based on a second luminescence value RLU, obtaining the creatinine content in the sample to be detected;

determining the relative content of 11-dehydrothromboxane B2 in the sample to be tested based on the following formula

19. The method of claim 18, wherein the detecting process further comprises:

Respectively drawing a standard curve of 11-dehydrothromboxane B2 concentration-luminescence value and a standard curve of creatinine concentration-luminescence value by using the 11-dehydrothromboxane B2 calibrator and the creatinine calibrator; wherein,

The creatinine calibrator comprises a predetermined concentration gradient of creatinine;

Optionally, the step of obtaining the creatinine content in the sample to be measured based on the second luminescence value RLU includes:

Calculating the creatinine content in the sample to be detected based on the creatinine concentration-luminescence value standard curve and the second luminescence value RLU;

optionally, the sample to be tested is urine.