Method for detecting non-carboxylated osteocalcin and detection kit

Abstract

The application belongs to the field of biological detection. Specifically, it relates to a non-carboxylated osteocalcin detection method and a detection kit. In the application, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region, and the CDR sequence of the heavy chain variable region has an amino acid sequence as shown in SEQ ID NO: 1-3 or an amino acid sequence with at least 80% identity thereto. The antibody or antigen-binding fragment thereof can be used for specifically detecting non-carboxylated osteocalcin. The detection kit prepared based on the antibody or antigen-binding fragment thereof can rapidly detect the content of non-carboxylated osteocalcin in a serum or plasma sample, has high sensitivity and specificity, is simple to operate, has low requirements for sample pretreatment, and is suitable for rapid high-throughput detection of a large number of samples.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biological detection, and particularly relates to a non-carboxylated osteocalcin detection method and a detection kit. BACKGROUND

[0002] Osteocalcin (OC) is a non-collagenous bone matrix protein synthesized and secreted by non-proliferative osteoblasts in bone. The precursor protein is composed of a 23-amino acid signal peptide, a 28-amino acid propeptide, and a 49-amino acid mature protein. During the modification stage after expression, the signal peptide is hydrolyzed, and the propeptide recognition site binds to carboxylase to carboxylate Glu at positions 17, 21, and 24 into Gla, also known as bone gamma-carboxyglutamic acid protein. The mature osteocalcin molecule contains 49 amino acids and has a molecular weight of about 5800 Da. Osteocalcin accounts for about 1% to 2% of the total protein in the human body, and its expression is affected by age, gender, and menopausal status. Osteocalcin has the effect of promoting bone mineralization and is an important component of bone mineralization matrix. There are various OCs in the blood, including uncarboxylated or decarboxylated osteocalcin, fully carboxylated osteocalcin, and osteocalcin fragments. Determining the content of serum osteocalcin can reflect the activity of osteoblasts and the degree of bone turnover. The faster the bone turnover rate, the higher the OC value, and vice versa. Therefore, osteocalcin is used as a common marker of bone turnover and a physiological indicator of bone transformation in clinical practice.

[0003] Osteocalcin is also involved in energy metabolism of sugar, fat, and the like. Under the acidification effect of the bone resorption process, the osteocalcin molecule loses the glutamic acid group, and its carboxylation degree decreases, increasing the content of incompletely carboxylated osteocalcin in the circulation. Such incompletely carboxylated osteocalcin is considered to be an active endocrine hormone and a bridge for the skeleton to participate in endocrine regulation activities in the human body. Uncarboxylated osteocalcin can promote islet cell proliferation and insulin expression, reduce blood glucose levels, improve glucose tolerance, and increase insulin sensitivity, while also increasing adiponectin content in the body and reducing serum triglycerides, thereby avoiding diet-induced obesity. Low-carboxylated osteocalcin can act on adipose tissue to inhibit hepatic glycogen synthesis, and then promote fatty acid oxidation in skeletal muscle and liver, increase glucose uptake in skeletal muscle to improve the sensitivity of peripheral tissues to glucose, and has important significance for the study of endocrine diseases such as obesity, diabetes, and other metabolic syndromes that are currently prevalent. In addition, non-carboxylated osteocalcin can act on the testis and Leydig cells, thereby promoting the biosynthesis of testosterone and having an impact on male reproduction.

[0004] Osteocalcin exists in two forms, namely carboxylated osteocalcin and non-carboxylated osteocalcin. It is difficult to screen an antibody with good specificity that can distinguish between the two forms, which in turn makes it difficult to determine positive serum of non-carboxylated osteocalcin in clinical practice. The diagnostic reagents for non-carboxylated osteocalcin in the industry mostly rely on imports, which are expensive.

[0005] The known non-carboxylated osteocalcin determination methods include radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), latex-enhanced immunoturbidimetry, etc. The radioimmunoassay has complicated steps, expensive reagents, requires a matched instrument and has radioactive pollution. The enzyme-linked immunosorbent assay has long detection time, complicated operation and poor repeatability. The latex-enhanced immunoturbidimetry has low sensitivity and poor repeatability of low values.

[0006] At present, it is urgent to develop a non-carboxylated osteocalcin antibody with high specificity and sensitivity and a rapid quantitative detection method for non-carboxylated osteocalcin. SUMMARY

[0007] The present application aims to solve one of the problems in the related art to some extent. To this end, the present application develops a non-carboxylated osteocalcin antibody with high specificity and sensitivity and the use of the antibody in specific detection of non-carboxylated osteocalcin.

[0008] Specifically, the present application provides the following technical solutions:

[0009] In the first aspect of the present application, an antibody or an antigen binding fragment thereof is provided. According to the embodiments of the present application, it comprises a heavy chain variable region, and the CDR sequence of the heavy chain variable region has an amino acid sequence as shown in SEQ ID NO: 1-3 or an amino acid sequence with at least 80% identity thereto. In some examples of the present application, the antibody or the antigen binding fragment thereof can be used for specific detection of non-carboxylated osteocalcin.

[0010] In the second aspect of the present application, a nucleic acid molecule is provided. According to the embodiments of the present application, the nucleic acid molecule encodes the antibody or the antigen binding fragment thereof of the first aspect of the present application. In some examples of the present application, the antibody or the antigen binding fragment thereof encoded by the nucleic acid molecule can be used for specific detection of non-carboxylated osteocalcin.

[0011] In the third aspect of the present application, an expression vector is provided. According to the embodiments of the present application, the expression vector carries the nucleic acid molecule of the second aspect of the present application. In some examples of the present application, the expression vector can efficiently express the antibody or the antigen binding fragment thereof in a suitable host cell, so as to be used for specific detection of non-carboxylated osteocalcin or preparation of a drug related to non-carboxylated osteocalcin disease.

[0012] In a fourth aspect, the present application provides a method for preparing the antibody or antigen-binding fragment thereof of the first aspect. According to embodiments of the present application, the method comprises: introducing the expression vector of the third aspect into a cell; culturing the cell under conditions suitable for protein expression and secretion, so as to obtain the antibody or antigen-binding fragment thereof. In some examples of the present application, the antibody or antigen-binding fragment thereof can be prepared in large quantities in vitro by the method.

[0013] In a fifth aspect, the present application provides a recombinant cell. According to embodiments of the present application, the recombinant cell carries the antibody or antigen-binding fragment thereof of the first aspect, the nucleic acid molecule of the second aspect, or the expression vector of the third aspect. In some examples of the present application, the recombinant cell can efficiently express the above-mentioned antibody or antigen-binding fragment thereof under suitable conditions, and the antibody or antigen-binding fragment thereof can specifically bind to uncarboxylated osteocalcin.

[0014] In a sixth aspect, the present application provides an immunoconjugate. According to embodiments of the present application, the immunoconjugate comprises: the antibody or antigen-binding fragment thereof of the first aspect, the nucleic acid molecule of the second aspect, the expression vector of the third aspect, or the recombinant cell of the fifth aspect. In some examples of the present application, the antibody or antigen-binding fragment thereof can specifically bind to uncarboxylated osteocalcin, and can be used to prepare a prophylactic and / or therapeutic agent for preventing and / or treating diseases related to uncarboxylated osteocalcin.

[0015] In a seventh aspect, the present application provides a composition. According to embodiments of the present application, the composition comprises: the antibody or antigen-binding fragment thereof of the first aspect, the nucleic acid molecule of the second aspect, the expression vector of the third aspect, the recombinant cell of the fifth aspect, or the immunoconjugate of the sixth aspect. In some examples of the present application, the antibody or antigen-binding fragment thereof can specifically bind to uncarboxylated osteocalcin. In some examples of the present application, the composition comprising the antibody or antigen-binding fragment thereof, such as a food composition, a pharmaceutical composition, etc., also has a significant effect on preventing and / or treating diseases related to uncarboxylated osteocalcin.

[0016] In an eighth aspect of the present application, the present application provides use of the antibody or antigen binding fragment thereof of the first aspect, the nucleic acid molecule of the second aspect, the expression vector of the third aspect, the recombinant cell of the fifth aspect, the immunoconjugate of the sixth aspect or the composition of the seventh aspect in the preparation of a medicament for treating or preventing a disease associated with non-carboxylated osteocalcin. In some embodiments of the present application, the antibody or antigen binding fragment thereof is capable of specifically binding to non-carboxylated osteocalcin. It is understood that the medicament comprising the antibody or antigen binding fragment thereof and the like has a significant effect on treating or preventing a disease associated with non-carboxylated osteocalcin, such as bone and joint disease.

[0017] In a ninth aspect of the present application, the present application provides a medicament. According to embodiments of the present application, the medicament comprises the antibody or antigen binding fragment thereof of the first aspect, the nucleic acid molecule of the second aspect, the expression vector of the third aspect, the recombinant cell of the fifth aspect, the immunoconjugate of the sixth aspect or the composition of the seventh aspect, and the medicament is used for treating or preventing a disease associated with non-carboxylated osteocalcin. In some embodiments of the present application, the antibody or antigen binding fragment thereof is capable of specifically binding to non-carboxylated osteocalcin. It is understood that the medicament comprising the antibody or antigen binding fragment thereof and the like has a significant effect on treating or preventing a disease associated with non-carboxylated osteocalcin, such as bone and joint disease.

[0018] In a tenth aspect of the present application, the present application provides use of the antibody or antigen binding fragment thereof of the first aspect in the preparation of a kit for detecting non-carboxylated osteocalcin. In some embodiments of the present application, the antibody or antigen binding fragment thereof is capable of specifically binding to non-carboxylated osteocalcin. Therefore, the kit comprising the antibody or antigen binding fragment thereof can be used for specifically and sensitively detecting non-carboxylated osteocalcin.

[0019] In an eleventh aspect of the present application, the present application provides a kit. According to embodiments of the present application, the kit comprises the antibody or antigen binding fragment thereof of the first aspect. In some embodiments of the present application, the antibody or antigen binding fragment thereof is capable of specifically binding to non-carboxylated osteocalcin. In some scientific researches, the kit can be used for qualitatively or quantitatively detecting non-carboxylated osteocalcin protein in a biological sample, and can also be used for judging the state of an individual, such as judging whether the non-carboxylated osteocalcin level of the individual is higher or lower than the normal level after obtaining the non-carboxylated osteocalcin level of the individual.

[0020] In a twelfth aspect, the present application provides a method for detecting non-carboxylated osteocalcin. According to an embodiment of the present application, the method comprises: using the kit of the eleventh aspect of the present application to detect a sample to be tested, so as to determine the content of the non-carboxylated osteocalcin in the sample to be tested. In some examples of the present application, the above method is used for high-sensitivity detection of non-carboxylated osteocalcin, and is environmentally friendly, specific, simple to operate, and requires low sample pretreatment. In clinical practice, a large number of samples can be rapidly and high-throughput detected by means of a chemiluminescence instrument.

[0021] Additional aspects and advantages of the present application will be in part apparent and in part pointed out hereinafter. BRIEF DESCRIPTION OF DRAWINGS

[0022] The above and / or additional aspects and advantages of the present application will become apparent and be readily appreciated from the following description, including the appended drawings, wherein:

[0023] Figure 1 is a schematic diagram of the detection results of antibody subtypes according to an embodiment of the present application;

[0024] Figure 2 is a schematic diagram of the process of the method for detecting non-carboxylated osteocalcin by magnetic micro-particle separation and chemiluminescence immunoassay according to an embodiment of the present application. DETAILED DESCRIPTION

[0025] Embodiments of the present application are described in detail below with reference to the attached drawings. The embodiments described below are examples for explaining the present application and are not to be construed as limiting the present application.

[0026] In order to make the present application more easily understood, certain technical and scientific terms are specifically defined below. Unless otherwise clearly indicated otherwise in the present document, all other technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which the present application belongs.

[0027] In the present application, unless otherwise specified, the term "comprising" or "including" is an open-ended expression, i.e., it includes the contents indicated in the present application, but does not exclude other contents.

[0028] In the present application, unless otherwise specified, the term "optionally", "optional" or "optional" generally means that the event or condition described subsequently can but does not necessarily occur, and the description includes both cases where the event or condition occurs and cases where the event or condition does not occur.

[0029] In this application, unless otherwise stated, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one of that feature. In the description of this application, "multiple" means at least two, such as two, three, etc., unless otherwise explicitly specified.

[0030] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0031] In this application, unless otherwise stated, the term "antibody or antigen-binding fragment thereof" refers to a protein encoded by an animal gene and biosynthesized. In some examples of this application, the antibody is selected from polyclonal antibodies or monoclonal antibodies, and the antigen-binding fragment is selected from F(ab')2 fragment, Fab' fragment, Fab fragment, F(ab)2 fragment, Fv fragment, scFv fragment, scFv-Fc fusion protein, scFv-Fv fusion protein, or the smallest recognition unit.

[0032] The term "monoclonal antibody" (or monoclonal antibody) mentioned above refers to an antibody that can recognize only one specific antigenic epitope. Common monoclonal antibodies consist of two lighter light chains and two heavier heavy chains. The heavy chain (H chain) and light chain (L chain) are linked by disulfide bonds to form a tetrapeptide molecule. The amino-terminal (N-terminus) amino acid sequence of this heavy or light chain varies considerably and is called the variable region (V region). The carboxyl-terminus (C-terminus) of this heavy or light chain is relatively stable and varies little, and is called the constant region (C region). The V regions of the L chain and H chain are called VL and VH, respectively. The amino acid sequences of the variable regions are highly different between different antibodies (while the amino acid sequences of other regions of the antibody are relatively highly similar), and these regions are responsible for recognizing and binding to specific antigenic determinants. The antibody variable region can be further subdivided into the backbone region and the CDR region (completional determination region). A typical antibody variable region has three backbone regions and three CDR regions (interleaved with each other). The backbone region mainly plays a role in the formation of protein structural domains, while the CDR region mainly plays a role in the specific recognition and binding of antigens and antibodies.

[0033] The "polyclonal antibody" (or "polyclonal antibody") as referred to above means an antibody that can recognize multiple epitopes, for example, an antibody that can recognize two epitopes (referred to as a bispecific antibody), an antibody that can recognize three epitopes, or an antibody that can recognize four epitopes, which is a broad understanding and the specific structure is not limited, as long as it can recognize multiple epitopes.

[0034] The "antigen binding fragment" as referred to above means an antibody fragment that retains the ability to specifically bind to an antigen.

[0035] The "Fab fragment" as referred to above generally refers to an antibody fragment that binds to an antigen, which is composed of VH and CH1 of the heavy chain and the complete light chain, and the light chain and the heavy chain are connected by a disulfide bond.

[0036] The "Fv fragment" as referred to above generally refers to an antibody fragment that is only connected by a non-covalent bond between the light chain variable region (VL) and the heavy chain variable region (VH), which is the smallest functional fragment of an antibody that retains the complete antigen binding site.

[0037] The "scFv fragment" as referred to above refers to an antibody fragment connected by a connecting peptide between the heavy chain variable region (VH) and the light chain variable region (VL).

[0038] The "minimal recognition unit" as referred to above means an antibody that is only composed of one CDR, and the molecular weight is very small, accounting for only about 1% of the complete antibody.

[0039] In the present application, unless otherwise specified, the amino acid sequences of the CDRs listed in the present application are shown according to the IMGT definition rule. However, it is well known to those skilled in the art that the CDR of an antibody can be defined by various methods in the art, such as the Kabat rule, the Chothia rule, etc. Those skilled in the art should understand that, unless otherwise specified, the terms "CDR" and "complementary determining region" of a given antibody or region (such as a variable region) thereof are understood to encompass the complementary determining region defined by any of the above known schemes described in the present application. Although the scope claimed in the present application is based on the sequence shown according to the IMGT definition rule, the amino acid sequences corresponding to the definition of other CDRs should also be included in the scope of protection of the present application.

[0040] As described herein, the "IMGT definition", Lefranc et al. introduced a new standardized numbering system for all protein sequences of the immunoglobulin superfamily, see Lefranc, et al., The Immunologist, 7, 132-136 (1999). The "Kabat definition" refers to the system of definitions described by Kabat et al., U.S. Dept. of Health and Human Services, "Sequences of Proteins of Immunological Interest" (1983). The "Chothia definition" see Chothia et al., J Mol Biol 196:901-917 (1987).

[0041] In the present application, the abbreviations for amino acid residues are the standard 3-letter and / or 1-letter codes used in the art to designate one of the 20 common L-amino acids, having the following meanings: A: Ala (alanine); R: Arg (arginine); N: Asn (asparagine); D: Asp (aspartic acid); C: Cys (cysteine); Q: Gin (glutamine); E: Glu (glutamic acid); G: Gly (glycine); H: His (histidine); I: He (isoleucine); L: Leu (leucine); K: Lys (lysine); M: Met (methionine); F: pHe (phenylalanine); P: Pro (proline); S: Ser (serine); T: Thr (threonine); W: Trp (tryptophan); Y: Tyr (tyrosine); V: Val (valine).

[0042] In the present application, the term "identity" when used to describe an amino acid sequence or a nucleic acid sequence relative to a reference sequence, is determined by the percent of identical amino acids or nucleotides between two amino acid sequences or nucleic acid sequences by conventional methods, for example, see, Ausubel et al., eds. (1995), Current Protocols in Molecular Biology, Chapter 19 (Greene Publishing and Wiley-Interscience, New York); and the ALIGN program (Dayhoff (1978), Atlas of Protein Sequence and Structure 5: Suppl. 3 (National Biomedical Research Foundation, Washington, D.C.). There are many algorithms for aligning sequences and determining sequence identity, including the homology alignment algorithm of Needleman et al. (1970) J. Mol. Biol. 48:443; the local homology algorithm of Smith et al. (1981) Adv. Appl. Math. 2:482; the search for similarity method of Pearson et al. (1988) Proc. Natl. Acad. Sci. 85:2444; the Smith-Waterman algorithm (Meth. Mol. Biol. 70:173-187 (1997); and the BLAST family of algorithms (see Altschul et al. (1990) J. Mol. Biol. 215:403-410). Computer programs are also available that utilize these algorithms, and include, but are not limited to: the ALIGN or Megalign (DNASTAR) software, or the WU-BLAST-2 (Altschul et al., Meth. Enzym., 266:460-480 (1996)); or GAP, BESTFIT, BLAST Altschul et al., supra, FASTA, and TFASTA, available in the Genetics Computing Group (GCG) package, Version 8, Madison, Wisconsin, USA; and CLUSTAL in the PC / Gene program from Intelligenetics, Mountain View, California.

[0043] In the present application, one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) amino acids can be substituted, added, and / or deleted from the sequences of the present application by one skilled in the art without substantially affecting the activity of the antibody (retaining at least 95% activity) to obtain variants of the sequences of the antibody or functional fragments thereof. They are all considered to be included in the scope of protection of the present application. Amino acids with similar properties are substituted in the variable region. The variant sequences described in the present application can have at least 80% identity (or homology) with the reference sequences, which means at least 80%, which can 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 with each reference sequence. The sequence identity described in the present application can be measured using sequence analysis software. For example, using the computer program BLAST, especially BLASTP or TBLASTN with default parameters. The amino acid sequences referred to in the present application are shown in the order of N-terminal to C-terminal.

[0044] In the present application, unless otherwise specified, the term "conservative amino acid substitution" refers to the substitution of an amino acid by another amino acid which is biologically, chemically or structurally similar. Biologically similar means that the substitution does not destroy the biological activity of the non-carboxylated osteocalcin antibody or the non-carboxylated osteocalcin antigen. Structurally similar means that the amino acids have similar length side chains, such as alanine, glycine or serine, or have similar size side chains. Chemical similarity means that the amino acids have the same charge or are both hydrophilic or hydrophobic. For example, hydrophobic residues isoleucine, valine, leucine or methionine are substituted for each other. Or use polar amino acids such as arginine to replace lysine, glutamic acid to replace aspartic acid, glutamine to replace asparagine, serine to replace threonine, etc.

[0045] In one aspect of the present application, an antibody or antigen-binding fragment thereof is provided, comprising: a heavy chain variable region; the CDR sequences of the heavy chain variable region have the amino acid sequences as shown in SEQ ID NO: 1-3 or amino acid sequences having at least 80% identity thereto. In some examples of the present application, the antibody or antigen-binding fragment thereof can be used for specific detection of non-carboxylated osteocalcin. Among them, the amino acid sequences are shown in Table 1.

[0046] In some examples of the present application, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region CDR1 sequence as set forth in the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having at least 80% identity to SEQ ID NO: 1, respectively. In one specific example of the present application, the heavy chain variable region CDR1 sequence of the antibody or antigen-binding fragment thereof is as set forth in SEQ ID NO: 1. In some examples of the present application, the antibody has higher affinity and specificity by using the heavy chain variable region CDR1 sequence as set forth in SEQ ID NO: 1.

[0047] In some examples of the present application, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region CDR2 sequence as set forth in the amino acid sequence of SEQ ID NO: 2 or an amino acid sequence having at least 80% identity to SEQ ID NO: 2, respectively. In one specific example of the present application, the heavy chain variable region CDR2 sequence of the antibody or antigen-binding fragment thereof is as set forth in SEQ ID NO: 2. In some examples of the present application, the antibody has higher affinity and specificity by using the heavy chain variable region CDR2 sequence as set forth in SEQ ID NO: 2.

[0048] In some examples of the present application, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region CDR3 sequence as set forth in the amino acid sequence of SEQ ID NO: 3 or an amino acid sequence having at least 80% identity to SEQ ID NO: 3, respectively. In one specific example of the present application, the heavy chain variable region CDR3 sequence of the antibody or antigen-binding fragment thereof is as set forth in SEQ ID NO: 3. In some examples of the present application, the antibody has higher affinity and specificity by using the heavy chain variable region CDR3 sequence as set forth in SEQ ID NO: 3.

[0049] In some examples of the present application, the antibody or antigen-binding fragment thereof further comprises a light chain variable region; the light chain variable region CDR sequence has an amino acid sequence as set forth in SEQ ID NO: 4-6 or an amino acid sequence having at least 80% identity thereto.

[0050] In some examples of the present application, the antibody or antigen-binding fragment thereof comprises a light chain variable region CDR1 sequence as set forth in the amino acid sequence of SEQ ID NO: 4 or an amino acid sequence having at least 80% identity to SEQ ID NO: 4, respectively. In one specific example of the present application, the light chain variable region CDR1 sequence of the antibody or antigen-binding fragment thereof is as set forth in SEQ ID NO: 4. In some examples of the present application, the antibody has high specific binding and recognition to different antigens by synergistic effect with the heavy chain variable region by using the light chain variable region CDR1 sequence as set forth in SEQ ID NO: 4.

[0051] In some examples of the application, the antibody or antigen-binding fragment thereof comprises a light chain variable region CDR2 sequence as set forth in, or having at least 80% identity to, the amino acid sequence set forth in SEQ ID NO: 5, respectively. In one specific example of the application, the antibody or antigen-binding fragment thereof light chain variable region CDR2 sequence is as set forth in SEQ ID NO: 7. In some examples of the application, the light chain variable region CDR2 sequence as set forth in SEQ ID NO: 5, in cooperation with the heavy chain variable region, achieves highly specific binding and recognition of different antigens by the antibody.

[0052] In some examples of the application, the antibody or antigen-binding fragment thereof comprises a light chain variable region CDR3 sequence as set forth in, or having at least 80% identity to, the amino acid sequence set forth in SEQ ID NO: 6, respectively. In one specific example of the application, the antibody or antigen-binding fragment thereof light chain variable region CDR3 sequence is as set forth in SEQ ID NO: 6. In some examples of the application, the light chain variable region CDR3 sequence as set forth in SEQ ID NO: 6, in cooperation with the heavy chain variable region, achieves highly specific binding and recognition of different antigens by the antibody.

[0053] In some examples of the application, the antibody or antigen-binding fragment thereof further comprises a heavy chain framework region sequence, at least a portion of which is derived from at least one of a murine antibody, a rabbit antibody, a primate antibody, or a mutant thereof. In some examples of the application, the antibody comprising the framework region helps to increase the structural stability of the antibody.

[0054] In some preferred examples of the application, at least a portion of the heavy chain framework region sequence is derived from a murine antibody.

[0055] In some examples of the application, the heavy chain variable region amino acid sequence is as set forth in SEQ ID NO: 7.

[0056] In some examples of the application, the antibody or antigen-binding fragment thereof amino acid sequence has one or more conservative amino acid substitutions compared to SEQ ID NO: 7. Alternatively, two or three conservative amino acid substitutions. Of course, these conservative amino acid substitutions do not alter the biological function of the antibody or antigen-binding fragment. In some specific examples of the application, these conservative amino acid substitutions can occur in the amino acids of the heavy chain variable region and the light chain variable region, except for the CDR regions.

[0057] In some examples of the present application, the antibody or antigen-binding fragment thereof further contains a light chain framework region sequence, at least a portion of which is derived from at least one of a murine antibody, a rabbit antibody, a primate antibody, or a mutant thereof. In some examples of the present application, the antibody containing the framework region helps to increase the structural stability of the antibody.

[0058] In some examples of the present application, the light chain variable region amino acid sequence is as shown in SEQ ID NO: 8.

[0059] In some examples of the present application, the antibody or antigen-binding fragment thereof further contains a heavy chain constant region, at least a portion of which is derived from at least one of a murine antibody, a rabbit antibody, a primate antibody, or a mutant thereof. By increasing the constant region sequence, it helps to maintain the structural stability of the entire antibody molecule.

[0060] In some examples of the present application, at least a portion of the heavy chain constant region sequence is derived from a murine antibody.

[0061] In some examples of the present application, the heavy chain constant region amino acid sequence is as shown in SEQ ID NO: 9.

[0062] In some examples of the present application, the antibody or antigen-binding fragment thereof further contains a light chain constant region sequence, at least a portion of which is derived from at least one of a murine antibody, a rabbit antibody, a primate antibody, or a mutant thereof. By increasing the constant region sequence, it helps to maintain the structural stability of the entire antibody molecule.

[0063] In some examples of the present application, the light chain constant region is selected from a murine antibody.

[0064] In some examples of the present application, the light chain constant region amino acid sequence is as shown in SEQ ID NO: 10.

[0065] In some examples of the present application, the antibody includes at least one selected from the group consisting of a polyclonal antibody and a monoclonal antibody; or the antigen-binding fragment includes at least one selected from the group consisting of a F(ab')2 fragment, a Fab' fragment, a Fab fragment, a F(ab)2 fragment, a Fv fragment, a scFv fragment, a scFv-Fc fusion protein, a scFv-Fv fusion protein, and a minimal recognition unit. In some preferred examples of the present application, the antibody is a monoclonal antibody.

[0066] It should be noted that the antibody of any of the above examples of the present application can be directly synthesized according to the antibody variable region amino acid sequence provided by the present application. In addition, different antibody analogs can also be derived by further chemical modification.

[0067] In another aspect of the present application, an antibody analogue is provided, which comprises the antibody or antigen-binding fragment thereof of any of the preceding examples.

[0068] It should be noted that the antibody analogue mentioned in the present application specification and claims refers to a derivative obtained by deleting, adding, or modifying a chemical group (e.g., an amino acid) on the basis of an antibody structure by biological or chemical methods. The derivative still contains a structure similar to an antibody variable region (or a CDR region in the antibody variable region) and can react with a structure similar to antigen-antibody binding.

[0069] In another aspect of the present application, a nucleic acid molecule is provided, which encodes the antibody or antigen-binding fragment thereof of any of the preceding examples. In some examples of the present application, the nucleic acid molecule encodes an antibody or antigen-binding fragment thereof that can be used for specifically detecting uncarboxylated osteocalcin.

[0070] In some examples of the present application, the nucleic acid molecule is DNA.

[0071] It should be noted that for the nucleic acid molecule mentioned in the present application specification and claims, those skilled in the art should understand that it actually includes any one of the complementary double strands, or both. For convenience, in the present specification and claims, although only one strand is given in most cases, the other complementary strand is actually disclosed. In addition, the nucleic acid sequence in the present application includes DNA or RNA form, and the disclosure of one means the disclosure of the other.

[0072] The nucleotide sequence of the nucleic acid molecule mentioned in the present application can be obtained by the commonly known technology of those skilled in the art. Moreover, it can also be species-optimized to make it more easily expressed in mammalian cells.

[0073] In another aspect of the present application, an expression vector is provided, which carries the nucleic acid molecule of any of the preceding examples. In some examples of the present application, the expression vector can efficiently express the antibody or antigen-binding fragment thereof in a suitable host cell, so as to be used for specifically detecting uncarboxylated osteocalcin or preparing a drug related to uncarboxylated osteocalcin disease.

[0074] It should be noted that when the above nucleic acid molecule is connected to the vector, the nucleic acid molecule can be directly or indirectly connected to the control elements on the vector, as long as the control elements can control the translation and expression of the nucleic acid molecule. Of course, these control elements can be directly from the vector itself, or can be exogenous, i.e., not from the vector itself. Of course, the nucleic acid molecule can be operably linked to the control elements. "Operably linked" herein means that the exogenous gene is connected to the vector, so that the control elements in the vector, such as transcription control sequences and translation control sequences, etc., can play their expected functions of regulating the transcription and translation of the exogenous gene. Common vectors can be plasmids, bacteriophages, etc., such as pcDNA plasmids.

[0075] In another aspect of the present application, a method for preparing the antibody or antigen-binding fragment thereof of any of the preceding examples is provided, comprising: introducing the expression vector of any of the preceding examples into a cell; and culturing the cell under conditions suitable for protein expression and secretion, so as to obtain the antibody or antigen-binding fragment thereof. In some examples of the present application, the antibody or antigen-binding fragment thereof can be prepared in vitro in large quantities by the method.

[0076] In some examples of the present application, the cell is a eukaryotic cell. In some specific examples of the present application, the eukaryotic cell is a mammalian cell, such as 293F cells, CHO cells, etc. When the cell is a mammalian cell, the expression efficiency of the antibody or antigen-binding fragment thereof is higher.

[0077] It should be noted that the eukaryotic cell does not include an animal reproductive cell, a fertilized egg, or an embryonic stem cell.

[0078] In another aspect of the present application, a recombinant cell carrying the antibody or antigen-binding fragment thereof, the nucleic acid molecule, or the expression vector of any of the preceding examples is provided. In some examples of the present application, the recombinant cell is obtained by transfection or transformation of the expression vector, and the recombinant cell can efficiently express the above-mentioned antibody or antigen-binding fragment thereof under suitable conditions, and the antibody or antigen-binding fragment thereof can specifically bind to non-carboxylated osteocalcin.

[0079] It should be noted that the recombinant cell of the present application is not particularly limited, and can be a prokaryotic cell, a eukaryotic cell, or a bacteriophage. The prokaryotic cell can be Escherichia coli, Bacillus subtilis, Streptomyces, or Proteus mirabilis, etc. The eukaryotic cell can be fungi including Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces, Trichoderma, etc., insect cells such as Spodoptera exigua, plant cells such as tobacco, mammalian cells such as BHK cells, CHO cells, COS cells, myeloma cells, etc. In some embodiments, the recombinant cell of the present application is preferably a mammalian cell, including BHK cells, CHO cells, NSO cells, or COS cells, and does not include animal reproductive cells, fertilized eggs, or embryonic stem cells.

[0080] It is noted that "appropriate conditions" in the present application refer to conditions suitable for expression of the antibodies or antigen-binding fragments thereof of the present application. Those skilled in the art will readily appreciate that conditions suitable for expression of recombinant antibodies include, but are not limited to, appropriate transformation or transfection means, appropriate transformation or transfection conditions, healthy host cell status, appropriate host cell density, appropriate cell culture environment, and appropriate cell culture time. "Appropriate conditions" are not particularly limited and those skilled in the art can optimize the conditions for optimal expression of recombinant antibodies according to the specific environment of the laboratory.

[0081] In yet another aspect of the present application, a hybridoma cell is provided. The hybridoma cell can produce the antibodies or antigen-binding fragments thereof of any of the preceding examples.

[0082] In some examples of the present application, the hybridoma cell can be produced by a classical method for preparing monoclonal antibodies.

[0083] In a classical method for preparing monoclonal antibodies, a suitable antigen is first needed and used to immunize an animal. In order to prepare an anti-uncarboxylated osteocalcin antibody (or antibody analog), the suitable antigen must contain uncarboxylated osteocalcin. This antigen can be obtained by isolation and purification from natural human tissues or blood, chemical synthesis, or a combination of these methods, for use in animal immunization and antibody screening.

[0084] Illustratively, in a classical method for preparing monoclonal antibodies, an animal is first immunized with an uncarboxylated osteocalcin antigen, and blood is collected at intervals to verify whether the animal has produced an antibody response to the uncarboxylated osteocalcin antigen. B cells are then isolated from the spleen of the animal with an antibody response and fused with immortal myeloma cells in vitro to obtain hybridoma cells. These hybridoma cells are then limit-diluted and regrown in culture plates (monoclonal hybridoma cell lines), and the culture supernatant of these hybridoma cell lines is collected and tested for the presence of specific antibodies against the antigen. According to the yield, quality, and cell line growth characteristics of the antibodies, the best monoclonal antibody-producing cell line can be selected for subsequent monoclonal antibody production.

[0085] Other methods can also be used to obtain monoclonal antibodies. For example, the spleen cells of the above-mentioned animals can be isolated and then incubated with labeled antigens (such as fluorescein-labeled non-carboxylated osteocalcin). Since the B cells producing antibodies usually have antibody molecules present on the cell membrane surface, these cells will be bound (stained) by the labeled antigens, and can be sorted out by a fluorescent flow cytometer. The mRNA of these sorted B cells can be isolated, and through in vitro reverse transcription and specific PCR reactions, a library of variable region cDNA of the antibodies can be obtained. This cDNA library can be inserted into an expression plasmid (such as an antibody expression plasmid suitable for expression in mammalian cells, or a phage expression plasmid suitable for expression in bacterial cells), and expressed in host cells suitable for this expression plasmid. These host cells (or phages) can be isolated and purified (cloned) by different methods (such as the cell limiting dilution culture method described above, or the phage plaque method). These cell strains or phage clones can be used to produce antibodies, and the antibodies can be analyzed and identified.

[0086] In some preferred examples of the present application, subcutaneous multiple and multi-point injections are used during the immunization of animals. This improves the titer of the final animal serum and improves the positive cloning rate.

[0087] In some preferred examples of the present application, the inventors delayed the addition of HAT screening reagents from the day of fusion to the second day, which significantly improved the positive rate of cells.

[0088] In some preferred examples of the present application, the inventors selected a variety of non-carboxylated osteocalcin structural analogs for comparison of antibody specific detection. After several rounds of screening, antibodies with better specificity for non-carboxylated osteocalcin were obtained (see Table 1 for sequences).

[0089] The non-carboxylated osteocalcin antibodies of the present application can be used to detect non-carboxylated osteocalcin in human serum or body fluids, or substances containing non-carboxylated osteocalcin, and substances that can specifically bind to non-carboxylated osteocalcin (such as non-carboxylated osteocalcin antibodies). The principle of detection is mainly based on the specific recognition and binding of the non-carboxylated osteocalcin antibodies of the present application to non-carboxylated osteocalcin, and the use of chemical labeling techniques (such as labeled antibodies, or labeled specific secondary antibodies) or physical detection techniques (such as light scattering techniques, plasmon resonance techniques, etc.) for detection.

[0090] In another aspect of the present application, an immunoconjugate is provided, comprising: the antibody or antigen binding fragment thereof, nucleic acid molecule, expression vector or recombinant cell of any of the preceding examples. In some examples of the present application, the antibody or antigen binding fragment can specifically bind to non-carboxylated osteocalcin, and can be used to prepare a prophylactic and / or therapeutic agent for preventing and / or treating non-carboxylated osteocalcin-mediated related diseases.

[0091] In some examples of the present application, the immunoconjugate can further comprise a therapeutic agent. In some examples, the therapeutic agent is selected from at least one of a cytokine, an immunomodulator, and a small molecule drug.

[0092] In another aspect of the present application, the present application provides a composition comprising the antibody or antigen-binding fragment thereof, the nucleic acid molecule, the expression vector, the recombinant cell, or the immunoconjugate of any of the preceding examples. In some examples of the present application, the antibody or antigen-binding fragment thereof is capable of specifically binding to uncarboxylated osteocalcin. In some examples of the present application, the composition comprising the antibody or antigen-binding fragment thereof, such as a food composition, a pharmaceutical composition, and the like, is also capable of significantly treating and / or preventing the relevant diseases mediated by uncarboxylated osteocalcin.

[0093] It should be noted that the composition comprises components separated in time and / or space, as long as they can act together to achieve the purpose of the present application. For example, the components contained in the composition can be administered to the subject as a whole, or administered to the subject separately. When the components contained in the composition are administered to the subject separately, each component can be administered to the subject simultaneously or sequentially.

[0094] In another aspect of the present application, the present application provides the use of the antibody or antigen-binding fragment thereof, the nucleic acid molecule, the expression vector, the recombinant cell, the immunoconjugate, or the composition of any of the preceding examples in the preparation of a medicament for treating or preventing the relevant diseases mediated by uncarboxylated osteocalcin. In some examples of the present application, the antibody or antigen-binding fragment thereof is capable of specifically binding to uncarboxylated osteocalcin. In some examples of the present application, the antibody or antigen-binding fragment thereof is capable of specifically binding to uncarboxylated osteocalcin. It can be understood that the medicament comprising the antibody or antigen-binding fragment thereof and the like is also capable of significantly treating or preventing the relevant diseases mediated by uncarboxylated osteocalcin, such as bone and joint diseases.

[0095] In some examples of the present application, the relevant diseases mediated by uncarboxylated osteocalcin include liver disease, diabetes, rheumatoid arthritis, hypoparathyroidism peripheral arterial and coronary sclerosis, bone trauma, osteomalacia, primary hyperthyroidism, menopausal syndrome, premature ovarian failure, osteoporosis, abnormal calcium metabolism, calcium deficiency, obesity, and bone tumors.

[0096] In yet another aspect of the present application, the present application provides a medicament comprising the antibody or antigen-binding fragment thereof, nucleic acid molecule, expression vector, recombinant cell, immunoconjugate or composition of any of the preceding examples, for use in the treatment or prevention of a disease associated with non-carboxylated osteocalcin. In some examples of the present application, the antibody or antigen-binding fragment thereof is capable of specifically binding to non-carboxylated osteocalcin. It will be appreciated that the medicament comprising the antibody or antigen-binding fragment thereof and the like has a significant effect on the treatment or prevention of a disease associated with non-carboxylated osteocalcin, such as a bone and joint disease.

[0097] In some examples of the present application, the medicament comprises a pharmaceutically acceptable carrier and an effective amount of the antibody active ingredient.

[0098] As used herein, the term "effective amount" or "effective dose" means an amount that is functional or active and acceptable to a human and / or an animal.

[0099] As used herein, a "pharmaceutically acceptable" ingredient is one that is suitable for use with humans and / or animals without undue adverse side effects (such as toxicity, irritation, and allergic response) commensurate with a reasonable benefit / risk ratio, i.e., the

[0100] The medicament of the present application contains a safe and effective amount of the active ingredient of the present application and a pharmaceutically acceptable carrier. Such carriers include, but are not limited to, saline, buffers, dextrose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical preparation is generally matched to the mode of administration, and the dosage form of the medicament of the present application is an injection, an oral preparation (tablet, capsule, oral solution), a transdermal preparation, a sustained-release preparation. For example, it is prepared by a conventional method using physiological saline or an aqueous solution containing dextrose and other auxiliary agents. The medicament of the present application is preferably manufactured under sterile conditions.

[0101] The effective amount of the active ingredient of the present application can vary depending on the mode of administration and the severity of the disease to be treated. The selection of the preferred effective amount can be determined by a person of ordinary skill in the art (e.g., by clinical trials) according to various factors. The factors include, but are not limited to, pharmacokinetic parameters of the active ingredient of the present application such as bioavailability, metabolism, half-life, etc.; the severity of the disease to be treated in the patient, the body weight of the patient, the immune status of the patient, the route of administration, etc. For example, several divided doses can be administered daily, or the dose can be proportionally reduced as indicated by the exigencies of the therapeutic situation.

[0102] The pharmaceutically acceptable carrier of the present application includes, but is not limited to, water, saline, liposome, lipid, protein, protein-antibody conjugate, peptide substance, cellulose, nanogel, or a combination thereof. The selection of the carrier should be matched with the administration mode, which is well known to those skilled in the art.

[0103] In still another aspect of the present application, the present application provides the use of the antibody or antigen-binding fragment thereof of any of the foregoing examples in the preparation of a kit for detecting non-carboxylated osteocalcin. In some examples of the present application, the antibody or antigen-binding fragment thereof can specifically bind to non-carboxylated osteocalcin. Therefore, the kit comprising the antibody or antigen-binding fragment thereof can be used to specifically and sensitively detect non-carboxylated osteocalcin.

[0104] In still another aspect of the present application, the present application provides a kit comprising the antibody or antigen-binding fragment thereof of any of the foregoing examples. In some examples of the present application, the antibody or antigen-binding fragment thereof has high sensitivity and specificity in detecting non-carboxylated osteocalcin protein. In addition, the kit can be used with a fully automatic chemiluminescence analyzer, which greatly simplifies the operation steps, increases the detection speed and throughput, improves the detection efficiency, and avoids errors caused by human operation. By using the principle of combining chemiluminescence detection technology with magnetic microsphere immunoseparation technology, the content of non-carboxylated osteocalcin in human serum or plasma samples can be quantitatively detected, ensuring the sensitivity of the detection. Since the kit uses a sandwich method, it has no pollution, high specificity, simple operation, low requirements for sample pretreatment, and can quickly and high-throughput detect a large number of samples, which is convenient for clinical reagent application. The kit provided by the present application provides a more accurate, precise, convenient, fast and simple method for detecting non-carboxylated osteocalcin in human serum.

[0105] In some examples of the present application, the kit further comprises at least one of a magnetic microsphere, a biomarker, a biomarker substrate, a buffer, a calibrator, and a quality control.

[0106] In some examples of the present application, the magnetic microsphere has a particle size selected from 0.5-10 μm, preferably 3-8 μm. Alternatively, it is 1 μm, 2 μm, 3 μm, 4 μm, 5 μm, 6 μm, 7 μm, 8 μm, 9 μm, or 10 μm.

[0107] In some examples of the present application, the biomarker comprises at least one of a biological enzyme, a fluorescein, and a chemiluminescent marker.

[0108] In some examples of the present application, the biological enzyme is selected from at least one of horseradish peroxidase and alkaline phosphatase. In actual application, the biological enzyme is adapted to the biomarker substrate, i.e., the biological enzyme has the activity of catalyzing the luminescence of the biomarker substrate.

[0109] In some examples of the present application, the fluorescein is selected from at least one of fluorescein isothiocyanate, tetraethyl rhodamine and tetramethyl rhodamine isothiocyanate.

[0110] In some examples of the present application, the chemiluminescent label is selected from at least one of acridinium ester, trispyridyl ruthenium, isoluminol or derivatives thereof.

[0111] In some examples of the present application, the buffer solution comprises at least one selected from phosphate buffer, carbonate buffer, tris buffer and citrate buffer. The concentration of the buffer solution is selected from 10-100 mM, optionally 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM or 100 mM; and the pH of the buffer solution is selected from 6.0-9.0, optionally 6.0, 7.0, 8.0 or 9.0.

[0112] In some examples of the present application, the calibrator contains non-carboxylated osteocalcin with different concentration gradients.

[0113] In some examples of the present application, the quality control sample comprises a non-carboxylated osteocalcin buffer solution with a determined concentration.

[0114] In another aspect of the present application, a method for detecting non-carboxylated osteocalcin is provided, which comprises: detecting a sample to be tested by using the kit of any one of the preceding examples, so as to determine the content of non-carboxylated osteocalcin in the sample to be tested. In some examples of the present application, the above method is used for high-sensitivity detection of non-carboxylated osteocalcin, and is environmentally friendly, specific, simple to operate, and requires low sample pretreatment. In clinical practice, a large number of samples can be rapidly and high-throughput detected by means of a chemiluminescence instrument. The detection method according to the embodiments of the present application can be used for non-clinical diagnostic purposes. For example, when a researcher wants to screen an intervention factor of non-carboxylated osteocalcin, the researcher can apply the intervention factor to be screened to the sample to be tested, and then detect whether the content of non-carboxylated osteocalcin in the sample to be tested changes before and after the application of the intervention factor by using the method according to the embodiments of the present application, so as to determine the target non-carboxylated osteocalcin intervention factor.

[0115] In some examples of the present application, the detection process comprises: coupling the magnetic microspheres with the first antibody to obtain a first antibody complex; labeling the second antibody with a biological marker to obtain a second antibody complex; mixing the sample to be tested, the first antibody complex and the second antibody complex; contacting the mixing product with a biological marker substrate, the biological marker having the activity of catalyzing the luminescence of the biological marker substrate; and determining the content of non-carboxylated osteocalcin in the sample to be tested based on the luminescence intensity obtained after the contacting process. The first antibody and the second antibody independently recognize non-carboxylated osteocalcin and osteocalcin, respectively. It can be understood that the first antibody is selected from an antibody that recognizes non-carboxylated osteocalcin, and the second antibody is selected from an antibody that recognizes osteocalcin; or the first antibody is selected from an antibody that recognizes osteocalcin, and the second antibody is selected from an antibody that recognizes non-carboxylated osteocalcin. The present application uses an immunological sandwich method to detect non-carboxylated osteocalcin, so the first antibody and the second antibody should not be the same to prevent the two antibodies from recognizing the same antigen epitope, thereby causing the detection to fail.

[0116] In some examples of the present application, the antibody that recognizes non-carboxylated osteocalcin is from the antibody or antigen-binding fragment thereof of any example of the present application.

[0117] In some examples of the present application, the antibody that recognizes osteocalcin is from a commercially available osteocalcin antibody, such as R&D Systems, Inc., Catalog No. MAB11327.

[0118] In some examples of the present application, the first antibody is selected from an antibody that recognizes non-carboxylated osteocalcin. As described previously, the first antibody complex refers to the antibody or antigen-binding fragment thereof of any of the preceding examples being bound to the magnetic microspheres through a functional group to form an immunomagnetic bead. Because the antibody has a very high affinity for its corresponding antigen, it can be fixed to the magnetic microspheres for detection together with the antigen.

[0119] In some examples of the present application, the magnetic microspheres are selected from composite microspheres formed by ferroferric oxide or magnetite magnetic nanoparticles and organic high polymer materials. Because they have superparamagnetic properties, high specific surface area, and can be modified with functional groups, etc. Therefore, the inventors chose to immobilize the antibody or antigen-binding fragment thereof on their surface.

[0120] In some examples of the present application, the surface of the composite microspheres is modified with one or more active groups that can cross-link with the amino or carboxyl groups of the non-carboxylated osteocalcin antibody through amide bonds to form non-carboxylated osteocalcin antibody-coated magnetic microspheres.

[0121] In some examples of the present application, the non-carboxylated osteocalcin antibody (or osteocalcin antibody) can also be directly or indirectly coupled to the surface of the magnetic beads by its own amino or carboxyl group, such as the method of indirect coating of magnetic microspheres by magnetic bead-streptavidin coupling-biotin-biotin carrier protein, or can be connected to the magnetic microparticles in the reaction in the form of an antibody solution.

[0122] In some examples of the present application, the second antibody is selected from an antibody that recognizes osteocalcin. As previously described, the second antibody complex refers to an antibody complex obtained by immunolabeling technology (such as enzyme labeling, ferritin labeling, or labeling by colloidal gold) of a commercial osteocalcin antibody. In one example of the present application, the second antibody complex is obtained by enzyme labeling.

[0123] In some examples of the present application, the label is coupled to the osteocalcin antibody by an amide bond to form a label-osteocalcin antibody conjugate.

[0124] In some examples of the present application, the coupling method of the label and the osteocalcin antibody can be obtained based on any method known in the art, for example, by adding 2-Iminothiolane hydrochloride (2-IT) to the osteocalcin antibody, which is a commonly used activator in the art, adding Succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC) to the label, which is a commonly used activator in the art, and the mixing ratio of the activated label and the osteocalcin antibody is preferably 1:0.5-2, and can be 1:0.5; 1:1; 1:1.5 or 1:2.

[0125] As previously described, immunolabeling technology is to label some substances that are easy to determine and have high sensitivity to specific antigen or antibody molecules, and to display the nature and content of antigens or antibodies in the reaction system through the amplification effect of these labels. In some examples of the present application, the second antibody complex is mainly used for localization analysis of antigens, and in some cases, it can also be used for quantitative detection of antigens in samples mixed with a large number of other molecules. Because the antibody has a very high affinity for its corresponding antigen, the antibody with an easily recognizable label can be used for localization analysis of antigens, and is an ideal rapid and inexpensive quantitative determination method.

[0126] In some examples of the present application, the coupling process is carried out by the following steps: coupling a predetermined mass ratio of magnetic microspheres and the first antibody in an MES buffer system to obtain a first antibody complex.

[0127] In some examples of the present application, the mass ratio of the first antibody to the magnetic microspheres is 1-20:1000. Specifically, the mass ratio can be 1:1000, 1:500 or 1:50. In some preferred examples of the present application, the concentration of the magnetic microspheres is 0.1-2 mg / ml, preferably 1 mg / ml.

[0128] In some examples of the present application, the coupling reaction time is selected from 5-8 hours (h), and can be 5 h, 6 h, 6.5 h, 7 h or 8 h. In one preferred example of the present application, the coupling reaction time is 6.5 hours.

[0129] In some examples of the present application, the coupling reaction temperature is selected from 4-8°C, and can be 4°C, 5°C, 6°C, 7°C or 8°C. In one preferred example of the present application, the coupling reaction temperature is 4°C.

[0130] In some examples of the present application, the detection method further comprises: preparing a non-carboxylated osteocalcin concentration-luminous value standard curve using a calibration sample, wherein the calibration sample comprises a predetermined concentration gradient of non-carboxylated osteocalcin.

[0131] For example, the first antibody complex is prepared by coupling the antibody or antigen-binding fragment thereof of any of the preceding examples with magnetic microspheres, the second complex is prepared by labeling the antibody or antigen-binding fragment thereof of any of the preceding examples with alkaline phosphatase (ALP), and 9,10-dihydroacridine derivative or adamantane derivative at a concentration of 0.005%-0.05% is used as a substrate solution. When the reaction occurs, the aforementioned substrate is catalytically cleaved under the action of alkaline phosphatase (ALP) to form an unstable excited state intermediate. When the excited state intermediate returns to the ground state, it emits photons to form a luminescence reaction. The luminescence intensity of the reaction can be detected using a luminometer.

[0132] In some examples of the present application, based on the luminescence intensity obtained after the contact treatment, the step of determining the content of non-carboxylated osteocalcin comprises: detecting and treating the standard sample to determine the luminescence intensity corresponding to different concentrations; wherein the calibration sample comprises a predetermined concentration gradient of non-carboxylated osteocalcin; based on the luminescence intensity corresponding to different concentrations, a non-carboxylated osteocalcin concentration-luminous value standard curve is drawn; based on the non-carboxylated osteocalcin concentration-luminous value standard curve and the luminescence intensity obtained after the contact treatment, the content of non-carboxylated osteocalcin in the sample to be tested is obtained.

[0133] In some other examples of the present application, the first antibody is selected from an antibody that recognizes osteocalcin. The preparation process of the first antibody complex is as described above, and will not be repeated here.

[0134] In some other examples of the present application, the second antibody is selected from an antibody that recognizes non-carboxylated osteocalcin. The preparation process of the second antibody complex is as described above, and will not be repeated here.

[0135] In one example of the present application, the non-carboxylated osteocalcin concentration-luminous value standard curve is obtained by the following method, comprising:

[0136] (1) Immune response: 50 μl of different concentrations of calibrators or quality control samples are sequentially added to the reaction tube with 50 μl of the first antibody complex and 50 μl of the second antibody complex, respectively, and mixed and incubated at 37°C for 10 min;

[0137] (2) Magnetic separation: The magnetic microspheres are allowed to settle in the magnetic field, the supernatant is removed, 200-500 μl of washing solution is added, the magnetic field is removed, and then the magnetic microspheres are again allowed to settle in the magnetic field, and the supernatant is removed; repeat 2-4 times to remove unbound antibodies and impurities;

[0138] (3) Reading: 200 μl of luminescent substrate solution is added, and the relative luminescence intensity is measured by the full-automatic chemiluminescence immunoassay analyzer SMART 500H after the alkaline phosphatase catalyzes the substrate to emit light;

[0139] (4) The non-carboxylated osteocalcin concentration-luminous value standard curve is obtained by fitting the detected values using a four-parameter equation.

[0140] For ease of understanding, the above detection method is described in detail taking the non-carboxylated osteocalcin chemiluminescence immunoassay method as an example.

[0141] Step one: coat the non-carboxylated osteocalcin antibody of any of the preceding embodiments on the surface of the magnetic microspheres to prepare a non-carboxylated osteocalcin antibody magnetic microsphere buffer solution;

[0142] Step two: prepare a marker-labeled osteocalcin solution;

[0143] Step three: mix the sample to be tested, the solution in step one, and the solution in step two in proportion, and then perform immune response;

[0144] Step four: after the reaction is complete, collect the magnetic microspheres precipitate with a magnetic field, and add a substrate that causes the marker in step two to emit light, to obtain a non-carboxylated osteocalcin concentration-luminous value standard curve;

[0145] Step five: calculate the content of the non-carboxylated osteocalcin according to the standard curve.

[0146] The above detection kit and its detection method have the following beneficial effects:

[0147] This application employs a sandwich reaction mode, utilizing a combination of chemiluminescence detection technology and magnetic microsphere immunoassay technology to quantitatively detect the content of non-carboxylated osteocalcin in human serum or plasma samples. This ensures high detection sensitivity. Because the experiment uses a sandwich method, it is pollution-free, highly specific, simple to operate, requires minimal sample pretreatment, and allows for rapid, high-throughput detection of large batches of samples, facilitating clinical reagent application. This application provides a more accurate, convenient, and rapid method for clinical detection of non-carboxylated osteocalcin in samples.

[0148] Table 1

[0149]

[0150]

[0151]

[0152] The present invention will be explained below with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0153] Example 1: Synthesis of non-carboxylated osteocalcin peptide, carboxylated osteocalcin peptide, and full-length osteocalcin antigen

[0154] The full-length osteocalcin antigen sequence (Table 1, SEQ ID NO:15), carboxylated osteocalcin, and non-carboxylated osteocalcin protein sequences (Table 1, SEQ ID NO:16-18) were synthesized using Haibotai Biotechnology Co., Ltd.

[0155] Example 2: Identification of non-carboxylated osteocalcin

[0156] This embodiment uses standard laboratory methods to detect the antigenic activity of non-carboxylated osteocalcin. The specific steps are as follows:

[0157] Non-carboxylated osteocalcin protein was coated onto an ELISA plate at a concentration of 1 μg / ml. Purified non-carboxylated osteocalcin antibody was used as the primary antibody in the indirect ELISA method, and HRP-labeled goat anti-rat antibody was used as the secondary antibody. TMB was used as the chromogenic buffer, and the reaction was terminated with 1M H₂SO₄. The OD value of the ELISA reader was measured. 450 Reading values ​​were obtained. The antigenic activity of non-carboxylated osteocalcin was compared, and it was found that the antigenic activity of non-carboxylated osteocalcin was higher (Table 2).

[0158] Table 2

[0159] Sample OD 450 ]] Non-carboxylated osteocalcin concentration (1 μg / ml) 0.928 Blank 0.028

[0160] Example 3: Immunization of BALB / c mice with non-carboxylated osteocalcin protein

[0161] In this example, BALB / c mice were immunized with the non-carboxylated osteocalcin protein synthesized in Example 1, and the titer of the mouse antibody serum was detected.

[0162] The specific steps are as follows:

[0163] BALB / c mice aged 6-8 weeks were selected for the following immunization procedure: for the first immunization, 25 μg of non-carboxylated osteocalcin was mixed with an equal amount of Freund's complete adjuvant, emulsified, and injected subcutaneously in multiple points; 14 days after the first immunization, 12.5 μg of non-carboxylated osteocalcin was mixed with Freund's incomplete adjuvant, emulsified, and used for booster immunization. 14 days after the second immunization, 12.5 μg of non-carboxylated osteocalcin was mixed with Freund's incomplete adjuvant, emulsified, and used for booster immunization. 14 days after the third immunization, blood was collected and the serum was separated, an ELISA plate was coated with 1 μg / mL of non-carboxylated osteocalcin, and indirect ELISA was performed to determine the titer of the serum. The results showed that the titer of the prepared mouse antiserum was 1:72,900. The results of the detection of serum from a typical immunized mouse are shown in Table 3.

[0164] Table 3

[0165]

[0166]

[0167] Example 4: Cell fusion

[0168] Cell fusion was performed 3 days after booster immunization in Example 3. After blood was collected from the eye of the mouse, the mouse was sacrificed by dislocation, placed in a 70% alcohol bottle for 2 minutes, fixed on a foam board in a biological safety cabinet, the abdominal skin was unfastened to find the spleen, the spleen was taken out with tweezers and placed in a 200-mesh stainless steel filter membrane to be gently ground, the cells were gently washed with DMEM medium (Thermo, 11965092), then centrifuged at 200g for 10 minutes at room temperature, and the supernatant was discarded for standby; for preparation of feeder cells, the mouse was sacrificed by dislocation, placed in a 70% alcohol bottle for 2 minutes, fixed on a foam board in a biological safety cabinet, the abdominal skin was unfastened, PBS was sucked with a syringe and injected subcutaneously, and the liquid containing feeder cells was washed out from the other side, then centrifuged at 200g for 10 minutes at room temperature, and the supernatant was discarded for standby. 2.0 x 10 7 8 ​The spleen cells were mixed, centrifuged at 200 g for 10 minutes, the supernatant was discarded, and the cells were mixed gently. The cells were placed in a 37°C water bath, and 1 mL of a 50% PEG-1450 (Merk, P1458) aqueous solution was added dropwise over 90 seconds. Then, 20 mL of DMEM medium was added dropwise, and the cells were centrifuged at 200 g for 10 minutes. The supernatant was discarded, and the cells were washed once more by centrifugation at 200 g for 10 minutes. The supernatant was discarded, and the hybridoma cells were obtained. The cells were plated in 10 96-well plates, 150 μL per well. Feeder cells (10000 cells / well) were added to the 10 96-well plates, 100 μL per well. The plates were labeled and placed in a 37°C cell culture incubator containing 5% CO2. The next day, HAT selection medium (Merk, H0262) was added, and the cells were cultured for 1-2 days. A large number of tumor cells died, and the tumor cells disappeared after 3-4 days. Hybridoma cells formed small colonies, and the HAT selection medium was replaced with HT medium (Merk, H0137) after 7-10 days. The cells were cultured for another 2 weeks, and then the medium was replaced with DMEM medium containing 20% FBS (ExCell, FSP500). During the selection and culture, the hybridoma cells were detected for specific antibodies when the cells covered 1 / 10 of the area of the well bottom. The desired hybridoma cell line was selected. The medium was replaced every 2-3 days during the selection and culture.

[0169] Example 5: Screening and subcloning of positive hybridoma cell lines

[0170] First, the optimal coating amount of non-carboxylated osteocalcin as antigen was determined using a squared titration. 0.5, 1.0, 2.0, and 4.0 μg of non-carboxylated osteocalcin were coated onto 96-well plates, with each concentration represented by 6 wells (3 positive and 3 negative). Squared titrations were performed using positive mouse serum immunized with different dilutions of non-carboxylated osteocalcin, with negative serum from unimmunized mice serving as a negative control. 0.5 μg of purified non-carboxylated osteocalcin was coated onto each well of a 96-well ELISA plate and incubated overnight at 4°C. The plates were washed twice with PBST. 200 μL of 1% BSA in PBS was added to each well, and the plates were blocked at room temperature for 2 hours, then patted dry on folded paper. Sample loading: 0.1 mL of the test sample was added to each well, and the plates were incubated at 37°C for 1 hour, followed by washing. Blank wells (without sample), negative control wells, and positive control wells were also prepared. 0.1 mL of freshly diluted antibody was added to each well, and the plates were incubated at 37°C for 1 hour, followed by washing three times. Add enzyme-labeled secondary antibody: Add 0.1 mL of freshly diluted enzyme-labeled antibody to each reaction well. Incubate at 37°C for 1 hour, then wash 3 times. Add substrate solution for color development: Add 0.1 mL of TMB substrate solution to each reaction well and incubate at room temperature for 10 minutes. Add 0.1 mL of 1M H₂SO₄ to each reaction well. Measure the OD value to determine the result: Use an ELISA reader to measure the absorbance at 450 nm (A450). A positive result is defined as an OD value greater than 2.1 times that of the negative control (calculated after zeroing the blank control well). Select hybridoma cell clones resistant to non-carboxylated osteocalcin.

[0171] Following the above method, the selected positive hybridoma cells were subcloned. The original wells were diluted with HAT selective medium using a limiting dilution method and then re-distributed into 96-well culture plates. Cell morphology and quantity were then observed. The cell density was adjusted to 3–10 cells / mL. 100 μL of diluted cells was added to each well of a cell culture plate containing a feeder cell layer prepared the previous day. The plates were incubated statically at 37°C with 5% CO2. The medium was changed on day 7, and thereafter every 2–3 days. Cell clone formation was observed on days 8–9, and antibody activity was promptly assessed. Cells from the positive wells were transferred to 24-well plates for further culture. Each clone was cryopreserved as soon as possible, and the hybridoma cell line with clone number G069 was ultimately selected for antibody production.

[0172] Example 6: Large-scale preparation of monoclonal antibodies and determination of antibody titer

[0173] (1) Large-scale preparation of monoclonal antibodies

[0174] Eight-week-old BALB / c mice were injected intraperitoneally with 0.5 mL of Freund's incomplete adjuvant, followed by an intraperitoneal injection of 1 × 10⁻⁶ mg / mL two weeks later. 6The hybridoma cells were inoculated and 7-10 days later ascites were produced. The health status and ascites signs of the animals were observed closely. The mice were sacrificed when the ascites were as much as possible and the mice were not likely to die. The ascites were collected in a test tube by using a dropper. One mouse could produce 5-10 ml ascites. The ascites could be repeatedly collected several times by using a syringe. The ascites were centrifuged at 3000 g for 10 minutes. The upper oil and the bottom precipitate were discarded. The supernatant was collected and stored at -20°C. The ascites supernatant was thawed and equilibrated to room temperature. One-tenth volume of 1M Tris-HCl pH 8.0 was added to adjust the pH of the sample to 8.0. The ascites supernatant with pH adjusted to 8.0 was loaded onto a protein G affinity column equilibrated with 20 column volumes of 100 mM Tris-HCl pH 8.0. Then the column was washed with 20 column volumes of 100 mM Tris-HCl pH 8.0. Finally, the antibody was eluted with 100 mM Glycine-HCL pH 2.5. The antibody eluate was centrifuged in a concentrator (Xiangyi, L550) at 3000 x g for 20 minutes at room temperature. The solution was centrifuged in batches until the volume was 1 ml / concentrator (2 tubes). 4 mL of 10 mM PBS pH 7.4 buffer was added. The centrifugation was continued at 3000 x g for 20 minutes at room temperature. The centrifugation was repeated 3 times. The buffer of the antibody was 10 mM PBS pH 7.4. 10 mM PBS pH 7.4 was added to a total volume of 10 mL. Finally, the concentrated antibody solution 2 mL / tube was aliquoted in centrifuge tubes and stored at -80°C. The antibody concentration was determined by using a BCA kit (Solebo, PC0020). The concentration of the purified monoclonal antibody was 2.9 mg / mL.

[0175] (2) Antibody titer determination

[0176] The titer of non-carboxylated osteocalcin antibody G069 was detected by indirect ELISA. Non-carboxylated osteocalcin was diluted with PBS to 0.2 μg / mL, 100 μL / well, coated in a 96-well enzyme-labeled plate, and incubated at 4°C overnight. The enzyme-labeled plate was washed twice with 300 μL / well of PBST solution, and then dried. The enzyme-labeled plate was blocked with 1% BSA in PBS solution, 200 μL / well, at room temperature for 2 hours, and then dried. Non-carboxylated osteocalcin antibody G069 diluted with PTB to 20 ng / mL was added, and incubated at 37°C for 1 hour. The enzyme-labeled plate was washed twice with 300 μL / well of PBST solution. After washing, the plate was dried. HRP-labeled goat anti-mouse antibody diluted 5000-fold with PTB was added, and incubated at 37°C for 1 hour. The enzyme-labeled plate was washed twice with 300 μL / well of PBST solution. After washing, the plate was dried. TMB substrate solution, 100 μL / well, was added, and incubated at room temperature for 10 minutes. The reaction was terminated by adding 100 μL / well of 1 M H2SO4. The absorbance (A450) was measured at 450 nm using an enzyme-labeled instrument. The results are shown in Table 4, and it can be seen that the titer of the purified antibody was 1:729,000.

[0177] Table 4

[0178] Antibody dilution factor OD450 1000 1.530 3000 1.393 9000 1.283 27000 1.169 81000 0.904 243000 0.572 729000 0.262 Background 0.048

[0179] Example 7: Sequence analysis of monoclonal antibody

[0180] (1) Identification of monoclonal antibody subtype

[0181] The hybridoma cell line G069 was cultured in a 10 cm diameter cell culture dish (37°C, 5% CO2) using DMEM medium (GIBCO, #C11995500BT) with 10% serum. After 7 days of culture, the cells were transferred to a 15 ml centrifuge tube, counted using a hemocytometer, and 4 x 10 6 After centrifugation at 200 g for 5 minutes, the supernatant was discarded, and the tube was inverted to dry the liquid inside. The cells in the tube were used to synthesize cDNA using a reverse transcription kit (Qiagen, 74134).

[0182] The antibody subtype was determined by PCR using primers specific to the antibody subtype. The synthesized cDNA described above was used as a template for the PCR reaction. The PCR reaction solution system: TAKARA Ex Taq (5 U / μL, TAKARA, RR001B), 0.25 μL; 10 x Ex Taq Buffer, 5 μL; dNTP mixture (2.5 mM each), 4 μL; template cDNA, 1 μL; upstream primer (100 μM), 1 μL; downstream primer (100 μM), 1 μL; add double distilled water to a total volume of 50 μL. The PCR reaction temperature program: pre-denaturation at 94°C for 5 minutes, temperature cycling 30 times (94°C for 1 minute, 57°C for 1 minute, 72°C for 1 minute), and extension at 72°C for 10 minutes. After the reaction, 10 μL of the PCR product was loaded on a 1% agarose gel for electrophoresis, and the electrophoresis pattern is shown in Figure 1 Table 6. According to the PCR product results, the antibody subtype can be inferred (Table 5). The heavy chain of the monoclonal antibody G069 obtained in the present application is IgG1, and the light chain is kappa.

[0183] Table 5

[0184]

[0185]

[0186] Note: S = C or G, M = A or C, R = A or G, and W = A or T.

[0187] Table 6

[0188] Lane PCR primer Antibody isotype specificity 1 PCR product of primers F-VH and R-VH 1 IgGl heavy chain 2 PCR product of primers F-VH and R-VH 2A IgG2A heavy chain 3 PCR product of primers F-VH and R-VH 2B IgG2B heavy chain 4 PCR product of primers F-VH and R-VH 3 IgG3 heavy chain 5 PCR product of primers F-VH and R-VK Ig Kappa light chain 6 PCR product of primers F-VL and R-VL 1 Ig Lambda 1 light chain 7 PCR product of primers F-VL and R-VL 2 Ig Lambda 2 light chain 8 DNA molecular weight standard (Full-length Gold, DNA Marker II, #BM411) N / A

[0189] (2) Sequencing of the antibody variable region (V region) of the hybridoma cell strain G069

[0190] The V region of the antibody of the cell strain G069 was cut from the agarose gel after PCR amplification (see above) and extracted using a DNA extraction kit (Qiagen, 74134). The extracted DNA fragment was linked to a pEASY-T1 cloning vector and transformed into Trans1-T1 competent cells (Transgen, CT101-1). The transformed bacterial colonies were picked into LB medium and subjected to DNA sequencing after overnight culture. The nucleotide sequence of the light chain variable region (V region) of the non-carboxylated osteocalcin antibody (G069) provided in the present application is shown in SEQ ID NO: 12, and the nucleotide sequence of the heavy chain variable region (V region) is shown in SEQ ID NO: 11.

[0191] Example 8: Identification of the specificity of antibody G069 in detecting non-carboxylated osteocalcin

[0192] The ability of antibody G069 to detect the specificity of free non-carboxylated osteocalcin was evaluated by ELISA. In this experiment, non-carboxylated osteocalcin peptide and carboxylated osteocalcin peptide were diluted in PBS and coated in 96-well microplates at a concentration of 1 μg / mL and 0.5 μg / mL, respectively, in a volume of 100 μL / well. After coating at 4°C overnight, the microplates were washed twice with 300 μL / well of PBST. After washing, the microplates were dried, and the microplates were blocked with 200 μL / well of PBS containing 1% BSA at room temperature for 2 hours. After drying, antibody G069 (diluted with PTB to 10 ng / mL) was added to the blocked microplates at a volume of 100 μL / well, and the microplates were incubated in a 37°C incubator for 1 hour. After drying, the microplates were washed three times with 300 μL / well of PBST. After drying, the microplates were added with 100 μL / well of HRP-labeled goat anti-mouse antibody diluted 5000-fold with PTB, and the microplates were incubated in a 37°C incubator for 1 hour. The microplates were washed three times with 300 μL / well of PBST. After drying, the microplates were added with 100 μL / well of TMB substrate solution, and the microplates were incubated at room temperature for 10 minutes. Then, 100 μL / well of 1 M H2SO4 was added to stop the reaction. The absorbance (A450) was measured at 450 nm using a microplate reader. The results of the experiment are shown in Table 7, which indicates that antibody G069 has good specificity in detecting non-carboxylated osteocalcin.

[0193] Table 7

[0194] Antigen concentration (ng / ml) Non-carboxylated osteocalcin peptide Carboxylated osteocalcin peptide 1000 1.683 0.098 500 0.935 0.086 Background 0.061 0.063

[0195] Example 9: Application of an enzyme immunoassay kit prepared using antibody G069 in detecting non-carboxylated osteocalcin in human serum

[0196] (1) Labeling of antibody G069 with horseradish peroxidase (HRP) and identification of the labeled product

[0197] Antibody G069 (20 nmol, 3 mg) was taken and added to a dialysis bag (10 KDa, width 1 cm), and dialyzed in 2 L of 10 mM PBS solution (pH 7.4) at 4°C overnight. The next day, the dialysis bag containing the antibody solution was placed in 1 L of 10 mM carbonate buffer (pH 9.5) and dialyzed at room temperature for 2 hours with stirring, in preparation for coupling with activated HRP.

[0198] Meanwhile, 1 mg of HRP was accurately weighed using an analytical balance, and 0.2 mL of ultrapure water was added to dissolve the HRP, so that the concentration of the HRP was 5 mg / mL. To the HRP solution, 40 μl of 0.1 M NaIO4was added, and the mixture was placed on a horizontal shaker and reacted (activated) at room temperature in the dark for 20 minutes. The activated HRP solution was added to a dialysis bag (10 KDa, 1 cm wide), and dialyzed overnight at 4°C in 2 L of 1 mM sodium acetate buffer (pH 4.4). The dialyzed HRP solution was carefully aspirated and transferred to a new 1.5 mL centrifuge tube, and 1 / 10 volume of 0.2 M carbonate buffer was added to raise the pH of the activated HRP solution to 9.0-9.5.

[0199] The above antibody and HRP were mixed (coupling reaction was performed), and the mixture was placed on a horizontal shaker and reacted at room temperature in the dark for 4 hours. After the coupling reaction was completed, 10 ul of freshly prepared NaBH4(pre-cooled with ultrapure water) was added, and the reaction was terminated by dialysis overnight at 4°C in the dark. The antibody solution after the coupling reaction was terminated was moved to a dialysis bag (10 KDa, 1 cm wide), and dialyzed at room temperature for 2 hours. Finally, the solution was transferred to a brown centrifuge tube and stored at 4°C.

[0200] (2) Enzyme immunoassay kit prepared using antibody G069 for detecting non-carboxylated osteocalcin in human serum

[0201] In this experiment, the osteocalcin antibody (SCBT, sc-74495) was diluted to 1 μg / mL with PBS, and then 100 μL / well was coated in a 96-well enzyme plate (4°C overnight). The plate was washed twice with 300 μL / well of PBST. After drying, the enzyme plate was blocked with a PBS solution containing 1% BSA, 200 μL / well, at room temperature for 2 hours. After drying, 25 ul / well of a 1000-fold diluted HRP-labeled antibody G069 (diluted with PTB) solution was added to the enzyme plate, and then 75 μL / well of a human serum sample of different concentrations (serum samples from patients diagnosed with bone-related diseases were selected as test samples, and serum samples from healthy people were used as negative samples) were added, and the mixture was reacted in a 37°C incubator for 45 minutes. The plate was washed with PBST three times, 300 μL / well each time. After drying, 100 μL / well of TMB substrate solution was added, and the mixture was reacted at room temperature for 10 minutes. Then, 100 μL / well of 1 M H2SO4was added to terminate the reaction, and the absorbance (A450) was measured at 450 nm using an enzyme marker. The results are shown in Table 8, which shows that the enzyme immunoassay kit prepared using antibody G069 can efficiently detect non-carboxylated osteocalcin in human serum.

[0202] Table 8

[0203] Patient serum sample OD 450 ]]> OD 450(空白) ]] Patient 1 0.913 0.059 Patient 2 0.845 0.037 Patient 3 0.986 0.082 Patient 4 0.275 0.093 Patient 5 0.485 0.081

[0204] Example 10: Detection of non-carboxylated osteocalcin based on chemiluminescence

[0205] The present embodiment uses the non-carboxylated osteocalcin antibody prepared in the foregoing embodiment to design a detection kit. Taking a magnetic microsphere separation chemiluminescence immunoassay method as an example, the detection principle is as shown in Figure 2 The kit shows the specific steps of detecting the content of non-carboxylated osteocalcin in the sample to be detected.

[0206] Instrument: self-developed chemiluminescence detector of the company;

[0207] Reagents: labeled alkaline phosphatase (ALP) was purchased from BBI Company in the United Kingdom, model number ALPI12G; SMCC was purchased from thermofisher scientific company, product number 22360; 2-IT was purchased from thermofisher scientific company, CAS: 4781-83-3; product number: 26101; carboxyl-modified microparticles were purchased from thermofisher scientific company; EDC was purchased from SIMGA company, CAS: 25952-53-8, product number: E7750; the antibody was the G069 antibody prepared in the foregoing embodiment and the commercial osteocalcin antibody purchased from R&D Systems, Inc., product number MAB11327; the luminescent substrate was a self-developed reagent of the company; the remaining reagents were conventional products obtained by purchase.

[0208] Experiment one: magnetic microsphere labeled non-carboxylated osteocalcin antibody

[0209] Take 20 mg of carboxyl-modified microparticle solution, and the magnetic microspheres with superparamagnetic properties, uniform particle size, and carboxyl (COOH-) active groups on the surface are settled (magnetic separation) under the action of a magnetic field for 10 minutes. The supernatant is removed, and the settled magnetic microspheres are washed with an activation buffer solution (2-(N-morpholine) ethanesulfonic acid) MES buffer solution with a molar concentration of 0.05 M and a pH of 6.0 for 3 times, each time with a volume of 2 ml.

[0210] After washing, the magnetic microspheres are fully suspended in 1.0 ml of the activation buffer solution (2-(N-morpholine) ethanesulfonic acid) MES buffer solution with a molar concentration of 0.05 M and a pH of 6.0, and then 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride (EDC) is added. The suspension is reacted at room temperature for 30 minutes, and the EDC reaction molar concentration is 7.5 mM.

[0211] Take 1.0 mg of non-carboxylated osteocalcin antibody and concentrate to 2.5 mg / mL.

[0212] The non-carboxylated osteocalcin antibody was concentrated to 2.5 mg / mL, and 1.0 mg of the non-carboxylated osteocalcin conjugated carrier protein antigen was added to 20 mg of the activated magnetic microspheres solution, mixed by shaking, and reacted for 6.5 hours under suspension at 4°C, so that the non-carboxylated osteocalcin antibody was covalently coupled to the surface of the magnetic microspheres, and was named reagent A.

[0213] Experiment two: alkaline phosphatase-labeled osteocalcin antibody

[0214] 1.0 mg of the osteocalcin antibody was concentrated to 2.5 mg / mL, and 5 μL of an activated agent 2-Iminothiolane hydrochloride (2-IT) solution with a concentration of 13.76 mg / mL was added, and reacted for 15 minutes at room temperature. The activated antibody was collected by desalting using a Sephadex G25 gel column.

[0215] 1.2 mg of alkaline phosphatase was concentrated to 2.5 mg / mL, and 12 μL of an activated agent Succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC) solution with a concentration of 6.69 mg / mL was added, and reacted for 15 minutes at room temperature. The activated alkaline phosphatase was collected by desalting using a Sephadex G25 gel column.

[0216] The activated osteocalcin antibody and alkaline phosphatase were mixed at a ratio of 1.0 mg of the osteocalcin antibody to 1.0 mg of the alkaline phosphatase, and reacted for 18 hours at 4°C.

[0217] The unlinked osteocalcin antibody 2 and alkaline phosphatase were removed by separation and purification using a Supperdex 200 gel chromatography column, and the linker was stored at 4°C, and was named reagent B.

[0218] Experiment three: non-carboxylated osteocalcin magnetic microsphere separation chemiluminescence immunoassay method

[0219] (1) Immune reaction: 50 μL of the calibrators or quality control samples were added to reaction tubes, followed by 50 μL of reagent A prepared in experiment one and 50 μL of reagent B prepared in experiment two, and mixed and incubated at 37°C for 10 minutes;

[0220] (2) Magnetic separation: the magnetic microspheres were allowed to settle in a magnetic field, the supernatant was removed, 200-500 μL of washing solution was added, the magnetic field was removed, and then the magnetic microspheres were again allowed to settle in a magnetic field, and the supernatant was removed; this was repeated 2-4 times to remove unbound antibodies and impurities;

[0221] (3) Reading: 200 μl of luminescent substrate solution was added, and the relative luminescent intensity (RLU) was measured by using a self-developed chemiluminescence detector after the luminescence of the ALP catalyzed substrate;

[0222] (4) The non-carboxylated osteocalcin concentration-luminescent value standard curve was obtained by using a four-parameter equation fitting according to the detected values.

[0223] (5) 50 μl of the sample to be detected was added into the reaction tube in sequence with 50 μl of reagent A prepared in experiment one and 50 μl of reagent B prepared in experiment two, and mixed and incubated at 37°C for 15 min;

[0224] (6) Magnetic separation: the magnetic microspheres were allowed to settle in the magnetic field, the supernatant was removed, 200-500 μl of washing solution was added, the magnetic field was removed, and then the magnetic microspheres were allowed to settle in the magnetic field again, and the supernatant was removed; this was repeated for 2-4 times to remove the unbound antibodies and impurities;

[0225] (7) Reading: 200 μl of luminescent substrate solution was added, and the relative luminescent intensity (RLU) was measured by using a self-developed chemiluminescence detector after the luminescence of the ALP catalyzed substrate;

[0226] (8) The luminescent intensity of the sample to be detected was compared with the standard curve of step (4), and the content of the non-carboxylated osteocalcin in the sample to be detected was calculated by using a four-parameter equation fitting.

[0227] In summary, the monoclonal antibody of the non-carboxylated osteocalcin prepared in the present application has the characteristics of high affinity and specificity. The non-carboxylated osteocalcin detection kit prepared based on the antibody can be used for rapidly detecting the content of the non-carboxylated osteocalcin in serum or plasma samples, has high sensitivity, strong specificity, simple operation, and low requirement for sample pretreatment. By being combined with a full-automatic chemiluminescence analyzer, the operation steps are greatly simplified, the detection speed and throughput are increased, the detection efficiency is improved, and errors caused by human operation are avoided.

[0228] In the description of the present specification, the description of the terms "one embodiment", "some embodiments", "an example", "a specific example", or "some examples" and the like means that the specific features, structures, materials or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present application. In the present specification, the illustrative description of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any appropriate manner in any one or more embodiments or examples. In addition, the different embodiments or examples described in the present specification and the features of the different embodiments or examples can be combined and combined by those skilled in the art without contradiction.

[0229] Although the embodiments of the present application have been shown and described above, it is understood that the above-described embodiments are exemplary and are not to be construed as limiting the present application, and that variations, modifications, substitutions and changes can be made by those skilled in the art without departing from the scope of the present application.

Claims

1. An antibody or antigen-binding fragment thereof targeting uncarboxylated osteocalcin, characterized in that, include: The heavy chain variable region CDR1 sequence is shown in the amino acid sequence of SEQ ID NO: 1; The heavy chain variable region CDR2 sequence is shown in the amino acid sequence of SEQ ID NO: 2; The heavy chain variable region CDR3 sequence is shown in the amino acid sequence of SEQ ID NO: 3; The light chain variable region CDR1 sequence is shown in the amino acid sequence of SEQ ID NO: 4; The light chain variable region CDR2 sequence is shown in the amino acid sequence of SEQ ID NO: 5; The light chain variable region CDR3 sequence is shown in the amino acid sequence of SEQ ID NO:

6.

2. The antibody or its antigen-binding fragment according to claim 1, characterized in that, The antibody or its antigen-binding fragment further contains a heavy chain framework region sequence, at least a portion of which is derived from at least one of a murine antibody, a rabbit antibody, a primate antibody, or a mutant thereof.

3. The antibody or its antigen-binding fragment according to claim 2, characterized in that, At least a portion of the heavy chain framework region sequence is derived from a murine antibody.

4. The antibody or its antigen-binding fragment according to claim 3, characterized in that, The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:

7.

5. The antibody or its antigen-binding fragment according to claim 1, characterized in that, The antibody or its antigen-binding fragment further contains a light chain framework region sequence, at least a portion of which is derived from at least one of a mouse antibody, a rabbit antibody, a primate antibody, or a mutant thereof.

6. The antibody or its antigen-binding fragment according to claim 5, characterized in that, At least a portion of the light chain framework region sequence is derived from a murine antibody.

7. The antibody or its antigen-binding fragment according to claim 6, characterized in that, The amino acid sequence of the light chain variable region is shown in SEQ ID NO:

8.

8. The antibody or antigen-binding fragment thereof according to any one of claims 1-7, characterized in that, The antibody or its antigen-binding fragment further comprises a heavy chain constant region, at least a portion of which is derived from at least one of a murine antibody, a rabbit antibody, a primate antibody, or a mutant thereof.

9. The antibody or its antigen-binding fragment according to claim 8, characterized in that, At least a portion of the heavy chain constant region sequence is derived from a murine antibody.

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

9.

11. The antibody or antigen-binding fragment thereof according to any one of claims 1-7, characterized in that, The antibody or its antigen-binding fragment further contains a light chain constant region sequence, at least a portion of which is derived from at least one of a murine antibody, a rabbit antibody, a primate antibody, or a mutant thereof.

12. The antibody or its antigen-binding fragment according to claim 11, characterized in that, At least a portion of the light chain constant region sequence is derived from a murine antibody.

13. The antibody or its antigen-binding fragment according to claim 12, characterized in that, The amino acid sequence of the light chain constant region is shown in SEQ ID NO:

10.

14. The antibody or its antigen-binding fragment according to claim 1, characterized in that, The antibody is a monoclonal antibody.

15. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the antibody or antigen-binding fragment thereof as described in any one of claims 1 to 14.

16. The nucleic acid molecule according to claim 15, characterized in that, The nucleic acid molecule is DNA.

17. An expression carrier, characterized in that, Carrying the nucleic acid molecule as described in claim 15 or 16.

18. A method for preparing the antibody or antigen-binding fragment thereof according to any one of claims 1 to 14, characterized in that, include: The expression vector of claim 17 is introduced into cells; The cells are cultured under conditions suitable for protein expression and secretion in order to obtain the antibody or its antigen-binding fragment.

19. The method according to claim 18, characterized in that, The cells in question are eukaryotic cells.

20. A recombinant cell, characterized in that, The recombinant cells carry the nucleic acid molecule of claim 15 or 16 or the expression vector of claim 17.

21. A composition, characterized in that, include: The antibody or its antigen-binding fragment as described in any one of claims 1 to 14, the nucleic acid molecule carrying the nucleic acid molecule as described in claim 15 or 16, the expression vector as described in claim 17, or the recombinant cell as described in claim 20.

22. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 14 in the preparation of a kit for detecting uncarboxylated osteocalcin.

23. A reagent kit, characterized in that, The kit includes: The antibody or antigen-binding fragment thereof as described in any one of claims 1 to 14.

24. The reagent kit according to claim 23, characterized in that, Further includes: At least one of the following: magnetic microspheres, biomarkers, biomarker substrates, chemiluminescent labels, buffer solutions, calibrators, and quality control products.

25. The kit according to claim 24, characterized in that, The magnetic microspheres are selected from 0.5-10 μm in size.

26. The reagent kit according to claim 25, characterized in that, The magnetic microspheres are selected from 3-8 μm in size.

27. The kit according to claim 24, characterized in that, The biomarkers include biological enzymes.

28. The reagent kit according to claim 27, characterized in that, The bioenzyme is selected from at least one of horseradish peroxidase and alkaline phosphatase.

29. The reagent kit according to claim 27, characterized in that, The biomarker substrate includes fluorescein, which is selected from at least one of fluorescein isothiocyanate, tetraethylrhodamine, and tetramethylrhodamine isothiocyanate.

30. The kit according to claim 27, characterized in that, The chemiluminescent label is selected from at least one of acridine ester, ruthenium terpyridine, isoluminol, or their derivatives.

31. The reagent kit according to claim 24, characterized in that, The buffer solution includes at least one selected from phosphate buffer, carbonate buffer, tris(hydroxymethyl)aminomethane buffer, and citrate buffer.

32. A method for detecting non-carboxylated osteocalcin for non-clinical diagnostic purposes, characterized in that, include: The test sample is processed using the kit according to any one of claims 23-31 to determine the content of the uncarboxylated osteocalcin in the test sample.

33. The method according to claim 32, characterized in that, The detection process includes: The magnetic microspheres were coupled with the first antibody to obtain the first antibody complex. A second antibody is labeled with a biomarker to obtain a second antibody complex. The test sample, the first antibody complex, and the second antibody complex are mixed and processed. The mixed treatment product is contacted with a biomarker substrate, wherein the biomarker has the activity of catalyzing the luminescence of the biomarker substrate; The content of non-carboxylated osteocalcin in the sample to be tested was determined based on the luminescence intensity obtained after contact treatment. in, The first antibody and the second antibody independently recognize uncarboxylated osteocalcin and osteocalcin, respectively; The antibody that recognizes non-carboxylated osteocalcin is selected from the antibody or antigen-binding fragment thereof as described in any one of claims 1 to 14.

34. The method according to claim 33, characterized in that, The coupling process is performed through the following steps: The magnetic microspheres in a predetermined mass ratio are coupled with the first antibody in an MES buffer system to obtain the first antibody complex.

35. The method according to claim 33, characterized in that, The mass ratio of the first antibody to the magnetic microspheres is 1~20:1000.

36. The method according to claim 34 or 35, characterized in that, The coupling reaction time is selected from 5 to 8 hours.

37. The method according to claim 36, characterized in that, The coupling reaction time is selected from 6.5 hours.

38. The method according to claim 34 or 35, characterized in that, The coupling reaction temperature is selected from 4~8℃.

39. The method according to claim 38, characterized in that, The coupling reaction temperature is selected from 4℃.

40. The method according to claim 33, characterized in that, The detection method further includes: A standard curve of non-carboxylated osteocalcin concentration-luminescence value was plotted using calibrators, wherein the calibrators contained non-carboxylated osteocalcin at predetermined concentration gradients.

41. The method according to claim 33, characterized in that, The step of determining the content of the non-carboxylated osteocalcin based on the luminescence intensity obtained after contact treatment includes: The standard is subjected to detection processing to determine the luminescence intensity corresponding to different concentrations; wherein, the calibrator contains non-carboxylated osteocalcin at a predetermined concentration gradient; A standard curve of non-carboxylated osteocalcin concentration-luminescence value was plotted based on the luminescence intensity corresponding to different concentrations. The content of non-carboxylated osteocalcin in the sample to be tested is obtained based on the standard curve of non-carboxylated osteocalcin concentration-luminescence value and the luminescence intensity obtained after the contact treatment.

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