Antibodies or antigen-binding fragments thereof specifically binding to a PG II antigen, antibody combinations containing the same and their use in the detection of PG II

Abstract

The application discloses an antibody or antigen-binding fragment thereof specifically binding to a PG II antigen, an antibody combination containing the same and application thereof in detecting the PG II. The antibody or antigen-binding fragment thereof specifically binding to the PG II antigen comprises HCDR1, HCDR2 and HCDR3 included in a heavy chain variable region with a sequence as shown in any one of SEQ ID NO: 19, SEQ ID NO: 21 and SEQ ID NO: 23, and comprises LCDR1, LCDR2 and LCDR3 included in a light chain variable region with a sequence as shown in any one of SEQ ID NO: 20, SEQ ID NO: 22 and SEQ ID NO: 24. A PG II protein detection kit is prepared by using the same, and sensitivity and detection rate of reagents can be effectively improved.

Description

Technical Field

[0001] This application relates to the field of structural immunology detection application technology, specifically to antibodies that specifically bind to PG II antigen or their antigen-binding fragments, antibody combinations containing them, and their application in the detection of PG II. Background Technology

[0002] Tumor markers are detectable substances in blood, body fluids, and tissues that are associated with the occurrence and development of tumors. Their production in tumor tissues is far greater than in normal tissues. The presence and changes in their quantities can indicate the nature of a tumor and have important auxiliary value in tumor diagnosis, classification, prognosis, and clinical treatment guidance.

[0003] Gastric cancer is the fourth most common cancer worldwide and the second leading cause of cancer death, with its incidence rate showing an upward trend year by year. Early diagnosis and early treatment are the only effective ways to improve patients' quality of life and reduce mortality. If gastric cancer can be diagnosed early, the 5-year survival rate can exceed 90%, while the 5-year survival rate for those diagnosed at a late stage is only 10-20%. Retrospective studies have shown that more than 90% of gastric cancer patients also have atrophic gastritis; therefore, atrophic gastritis is widely considered an important precancerous lesion and plays a key role in the pathogenesis of gastric cancer.

[0004] Pepsinogen (PG), a precursor to digestive enzymes secreted by the stomach, is the inactive precursor of pepsin in gastric juice. Based on biochemical properties, immunogenicity, cellular origin, and tissue distribution, PG can be divided into two subgroups: pepsinogen I (PG I) and pepsinogen II (PG II). PG I is mainly secreted by the chief cells and mucus neck cells of gastric glands, while PG II, in addition to these cells, can also be produced by the cardiac glands, pyloric glands, and Brunner's glands in the duodenum. Studies have shown that the PG I / PG II ratio serves as a marker for atrophic gastritis and has certain identification value in high-risk groups for gastric cancer. In patients with gastric cancer, serum PG I levels are usually decreased, while PG II levels remain normal or elevated. Therefore, measuring serum PG I levels and the PG I / PG II ratio is helpful in the differential diagnosis of gastric cancer. Serum PG protein content directly reflects gastric mucosal function; a significant decrease in PG I levels in gastric cancer patients indicates a reduced gastric mucosal secretory capacity. Since over 80% of gastric cancers are accompanied by atrophic gastritis, which can lead to the loss of chief cells in the gastric mucosa and thus affect secretory function, a significant decrease in the PG I / PG II ratio is of great clinical significance for early gastric cancer detection, especially for initial screening in populations in high-incidence areas of gastric cancer. PG testing, as a non-invasive method, has advantages such as simplicity, economy, and minimal discomfort, and has significant application value in early gastric cancer screening and high-risk population monitoring.

[0005] In recent years, methods for detecting tumor markers have been continuously developed, with common techniques including enzyme-linked immunosorbent assay (ELISA), chemiluminescent immunoassay (CLIA), and radioimmunoassay (RIA). Currently, the detection of PG II levels mainly relies on chemiluminescent immunoassay and ELISA, both of which require the use of specific antibodies against the PG II protein.

[0006] Some existing technologies employ two mouse monoclonal antibodies targeting PG II protein to detect serum PG II levels using a double-antibody sandwich assay. However, the antibodies used in this technology have insufficient sensitivity and specificity for detecting PG II protein, leading to false negatives or false positives in practical applications. Furthermore, the use of mouse-derived antibodies can easily trigger the human anti-animal antibody (HAAA) effect in human testing, particularly the human anti-mouse antibody (HAMA) effect, potentially distorting test results or interfering with subsequent treatment monitoring. Other existing technologies use a mouse monoclonal antibody targeting PG II protein as a capture antibody, combined with a polyclonal antibody (usually derived from rabbits) targeting PG II protein as a detection antibody to detect serum PG II levels. However, the antibody combination used in this technology still has limited sensitivity for detecting serum PG II, easily leading to false negatives, especially in low-concentration samples. In addition, significant batch-to-batch variability exists in the polyclonal antibodies used, resulting in poor consistency and repeatability of test results, affecting their reliability and standardized application in large-scale clinical screening.

[0007] In summary, existing antibodies used for PG II detection suffer from low sensitivity, insufficient specificity, susceptibility to interfering antibodies, and significant batch-to-batch variability, severely limiting the clinical application of PG II as a biomarker for early gastric cancer screening. Therefore, there is an urgent need to develop a PG II protein-specific antibody and its detection system with higher sensitivity, stronger specificity, lower immunogenicity, and better stability to improve the accuracy and reliability of early gastric cancer diagnosis. Summary of the Invention

[0008] Based on this, this application provides at least one antibody that specifically binds to PG II antigen or its antigen-binding fragment, an antibody combination containing the same, and its application in the detection of PG II.

[0009] One objective of this application is to design a chimeric antibody specifically targeting PG II protein as a capture antibody for the detection of PG II protein. This approach can improve sensitivity and increase detection rate, while also enhancing specificity and reducing false positives. The expression and purification of the recombinant antibody can be controlled through batch-to-batch variations in the manufacturing process.

[0010] In a first aspect of this application, an antibody or antigen-binding fragment thereof that specifically binds to PG II is provided, comprising HCDR1, HCDR2 and HCDR3 in the heavy chain variable region shown in any of SEQ ID NO: 19, SEQ ID NO: 21 and SEQ ID NO: 23, and comprising LCDR1, LCDR2 and LCDR3 in the light chain variable region shown in any of SEQ ID NO: 20, SEQ ID NO: 22 and SEQ ID NO: 24.

[0011] In a second aspect of this application, an antibody combination is provided, comprising an antibody or antigen-binding fragment thereof that specifically binds to PG II as described in the first aspect, serving as a capture antibody and a detection antibody, respectively.

[0012] The capture antibody and the detection antibody are different.

[0013] In a third aspect of this application, a nucleic acid molecule is provided that encodes an antibody or antigen-binding fragment thereof that specifically binds to PG II as described in the first aspect, or a combination of antibodies as described in the second aspect.

[0014] In a fourth aspect of this application, a recombinant expression vector is provided, comprising the nucleic acid molecule as described in the third aspect.

[0015] In a fifth aspect of this application, a host cell is provided which is transformed with the recombinant expression vector as described in the fourth aspect.

[0016] In a sixth aspect of this application, a kit for detecting PG II is provided, comprising an antibody or antigen-binding fragment thereof that specifically binds to PG II as described in the first aspect, or a combination of antibodies as described in the second aspect.

[0017] In a seventh aspect of this application, a method is provided for preparing an antibody or antigen-binding fragment thereof that specifically binds to PG II as described in the first aspect, or an antibody combination as described in the second aspect, comprising:

[0018] Culture the host cells as described in the fifth aspect and prepare the culture medium;

[0019] Isolate the antibody that specifically binds to PG II or its antigen-binding fragment or antibody combination from the culture medium.

[0020] In an eighth aspect of this application, the use of an antibody that specifically binds to PG II as described in the first aspect, or an antigen-binding fragment thereof, or an antibody combination as described in the second aspect, in the preparation of a product for detecting PG II is provided.

[0021] Using the antibodies or antigen-binding fragments of PG II that specifically bind as described above, a PG II protein detection kit can be prepared, which can effectively improve the sensitivity and detection rate of the reagent. Expression and purification via a recombinant platform can control batch-to-batch variability of raw materials and reduce costs. Attached Figure Description

[0022] To more clearly illustrate the technical solutions in the embodiments and examples of this application, and to more completely understand this application and its beneficial effects, the drawings used in the description of the embodiments or examples will be briefly introduced below. Obviously, the drawings described below are merely some embodiments of this application. Those skilled in the art can obtain other drawings based on these drawings without creative effort. It should also be noted that the drawings are all drawn in a simplified form and are only used to conveniently and clearly assist in illustrating this application.

[0023] Figure 1 This is a gel image of the full-length PG II protein in one embodiment of this application.

[0024] Figure 2 This is a gel image schematic diagram of the PG II specific antibody in one embodiment of this application.

[0025] Figure 3 This shows a schematic diagram of the heavy chain expression vector pFUSE-CHIg-mG1 according to one embodiment of this application.

[0026] Figure 4 This image shows a schematic diagram of the light chain expression vector pFUSE2-CLIg-mk according to one embodiment of this application. Detailed Implementation

[0027] To facilitate understanding of this application, a more complete description will be provided below with reference to the accompanying drawings. Preferred embodiments of this application are shown in the drawings. However, this application can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of this application.

[0028] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0029] In this application, unless otherwise specified, "one or more" means any one of the listed items or any combination of the listed items. Similarly, "one or more" and other instances that otherwise indicate "one or more" shall be understood in the same way unless otherwise specified.

[0030] The terms “combinations thereof,” “any combination thereof,” and “any combination thereof” as used in this application include all suitable combinations of any two or more of the listed items.

[0031] In this application, the word "suitable" in "suitable combination", "suitable method", "any suitable method" etc., shall be defined as being able to implement the technical solution of this application, solve the technical problem of this application, and achieve the expected technical effect of this application.

[0032] In this application, terms such as "further," "even more," "particularly," "for example," "like," "example," and "exemplary" are used for descriptive purposes to indicate that different technical solutions preceding and following each other are related in terms of their coverage, but should not be construed as limiting the preceding technical solution or restricting the scope of protection of this application. In this application, unless otherwise specified, A (e.g., B) indicates that B is a non-limiting example of A, and it can be understood that A is not limited to B.

[0033] In this application, "optionally," "optionally," and "optional" mean that something is optional, that is, it refers to either "with" or "without" a parallel solution. If multiple "options" appear in a technical solution, unless otherwise specified and there are no contradictions or mutual constraints, each "option" is independent. Unless otherwise specified, the descriptions such as "optionally include" and "optionally contain" in this application, taking "optionally include" as an example, mean "may include or not include."

[0034] The terms “containing,” “comprising,” and “including” as used in this application are synonyms and are inclusive or open-ended, not excluding additional, uncited members or features. Members or features include, for example, materials or components, structures, elements, instruments, etc.; non-limiting examples of members or features include actions, conditions under which actions occur, timing, states, etc.

[0035] In this application, the technical features or solutions described in open-ended language include both closed-ended technical features or solutions consisting of the listed contents and open-ended technical features or solutions that include the listed contents.

[0036] In this application, the exemplary descriptions such as "in some implementations (or embodiments)" and "in one implementation (or embodiment)" may cover, but are not limited to, the following meanings: these solutions can be combined with other solutions in a suitable manner to form new technical solutions.

[0037] In this application, the terms "first aspect," "second aspect," etc., are used for descriptive purposes only and should not be construed as indicating or implying relative importance or quantity, nor should they be construed as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first aspect," "second aspect," etc., serve only as a non-exhaustive enumeration and should be understood not to constitute a closed limitation on quantity.

[0038] In this application, when numerical intervals (i.e., numerical ranges) are involved, unless otherwise specified, the distribution of selectable numerical values ​​within the numerical interval is considered continuous, and includes the two endpoints of the numerical interval (i.e., the minimum and maximum values), as well as every numerical value between these two endpoints. Unless otherwise specified, when a numerical interval refers only to integers within that numerical interval, it includes the two endpoint integers of the numerical range, as well as every integer between the two endpoints, which is equivalent to directly listing every integer. When multiple numerical ranges are provided to describe features or characteristics, these numerical ranges can be merged. In other words, unless otherwise specified, the numerical ranges disclosed herein should be understood to include any and all subranges included therein. The "numerical value" in the numerical interval can be any quantitative value, such as a number, percentage, ratio, etc. The term "numerical interval" can be broadly included to include numerical interval types such as percentage intervals, ratio intervals, and proportion intervals.

[0039] In this application, where the method flow involves multiple steps, unless otherwise explicitly stated herein, there is no strict order restriction on the execution of these steps; they can be executed in any order other than those described. Moreover, any step may include multiple sub-steps or multiple stages, which are not necessarily completed at the same time, but can be executed at different times, and their execution order is not necessarily sequential, but can be performed alternately or simultaneously with other steps or parts of the sub-steps or stages of other steps.

[0040] The "CDR region" of heavy chain antibodies, as described in this article, refers to the "Complementarity Determining Region" within the variable domain of a heavy chain antibody. The CDR region is the most variable part of the heavy chain antibody and contains antigen-binding sites. The "FR region" of heavy chain antibodies, on the other hand, refers to the "Framework Region" within the variable domain. The non-CDR portion of the variable region is called the Framework Region (FR), and its amino acid composition and arrangement vary less compared to the CDR.

[0041] The "ELISA" mentioned in this article refers to the "Enzyme-Linked Immunosorbent Assay (ELISA)," which is currently the most widely used immunoassay method. This method involves labeling antibodies with enzymes, combining the specificity of the antigen-antibody reaction with the enzyme's catalytic action on the substrate, and interpreting the test results based on the color change after the enzyme reacts with the substrate.

[0042] The "CLIA" mentioned in this article refers to "Chemiluminescence Immunoassay (CLIA)". This method involves directly labeling antibody 1 with a chemiluminescent agent and magnetically conjugating antibody 2. The sample to be tested is then reacted with antibody 1 and antibody 2. A magnetic field is used to separate the bound (precipitated) and free chemiluminescent agent labels. A luminescence promoter is then added to initiate the luminescence reaction. Quantitative or qualitative detection is performed by measuring the intensity of the luminescence.

[0043] One aspect of this application provides an antibody or antigen-binding fragment thereof that specifically binds to pepsinogen II (PG II), comprising HCDR1, HCDR2, and HCDR3 in the heavy chain variable region shown in any of SEQ ID NO: 19, SEQ ID NO: 21, and SEQ ID NO: 23, and comprising LCDR1, LCDR2, and LCDR3 in the light chain variable region shown in any of SEQ ID NO: 20, SEQ ID NO: 22, and SEQ ID NO: 24.

[0044] In some embodiments, the antibody or its antigen-binding fragment includes HCDR1, HCDR2 and HCDR3 in the heavy chain variable region as shown in SEQ ID NO: 19, and LCDR1, LCDR2 and LCDR3 in the light chain variable region as shown in SEQ ID NO: 20.

[0045] In some embodiments, the antibody or its antigen-binding fragment includes HCDR1, HCDR2 and HCDR3 in the heavy chain variable region as shown in SEQ ID NO: 21, and LCDR1, LCDR2 and LCDR3 in the light chain variable region as shown in SEQ ID NO: 22.

[0046] In some embodiments, the antibody or its antigen-binding fragment includes HCDR1, HCDR2 and HCDR3 in the heavy chain variable region as shown in SEQ ID NO: 23, and LCDR1, LCDR2 and LCDR3 in the light chain variable region as shown in SEQ ID NO: 24.

[0047] The monoclonal antibody of this application may have the aforementioned CDRs, or a derived fragment having the aforementioned CDRs. The derived fragment is formed by replacing amino acids at no more than six sites relative to its corresponding CDR (“conservative modification” or “conservative substitution”), retaining the biological activity consistent with its corresponding complementarity-determining region. For example, the derivative fragment may undergo substitutions at sites 1, 2, 3, 4, 5, or 6 of its corresponding complementarity-determining region, which may involve replacing one amino acid with another, or replacing one amino acid with multiple amino acids (e.g., two).

[0048] In the CDRs (Kabat numbering rules) provided in this application, derived fragments (conserved variants) refer to polypeptides formed by replacing one, two, or three amino acids with amino acids of similar or related properties compared to the amino acid sequence of the antibody in this application. These conserved variant polypeptides are preferably generated by amino acid substitutions according to Table A.

[0049] Table A

[0050]

[0051] "Conservative modification" or "conservative substitution" refers to the replacement of an amino acid in a protein with another amino acid having similar characteristics (e.g., charge, side chain size, hydrophobicity / hydrophilicity, main chain conformation, and rigidity), allowing for frequent alterations without changing the protein's biological activity. Those skilled in the art will recognize that, in general, the substitution of a single amino acid in a non-essential region of a polypeptide does not substantially alter its biological activity (see, for example, Watson et al. (1987), Molecular Biology of the Gene, The Benjamin / Cummings Pub. Co., p. 224, (4th edition)). Furthermore, substitutions of structurally or functionally similar amino acids are unlikely to disrupt biological activity.

[0052] In some embodiments, HCDR1 comprises an amino acid fragment having at least 85%, at least 90%, at least 95%, or 100% identity with the amino acid sequence shown in SEQ ID NO: 1; HCDR2 comprises an amino acid fragment having at least 85%, at least 90%, at least 95%, or 100% identity with the amino acid sequence shown in SEQ ID NO: 2; HCDR3 comprises an amino acid fragment having at least 85%, at least 90%, at least 95%, or 100% identity with the amino acid sequence shown in SEQ ID NO: 3; LCDR1 comprises an amino acid fragment having at least 85%, at least 90%, at least 95%, or 100% identity with the amino acid sequence shown in SEQ ID NO: 4; LCDR2 comprises an amino acid fragment having at least 85%, at least 90%, at least 95%, or 100% identity with the amino acid sequence shown in SEQ ID NO: 5; and LCDR3 comprises an amino acid fragment having at least 85%, at least 90%, at least 95%, or 100% identity with the amino acid sequence shown in SEQ ID NO: 6.

[0053] In some embodiments, HCDR1 comprises an amino acid fragment having at least 85%, at least 90%, at least 95%, or 100% identity with the amino acid sequence shown in SEQ ID NO: 7; HCDR2 comprises an amino acid fragment having at least 85%, at least 90%, at least 95%, or 100% identity with the amino acid sequence shown in SEQ ID NO: 8; HCDR3 comprises an amino acid fragment having at least 85%, at least 90%, at least 95%, or 100% identity with the amino acid sequence shown in SEQ ID NO: 9; LCDR1 comprises an amino acid fragment having at least 85%, at least 90%, at least 95%, or 100% identity with the amino acid sequence shown in SEQ ID NO: 10; LCDR2 comprises an amino acid fragment having at least 85%, at least 90%, at least 95%, or 100% identity with the amino acid sequence shown in SEQ ID NO: 11; and LCDR3 comprises an amino acid fragment having at least 85%, at least 90%, at least 95%, or 100% identity with the amino acid sequence shown in SEQ ID NO: 12.

[0054] In some embodiments, HCDR1 comprises an amino acid fragment having at least 85%, at least 90%, at least 95%, or 100% identity with the amino acid sequence shown in SEQ ID NO: 13; HCDR2 comprises an amino acid fragment having at least 85%, at least 90%, at least 95%, or 100% identity with the amino acid sequence shown in SEQ ID NO: 14; HCDR3 comprises an amino acid fragment having at least 85%, at least 90%, at least 95%, or 100% identity with the amino acid sequence shown in SEQ ID NO: 15; LCDR1 comprises an amino acid fragment having at least 85%, at least 90%, at least 95%, or 100% identity with the amino acid sequence shown in SEQ ID NO: 16; LCDR2 comprises an amino acid fragment having at least 85%, at least 90%, at least 95%, or 100% identity with the amino acid sequence shown in SEQ ID NO: 17; and LCDR3 comprises an amino acid fragment having at least 85%, at least 90%, at least 95%, or 100% identity with the amino acid sequence shown in SEQ ID NO: 18.

[0055] In some embodiments, the heavy chain variable region of the antibody that specifically binds to PG II or its antigen-binding fragment includes an amino acid fragment having at least 85%, at least 90%, at least 95%, or 100% identity with the amino acid sequence shown in any of SEQ ID NO: 19, SEQ ID NO: 21, and SEQ ID NO: 23, and the light chain variable region includes an amino acid fragment having at least 85%, at least 90%, at least 95%, or 100% identity with the amino acid sequence shown in any of SEQ ID NO: 20, SEQ ID NO: 22, and SEQ ID NO: 24.

[0056] In some embodiments, the heavy chain variable region includes an amino acid fragment having at least 85%, at least 90%, at least 95%, or 100% identity with the amino acid sequence shown in SEQ ID NO: 19, and the light chain variable region includes an amino acid fragment having at least 85%, at least 90%, at least 95%, or 100% identity with the amino acid sequence shown in SEQ ID NO: 20.

[0057] In some embodiments, the heavy chain variable region includes an amino acid fragment having at least 85%, at least 90%, at least 95%, or 100% identity with the amino acid sequence shown in SEQ ID NO: 21, and the light chain variable region includes an amino acid fragment having at least 85%, at least 90%, at least 95%, or 100% identity with the amino acid sequence shown in SEQ ID NO: 22.

[0058] In some embodiments, the heavy chain variable region includes an amino acid fragment having at least 85%, at least 90%, at least 95%, or 100% identity with the amino acid sequence shown in SEQ ID NO: 23, and the light chain variable region includes an amino acid fragment having at least 85%, at least 90%, at least 95%, or 100% identity with the amino acid sequence shown in SEQ ID NO: 24.

[0059] Amino acid sequence “identity” refers to the percentage of amino acid residues in a first sequence that are identical to those in a second sequence when aligning amino acid sequences (introducing gaps where necessary) to achieve the maximum percentage of sequence identity, without considering any conserved substitutions as part of the sequence identity. To determine the percentage of amino acid sequence identity, alignment can be performed in a variety of ways within the scope of the art, such as using publicly available computer software like BLAST, BLAST 2, ALIGN, ALIGN 2, or Megalign (DNASTAR) software. Those skilled in the art can determine the parameters suitable for measuring alignment, including any algorithms required to achieve maximum alignment across the full length of the sequences being compared.

[0060] In some embodiments, the antibody or its antigen-binding fragment is monovalent, bivalent, or multivalent.

[0061] In some embodiments, the antibody or its antigen-binding fragment is single-specific, bispecific, or multispecific.

[0062] In some embodiments, the antibody or its antigen-binding fragment is a heavy chain antibody.

[0063] In some embodiments, the antibody or its antigen-binding fragment is a chimeric antibody or a humanized antibody.

[0064] In some embodiments, the heavy chain constant region of the chimeric antibody comprises human IgG1, and the light chain constant region comprises human Kappa type.

[0065] Another aspect of this application provides an antibody combination comprising an antibody or an antigen-binding fragment thereof that specifically binds to PG II as described in the first aspect, serving as a capture antibody and a detection antibody, respectively.

[0066] The capture antibody and the detection antibody are different.

[0067] For example, the capture antibody may be an antibody or its antigen-binding fragment comprising HCDR1, HCDR2, and HCDR3 in the heavy chain variable region as shown in SEQ ID NO: 19, and LCDR1, LCDR2, and LCDR3 in the light chain variable region as shown in SEQ ID NO: 20 (hereinafter referred to as "antibody A"); an antibody or its antigen-binding fragment comprising HCDR1, HCDR2, and HCDR3 in the heavy chain variable region as shown in SEQ ID NO: 21, and LCDR1, LCDR2, and LCDR3 in the light chain variable region as shown in SEQ ID NO: 22 (hereinafter referred to as "antibody B"); or an antibody or its antigen-binding fragment comprising HCDR1, HCDR2, and HCDR3 in the heavy chain variable region as shown in SEQ ID NO: 23, and LCDR1, LCDR2, and LCDR3 in the light chain variable region as shown in SEQ ID NO: 24 (hereinafter referred to as "antibody C").

[0068] Meanwhile, the detection antibody can also be any one of antibody A, antibody B, and antibody C. However, the capture antibody and the detection antibody cannot be antibody A, antibody B, or antibody C at the same time.

[0069] In some embodiments, the detection antibody is antibody A, and the capture antibody is antibody B.

[0070] In some embodiments, the detection antibody is antibody A, and the capture antibody is antibody C.

[0071] In some embodiments, the detection antibody is antibody B, and the capture antibody is antibody C.

[0072] In some embodiments, the detection antibody is antibody B, and the capture antibody is antibody A.

[0073] In some embodiments, the detection antibody is antibody B, and the capture antibody is antibody C.

[0074] In some embodiments, the detection antibody is antibody C, and the capture antibody is antibody B.

[0075] In some embodiments, the capture antibody and the detection are each independently selected from antibodies 1 to 3, wherein:

[0076] The amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of antibody 1 are shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively.

[0077] The amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of antibody 2 are shown in SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12, respectively.

[0078] The amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of antibody 3 are shown in SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18, respectively.

[0079] In some embodiments, the amino acid sequences of the heavy chain variable region and the light chain variable region of antibody 1 are shown in SEQ ID NO: 19 and SEQ ID NO: 20, respectively.

[0080] In some embodiments, the amino acid sequences of the heavy chain variable region and the light chain variable region of the antibody 2 are shown in SEQ ID NO: 21 and SEQ ID NO: 22, respectively.

[0081] In some embodiments, the amino acid sequences of the heavy chain variable region and the light chain variable region of the antibody 3 are shown in SEQ ID NO: 23 and SEQ ID NO: 24, respectively.

[0082] I do not wish to be limited by any theory, but I believe that the role of capture antibodies includes at least immobilizing on the surface of a solid-phase carrier (such as an ELISA plate) to specifically "capture" the target antigen in the sample to be tested.

[0083] Another aspect of this application provides a nucleic acid molecule that encodes an antibody or antigen-binding fragment thereof that specifically binds to PGII as described above, or a combination of antibodies as described above.

[0084] Unless otherwise specified, the nucleic acid molecules in this application may be isolated nucleic acid molecules. The nucleic acid molecules in this application primarily refer to isolated nucleic acid molecules. "Isolated" means that the molecule is substantially free of other biomolecules, such as nucleic acids, proteins, lipids, carbohydrates, or other materials, such as cell debris and growth media. Generally, the term "isolated" is not intended to mean the complete absence of these materials or the absence of water, buffer solutions, or salts.

[0085] In some embodiments, the nucleic acid molecule may be a combination product when it encodes the antibody combination. For example, it may comprise nucleic acid molecule 1 encoding a capture antibody and nucleic acid molecule 2 encoding a detection antibody.

[0086] Another aspect of this application provides a recombinant expression vector comprising the nucleic acid molecules described above.

[0087] The term "vector," also known as a "nucleic acid construct," refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. One type of vector is the "plasmid," which is a circular double-stranded DNA loop in which an additional DNA segment can be linked. Another type of vector is a viral vector, in which an additional DNA segment can be linked to a viral genome. Some vectors are capable of autonomous replication in the host cells to which they are introduced (e.g., bacterial vectors with bacterial origins of replication and free mammalian vectors). Other vectors (e.g., non-attached mammalian vectors) can integrate into the host cell's genome after introduction into the host cell and thereby replicate along with the host genome. Furthermore, some vectors are capable of directing the expression of genes to which they are effectively linked. Such vectors are referred to in this application as "recombinant expression vectors" (or simply "expression vectors"). Typically, expression vectors useful in recombinant DNA technology are in the form of plasmids. However, other forms of expression vectors, such as viral vectors (e.g., replication-defective retroviruses, adenoviruses, and adeno-associated viruses), also serve equivalent functions.

[0088] In some embodiments, the recombinant expression vector may be a combination product. Exemplarily, it may include a recombinant expression vector 1 containing the nucleic acid molecule 1 as described above, and a recombinant expression vector 2 containing the nucleic acid molecule 2 as described above.

[0089] Another aspect of this application provides a host cell that expresses an antibody or antigen-binding fragment thereof that specifically binds to PGII as described above, or a combination of antibodies as described above.

[0090] In some embodiments, the host cell is transformed with the recombinant expression vector described above.

[0091] The term "cell," also known as "host cell," refers to a cell into which a recombinant expression vector has been introduced. Host cells can include bacterial, microbial, plant, or animal cells. Easily transformable bacteria include members of the Enterobacteriaceae family, such as strains of Escherichia coli or Salmonella; members of the Bacillaceae family, such as Bacillus subtilis; Pneumococcus; Streptococcus; and Haemophilus influenzae. Suitable microorganisms include Saccharomyces cerevisiae and Pichia pastoris. Suitable animal host cell lines include CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, or HEK293 cells.

[0092] In some embodiments, the host cell includes a non-human mammalian cell.

[0093] Another aspect of this application provides a method for preparing the aforementioned host cells, including the step of introducing the aforementioned or recombinant expression vector into target cells.

[0094] In one example, the import method uses transfection.

[0095] The term “transfection” refers to the process of introducing nucleic acids into cells, such as non-human mammalian cells. Protocols and techniques used for transfection include, but are not limited to, lipid transfection and chemical and physical methods such as electroporation. Many transfection techniques are well known in the art and are disclosed herein. See, for example, Graham et al., 1973, Virology 52:456; Sambrook et al., 2001, Molecular Cloning: A Laboratory Manual, ibid.; Davis et al., 1986, Basic Methods in Molecular Biology, Elsevier; Chu et al., 1981, Gene 13:197.

[0096] Another aspect of this application provides a kit for detecting PG II, comprising an antibody that specifically binds to PG II as described above, or an antigen-binding fragment thereof, or a combination of antibodies as described above.

[0097] In some embodiments, the kit includes the antibody combination, wherein the detection antibody is biolabeled or chemically labeled; the chemical label includes horseradish peroxidase.

[0098] In some embodiments, the kit further includes one or more of coating solution, blocking solution, diluent, buffer solution, washing solution, chromogenic solution and stop solution.

[0099] Another aspect of this application provides a method for preparing an antibody or antigen-binding fragment thereof that specifically binds to PG II as described above, or an antibody combination as described above, comprising:

[0100] Culture the host cells as described above and prepare the culture medium;

[0101] Isolate the antibody that specifically binds to PG II or its antigen-binding fragment or antibody combination from the culture medium.

[0102] Another aspect of this application provides the use of antibodies that specifically bind to PG II as described above, or antigen-binding fragments thereof, or combinations of antibodies as described above, in the preparation of products for detecting PG II.

[0103] Another aspect of this application provides a method for detecting PG II using an antibody that specifically binds to PG II as described above, or an antigen-binding fragment thereof, or a combination of antibodies as described above.

[0104] The following are some examples.

[0105] The embodiments of this application will be described in detail below with reference to examples. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of this application. For experimental methods in the following embodiments where conditions are not specified, reference should be made to the guidelines given in this application, or to experimental manuals or conventional conditions in the art, or to the conditions recommended by the manufacturer, or to experimental methods known in the art.

[0106] Example 1 Immunogen Preparation—Full-length PG II Protein

[0107] The full-length gene sequence of PG II protein was constructed into the pcDNA3.1(+) transvector, and after recombinant expression in 293F cells, the antigen was prepared using affinity chromatography and ion exchange chromatography. See the full-length gel image of PG II protein. Figure 1 .

[0108] The amino acid sequence is as follows:

[0109] Full-length PG II protein:

[0110] MKWMVVVLVCLQLLEAAVVKVPLKKFKSIRETMKEKGLLGEFLRTHKYDPAWKYRFGDLSVTYEPMAYMDAAYFGEISIGTPPQNFLVLFDTGSSNLWVPSVYCQSQACTSHSRFNPSESSTYSTNGQTFSLQYGSGSLTGFFGYDTLTVQSIQVPNQEFGLSENEPGTNFVYAQFDGIMGLAYPALSVDEATTAMQGM VQEGALTSPVFSVYLSNQQGSSGGAVVFGGVDSSLYTGQIYWAPVTQELYWQIGIEEFLIGGQASGWCSEGCQAIVDTGTSLLTVPQQYMSALLQATGAQ EDEYGQFLVNCNSIQNLPSLTFIINGVEFPLPPSSYILSNNGYCTVGVEPTYLSSQNGQPLWILGDVFLRSYYSVYDLGNNRVGFATAAHHHHHH* (SEQ ID NO: 25).

[0111] Example 2: Construction of PG II antibody phage library

[0112] 1. Animal immunization

[0113] BalB / C mice aged 6-8 weeks were immunized with the full-length PG II protein. For the initial immunization, 50 μg of protein was administered per mouse via a 1:1 mixture of Freund's complete adjuvant and PG II monoclonal antibody, emulsified and administered subcutaneously at multiple sites. Subsequent booster immunizations were performed using a 1:1 mixture of Freund's incomplete adjuvant and antigen, 20 μg per mouse, emulsified and administered subcutaneously at multiple sites. Each immunization cycle was spaced 14 days apart. After three immunizations, the mouse tail blood coated with the full-length PG II protein was analyzed by ELISA until a titer of 10 was achieved. 5 Stop immunization at that time.

[0114] 2. Gene amplification and bacterial library construction

[0115] After immunization, mouse spleens were ground and mRNA was extracted from the spleen as a template. cDNA from spleen cells was obtained by reverse transcription using oligo(dT) primers. Then, antibody-specific primers were used to amplify the gene sequences of the light and heavy chain variable regions, respectively. The antibody light and heavy chains were ligated together using recombinant PCR, and the resulting scFv gene fragment mixture was purified and constructed into the pCNATAB5E vector. The product ligated into the vector was subjected to electroporation of TG1 strain. The electroporation mixture was then serially diluted and plated. Colony counts were performed the next day, and the stock was calculated based on the number of colonies at each dilution. Twenty-four clones were randomly selected for testing to analyze the diversity of the bacterial library.

[0116] 3. Phage library enrichment and selection

[0117] A primary phage library was obtained by infecting a bacterial library with M13KO7 helper phage at a multiplicity of infection ratio of 20:1. PG II antigen was coated onto 96-well microplates at four concentrations: 100 μg / mL, 25 μg / mL, 6.25 μg / mL, and 2 μg / mL, 100 μL / well, and incubated overnight at 4°C. The next day, the antigen solution was discarded, and 300 μL of PBS solution containing 5% skim milk powder (MPBS) was added, and the plates were blocked at room temperature for 2 hours. The microplates were washed three times with PBST solution (PBS containing 0.1% Tween 20), and the phage library was diluted to 10 cfu / mL with MPBS to obtain phage library R0. 100 μL of MPBS solution containing phage library R0 was added to each well of the microplate, and the plates were shaken on a horizontal shaker at room temperature for 1 hour, followed by incubation at room temperature for 1 hour. Wash 10 times with PBST solution. Add 100 μL of trypsin solution (1 mg / mL, dissolved in sterile PBS) to each well and incubate at room temperature for 10 min to elute phages bound to PG II antigen. Add 100 μL of glycine-hydrochloric acid buffer (GlycinHCl, pH=2.2) to each well and incubate at room temperature for 5 min to elute phages bound to PG II protein. Then add 50 μL of Tris-HCl buffer (pH=7.4) to each well for neutralization. Infect TG1 cells with the neutralized samples. After phage amplification and purification, perform enrichment and screening again, for a total of 4 rounds of screening.

[0118] Example 3: Screening of mouse monoclonal antibodies against PG II protein

[0119] After three rounds of screening and enrichment, polyclonal and monoclonal phages were detected using Phage-ELISA. For polyclonal detection, recombinant phages amplified after screening were used for PG II binding ELISA. Ninety-four monoclonal phages were randomly selected and cultured in TG1 plates, infected with M13KO7 helper phage, and cultured with shaking at 250 r / min for 36 h. The phage supernatant was collected by centrifugation. Helper phage M13KO7 was obtained using the same method as a negative control for PG II binding ELISA (coating amount 2 μg / mL). First, the phage supernatant was incubated at 37 ℃ for 2 h, then washed, and the secondary antibody HRP-anti-M13 antibody (diluted in 5% skim milk) was added and incubated at 37 ℃ for 1 h. Color development was performed using 3,3',5,5'-tetramethylbenzidine chromogenic solution, and the reaction was terminated with 2 mol / L concentrated sulfuric acid after color development. The A450 value was measured using an ELISA reader. Calculate the P / N ratio, where H11 is the OD value of the positive well and H12 is the OD value of the negative well. The positive determination criterion is P / N ≥ 2.5, and the negative determination criterion is P / N < 1.5. The results are shown in Table 1.

[0120] Table 1. Results of monoclonal antibody cell supernatant detection (coated with full-length PG II protein)

[0121]

[0122] The full-length PG II protein was coated onto an ELISA plate at a concentration of 1 μg / ml, and the signals of the nine highly positive clones selected in the first round of screening were tested in duplicate. The P / N ratio was calculated, where Pos. is the OD value of the positive wells and Neg. is the OD value of the negative wells (see Table 2). A positive result was defined as P / N ≥ 2.5, and a negative result as P / N < 1.5. The 63 clones with the highest positive count were amplified, and plasmids were extracted and sequenced, named Ab01-03. Sequence analysis yielded three completely different antibody sequences.

[0123] Table 2

[0124]

[0125] Example 3 Construction of a specific antibody against PG II protein

[0126] The corresponding positive hybridoma cell lines were amplified, and their variable region gene sequences were amplified using RT-PCR with appropriate primers. The sequences were named: VH1 (PG II-01 heavy chain variable region), VL1 (PG II-01 light chain variable region); VH2 (PG II-02 heavy chain variable region), VL2 (PG II-02 light chain variable region); VH3 (PG II-03 heavy chain variable region), VL3 (PG II-03 light chain variable region), as shown below:

[0127] VH1:EVQLVESGPELVKPGASVKVSCAASGYSFNDSNMHWVRQSHGKSPEWIGYLDPYNGATTYSQKFKGKATLTADTSSSTAFMQLNSLTSEDSAVYYCARSYYGAWFAYWGQGTLVTVSAAKTTPPSVYPLAP(SEQ IDNO: 19);

[0128] VL1:DIQMSQSPKSRYASLGERVTFTCKASEGISYLSWLQQKPGKSPKTLIYQVLKIVDGVPSRFPGSGSGQDYSLTISSLEYQDLGIYYCSQYAEFPFTFGGGTKLEIKRADAAPTVSIF(SEQ ID NO: 20)。

[0129] VH2:DVQLPESGAGLVQPAGSRKLSCRASGFTYSSRGMWWVRPAREKGLEWRAYLGSRSDRTYKADAVRGRFTISRDNPKNTLFLQMTSLRSEDTAMYFCARRDKYWFRYWGQGTLVTVSAAKTTAPSVYPLAP(SEQ ID NO:21);

[0130] VL2:DVVLTQSPASLAVSLGQRATISCKASQSVDYDASYMNWYQQKPGQPPKLLIYAASNIRESGIPARFSGSGSGTDFTLNIHPVEEEDAAIYYCQQSDEDAWTFGGGTKLEVKRADAAPTVSIF(SEQ ID NO: 22)。

[0131] VH3:QAQLVQSGPDLRKPGETVKISCKASGYTRTDYSMHWVKQAPGKGLKWMGWLNTYTGEPTYADAFKGRFAFSLETSASSAYIQLNNLKNEDTATYFCARRNAWIAYWGQGTLVTVSAAKTTPPSVYPLAP(SEQ ID NO:23);

[0132] VL3: DVVMTQTPSSLSASLGDRVSISCRASQDIGSNYNWLQQKPGGTVKLLIYYTSRLRSGVPSRFSGSGPSGTDYSITLSNLEQEDIATYFCQQGNTLTWTFGGGTNLEIKRADAAPTVSIF (SEQ ID NO: 24).

[0133] VH1, VH2, and VH3 were respectively constructed into the heavy chain expression vector pFUSE-CHIg-mG1 (vector map see below). Figure 3 VL1, VL2, and VL3 were constructed into the light chain expression vector pFUSE2-CLIg-mk (vector map see [link]). Figure 4 Plasmids containing light and heavy chains were obtained using an endotoxin-free plasmid extraction kit. These plasmids were then transiently transfected into 293F cells at an appropriate ratio. Approximately 48 hours later, the cell culture supernatant was collected, concentrated, and purified by Protein A affinity chromatography and ion exchange chromatography to obtain the specific antibodies PG II-Ab-01, PG II-Ab-02, and PG II-Ab-03. The SDS-PAGE electrophoresis images of the prepared specific antibodies are shown below. Figure 2 .

[0134] Example 4: Verification of bispecific antibody activity against PG II protein

[0135] PG II-Ab-01, PG II-Ab-02, and PG II-Ab-03 were coated onto an ELISA plate at a concentration of 1 μg / ml. After blocking with BSA, the plates were reacted with different concentrations of PG II protein, and then an appropriate concentration of HRP-labeled rabbit polyclonal antibody was added. Color development was performed using TMB. Sensitivity test results are shown in Table 3.

[0136] Table 3. Sensitivity test results for each PG II antibody antigen.

[0137]

[0138] PG II-Ab-01, PG II-Ab-02, and PG II-Ab-03 were coated onto an ELISA plate at a concentration of 1 μg / ml. After blocking with BSA, the plates were reacted with different concentrations of PG II protein, and then appropriate concentrations of HRP-labeled PG II-Ab-01, PG II-Ab-02, and PG II-Ab-03 antibodies were added. TMB was used for color development.

[0139] Table 4. Sensitivity test results for each PG II antibody-paired antigen.

[0140]

[0141] Antibody pairing 01: PG II-Ab01 (capture antibody) + PG II-Ab02-HRP;

[0142] Antibody pairing 02: PG II-Ab01 (capture antibody) + PG II-Ab03-HRP;

[0143] Antibody pairing 03: PG II-Ab02 (capture antibody) + PG II-Ab01-HRP;

[0144] Antibody pairing 04: PG II-Ab02 (capture antibody) + PG II-Ab03-HRP;

[0145] Antibody pairing 05: PG II-Ab03 (capture antibody) + PG II-Ab01-HRP;

[0146] Antibody pairing 06: PG II-Ab03 (capture antibody) + PG II-Ab02-HRP.

[0147] Table 4 shows that all paired groups can effectively test the PGII antigen. Among them, the antigen test sensitivity is the best when PGII-Ab02 is used as the capture end antibody and Ab03 is used as the labeling end antibody, which can detect up to 0.16 ng / mL.

[0148] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0149] The embodiments described above are merely illustrative of several implementation methods of this application, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the protection scope of this application. Therefore, the protection scope of this patent application should be determined by the appended claims, and the specification and drawings can be used to interpret the content of the claims.

Claims

1. An antibody or antigen-binding fragment thereof that specifically binds to pepsinogen II (PG II), characterized in that, It includes HCDR1, HCDR2, and HCDR3 in the heavy chain variable region shown in any of SEQ ID NO: 19, SEQ ID NO: 21, and SEQ ID NO: 23, and also includes LCDR1, LCDR2, and LCDR3 in the light chain variable region shown in any of SEQ ID NO: 20, SEQ ID NO: 22, and SEQ ID NO: 24; Optionally: The antibody or its antigen-binding fragment includes HCDR1, HCDR2 and HCDR3 in the heavy chain variable region as shown in SEQ ID NO: 19, and LCDR1, LCDR2 and LCDR3 in the light chain variable region as shown in SEQ ID NO:

20. The antibody or its antigen-binding fragment includes HCDR1, HCDR2, and HCDR3 in the heavy chain variable region as shown in SEQ ID NO: 21, and LCDR1, LCDR2, and LCDR3 in the light chain variable region as shown in SEQ ID NO: 22; or, The antibody or its antigen-binding fragment includes HCDR1, HCDR2 and HCDR3 in the heavy chain variable region as shown in SEQ ID NO: 23, and LCDR1, LCDR2 and LCDR3 in the light chain variable region as shown in SEQ ID NO:

24.

2. The antibody or antigen-binding fragment thereof that specifically binds to PG II as described in claim 1, characterized in that, CDR1, CDR2, and CDR3 are encoded according to Kabat, and: (1) The HCDR1 comprises an amino acid fragment having at least 85%, at least 90%, at least 95%, or 100% identity with the amino acid sequence shown in SEQ ID NO: 1; the HCDR2 comprises an amino acid fragment having at least 85%, at least 90%, at least 95%, or 100% identity with the amino acid sequence shown in SEQ ID NO: 2; the HCDR3 comprises an amino acid fragment having at least 85%, at least 90%, at least 95%, or 100% identity with the amino acid sequence shown in SEQ ID NO: 3; the LCDR1 comprises an amino acid fragment having at least 85%, at least 90%, at least 95%, or 100% identity with the amino acid sequence shown in SEQ ID NO: 4; the LCDR2 comprises an amino acid fragment having at least 85%, at least 90%, at least 95%, or 100% identity with the amino acid sequence shown in SEQ ID NO: 5; and the LCDR3 comprises an amino acid fragment having at least 85%, at least 90%, at least 95%, or 100% identity with the amino acid sequence shown in SEQ ID NO:

6. (2) The HCDR1 comprises an amino acid fragment having at least 85%, at least 90%, at least 95%, or 100% identity with the amino acid sequence shown in SEQ ID NO: 7; the HCDR2 comprises an amino acid fragment having at least 85%, at least 90%, at least 95%, or 100% identity with the amino acid sequence shown in SEQ ID NO: 8; the HCDR3 comprises an amino acid fragment having at least 85%, at least 90%, at least 95%, or 100% identity with the amino acid sequence shown in SEQ ID NO: 9; the LCDR1 comprises an amino acid fragment having at least 85%, at least 90%, at least 95%, or 100% identity with the amino acid sequence shown in SEQ ID NO: 10; the LCDR2 comprises an amino acid fragment having at least 85%, at least 90%, at least 95%, or 100% identity with the amino acid sequence shown in SEQ ID NO: 11; and the LCDR3 comprises an amino acid fragment having at least 85%, at least 90%, at least 95%, or 100% identity with the amino acid sequence shown in SEQ ID NO: 12; or, (3) The HCDR1 comprises an amino acid fragment having at least 85%, at least 90%, at least 95%, or 100% identity with the amino acid sequence shown in SEQ ID NO: 13; the HCDR2 comprises an amino acid fragment having at least 85%, at least 90%, at least 95%, or 100% identity with the amino acid sequence shown in SEQ ID NO: 14; the HCDR3 comprises an amino acid fragment having at least 85%, at least 90%, at least 95%, or 100% identity with the amino acid sequence shown in SEQ ID NO: 15; the LCDR1 comprises an amino acid fragment having at least 85%, at least 90%, at least 95%, or 100% identity with the amino acid sequence shown in SEQ ID NO: 16; the LCDR2 comprises an amino acid fragment having at least 85%, at least 90%, at least 95%, or 100% identity with the amino acid sequence shown in SEQ ID NO: 17; and the LCDR3 comprises an amino acid fragment having at least 85%, at least 90%, at least 95%, or 100% identity with the amino acid sequence shown in SEQ ID NO:

18.

3. The antibody or antigen-binding fragment thereof that specifically binds to PG II as described in claim 1, characterized in that, Its heavy chain variable region includes an amino acid fragment having at least 85%, at least 90%, at least 95%, or 100% identity with the amino acid sequence shown in any of SEQ ID NO: 19, SEQ ID NO: 21, and SEQ ID NO: 23; the light chain variable region includes an amino acid fragment having at least 85%, at least 90%, at least 95%, or 100% identity with the amino acid sequence shown in any of SEQ ID NO: 20, SEQ ID NO: 22, and SEQ ID NO: 24; optionally: The heavy chain variable region includes an amino acid fragment having at least 85%, at least 90%, at least 95%, or 100% identity with the amino acid sequence shown in SEQ ID NO: 19, and the light chain variable region includes an amino acid fragment having at least 85%, at least 90%, at least 95%, or 100% identity with the amino acid sequence shown in SEQ ID NO:

20. The heavy chain variable region comprises an amino acid fragment having at least 85%, at least 90%, at least 95%, or 100% identity with the amino acid sequence shown in SEQ ID NO: 21, and the light chain variable region comprises an amino acid fragment having at least 85%, at least 90%, at least 95%, or 100% identity with the amino acid sequence shown in SEQ ID NO: 22; or, The heavy chain variable region includes an amino acid fragment having at least 85%, at least 90%, at least 95%, or 100% identity with the amino acid sequence shown in SEQ ID NO: 23, and the light chain variable region includes an amino acid fragment having at least 85%, at least 90%, at least 95%, or 100% identity with the amino acid sequence shown in SEQ ID NO:

24.

4. The antibody or antigen-binding fragment thereof that specifically binds to PG II as described in any one of claims 1 to 3, characterized in that, It meets one or more of the following conditions: 1) The antibody or its antigen-binding fragment is monovalent, bivalent, or polyvalent; 2) The antibody or its antigen-binding fragment is monospecific, bispecific, or multispecific; 3) The antibody or its antigen-binding fragment is a heavy chain antibody; and, 4) The antibody or its antigen-binding fragment is a chimeric antibody or a humanized antibody.

5. An antibody combination, characterized in that, It includes antibodies that specifically bind to PG II as described in any one of claims 1 to 4, or their antigen-binding fragments, as capture antibodies and detection antibodies, respectively; The capture antibody and the detection antibody are different; Optionally, the capture antibody and the detection are each independently selected from antibodies 1 to 3, wherein: The amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of antibody 1 are shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively; optionally, the amino acid sequences of the heavy chain variable region and the light chain variable region of antibody 1 are shown in SEQ ID NO: 19 and SEQ ID NO: 20, respectively. The amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of antibody 2 are shown in SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12, respectively; optionally, the amino acid sequences of the heavy chain variable region and the light chain variable region of antibody 2 are shown in SEQ ID NO: 21 and SEQ ID NO: 22, respectively. The amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of antibody 3 are shown in SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18, respectively; optionally, the amino acid sequences of the heavy chain variable region and the light chain variable region of antibody 3 are shown in SEQ ID NO: 23 and SEQ ID NO: 24, respectively.

6. A nucleic acid molecule, characterized in that, It encodes an antibody or antigen-binding fragment thereof that specifically binds to PG II as described in any one of claims 1 to 4, or an antibody combination as described in claim 5.

7. A recombinant expression vector, characterized in that, It contains the nucleic acid molecule as described in claim 6.

8. A host cell, characterized in that, Its transformation can be performed using the recombinant expression vector as described in claim 7.

9. A kit for detecting PG II, characterized in that, It includes an antibody that specifically binds to PG II as described in any one of claims 1 to 4, or an antigen-binding fragment thereof, or an antibody combination as described in claim 5; Optionally, the kit includes the antibody combination, wherein the detection antibody is biolabeled or chemically labeled; the chemical label includes horseradish peroxidase; Optionally, the kit may further include one or more of the following: coating solution, blocking solution, diluent, buffer, washing solution, chromogenic solution, and stop solution.

10. A method for preparing an antibody or antigen-binding fragment thereof that specifically binds to PG II as described in any one of claims 1 to 4, or an antibody combination as described in claim 5, characterized in that, It includes: Culture medium is prepared by culturing the host cells as described in claim 8; Isolate the antibody that specifically binds to PG II or its antigen-binding fragment or antibody combination from the culture medium.

11. Use of the antibody that specifically binds to PG II as described in any one of claims 1 to 4, or the antigen-binding fragment thereof, or the antibody combination as described in claim 5, in the preparation of a product for detecting PG II.

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