A monoclonal antibody targeting PGLYRP1 and preparation method and application thereof

Abstract

The application discloses a monoclonal antibody targeting PGLYRP1 and a preparation method and application thereof. The monoclonal antibody capable of accurately recognizing PGLYRP1 is constructed, and the monoclonal antibody can stably recognize the conserved domain of PGLYRP1. The monoclonal antibody can significantly reduce cross-reaction and improve the accuracy of experimental results, and is particularly suitable for signal detection in complex tissues. In addition, the monoclonal antibody is derived from a stable cell clone, can be cultured and amplified for a long time, ensures the quality stability of the product, and overcomes the problems of large batch-to-batch difference of polyclonal antibodies and non-reproducibility.

Description

Technical Field

[0001] This invention belongs to the fields of molecular biology and immunology, and in particular relates to a monoclonal antibody targeting PGLYRP1, its preparation method and application. Background Technology

[0002] PGLYRP1 is a member of the peptidoglycan recognition protein (PGRP) family, which is highly conserved in insects and mammals. In mammals, PGLYRP1 is approximately 200 amino acids long, with a molecular weight of 18-20 kDa, and possesses a signal peptide. PGLYRP1 is expressed along the digestive tract, particularly in the granules of neutrophils and eosinophils, and its expression is lower in non-immune cells. PGLYRP1 is a key effector molecule in the innate immune system, and its core function is to recognize and bind to peptidoglycan, a major component of bacterial cell walls.

[0003] Currently, the detection of PGLYRP1 primarily utilizes polyclonal antibodies. These antibodies originate from different B cell clones and therefore recognize multiple epitopes of the antigen, resulting in lower antibody specificity. Polyclonal antibodies can bind to different parts of the target antigen or cross-react with similar molecules, leading to nonspecific binding. In complex samples (such as serum or cell lysates), nonspecific binding can interfere with result interpretation, affecting the accuracy and reproducibility of the experiment.

[0004] Therefore, there is an urgent need to develop a monoclonal antibody that can target PGLYRP1.

[0005] The information in the background section is merely intended to illustrate the general background of the invention and should not be construed as an admission or implication in any way that such information constitutes prior art known to those skilled in the art. Summary of the Invention

[0006] To address at least some of the technical problems in the prior art, this invention provides a monoclonal antibody targeting PGLYRP1, its preparation method, and its applications. Specifically, this invention includes the following:

[0007] In a first aspect, the present invention provides an antibody or an antigen-binding fragment thereof capable of targeting peptidoglycan recognition protein 1, said antibody or antigen-binding fragment thereof comprising the heavy chain CDR1-3 shown in SEQ ID NO. 1-3 and / or the light chain CDR1-3 shown in SEQ ID NO. 4-6.

[0008] In some embodiments, the antibody or antigen-binding fragment thereof according to the present invention has any one of the amino acid sequences shown in (I)-(III):

[0009] (I) The heavy chain variable region sequence shown in SEQ ID NO.7 and / or the light chain variable region sequence shown in SEQ ID NO.8;

[0010] (II) An amino acid sequence that has at least 90% homology with the amino acid sequence shown in (I) and has the same function;

[0011] (III) An amino acid sequence that has the same function as the amino acid sequence shown in (I) or (II) obtained by modifying, substituting, deleting or adding one or more amino acids.

[0012] In some embodiments, the antibody or antigen-binding fragment thereof according to the present invention includes a chimeric antibody, a humanized antibody or a murine antibody, and the antigen-binding fragment includes Fab, Fab', F(ab')2, scFv or scFv Fc fragments.

[0013] In a second aspect, the invention provides a nucleic acid molecule comprising a nucleotide sequence of an antibody or an antigen-binding fragment thereof as described in the first aspect of the invention.

[0014] A third aspect of the invention provides a carrier molecule comprising the nucleic acid molecule described in the second aspect of the invention.

[0015] In a fourth aspect, a host cell is provided that comprises a nucleic acid molecule as described in the second aspect of the invention or a carrier molecule as described in the third aspect of the invention.

[0016] A fifth aspect of the present invention provides a method for preparing an antibody or an antigen-binding fragment thereof according to a first aspect of the present invention, wherein the antibody or the antigen-binding fragment thereof is prepared by artificial synthesis or genetic engineering.

[0017] A sixth aspect of the invention provides a detection product for peptidoglycan recognition protein 1, comprising the antibody or antigen-binding fragment thereof described in the first aspect of the invention.

[0018] In some embodiments, the detection product according to the present invention includes a reagent kit, test strip, or protein chip.

[0019] A seventh aspect of the present invention provides the use of the antibody or antigen-binding fragment thereof described in the first aspect of the present invention in the preparation of a peptidoglycan recognition protein 1 detection product.

[0020] This invention constructs a monoclonal antibody capable of accurately recognizing PGLYRP1, ensuring its stable recognition of the conserved domains of PGLYRP1. Its binding efficacy is significantly superior to polyclonal antibodies recognizing multiple non-specific epitopes. The monoclonal antibody of this invention significantly reduces cross-reactivity, improving the accuracy of experimental results, and is particularly suitable for signal detection in complex tissues. Furthermore, the monoclonal antibody of this invention is derived from stable cell clones, allowing for long-term culture and amplification, ensuring product quality stability and overcoming the problems of large batch-to-batch variability and non-reproducibility associated with polyclonal antibodies. Attached Figure Description

[0021] Figure 1 The results of plasmid digestion verification after constructing the target gene are shown. In the figure, M is the DNA marker, 1 is the XbaI and HindIII digested plasmid, and 2 is the plasmid DNA.

[0022] Figure 2 The results of antigen protein expression purification (Coomassie Brilliant Blue staining) are shown. In the figure, A is the purification test diagram, B is the final concentration diagram, M is the protein marker, IN is the original sample, FT is the flow-through solution, W is the washing solution, and E is the elution solution.

[0023] Figure 3 The results of antibody expression purification (Coomassie Brilliant Blue staining) are shown, where A is the purification test image, B is the final concentration image, M is the protein marker, IN is the original sample, FT is the flow-through buffer, W is the washing buffer, and E is the elution buffer. Detailed Implementation

[0024] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0025] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that the upper and lower limits of the range and each intermediate value between them are specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, are also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0026] Unless otherwise stated, 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 invention pertains. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0027] Antibody or its antigen-binding fragment

[0028] In one aspect, the present invention provides an antibody or antigen-binding fragment thereof capable of binding peptidoglycan recognize protein 1 with high affinity and targeting, without being bound by any theory. The antibody or antigen-binding fragment thereof of the present invention comprises heavy chain CDR1-3 shown in SEQ ID NO. 1-3 and / or light chain CDR1-3 shown in SEQ ID NO. 4-6, wherein the amino acid sequences of the heavy chain CDR1-3 are GFSLSSY (SEQ ID NO. 1), GNYGN (SEQ ID NO. 2) and GDYYIYGYSQNGFDP (SEQ ID NO. 3), respectively; and the amino acid sequences of the light chain CDR1-3 are QASQSIGSYLN (SEQ ID NO. 4), SASTLAS (SEQ ID NO. 5) and QQGYSSTDIANA (SEQ ID NO. 6), respectively.

[0029] In one specific embodiment, the antibody or its antigen-binding fragment has any one of the amino acid sequences shown in (I)-(III):

[0030] (I) The heavy chain variable region sequence shown in SEQ ID NO.7 and / or the light chain variable region sequence shown in SEQ ID NO.8;

[0031] (II) An amino acid sequence that has at least 90% homology with the amino acid sequence shown in (I) and has the same function;

[0032] (III) An amino acid sequence that has the same function as the amino acid sequence shown in (I) or (II) obtained by modifying, substituting, deleting or adding one or more amino acids.

[0033] In this article, the term "antibody" refers to an immunoglobulin molecule that has the ability to specifically bind to a particular antigen. Antibodies typically contain variable and constant regions in each of their heavy and light chains. The variable regions of the antibody heavy and light chains contain binding domains that interact with the antigen. The constant regions of the antibody mediate the binding of the immunoglobulin to host tissues or factors. Therefore, most antibodies have a heavy chain variable region (VH) and a light chain variable region (VL), which together form the antibody moiety that binds to the antigen.

[0034] The "light chain variable region (VL)" or "heavy chain variable region (VH)" of this invention consists of a "framework" region interspersed with three "complementarity-determining regions (CDRs)". The framework regions are used to modulate the CDRs for specific binding to antigenic epitopes. The CDRs contain the amino acid residues in the antibody that are primarily responsible for antigen binding. From the amino terminus to the carboxyl terminus, both the VL and VH domains contain the following framework (FR) regions and CDR regions: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.

[0035] In this document, the terms "homology" and "identity" are used interchangeably. Homologous sequences include amino acid sequences that are at least 90%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to the sequences of this invention. To determine sequence identity, sequence alignment can be performed, which can be done in various ways known to those skilled in the art, such as using BLAST, BLAST-2, ALIGN, NEEDLE, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for alignment, including any algorithms required to achieve optimal alignment across the full-length sequences being compared.

[0036] In this document, the modified antibody sequences also fall within the scope of protection of this invention. The term "modification" refers to any chemical modification of an amino acid sequence, such as substitution, deletion, insertion, and / or addition of amino acids. The term "substitution" refers to replacing one or more amino acids with different amino acids. "Deletion" refers to the reduction of one or more amino acids in the amino acid sequence. "Insertion" or "addition" refers to a change in the amino acid sequence resulting in an increase of one or more amino acids compared to a naturally occurring molecule. It should be noted that in the modified antibodies provided by this invention, the modification preferably occurs in regions other than the variable region, such as the frame region or constant region of the antibody, and the modified antibody still retains the desired functional properties of the antibody or its antigen-binding fragment of this invention, or has improved antigen-binding properties.

[0037] In this invention, the antibody includes chimeric antibodies, humanized antibodies, or murine antibodies.

[0038] The term "monoclonal antibody," as used in this article, sometimes also called "monoclonal antibody" or "Ab," refers to an immunoglobulin derived from a pure cell line, possessing the same structure and chemical properties, and specific for a single antigenic determinant. Monoclonal antibodies differ from conventional polyclonal antibody preparations (which typically contain different antibodies targeting different determinants); each monoclonal antibody targets a single determinant on an antigen. In addition to their specificity, the advantage of monoclonal antibodies is that they are obtained through hybridoma or recombinant engineered cell culture, free from contamination by other immunoglobulins. This characteristic contrasts with polyclonal antibody products, which generally consist of antibodies targeting different antigenic determinants. The modifier "monoclonal" indicates the antibody's characteristic of being obtained from a homogeneous group of antibodies, but this should not be interpreted as requiring any special methods to produce the antibody.

[0039] In this article, the term "chimeric antibody" generally refers to an antibody in which a portion of the amino acid sequence of each heavy or light chain is homologous to, or belongs to, a corresponding amino acid sequence from an antibody of a specific species, while the remaining segments of that chain are homologous to a corresponding sequence from another species. For example, the variable regions of both the light and heavy chains may originate from the variable region of an antibody from one animal species (e.g., mouse, rat, etc.), while the constant region is homologous to an antibody sequence from another species (e.g., human). For instance, to obtain a chimeric antibody, the variable region can be generated using non-human B cells or hybridoma cells, with the constant region combined with it derived from human cells. The variable region has the advantage of being easy to prepare, and its specificity is not affected by the source of the constant region it is combined with. Furthermore, because the constant region of a chimeric antibody can be derived from humans, the likelihood of the chimeric antibody eliciting an immune response upon injection is lower than with antibodies using a non-human-derived constant region.

[0040] In this article, the term "humanized antibody" generally refers to a chimeric antibody that contains fewer sequences derived from non-human immunoglobulins, thereby reducing the immunogenicity of the xenobiotic antibody when introduced into humans, while maintaining the antibody's complete antigen-binding affinity and specificity.

[0041] In this document, the term "mouse antibody" generally refers to an antibody whose variable region framework and CDR region are derived from mouse germline immunoglobulin sequences. Additionally, if the antibody contains a constant region, it is also derived from mouse germline immunoglobulin sequences. In this invention, mouse antibodies may contain amino acid residues not encoded by mouse germline immunoglobulin sequences, for example, mutations introduced through in vitro random or point mutations or through in vivo somatic mutations.

[0042] In this article, the term "antigen-binding fragment" generally refers to one or more fragments in an antibody that perform the function of specifically binding antigens. The antigen-binding function of an antibody can be achieved through the full-length fragment of the antibody. The antigen-binding function of an antibody can also be achieved through a heavy chain containing fragments including Fv, scFv, dsFv, Fab, Fab', or F(ab')2, or a light chain containing fragments including Fv, scFv, dsFv, Fab, Fab', or F(ab')2.

[0043] In this invention, the terms “targeting,” “binding,” “immunobinding,” “specific binding,” and “targeting” are used interchangeably and generally refer to a non-covalent interaction occurring between an immunoglobulin molecule and an antigen specific to said immunoglobulin. The strength or affinity of an immunobinding interaction can be expressed as a dissociation constant (Kd), where a smaller Kd represents a higher affinity. “Affinity” refers to the strength of the sum of all non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise stated, when used herein, “binding affinity” refers to the intrinsic binding affinity reflecting a 1:1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of molecule X for its partner Y is generally expressed as a binding dissociation equilibrium constant. Affinity can be measured by methods commonly known in the art, including those known in the prior art and those described herein.

[0044] Unless otherwise stated, the antibodies or antigen-binding fragments thereof described herein are isolated antibodies or antigen-binding fragments thereof. The term "isolated" as used herein means an antibody or antigen-binding fragment thereof that has been extracted from its natural environment. "Isolated" antibodies or fragments thereof therefore include antibodies or fragments thereof purified by standard purification methods. This term also includes antibodies or fragments thereof prepared through recombinant expression in host cells and chemically synthesized antibodies or fragments thereof.

[0045] Nucleic acid molecules

[0046] In one aspect, the present invention provides a nucleic acid molecule comprising a nucleotide sequence encoding an antibody or an antigen-binding fragment thereof described in the present invention.

[0047] As used in this invention, the term "nucleic acid" is intended to include polymeric forms of nucleotides of any length containing deoxyribonucleotides, ribonucleotides, and / or their analogues, including DNA, RNA, and DNA / RNA hybrids, and also including DNA or RNA analogues, such as those containing a modified backbone (e.g., peptide nucleic acid (PNA) or phosphate thioester) or modified bases. Therefore, the nucleic acids of this invention include DNA, cDNA, mRNA, recombinant nucleic acids, etc.

[0048] Once the coding sequence of the antibody described in this invention is obtained, the antibody can be obtained in large quantities using recombinant technology. An exemplary method is to clone its coding gene into a vector, transform it into cells, and then isolate it from the proliferated host cells using conventional methods.

[0049] carrier molecules

[0050] In one aspect, the present invention provides a carrier molecule comprising the nucleic acid molecule described herein.

[0051] The vector of this invention refers to an artificial construct capable of delivering and preferably expressing one or more target genes or sequences in a host cell. The vector of this invention is not limited and can be an expression vector, viral vector, etc. In some embodiments, the vector contains a target gene encoding the antibody of this invention, a promoter, a terminator, or optionally a marker gene. The vector can be a known vector or a self-constructed vector. Known vectors include plasmid vectors, lentiviral vectors, adenovirus vectors, AAV viral vectors, etc.

[0052] host cells

[0053] In one aspect, the present invention provides a host cell comprising the nucleic acid molecule or the carrier molecule described in the present invention.

[0054] The host cell of this invention refers to any cell type suitable for transformation, transfection, transduction, etc., using a nucleic acid construct or expression vector containing the nucleic acid molecules of this invention. The host cell includes any offspring of the parent cell that differs from the parent cell due to mutations occurring during replication.

[0055] Preparation method

[0056] In one aspect, this invention provides a method for preparing the antibody or its antigen-binding fragment described herein. The preparation method is not particularly limited, and includes preparation by artificial synthesis or genetic engineering.

[0057] In some embodiments, the antibodies of the present invention are obtained by artificial synthesis. Methods for artificially synthesizing antibodies are known in the art, for example, the antibodies or antigen-binding fragments of the present invention are obtained by direct amino acid synthesis.

[0058] In some embodiments, the antibodies of the present invention are obtained through genetic engineering expression. Genetic engineering expression systems for use include, but are not limited to, prokaryotic cell expression systems, eukaryotic cell expression systems, and cell-free expression systems. Examples of prokaryotic cell expression systems include *E. coli* expression systems. Eukaryotic cell expression systems include enzyme expression systems, insect cell expression systems, and mammalian cell expression systems. In a preferred embodiment, the antibodies of the present invention can be prepared by the following steps:

[0059] (1) Construct a recombinant vector for antibody heavy chain expressing the amino acid sequence shown in SEQ ID NO.7 and / or a recombinant vector for antibody light chain expressing the amino acid sequence shown in SEQ ID NO.8;

[0060] (2) The vector is transformed into host cells and cultured under conditions suitable for antibody expression;

[0061] (3) Collect and purify the antibody.

[0062] In another preferred embodiment, the antibody of the present invention is prepared by immunization with an antigen having the amino acid sequence shown in SEQ ID No. 9.

[0063] Product testing

[0064] The present invention further provides a detection product for detecting (or quantifying) peptidoglycan recognition protein 1, comprising the antibody or its antigen-binding fragment described in this invention, and instructions on how to perform the detection method of this invention on peptidoglycan recognition protein 1.

[0065] In a preferred embodiment, the detection product includes a reagent kit, test strip, or protein chip.

[0066] In this document, the term "kit" refers to a combination of reagents and other materials. A kit is intended to contain reagents such as buffers, protein stabilizing agents, signal generation systems (e.g., fluorescence signal generation systems), antibodies or antigen-binding fragments thereof, control proteins, and test containers (e.g., microtiter plates, etc.). The term "kit" is not limited to a specific combination of reagents and / or other materials; for example, a kit may also include instructions for using the reagents. Kits can be packaged in any suitable manner, typically having components in a single container or (if necessary) in multiple containers, along with instructions for performing the assay. Kits can be prepared using a variety of methods known in the art.

[0067] In some embodiments, the kit may further include at least one of a washing solution, a substrate solution, a diluent, and a calibration solution. The washing solution is not particularly limited in composition, but examples include, but are not limited to, buffer solutions, surfactants, and preservatives. The substrate solution may use known substrates, including but not limited to chromogenic substrates, fluorescent substrates, and luminescent substrates. The diluent is not particularly limited in composition, but examples include, but are not limited to, buffer solutions and surfactants. The calibration solution is not particularly limited in composition, but examples include, but are not limited to, BSA solution, trehalose solution, and animal serum.

[0068] application

[0069] This invention further provides the application of the antibody or its antigen-binding fragment described herein in the preparation of a peptidoglycan recognition protein 1 (PGI1) detection product, wherein the detection product includes a kit, test strip, or protein chip. The detection product is used for the prediction, diagnosis, and / or prognosis of PGI1-related diseases.

[0070] Example

[0071] This embodiment illustrates the preparation and detection process of a monoclonal antibody targeting PGLYRP1.

[0072] 1. Antigen Design and Preparation

[0073] 1.1 Gene Synthesis and Vector Construction

[0074] The amino acid sequence of M40505-PGLYRP1 was used as the antigen sequence (as shown in SEQ ID NO. 9) for PCR amplification. The PCR amplification reaction system is shown in Table 1, and the PCR amplification reaction conditions are shown in Table 2. Electrophoretic detection and recovery of the amplified fragments were performed according to the instructions of the commercially available kit.

[0075] Table 1 PCR amplification reaction system

[0076]

[0077] Table 2 PCR amplification reaction conditions

[0078]

[0079] The vector and target gene were digested with enzymes at 37℃ for 1-2 h. The enzyme digestion reaction system is shown in Table 3. The enzyme digestion products were then detected by electrophoresis and recovered.

[0080] Table 3 Enzyme digestion reaction system of vector and target gene

[0081]

[0082] The vector and the target gene were ligated, and the reaction system is shown in Table 4.

[0083] Table 4. Ligation reaction system between vector and target gene

[0084]

[0085] Add the DNA fragment to be transformed into a tube containing TOP10 competent cells (50 μl of competent cells requires 25 ng of DNA), the volume should not exceed 5% of the competent cells, gently rotate a few times to mix the contents, and incubate on ice for 30 min. Place the centrifuge tube mixture in circulating water heated to 42°C and heat shock for 90 s without shaking the tube. Quickly transfer the tube to an ice bath to cool the cells for 1-2 min. Add 200 μl of SOC liquid medium to each tube, warm the medium to 37°C in a water bath, and then transfer the tube to a shaker set to 37°C and incubate at 220 rpm for 45 min to revive the cells and express the plasmid-encoded resistance marker gene. Transfer an appropriate volume (200 μl per 90 mm plate) of transformed competent cells to LB medium containing the corresponding antibiotic. Invert the plates and incubate at 37°C; plaques will appear after 12-16 hours.

[0086] Once colonies have grown on the plate, randomly select several for colony PCR verification to detect transformants. Positive clones are verified by sequencing and enzyme digestion. Figure 1 As shown.

[0087] 1.2 Protein Expression and Purification

[0088] Expression was tested using the HEK293 mammalian cell expression system. The target gene was transfected into HEK293 cells for protein expression and purification testing.

[0089] The expression conditions were as follows: HEK293 cell density: 1.5-2.0 × 10⁻⁶ 6 Samples / ml; culture temperature: 37°C; culture speed: 130 rpm / min; culture and sample collection time: 6 days.

[0090] Specific experimental procedure:

[0091] (1) According to 0.5×10 6 Inoculate cells at a rate of 300 ml per 1 L shake flask.

[0092] (2) Incubate at 37℃, 120 rpm, and 5% carbon dioxide concentration in a shaker incubator for 24 h until the cell density reaches 1×10⁻⁶ cells / h. 6 cells / ml (cells need to double every 24 hours).

[0093] (3) Add 300 μg of DNA (filtered and sterilized) to 30 ml of PBS, then vortex for 3 seconds to mix thoroughly.

[0094] (4) Add 1.2 ml of filtered sterile PEI solution (0.5 mg / ml) to the PBS / DNA mixture.

[0095] (5) Let the PEI-DNA mixture stand at room temperature for 20 min.

[0096] (6) Add the DNA / PEI mixture to the cells, and the cell density must reach 1.5-2.0 × 10⁶ cells / year. 6 cells / ml.

[0097] (7) After transfection, the cells were incubated for 6 days in a shaker incubator at 37°C, 120 rpm and 5% carbon dioxide concentration.

[0098] (8) Centrifuge at 3000 g for 5 min to collect the culture medium supernatant and cell pellet, and store at -80℃ or use for subsequent purification.

[0099] M40505 collected 1 L of culture medium supernatant, centrifuged at high speed, and filtered through a 0.22 μm filter membrane. The supernatant was then bound to resin for purification of the target protein. The resin was Protein A resin. The binding and equilibration solution was PBS at pH 7.4, the washing buffer was PBS at pH 7.4, the elution buffer was 0.1 M glycine at pH 3.0, and the neutralization buffer was 1 M Tris-HCl at pH 8.5. The expression and purification results are as follows. Figure 2 As shown, the theoretical size of the protein calculated is 21.5 kDa, which is consistent with the size of the SDS-PAGE result, indicating that the protein can be immunized.

[0100] 2. Animal immunization

[0101] 2.1 Immunity

[0102] Healthy female New Zealand White rabbits, 4 months old and weighing 2.1 kg, were selected. The primary immunization antigen was a recombinant protein mixed with an equal volume of Freund's complete adjuvant and emulsified. The secondary, tertiary, and quaternary immunization antigens were recombinant proteins mixed with an equal volume of Freund's incomplete adjuvant and emulsified.

[0103] First immunization: Day 1, the antigen used for immunization is Freund's complete adjuvant + recombinant protein;

[0104] Second immunization: On day 14, the antigen used for immunization is Freund's incomplete adjuvant + recombinant protein;

[0105] Third immunization: On day 28, the antigen used for immunization is Freund's incomplete adjuvant + recombinant protein;

[0106] Blood was collected after the third vaccination: On day 35, 1 ml of blood was collected from the ear vein and the antiserum titer was detected by ELISA;

[0107] Fourth immunization: On day 42, the antigen used for immunization is Freund's incomplete adjuvant + recombinant protein;

[0108] Final bloodletting: On day 49, the antiserum titer was found to be within the required range by ELISA, and whole blood was collected from the carotid artery.

[0109] 2.2 Indirect ELISA detection

[0110] Dilute the antigen to 6 μg / ml with 0.05 mol / L carbonate (pH=9.6), 100 μl / well, and incubate overnight at 4°C. Wash three times with 0.05% Tween-20 (PBST), 3 min / time. Add 150 μl of 5% skim milk powder (PBST) blocking buffer to each well and block at 37°C for 60 min. Wash three times with 0.05% Tween-20 (PBST), 3 min / time. Dilute the antiserum 1:1000, then serially dilute, and incubate at 37°C for 1 hour. Wash three times with 0.05% Tween-20 (PBST), 3 min / time. Horseradish enzyme-labeled goat anti-rabbit IgG (H+L) (catalog number: 116154, antibody company: Jackson), 1:8000, and incubate at 37°C for 45 min. Wash five times with 0.05% Tween-20 (PBST), 3 minutes each time. Add 100 μl of substrate solution (TMB) per well, react for 5-10 minutes, and finally stop the reaction by adding 100 μl of 2 mol / L sulfuric acid. Measure the OD value at 450 nm using a microplate reader (Kehua ST-360).

[0111] 2.3 Antibody affinity purification

[0112] Add an appropriate amount of affinity purification magnetic beads to the purification column. Wash three times with approximately 30 ml of 10 mM HCl. Then wash away any residual hydrochloric acid with binding buffer. Add the purified antigen dissolved in PBS to the magnetic beads. After adding buffer, incubate overnight at 4°C. Wash three times with alternating acid, water, and alkali for column connection. Finally, wash with PBS for later use. Identify the frozen antiserum and corresponding magnetic beads according to the antibody purification schedule and record them. Thaw the serum and transfer the magnetic beads to the purification column. Take the required amount of serum and return any excess serum. Centrifuge the serum at 3400 rpm for 20 minutes at 4-8°C. Use a pipette to remove lipids from the surface of the centrifuged serum and collect a small sample for ELISA testing. Incubate the centrifuged serum and magnetic beads together at room temperature for 2 hours (or 37°C on a shaker if the room temperature is low) or overnight at 4°C. After incubation, collect the flow-through (FT). Wash the magnetic beads three times with PBS, 10 column volumes each time. Wash the purification column with 5 ml of pre-chilled pH 5.0 wash buffer. Elute with pre-chilled pH 2.5 elution buffer, collecting 1 ml each time, with 50 μl of neutralization buffer pre-added to the EP tube. Detect the peak using Coomassie assay; add 10 μl of the collection buffer to 100 μl of Coomassie, and determine the required antibody based on the peak. Store at 4°C. Stop collection when no color is detected by Coomassie assay, and wash with 10 ml of PBS. Incubate the flow solution with magnetic beads again, repeating the above steps. Concentrate the collected antibody with PEG. Add 50% glycerol and 0.02% sodium azide according to the antibody volume, mix well, and store for later use.

[0113] 2.4 Valence Testing

[0114] The antibody ELISA test results for project M40505 are shown in Table 5. The purified antibody titers were: A ≥ 1024K and B ≥ 1024K. Rabbit A had the best titer, so rabbit A was selected for subsequent serum collection and B cell enrichment screening.

[0115] Table 5. Antibody ELISA Detection Results

[0116]

[0117] After sorting and culturing the B cells enriched from rabbit A, the supernatant was analyzed by ELISA to screen out candidate positive clones 1G3, 1B1, 1E4, 1E11, and 2E5 (Table 6). Among them, clone 2E5 was selected for subsequent antibody heavy and light chain gene amplification and vector construction for recombinant expression.

[0118] Table 6. Antibody titer test results

[0119]

[0120] The purification results of the recombinant expression antibody from the 2E5 positive clone are as follows: Figure 3 As shown.

[0121] The titer of the recombinant expression antibody from the 2E5 positive clone is shown in Table 7.

[0122] Table 7 Results of 2E5 antibody titer detection

[0123]

[0124] 3. Sequencing

[0125] The antibody expressed by the 2E5 clone was subjected to subsequent molecular sequencing, and the sequence is shown in Table 8:

[0126] Table 8. Amino acid sequence of 2E5 monoclonal antibody

[0127]

[0128] This embodiment constructs a monoclonal antibody capable of accurately recognizing PGLYRP1 by screening for specific epitopes and verifying their binding sites. This ensures stable recognition of the conserved domains of PGLYRP1, with binding efficacy significantly superior to polyclonal antibodies recognizing multiple non-specific epitopes. The monoclonal antibody of this invention significantly reduces cross-reactivity, improves the accuracy of experimental results, and is particularly suitable for signal detection in complex tissues. Furthermore, the monoclonal antibody of this invention is derived from stable cell clones, allowing for long-term culture and amplification, ensuring consistent product quality and overcoming the limitations of polyclonal antibodies, such as large batch-to-batch variability and lack of reproducibility.

[0129] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. These modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. An antibody or its antigen-binding fragment, characterized in that, It can target peptidoglycan recognition protein 1, and the antibody or its antigen-binding fragment includes the heavy chain CDR1-3 shown in SEQ ID NO.1-3 and the light chain CDR1-3 shown in SEQ ID NO.4-6.

2. The antibody or its antigen-binding fragment according to claim 1, characterized in that, The antibody or its antigen-binding fragment has the heavy chain variable region sequence shown in SEQ ID NO.7 and the light chain variable region sequence shown in SEQ ID NO.

8.

3. The antibody or its antigen-binding fragment according to claim 1, characterized in that, The antibody includes humanized antibody or mouse antibody, and the antigen-binding fragment includes Fab, Fab', F(ab')2, scFv or scFv Fc fragment.

4. A nucleic acid molecule, characterized in that, It consists of a nucleotide sequence encoding the antibody or antigen-binding fragment of any one of claims 1-3.

5. A carrier molecule, characterized in that, It comprises the nucleic acid molecule as described in claim 4.

6. A host cell, characterized in that, It comprises the nucleic acid molecule of claim 4 or the carrier molecule of claim 5.

7. The method for preparing the antibody or its antigen-binding fragment according to any one of claims 1-3, characterized in that, Prepared through artificial synthesis or genetic engineering.

8. A detection product for peptidoglycan recognition protein 1, characterized in that, It comprises the antibody or antigen-binding fragment thereof as described in any one of claims 1-3.

9. The testing product according to claim 8, characterized in that, The detection products include reagent kits, test strips, or protein chips.

10. The use of the antibody or antigen-binding fragment thereof according to any one of claims 1-3 in the preparation of a peptidoglycan recognition protein 1 detection product.

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