A monoclonal antibody specifically binding to PGLYRP1, and a preparation method and application thereof

Abstract

The application discloses a monoclonal antibody specifically combined with 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 conservative 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, and ensures the quality stability of the product, so that the problems of large batch-to-batch difference and non-reproducibility of polyclonal antibodies are overcome.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of molecular biology and immunology, and particularly relates to a monoclonal antibody specifically binding to PGLYRP1 and a preparation method and application thereof. BACKGROUND

[0002] PGLYRP1 is a member of the peptidoglycan recognition protein (PGRP) family, which is highly conserved in insects and mammals. The mammalian PGLYRP1 is about 200 amino acids long, with a molecular weight of 18-20 kDa, and has a signal peptide. PGLYRP1 is expressed along the digestive tract, especially in the granules of neutrophils and eosinophils, and is expressed to a lesser extent 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, the main component of bacterial cell walls.

[0003] Currently, the detection of PGLYRP1 mostly uses polyclonal antibodies, which are derived from different B cell clones, so they recognize multiple epitopes of the antigen, which results in low specificity of the antibodies. Polyclonal antibodies can bind to different parts of the target antigen, or cross-react with similar molecules, resulting in non-specific binding. In complex samples (such as serum or cell lysate), non-specific binding can interfere with the interpretation of results, affecting the accuracy and reproducibility of the experiment.

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

[0005] The information in the background art is merely intended to explain the general background of the application and should not be considered as admitting or in any form implying that these information constitutes prior art known to those skilled in the art. SUMMARY

[0006] To solve at least part of the technical problems in the prior art, the present application provides a monoclonal antibody specifically binding to PGLYRP1 and a preparation method and application thereof. Specifically, the present application includes the following contents.

[0007] In a first aspect of the present application, an antibody or antigen-binding fragment thereof capable of specifically binding to peptidoglycan recognition protein 1 is provided, the antibody or antigen-binding fragment thereof comprising heavy chain CDR1-3 with sequences of GFSLSSY, HFDSN and GDI and / or light chain CDR1-3 with sequences of GFSLSSY, HFDSN and GDI.

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

[0009] (I) the sequence of the heavy chain variable region shown in SEQ ID NO. 6 and / or the sequence of the light chain variable region shown in SEQ ID NO. 7;

[0010] (II) an amino acid sequence obtained by modification, substitution, deletion or addition of one or more amino acids to the amino acid sequence shown in (I) and having the same function.

[0011] In certain embodiments, the antibody or antigen-binding fragment thereof according to the present application, wherein the antibody comprises a chimeric antibody, a humanized antibody or a murine antibody, and the antigen-binding fragment comprises a Fab, a Fab', a F(ab')2, a scFv or a scFv Fc fragment.

[0012] In a second aspect of the present application, there is provided a nucleic acid molecule comprising a nucleotide sequence of the antibody or antigen-binding fragment thereof according to the first aspect of the present application.

[0013] In a third aspect of the present application, there is provided a vector molecule comprising the nucleic acid molecule according to the second aspect of the present application.

[0014] In a fourth aspect of the present application, there is provided a host cell comprising the nucleic acid molecule according to the second aspect of the present application or the vector molecule according to the third aspect of the present application.

[0015] In a fifth aspect of the present application, there is provided a method for preparing the antibody or antigen-binding fragment thereof according to the first aspect of the present application, wherein the antibody or antigen-binding fragment thereof is prepared by artificial synthesis or genetic engineering.

[0016] In a sixth aspect of the present application, there is provided a detection product for peptidoglycan recognition protein 1 comprising the antibody or antigen-binding fragment thereof according to the first aspect of the present application.

[0017] In certain embodiments, the detection product according to the present application, wherein the detection product comprises a kit, a test paper or a protein chip.

[0018] In a seventh aspect of the present application, there is provided use of the antibody or antigen-binding fragment thereof according to the first aspect of the present application in the preparation of a detection product for peptidoglycan recognition protein 1.

[0019] The application constructs a monoclonal antibody capable of accurately identifying PGLYRP1, ensuring that it can stably recognize the conserved domain of PGLYRP1, which is significantly better than the polyclonal antibody recognizing multiple non-specific epitopes. The monoclonal antibody of the application 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 of the application is derived from a stable cell clone, which can be cultured and expanded for a long time, ensuring that the product quality is stable and consistent, overcoming the limitations of large batch-to-batch differences and non-reproducibility of polyclonal antibodies. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 The plasmid enzyme digestion verification results after the construction of the target gene are shown, wherein M is a DNA marker, 1 is a plasmid digested by XbaI and HindIII, and 2 is a plasmid DNA.

[0021] Figure 2 The antigen protein expression and purification results (coomassie brilliant blue staining) are shown, wherein A is a purification test chart; B is a final concentration chart, M is a protein marker, IN is an original sample, FT is a flow-through liquid, W is a washing liquid, and E is an eluent.

[0022] Figure 3 The recombinant expression antibody purification results (coomassie brilliant blue staining) of the application are shown, wherein A is a purification test chart; B is a final concentration chart; M is a protein marker; IN is an original sample; FT is a flow-through liquid; W is a washing liquid; E is an eluent; NR is non-reduced; and R is reduced. DETAILED DESCRIPTION

[0023] The various exemplary embodiments of the application will now be described in detail, which should not be considered as limiting the application, but should be understood as a more detailed description of certain aspects, characteristics and embodiments of the application.

[0024] It should be understood that the terms described in the application are only for describing the specific embodiments, and are not used to limit the application. In addition, for the numerical range in the application, it should be understood that the upper limit and the lower limit of the range and every intermediate value between them are specifically disclosed. Each smaller range between any stated value or stated range and any other stated value or intermediate value in the stated range is also included in the application. The upper limit and the lower limit of these smaller ranges can be independently included or excluded from the range.

[0025] Unless otherwise indicated, all technical and scientific terms have the same meaning as those one of ordinary skill in the art of the application would understand. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, the preferred methods and materials are described. All documents mentioned herein are incorporated by reference to disclose and describe in full the methods and / or materials useful in connection to the documents. In case of conflict between the content of the specification and that of any document incorporated herein by reference, the content of the specification prevails.

[0026] Antibody or antigen-binding fragment thereof

[0027] In one aspect of the present application, there is provided an antibody or an antigen-binding fragment thereof capable of binding to Peptidoglycan Recognition Protein 1 with high affinity and target specificity, which comprises heavy chain CDR1-3 of the sequence GFSLSSY, HFDSN and GDI and / or light chain CDR1-3 of the sequence GFSLSSY, HFDSN and GDI, without being bound by any theory.

[0028] In one embodiment, the antibody or antigen-binding fragment thereof has any one of the amino acid sequences shown in (I)-(II):

[0029] (I) a heavy chain variable region sequence shown in SEQ ID NO. 6 and / or a light chain variable region sequence shown in SEQ ID NO. 7;

[0030] (II) an amino acid sequence modified, substituted, deleted or added with one or more amino acids from the amino acid sequence shown in (I) or (II) and having the same function.

[0031] Herein, the term "antibody" refers to an immunoglobulin molecule having the ability to specifically bind to a particular antigen. An antibody generally comprises a variable region and a constant region in each of a heavy chain and a light chain. The variable region of the heavy chain and the light chain of an antibody comprises a binding domain that interacts with an antigen. The constant region of an antibody can mediate the binding of the immunoglobulin to host tissues or factors. Thus, most antibodies have a heavy chain variable region (VH) and a light chain variable region (VL) that together form the portion of the antibody that binds to an antigen.

[0032] The "light chain variable region (VL)" or "heavy chain variable region (VH)" of the present application consists of "framework" regions interrupted by three "complementarity determining regions (CDRs)". The framework regions serve to adjust the CDRs for specific binding to an epitope of an antigen. The CDRs comprise amino acid residues in an antibody that are primarily responsible for antigen binding. From the amino-terminal to the carboxyl-terminal, both VL and VH domains comprise the following framework (FR) regions and CDR regions: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.

[0033] As used herein, the terms "homology" and "identity" are used interchangeably. A homologous sequence includes an amino acid sequence that is at least 90%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to a sequence of the present application. To determine sequence identity, a sequence alignment can be performed, which can be done in various ways known to those skilled in the art, e.g., using BLAST, BLAST-2, ALIGN, NEEDLE, or Megalign (DNASTAR) software, etc. Those skilled in the art will be able to determine appropriate parameters for alignment, including any algorithms needed to achieve optimal alignment over the full, or part, of the sequences being compared.

[0034] As used herein, modified antibody sequences are also within the scope of the present application. The term "modification" refers to any chemical modification of the amino acid sequence, such as substitution, deletion, insertion, and / or addition of amino acids. The term "substitution" refers to the replacement of one or more amino acids by 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 as compared to the naturally occurring molecule. It is noted that the modified antibodies provided herein preferably have modifications in regions other than the variable region, such as in the framework region or constant region of the antibody, and the modified antibodies retain the desired functional properties of the antibodies of the present application or antigen binding fragments thereof, or have improved properties for binding to the antigen.

[0035] In the present application, the antibodies include chimeric antibodies, humanized antibodies, or murine antibodies.

[0036] The term "monoclonal antibody", sometimes referred to as "monoclonal" or "Ab", as used herein, refers to an immunoglobulin produced by a single clone of cells, having identical structure and chemical properties, and specific for a single antigenic determinant. Monoclonal antibodies differ from conventional polyclonal antibody preparations, which typically include a mixture of different antibodies, each of which is directed against a different antigenic determinant. Each monoclonal antibody is directed against a single antigenic determinant. In addition to their specificity, the advantage of monoclonal antibodies is that they are produced by cell culture, either by hybridomas or recombinant engineered cells, and are not contaminated with other immunoglobulins. This contrasts with polyclonal antibody preparations, which generally include antibodies directed against different antigenic determinants. The modifier "monoclonal" indicates the character of the antibody as being obtained from a single clone, not to be construed as requiring any particular method for its production.

[0037] As used herein, the term "chimeric antibody" generally refers to an antibody in which a portion of each heavy or light chain amino acid sequence is homologous to the corresponding amino acid sequence in an antibody from a particular species, or belonging to a particular class, while the remainder of the chain is homologous to the corresponding sequence in another species. For example, the variable regions of both light and heavy chains are derived from the variable regions of an antibody of one animal species (e.g., mouse, rat, etc.), while the constant portions are homologous to antibody sequences from another species (e.g., human). For example, to obtain a chimeric antibody, a B cell or hybridoma cell of non-human origin can be used to produce the variable regions, while the constant regions combined therewith are derived from a human. The variable regions have the advantage of being readily prepared, and their specificity is not affected by the source of the constant regions combined therewith. At the same time, since the constant regions of the chimeric antibody can be derived from a human, the possibility of the antibody eliciting an immune response upon injection is lower than with an antibody whose constant regions are of non-human origin.

[0038] As used herein, the term "humanized antibody" generally refers to a chimeric antibody that contains fewer sequences from non-human immunoglobulins, thereby reducing the immunogenicity of the heterologous antibody when introduced into a human, while maintaining the full antigen binding affinity and specificity of the antibody.

[0039] As used herein, the term "murine antibody" generally refers to an antibody whose variable region framework and CDR regions are derived from mouse germline immunoglobulin sequences. In addition, if the antibody comprises a constant region, it is also derived from mouse germline immunoglobulin sequences. In the present invention, a murine antibody can comprise amino acid residues not encoded by mouse germline immunoglobulin sequences, for example, it can include mutations introduced by in vitro random or site-directed mutagenesis or by in vivo somatic mutation.

[0040] As used herein, the term "antigen binding fragment" generally refers to one or more fragments of an antibody that perform the function of specifically binding to an antigen. The antigen binding function of an antibody can be performed by full length fragments of an antibody. The antigen binding function of an antibody can also be performed by a heavy chain of a fragment including Fv, scFv, dsFv, Fab, Fab', or F(ab')2, or, alternatively, a light chain of a fragment including Fv, scFv, dsFv, Fab, Fab', or F(ab')2.

[0041] In the present application, the terms "against," "binds," "immunologically binds," "specifically binds," and "targets" are used interchangeably and generally refer to the noncovalent interactions between an immunoglobulin molecule and an antigen for which the immunoglobulin is specific. The strength or affinity of an immunological binding interaction can be expressed in terms of an dissociation constant (Kd), where a smaller Kdrepresents a higher affinity. "Affinity" refers to the strength of the total noncovalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, "binding affinity" refers to the intrinsic binding affinity reflecting the 1 : 1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y can be expressed in terms of the dissociation equilibrium constant for the binding. Affinity can be measured by common methods well known in the art, including those known in the art and described herein.

[0042] Unless otherwise indicated, the antibodies or antigen-binding fragments thereof described herein are isolated antibodies or antigen-binding fragments thereof. The term "isolated" as used herein with respect to an antibody or antigen-binding fragment thereof means an antibody or antigen-binding fragment thereof which has been removed from its natural milieu. Antibodies or fragments thereof which have been "isolated" thus include antibodies or fragments thereof purified by standard purification methods. The term also encompasses antibodies or fragments thereof prepared by recombinant expression in a host cell as well as chemically synthesized antibodies or fragments thereof.

[0043] Nucleic acid molecule

[0044] In one aspect of the application, there is provided a nucleic acid molecule comprising a nucleotide sequence encoding an antibody or antigen-binding fragment thereof according to the application.

[0045] The term "nucleic acid" as used herein is intended to include polymeric forms of nucleotides of any length, containing deoxyribonucleotides, ribonucleotides and / or their analogs, including DNA, RNA and hybrids thereof, which further include DNA or RNA analogs such as those containing modified backbones (e.g., peptide nucleic acids (PNAs)) or modified bases. Thus, nucleic acids of the application include DNA, cDNA, mRNA, recombinant nucleic acids, and the like.

[0046] Once the coding sequence of an antibody according to the application has been isolated, recombinant techniques can be employed to produce the antibody in large quantities. An exemplary method is to clone its encoding gene into a vector for introduction into cells, which are then propagated and the antibody isolated from the resulting host cells by conventional methods.

[0047] Vector molecule

[0048] In one aspect of the application, there is provided a vector molecule comprising a nucleic acid molecule according to the application.

[0049] 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.

[0050] Host cell

[0051] In one aspect, the present invention provides a host cell comprising the nucleic acid molecule or the carrier molecule described in the present invention.

[0052] 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.

[0053] Method of manufacture

[0054] 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.

[0055] 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.

[0056] 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:

[0057] (1) Construct a recombinant vector for antibody heavy chain expressing the amino acid sequence shown in SEQ ID NO.6 and / or a recombinant vector for antibody light chain expressing the amino acid sequence shown in SEQ ID NO.7;

[0058] (2) The vector is transformed into host cells and cultured under conditions suitable for antibody expression;

[0059] (3) Collect and purify the antibody.

[0060] 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. 8.

[0061] Detection product

[0062] 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.

[0063] In a preferred embodiment, the detection product includes a reagent kit, test strip, or protein chip.

[0064] 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.

[0065] 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.

[0066] Use

[0067] 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.

[0068] Example

[0069] This embodiment illustrates the preparation and detection process of a monoclonal antibody targeting PGLYRP1.

[0070] 1. Antigen Design and Preparation

[0071] 1.1 Gene Synthesis and Vector Construction

[0072] The amino acid sequence of M40505-PGLYRP1 was used as the antigen sequence (as shown in SEQ ID NO. 8) 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.

[0073] Table 1 PCR amplification reaction system

[0074]

[0075] Table 2 PCR amplification reaction conditions

[0076]

[0077] 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.

[0078] Table 3 Enzyme digestion reaction system of vector and target gene

[0079]

[0080] The vector and the target gene were ligated, and the reaction system is shown in Table 4.

[0081] Table 4. Ligation reaction system between vector and target gene

[0082]

[0083] 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 mixture in a circulating water bath 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.

[0084] 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.

[0085] 1.2 Protein Expression and Purification

[0086] Expression was tested using the HEK293 mammalian cell expression system. The target gene was transfected into HEK293 cells for protein expression and purification testing.

[0087] 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.

[0088] Specific experimental procedure:

[0089] (1) According to 0.5×10 6 Inoculate cells at a rate of 300 ml per 1 L shake flask.

[0090] (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).

[0091] (3) Add 300 μg of DNA (filtered and sterilized) to 30 ml of PBS, then vortex for 3 seconds to mix thoroughly.

[0092] (4) Add 1.2 ml of filtered sterile PEI solution (0.5 mg / ml) to the PBS / DNA mixture.

[0093] (5) Let the PEI-DNA mixture stand at room temperature for 20 min.

[0094] (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.

[0095] (7) After transfection, the cells were incubated for 6 days in a shaker incubator at 37°C, 120 rpm and 5% carbon dioxide concentration.

[0096] (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.

[0097] 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.

[0098] 2. Animal immunization

[0099] 2.1 Immunity

[0100] 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.

[0101] First immunization: Day 1, the antigen used for immunization is Freund's complete adjuvant + recombinant protein;

[0102] Second immunization: On day 14, the antigen used for immunization is Freund's incomplete adjuvant + recombinant protein;

[0103] Third immunization: On day 28, the antigen used for immunization is Freund's incomplete adjuvant + recombinant protein;

[0104] 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;

[0105] Fourth immunization: On day 42, the antigen used for immunization is Freund's incomplete adjuvant + recombinant protein;

[0106] 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.

[0107] 2.2 Indirect ELISA detection

[0108] 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).

[0109] 2.3 Antibody affinity purification

[0110] 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.

[0111] 2.4 Valence Testing

[0112] 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.

[0113] Table 5. Results of antibody ELISA detection for Project M40505

[0114]

[0115] 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 1B1 was selected for subsequent antibody heavy and light chain gene amplification and vector construction for recombinant expression.

[0116] Table 6. Antibody titer test results

[0117]

[0118] The purification results of the recombinant expression antibody from the 1B1 positive clone are as follows:Figure 3 Antibody or antigen-binding fragment thereof Nucleic acid molecule Vector molecule Host cell Method of manufacture Detection product Use Figure 1 Figure 2 Figure 3 Antibody or antigen-binding fragment thereof As shown.

[0119] The titer of the recombinant expression antibody from the 1B1 positive clone is shown in Table 7.

[0120] Table 7 Results of 1B1 antibody titer detection

[0121]

[0122] 3. Sequencing

[0123] The antibody expressed by the 1B1 clone recombinantly underwent subsequent molecular sequencing, and the sequence is shown in Table 8.

[0124] Table 8. Amino acid sequence of 1B1 monoclonal antibody

[0125]

[0126] 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, significantly outperforming polyclonal antibodies that recognize 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 problems of large batch-to-batch variability and non-reproducibility associated with polyclonal antibodies.

[0127] 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 specifically bind to peptidoglycan recognition protein 1, and the antibody or its antigen-binding fragment includes heavy chain CDR1-3 with sequences GFSLSSY, HFDSN and GDI respectively and light chain CDR1-3 with sequences QSSPSVYSNYLA, LSSTLAS and LGNYDCSSADCDA respectively.

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.6 and the light chain variable region sequence shown in SEQ ID NO.

7.

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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