An anti-PGLYRP1 antibody, its preparation method and application

Abstract

This invention discloses an anti-PGLYRP1 antibody, its preparation method, and its applications. This invention, using single-B cell cloning technology, is the first to construct a monoclonal antibody capable of accurately recognizing PGLYRP1. The constructed antibody, through screening for specific epitopes and verifying their binding sites, ensures stable recognition of the conserved domains of PGLYRP1, significantly outperforming polyclonal antibodies that recognize multiple non-specific epitopes. Furthermore, the monoclonal antibody of this invention is derived from stable cell clones, exhibiting strong batch-to-batch consistency, making it suitable for long-term standardized research and industrial-scale production.

Description

Technical Field

[0001] This invention relates to the fields of molecular biology and immunology, specifically to an anti-PGLYRP1 antibody, its preparation method, and its applications. Background Technology

[0002] PGLYRP1 is a member of the peptidoglycan recognition protein (PGRP) family, a pattern recognition protein that mediates antibacterial activity and innate immune responses. PGLYRP1 is highly expressed in immune cells such as neutrophils, macrophages, and eosinophils. Studies have found its association with cancer, coronary artery disease, and various inflammatory diseases, including rheumatoid arthritis, asthma, and oral and airway inflammation. Furthermore, PGLYRP1 therapy has been found to upregulate the expression of genes encoding pro-inflammatory cytokines in different cell types.

[0003] Currently, publicly available studies on PGLYRP1 detection or functional intervention generally rely on commercially available or self-made polyclonal antibodies, and there are no reports of specific monoclonal antibodies against PGLYRP1. However, polyclonal antibodies originate from different B cell clones and recognize multiple epitopes of antigens, resulting in low antibody specificity. Polyclonal antibodies may bind to different parts of the target antigen or cross-react with similar molecules, leading to nonspecific binding. Therefore, in complex samples (such as serum or cell lysates), nonspecific binding may interfere with the interpretation of results, affecting the accuracy and reproducibility of experiments.

[0004] There is an urgent need to develop an antibody against PGLYRP1 to specifically recognize the PGLYRP1 protein. Summary of the Invention

[0005] This application is the first to construct a monoclonal antibody capable of accurately recognizing PGLYRP1. Specifically, this invention includes the following:

[0006] In a first aspect, the present invention provides an antibody or an antigen-binding fragment thereof capable of targeting PGLYRP1, said antibody or antigen-binding fragment 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.

[0007] 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):

[0008] (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;

[0009] (II) An amino acid sequence that has at least 90% homology with the amino acid sequence shown in (I) and has the same function;

[0010] (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.

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

[0012] A second aspect of the present invention provides a nucleic acid molecule comprising a nucleotide sequence encoding an antibody or an antigen-binding fragment thereof as described in the present invention.

[0013] A third aspect of the present invention provides a carrier molecule comprising the nucleic acid molecule described herein.

[0014] In a fourth aspect, the present invention provides a host cell comprising the nucleic acid molecule or carrier molecule described herein.

[0015] In a fifth aspect, the present invention provides a method for preparing the antibody or antigen-binding fragment thereof described herein, which is prepared by artificial synthesis or genetic engineering.

[0016] In a sixth aspect, the present invention provides a detection product for PGLYRP1, comprising the antibody or antigen-binding fragment thereof described in the present invention.

[0017] In some embodiments, the detection product according to the present invention includes a reagent kit, test strip, or protein chip.

[0018] In some embodiments, the testing product according to the present invention includes a specification for implementing the PGLYRP1 testing method.

[0019] A seventh aspect of the present invention provides the use of the antibody or antigen-binding fragment thereof described herein in the preparation of PGLYRP1 detection products.

[0020] The beneficial technical effects of this invention include:

[0021] 1. This invention is the first to construct a monoclonal antibody that can accurately identify specific sites of PGLYRP1, filling a gap in this field.

[0022] 2. The antibody of this invention can recognize the key functional regions of the PGLYRP1 protein with high specificity. The constructed monoclonal antibody is screened for specific epitopes and its binding site is verified to ensure that it can stably recognize the conserved domain of PGLYRP1, which is significantly better than polyclonal antibodies that recognize multiple non-specific epitopes.

[0023] 3. High specificity, avoiding non-specific background interference. Compared with existing polyclonal antibodies, the monoclonal antibody of this invention recognizes only specific sites of PGLYRP1, significantly reducing cross-reactivity and improving the accuracy of experimental results, making it particularly suitable for signal detection in complex tissues.

[0024] 4. Strong batch consistency, suitable for long-term standardized research and industrial-scale preparation. The monoclonal antibody of this invention is derived from stable cell clones and can be cultured and amplified for a long time, ensuring stable and consistent product quality and overcoming the limitations of large batch-to-batch differences and non-replicability of polyclonal antibodies. Attached Figure Description

[0025] Figure 1 The results of plasmid digestion verification after construction according to the present invention are shown, wherein M is DNA marker, 1 is XbaI and HindIII digested plasmid, and 2 is plasmid DNA.

[0026] Figure 2 The results of protein expression and purification of the present invention (Coomassie Brilliant Blue staining) are shown, wherein 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.

[0027] Figure 3 The purification results of the recombinant expression antibody of the present invention (Coomassie brilliant blue staining) are shown, where 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; E is the elution solution; NR is non-reduced; and R is reduced. Detailed Implementation

[0028] 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.

[0029] 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.

[0030] 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.

[0031] Antibody or its antigen-binding fragment

[0032] In one aspect, the present invention provides an antibody or antigen-binding fragment thereof capable of binding PGLYRP1 with high affinity and targeting, unconstrained by any theoretical framework. The antibody or antigen-binding fragment of the present invention comprises heavy chain CDR1-3 as shown in SEQ ID NO. 1-3 and / or light chain CDR1-3 as shown in SEQ ID NO. 4-6. Specifically, the sequence of heavy chain CDR1 is GFDFSSNY (SEQ ID NO. 1), the sequence of heavy chain CDR2 is DAGSSGG (SEQ ID NO. 2), and the sequence of heavy chain CDR3 is ASFDLGYDHPCDL (SEQ ID NO. 3); the sequence of light chain CDR1 is QASQNIRSHLS (SEQ ID NO. 4), the sequence of light chain CDR2 is SSSTLAS (SEQ ID NO. 5), and the sequence of light chain CDR3 is QQGYSSRNVDNT (SEQ ID NO. 6).

[0033] In one specific embodiment, the antibody or its antigen-binding fragment has any one of the amino acid sequences shown in (I)-(III):

[0034] (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;

[0035] (II) An amino acid sequence that has at least 90% homology with the amino acid sequence shown in (I) and has the same function;

[0036] (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.

[0037] 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.

[0038] 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.

[0039] 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.

[0040] 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.

[0041] In this invention, the antibody includes monoclonal antibody, chimeric antibody, humanized antibody, or murine antibody.

[0042] 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.

[0043] 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.

[0044] 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.

[0045] 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.

[0046] 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.

[0047] 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.

[0048] 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.

[0049] Nucleic acid molecules

[0050] 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.

[0051] 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.

[0052] 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.

[0053] carrier molecules

[0054] In one aspect, the present invention provides a carrier molecule comprising the nucleic acid molecule described herein.

[0055] 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.

[0056] host cells

[0057] In one aspect, the present invention provides a host cell comprising the nucleic acid molecule or the carrier molecule described in the present invention.

[0058] 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.

[0059] Preparation method

[0060] 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.

[0061] 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.

[0062] 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:

[0063] (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;

[0064] (2) The vector is transformed into host cells and cultured under conditions suitable for antibody expression;

[0065] (3) Collect and purify the antibody.

[0066] In another preferred embodiment, the antibody of the present invention is prepared by immunization with a recombinant antigen, wherein the recombinant antigen has the amino acid sequence shown in SEQ ID No. 9.

[0067] Product testing

[0068] The present invention further provides a detection product for detecting (or quantifying) PGLYRP1, comprising the antibody or antigen-binding fragment thereof described in the present invention, and instructions on how to perform the detection method of the present invention on PGLYRP1.

[0069] In a preferred embodiment, the detection product includes a reagent kit, test strip, or protein chip.

[0070] 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.

[0071] 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.

[0072] use

[0073] This invention further provides the application of the antibody or its antigen-binding fragment described herein in the preparation of PGLYRP1 detection products, wherein the detection products include kits, test strips, or protein chips. The detection products are used for the prediction, diagnosis, and / or prognosis of PGLYRP1-related diseases.

[0074] Example

[0075] I. Experimental Methods

[0076] 1. Gene synthesis and vector construction

[0077] 1.1 Protein Name:

[0078] >M40505-Pglyrp1 amino acid sequence

[0079] MLFACALLALLGLATSCSFIVPRSEWRALPSECSSRLGHPVRYVVISHTAGSFCNSPDSCEQQARNVQHYHKNELGWCDVAYNFLIGEDGHVYEGRGWNIKGDHTGPIWNPMSIGITFMGNFMDRVPAKRALRAALNLLECGVSRGFLRSNYEVKGHRDVQSTLSPGDQLYQVIQSWEHYREGSHHHHHH* (SEQ ID NO. 9).

[0080] 1.2 Experimental Procedure:

[0081] (1) PCR amplification reaction system

[0082]

[0083] (2) PCR amplification reaction conditions

[0084]

[0085] Electrophoretic detection and recovery of the amplified fragments were performed according to the instructions of the commercially available kit.

[0086] 2. Enzyme digestion of vector and target gene

[0087]

[0088] Incubate at 37℃ for 1-2 hours; perform electrophoresis detection and recovery of the enzyme digestion products, following the instructions of the commercially available kit.

[0089] 3. Ligation of vector and target gene

[0090]

[0091] 4. Transformation

[0092] (1) Add the DNA fragment to be transformed into a tube containing TOP10 competent cells (50µl competent cells require 25ng DNA). The volume should not exceed 5% of the competent cells. Gently rotate the tube a few times to mix the contents and incubate on ice for 30 minutes.

[0093] (2) Place the mixture in the centrifuge tube into circulating water heated to 42°C and heat shock for 90 seconds without shaking the tube.

[0094] (3) Quickly transfer the tube to an ice bath to cool the cells for 1-2 minutes.

[0095] (4) Add 200µl of SOC liquid culture medium to each tube, heat the culture medium to 37°C in a water bath, and then transfer the tube to a shaker set to 37°C and incubate at 220rpm for 45min to allow the cells to recover and express the resistance marker gene encoded by the plasmid.

[0096] (5) Transfer an appropriate volume (200 µl per 90 mm plate) of the transformed competent cells onto LB medium containing the appropriate antibiotic.

[0097] (6) Invert the plate and incubate at 37°C. Plaques will appear after 12-16 hours.

[0098] 5. Colony PCR verification

[0099] Once colonies have grown on the plate, randomly select several for colony PCR verification to detect transformants.

[0100] 6. Sequencing verification

[0101] Positive clones were sent to our sequencing platform for sequencing verification. The enzyme digestion quality control results are attached. Figure 1 .

[0102] 7. Protein expression and purification

[0103] Expression was tested using the HEK293 mammalian cell expression system. The target gene was transfected into HEK293 cells for protein expression and purification testing.

[0104] 7.1 Expression Conditions

[0105] HEK293 cell density: 1.5-2.0 x 10⁻⁶ 6 pcs / ml;

[0106] -Incubation temperature: 37℃;

[0107] -Incubation rotation speed: 130 rpm / min;

[0108] - Sample collection time: Day 6 Detailed experimental procedure:

[0109] (1) According to 0.5X10 6 Inoculate cells at a rate of 300 ml per 1 L shake flask.

[0110] (2) Incubate at 37℃, 120 rpm, and 5% carbon dioxide concentration in a shaker incubator for 24 h until the cell density reaches 1 x 10⁻⁶ cells / year. 6 cells / ml (cells need to double every 24 hours).

[0111] (3) Add 300µg of DNA (filtered and sterilized) to 30ml of PBS, then vortex for 3 seconds to mix thoroughly.

[0112] (4) Add 1.2 ml of filtered sterilized PEI solution (0.5 mg / ml) to the PBS / DNA mixture.

[0113] (5) Let the PEI-DNA mixture stand at room temperature for 20 minutes.

[0114] (6) Add the DNA / PEI mixture to the cells, and the cell density must reach 1.5-2.0 x 10⁻⁶. 6 cells / ml.

[0115] (7) After transfection, the cells were cultured for 6 days in a shaker incubator at 37°C, 120 rpm and 5% carbon dioxide concentration.

[0116] (8) Centrifuge at 3000g for 5min to collect the culture medium supernatant and cell pellet, and store at -80℃ for later purification.

[0117] 7.2 Purification Protocol

[0118] -M40505 Collect 1L of culture medium supernatant, centrifuge at high speed, filter through a 0.22µm filter membrane, and purify the target protein by binding the supernatant with Protein A resin.

[0119] - Resin: Protein A resin;

[0120] - Binding and equilibrating resin solution: PBS, pH=7.4;

[0121] - Washing and elution solutions:

[0122] *Wash: PBS, pH=7.4;

[0123] *Elution: 0.1M glycine, pH=3.0;

[0124] *Neutralization: 1M Tris-HCl, pH=8.5.

[0125] 7.3 Test Results

[0126] The expression and purification results are attached. 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.

[0127] 8. Antigen treatment

[0128] The primary immunization antigen is a recombinant protein mixed with an equal volume of Freund's complete adjuvant and emulsified. The secondary, tertiary, and quaternary immunization antigens are recombinant proteins mixed with an equal volume of Freund's incomplete adjuvant and emulsified.

[0129] 9. Animal Immunization

[0130] 9.1 Animals

[0131] Healthy female New Zealand White rabbits, 4 months old and weighing 2.1kg, were selected.

[0132] 9.2 Immunity

[0133] First immunization: Day 1, the antigen used for immunization is Freund's complete adjuvant + recombinant protein;

[0134] Second immunization: On day 14, the antigen used for immunization is Freund's incomplete adjuvant + recombinant protein;

[0135] Third immunization: On day 28, the antigen used for immunization is Freund's incomplete adjuvant + recombinant protein;

[0136] 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;

[0137] Fourth immunization: On day 42, the antigen used for immunization is Freund's incomplete adjuvant + recombinant protein;

[0138] 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.

[0139] 10. Indirect ELISA detection

[0140] (1) Coating antigen

[0141] The antigen was diluted to 6 µg / ml with 0.05 mol / L carbonate (pH=9.6), 100 µl / well, and incubated overnight at 4°C.

[0142] (2) Washing the plate

[0143] Remove and wash three times with 0.05% Tween-20 (PBST), 3 minutes each time.

[0144] (3) Closed

[0145] Add 150µl of 5% skim milk powder (PBST) and blocking buffer to each well, and incubate at 37°C for 60 minutes.

[0146] (4) Washing the plate

[0147] Remove and wash three times with 0.05% Tween-20 (PBST), 3 minutes each time.

[0148] (5) Add a primary antibody

[0149] The antiserum was diluted 1:1000, then serially diluted, and incubated at 37°C for 1 hour.

[0150] (6) Washing the plate

[0151] Remove and wash three times with 0.05% Tween-20 (PBST), 3 minutes each time.

[0152] (7) Add secondary antibody

[0153] Horseradish enzyme-labeled goat anti-rabbit IgG (H+L), catalog number: 116154, antibody company: Jackson. Dilute 1:8000 and incubate at 37°C for 45 minutes.

[0154] (8) Washing the plate

[0155] Remove and wash five times with 0.05% Tween-20 (PBST), 3 minutes each time.

[0156] (9) Color development

[0157] Add 100 µl of substrate solution (TMB) per well and react for 5-10 minutes. Finally, add 100 µl of 2 mol / L sulfuric acid to terminate the reaction.

[0158] (10) Measure OD value

[0159] The OD value was measured at a wavelength of 450 nm using an ELISA reader (Kehua ST-360).

[0160] 11. Antibody affinity purification

[0161] (1) Take an appropriate amount of affinity-purified beads into the purification column, wash three times with 10mM HCl (about 30ml volume), wash with binding buffer to remove residual hydrochloric acid, add the purified antigen dissolved in PBS to the beads, add buffer and incubate overnight at 4°C, wash three times with acid, water and alkali in turn, and finally wash with PBS for later use.

[0162] (2) Locate the frozen antiserum and corresponding beads according to the antibody purification schedule, and record them.

[0163] (3) Thaw the serum and transfer the beads into the purification column. Take the required amount of serum and return any excess serum.

[0164] (4) Serum was kept at low temperature (4-8℃) and centrifuged at 3400 rpm for 20 minutes.

[0165] (5) Use a pipette to remove the lipids from the surface of the centrifuged serum, and at the same time keep a small sample of serum for ELISA testing.

[0166] (6) Incubate the centrifuged serum with the beads at room temperature for 2 hours (or at 37°C in a constant temperature shaker if the room temperature is low) or at 4°C overnight.

[0167] (7) After incubation, release the collected flow solution (FT), wash the beads three times with PBS, each time 10 column volumes.

[0168] (8) Wash the purification column with 5 ml of pre-cooled, pH 5.0 pre-wash solution.

[0169] (9) Elution: Elute with pre-cooled pH 2.5 elution buffer, collecting 1 ml each time. Add 50 µl of neutralization buffer to the EP tube beforehand.

[0170] (10) Detect the collection peak using Coomassie assay. Add 10µl of collection solution to 100µl of Coomassie assay and determine the required antibody based on the collection peak. Store at 4℃.

[0171] (11) Terminate collection when Coomassie assay shows no color and wash with 10 ml PBS.

[0172] (12) Incubate the flow solution with the beads again and repeat steps (6)-(10).

[0173] (13) The collected antibodies were concentrated using PEG.

[0174] (14) Antibody preservation: Add 50% glycerol and 0.02% sodium azide according to the antibody volume and mix well.

[0175] II. Experimental Results

[0176] 1. Results of antiserum titer test after final radiotherapy

[0177] Table 1. Antibody titer detection data obtained by the enzyme-linked immunosorbent assay (ELISA) reader.

[0178]

[0179] Note: The purified antibody titer is: A≥1024K, B≥1024K.

[0180] Based on the data in Table 1, the results of the rabbit serum titer test showed that rabbit A had the best titer. Therefore, rabbit A was selected for subsequent serum collection and enrichment screening of B cells.

[0181] 2. Detection of B cell culture supernatant

[0182] 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. Among them, clone 1G3 was selected for subsequent antibody heavy and light chain gene amplification and vector construction for recombinant expression.

[0183] Table 2 Results of B cell culture supernatant detection

[0184]

[0185]

[0186] Note: Original coating agent M40505 = 0.2 mg / ml; coating concentration 1 μg / ml; secondary antibody: goat anti-rabbit, cross-free.

[0187] As shown in Table 2, positive clone 1G3 showed the highest expression, with an OD value of up to 1.51.

[0188] 3. The purification results of the recombinant expression antibody from 1G3 positive clones are shown in the figure. Figure 3 .

[0189] 4. Detection of antibody titer for recombinant expression of 1G3 positive clones

[0190] Antibody titer was detected in the supernatant of 24 wells containing 1G3 positive clones for recombinant expression, followed by molecular sequencing.

[0191] Table 3. Results of antibody titer assay for recombinant expression in 1G3 positive clones.

[0192]

[0193] As shown in Table 3, the OD values ​​of 1G3 clones can reach 1.156-1.331, indicating that they have a strong ability to bind to the target.

[0194] The sequence list involved in this invention includes:

[0195]

[0196] Although the invention has been described with reference to exemplary embodiments, it should be understood that the invention is not limited to the disclosed exemplary embodiments. Various adjustments or changes may be made to the exemplary embodiments described in this specification without departing from the scope or spirit of the invention. The scope of the claims should be interpreted in the broadest possible sense to cover all modifications and equivalent structures and functions.

Claims

1. An antibody or its antigen-binding fragment, characterized in that, It can target PGLYRP1, 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 any one of the amino acid sequences shown in (I)-(III): (I) 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; (II) An amino acid sequence that has at least 90% homology with the amino acid sequence shown in (I) and has the same function; (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.

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

4. A nucleic acid molecule, characterized in that, It encodes the antibody or antigen-binding fragment thereof as described in 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 kit for detecting PGLYRP1, characterized in that, It comprises the antibody or antigen-binding fragment thereof as described in any one of claims 1-3.

9. The reagent kit according to claim 8, characterized in that, The kit includes instructions for performing the PGLYRP1 detection method.

10. The use of the antibody or antigen-binding fragment thereof according to any one of claims 1-3 in the preparation of PGLYRP1 detection products.

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