Anti-human P14 polypeptide rabbit monoclonal antibody and application thereof

Abstract

The invention discloses an anti-human P14 polypeptide rabbit monoclonal antibody and application thereof, and relates to the technical field of biological immunodetection, the anti-human P14 polypeptide rabbit monoclonal antibody comprises a first group: SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, DAS and SEQ ID NO.5; or, a second group: SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9, DAS, and SEQ ID NO. 10; or, a third group: SEQ ID NO.11, SEQ ID NO.12, SEQ ID NO.13, SEQ ID NO.14, DTS and SEQ ID NO.15. The invention also discloses a method for preparing the traditional Chinese medicine composition. The anti-human P14 polypeptide rabbit monoclonal antibody disclosed by the invention can specifically recognize the human P14 polypeptide and can be widely applied to detection of the human P14 polypeptide.

Description

Technical Field

[0001] This invention relates to the field of bioimmunoassay technology, and in particular to a rabbit monoclonal antibody against human P14 polypeptide and its application. Background Technology

[0002] Antiglomerular basement membrane (GBM) nephritis is a classic autoimmune disease caused by autoantibodies against the non-collagenous region 1 [α3(IV)NC1] of type IV collagen α3 chains in the basement membrane, resulting in severe damage to the kidneys and / or other tissues. In some patients, antiGBM antibodies cross-react with the capillary basement membrane of the alveolar walls, causing dual damage to the lungs and kidneys, known as Goodpasture syndrome. This disease is highly lethal, often asymptomatic, and is primarily characterized by antiGBM antibodies in serum and / or tissues. However, current treatments, such as immunosuppressive therapy and hemodialysis, have limited efficacy and poor prognosis. Literature indicates that the pathogenic antibody in antiGBM nephritis is a typical polyclonal antibody. This significantly limits research into the pathogenesis of antiGBM nephritis and its early diagnosis.

[0003] Human p14 peptide is a truncated polypeptide derived from human α3(IV)NC1, primarily found in the human glomerular basement membrane. It is a cryptic, immunodominant epitope recognized by both T and B cells. Under the influence of environmental and genetic factors, immune tolerance is broken, and human p14 peptide, as an immunodominant epitope, is recognized by immune cells, inducing autoreactive T and B cell responses, producing pathogenic antibodies, and causing epitope diffusion, ultimately leading to anti-glomerular basement membrane nephritis. Therefore, the autoimmune response induced by human p14 peptide is considered an important pathogenesis of anti-glomerular basement membrane nephritis.

[0004] Rabbit monoclonal antibody libraries are excellent resources for rabbit-derived polyclonal antibodies and monoclonal antibodies. Their antibody gene (VH / VL) diversity far exceeds that of mice (e.g., the VH gene family contains over 200 species, compared to only about 20 in mice), enabling the production of highly specific, highly active, and highly affinity antibodies, making them particularly suitable for the development of monoclonal antibodies against small molecule antigens. Therefore, the development of rabbit monoclonal antibodies that can specifically bind to human P14 peptides is of great significance. Summary of the Invention

[0005] In view of the shortcomings of the above-mentioned related technologies, the present invention provides a rabbit monoclonal antibody against human P14 polypeptide and its application.

[0006] In a first aspect, the present invention provides a rabbit monoclonal antibody against human P14 polypeptide using the following technical solution: A rabbit monoclonal antibody against human P14 peptide comprises a light chain variable region and a heavy chain variable region, wherein the heavy chain variable region includes three complementarity-determining regions (CDR1, CDR2, and CDR3), and the light chain variable region includes three CDR1, CDR2, and CDR3; the amino acid sequence of the rabbit monoclonal antibody against human P14 peptide includes: Group 1: The amino acid sequence of HCDR1 includes SEQ ID NO.1, the amino acid sequence of HCDR2 includes SEQ ID NO.2, the amino acid sequence of HCDR3 includes SEQ ID NO.3, the amino acid sequence of LCDR1 includes SEQ ID NO.4, the amino acid sequence of LCDR2 includes DAS, and the amino acid sequence of LCDR3 includes SEQ ID NO.5; Alternatively, the second group: the amino acid sequence of HCDR1 includes SEQ ID NO.6, the amino acid sequence of HCDR2 includes SEQ ID NO.7, the amino acid sequence of HCDR3 includes SEQ ID NO.8, the amino acid sequence of LCDR1 includes SEQ ID NO.9, the amino acid sequence of LCDR2 includes DAS, and the amino acid sequence of LCDR3 includes SEQ ID NO.10; Alternatively, the third group: the amino acid sequence of HCDR1 includes SEQ ID NO.11, the amino acid sequence of HCDR2 includes SEQ ID NO.12, the amino acid sequence of HCDR3 includes SEQ ID NO.13, the amino acid sequence of LCDR1 includes SEQ ID NO.14, the amino acid sequence of LCDR2 includes DTS, and the amino acid sequence of LCDR3 includes SEQ ID NO.15.

[0007] Preferably, the amino acid sequence of the anti-human P14 polypeptide rabbit monoclonal antibody includes: Group 1: includes the heavy chain variable region that is at least 90% homologous to the amino acid sequence of SEQ ID NO. 16 and the light chain variable region that is at least 90% homologous to the amino acid sequence of SEQ ID NO. 17; Alternatively, the second group includes a heavy chain variable region that is at least 90% homologous to the amino acid sequence of SEQ ID NO.18 and a light chain variable region that is at least 90% homologous to the amino acid sequence of SEQ ID NO.19; Alternatively, the third group includes a heavy chain variable region that is at least 90% homologous to the amino acid sequence of SEQ ID NO. 20 and a light chain variable region that is at least 90% homologous to the amino acid sequence of SEQ ID NO. 21.

[0008] Preferably, the amino acid sequence of the anti-human P14 polypeptide rabbit monoclonal antibody includes: Group 1: includes heavy chains with at least 90% homology to the amino acid sequence of SEQ ID NO. 22 and light chains with at least 90% homology to the amino acid sequence of SEQ ID NO. 23; Alternatively, the second group includes a heavy chain with at least 90% homology to the amino acid sequence of SEQ ID NO.24 and a light chain with at least 90% homology to the amino acid sequence of SEQ ID NO.25; Alternatively, the third group: includes a heavy chain with at least 90% homology to the amino acid sequence of SEQ ID NO.26 and a light chain with at least 90% homology to the amino acid sequence of SEQ ID NO.27.

[0009] Preferably, the amino acid sequence of the anti-human P14 polypeptide rabbit monoclonal antibody includes: Group 1: includes heavy chains obtained by modifying, substituting, deleting or adding one or more amino acids of the amino acid sequence shown in SEQ ID NO. 22, and light chains obtained by modifying, substituting, deleting or adding one or more amino acids of the amino acid sequence shown in SEQ ID NO. 23; Alternatively, the second group includes heavy chains obtained by modifying, substituting, deleting or adding one or more amino acids to the amino acid sequence shown in SEQ ID NO.24, and light chains obtained by modifying, substituting, deleting or adding one or more amino acids to the amino acid sequence shown in SEQ ID NO.25; Alternatively, the third group includes heavy chains obtained by modifying, substituting, deleting, or adding one or more amino acids to the amino acid sequence shown in SEQ ID NO.26, and light chains obtained by modifying, substituting, deleting, or adding one or more amino acids to the amino acid sequence shown in SEQ ID NO.27.

[0010] Secondly, the antigen-binding fragment provided by the present invention adopts the following technical solution: An antigen-binding fragment of a rabbit monoclonal antibody against human P14 peptide includes a Fab fragment, a Fab' fragment, an F(ab')2 fragment, a single-chain variable fragment scFv, and scFv. At least one of the Fc fragment and the single-chain antibody ScAb.

[0011] Thirdly, the present invention provides a nucleic acid molecule encoding the above-mentioned anti-human P14 polypeptide rabbit monoclonal antibody or the above-mentioned antigen-binding fragment.

[0012] Preferably, the nucleotide sequence of the nucleic acid molecule includes: Group 1: SEQ ID NO. 28 for encoding the heavy chain and SEQ ID NO. 29 for encoding the light chain; Alternatively, the second group: SEQ ID NO.30 for encoding the heavy chain and SEQ ID NO.31 for encoding the light chain; Alternatively, the third group: SEQ ID NO.32 for encoding the heavy chain and SEQ ID NO.33 for encoding the light chain.

[0013] Fourthly, the present invention provides a recombinant expression vector comprising the above-mentioned nucleic acid molecules.

[0014] Fifthly, the present invention provides a target cell, employing the following technical solution: A target cell is obtained by transfecting a host cell with the aforementioned recombinant expression vector, wherein the host cell includes one or more of human tumor cell lines, mouse tumor cell lines, immortalized human cell lines, and immortalized mouse cell lines.

[0015] Preferably, the host cell is a human chronic myeloid leukemia cell line (K562 cell line).

[0016] Sixthly, the present invention provides a rabbit monoclonal antibody conjugate using the following technical solution: A rabbit monoclonal antibody conjugate is prepared by reacting the above-mentioned anti-human P14 polypeptide rabbit monoclonal antibody and / or the above-mentioned antigen-binding fragment with an antigen and / or a fluorescently labeled molecule.

[0017] In a seventh aspect, the present invention provides the application of the above-mentioned rabbit monoclonal antibody against human P14 peptide or the above-mentioned antibody in the detection of human P14 peptide.

[0018] Eighthly, the present invention provides the application of the above-mentioned anti-human P14 peptide rabbit monoclonal antibody or the above-mentioned antibody in the detection of the positive rate of chimeric autoantibody receptor T cells developed based on human P14 peptide.

[0019] In a ninth aspect, the present invention provides the application of the above-described target cells in the in vitro activation, in vitro activation, proliferation, and killing detection of chimeric autoantibody receptor T cells developed based on human P14 peptide.

[0020] Preferably, the anti-human P14 polypeptide rabbit monoclonal antibody or the antigen-binding fragment is overexpressed on the cell membrane of the target cells.

[0021] In summary, the present invention has at least one of the following beneficial technical effects: The rabbit monoclonal antibody against human P14 peptide of the present invention can specifically recognize human P14 peptide and can be widely used for the detection of human P14 peptide; target cells expressing the scFv fragment of the rabbit monoclonal antibody against human P14 peptide can be constructed and can specifically bind to human P14 peptide. Attached Figure Description

[0022] Figure 1 This is a comparison chart of the background detection results of the serum of New Zealand rabbits R1, R2, R3 and R4 using enzyme-linked immunosorbent assay in Example 1 of the present invention. Figure 2 This is a comparison chart of the polyclonal antibody titers in the serum of R3, R4 and NC New Zealand rabbits in Example 1 of this invention; Figure 3 This is a comparison of the results of reduced 12% SDS-PAGE gel electrophoresis of three rabbit monoclonal antibodies against human P14 polypeptide in Example 1 of this invention. Figure 4 This is the ELISE binding curve of the anti-human P14 monoclonal antibody 5F6 in Example 1 of this invention; Figure 5 This is the ELISE binding curve of the anti-human P14 monoclonal antibody 7G5 in Example 1 of this invention; Figure 6 This is the ELISE binding curve of the anti-human P14 monoclonal antibody 8E1 in Example 1 of this invention; Figure 7 This is the result of the positive rate detection of target cells in Example 2 of the present invention; Figure 8 The results are from flow cytometry analysis of the target cells after co-incubation with FITC-P14 in Example 2 of this invention. Detailed Implementation

[0023] The present invention will be further described in detail below with reference to embodiments and accompanying drawings. The following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. Unless otherwise specified, specific conditions in the following embodiments were performed under conventional conditions or conditions recommended by the manufacturer. Unless otherwise specified, the methods used are conventional methods known in the art, and the consumables and reagents used are commercially available. Unless otherwise stated, the technical and scientific terms used herein have the same meaning as those familiar with the art. Furthermore, any methods or materials similar to or equivalent to those described herein may also be applied to the present invention.

[0024] All raw materials used in the examples are commercially available.

[0025] Example 1 Example 1 of this invention provides a rabbit monoclonal antibody against human P14 polypeptide, the steps of which are as follows: S1, Animal Background Detection: Serum samples from four New Zealand rabbits were collected and subjected to enzyme-linked immunosorbent assay (ELISA) for background detection. The four rabbits were numbered R1, R2, R3, and R4. The test results are as follows: Figure 1 As shown.

[0026] S2, Animal Immunization: Two New Zealand rabbits (R3 and R4) with lower test values ​​were selected for immunization. The rabbits were injected at multiple sites on their backs with complete adjuvant (CFA) and human P14 peptide (weight ratio 1:1). Repeat immunizations were performed every 2 weeks using incomplete Freund's adjuvant (IFA) and human P14 peptide (weight ratio 1:1), repeated 5 times. Simultaneously, another New Zealand rabbit, designated NC, was injected at multiple sites on its back with an equal volume of Freund's adjuvant without human P14 peptide, using the same method. The human P14 peptide was synthesized by Nanjing Genscript Biotech Co., Ltd., and its amino acid sequence is: TDIPPCPHGWISLWKGFSFIMF.

[0027] S3, Serum titer detection: After five immunizations, the polyclonal antibody titers in the serum of three rabbits (NC, R3, and R4) were detected using ELISA. The results are as follows: Figure 2 As shown, the polyclonal antibody titers in the serum of rabbits R3 and R4 were 1:27000 and 1:243000, respectively. The serum of rabbit R4 had a higher titer and met the requirements for shock immunization.

[0028] S4, Screening and culture of positive B cell clones: Referring to the method in paragraphs

[0030] -

[0044] of the patent document CN110016462B (Patent name: Method for efficiently isolating single antigen-specific B lymphocytes from spleen cells), B lymphocytes were sorted for rabbit number R4, and positive clones that specifically recognize antigens were screened out and cultured.

[0029] S5, VH / VL gene cloning: Positive clones were identified in the supernatant of cultured B cells using an antigen-coated ELISA. Cells from positive clones were collected, lysed, and RNA was extracted using the Quick-RNA™ MicroPrep kit (ZYMO), followed by reverse transcription into cDNA. Using the cDNA as a template, the naturally paired rabbit monoclonal antibody chain variable region (VL) and heavy chain variable region (VH) genes were amplified from the cDNA of the corresponding positive clones using PCR. Several clones were selected for sequencing.

[0030] The PCR reaction system consisted of the following: 4 μL cDNA, 1 μL 10 mM forward primer, 1 μL 10 mM reverse primer, 12.5 μL 2×Gloria HiFi (provided by Wuhan Aiboteke Biotechnology Co., Ltd.), and 6.5 μL NF-H2O. The PCR amplification program was as follows: pre-denaturation at 98℃ for 30 s, followed by 30 cycles of 98℃ for 10 s, 64℃ for 30 s, and 72℃ for 30 s, with a final incubation at 72℃ for 5 min. The resulting reaction solution was stored at 4℃.

[0031] S6, Recombinant Antibody Preparation and Identification: The heavy chain and light chain genes of several rabbit monoclonal antibodies selected in the above steps were loaded into expression vectors, using pcDNA3.4 as the mammalian expression vector. The mammalian cell (e.g., CHO) expression vectors containing the heavy chain constant region and light chain constant region of the rabbit monoclonal antibody were linearized using NheI and XbaI restriction endonucleases, respectively. After purifying the PCR products amplified in the above steps, homologous recombination was used to construct the heavy chain variable region gene and light chain variable region gene into the corresponding mammalian expression vectors. After sequencing verification, the expression vectors containing the corresponding rabbit monoclonal antibody light chain and heavy chain genes were transfected into 293F cells. After 72-96 hours of transfection, the transformants were cultured further, and the culture medium was collected. The cells were centrifuged using protein A affinity gel resin, and the rabbit monoclonal antibody recognizing the human P14 peptide was purified from the supernatant. The antibody purity was verified using 12% SDS-PAGE gel electrophoresis. The results are as follows: Figure 3 As shown in the figure, 1 is the marker, 2 is rabbit monoclonal antibody 8E1 containing human P14 peptide, 3 is rabbit monoclonal antibody 5F6 containing human P14 peptide, and 4 is rabbit monoclonal antibody 7G5 containing human P14 peptide. The results show that lanes 2, 3, and 4 each have two bands between 45-60 kDa and 15-25 kDa, respectively, with similar molecular weights and no obvious impurities, indicating that the three monoclonal antibodies were successfully purified and have high purity. Amino acid sequencing was performed on the purified anti-human P14 peptide rabbit monoclonal antibodies. The amino acid sequence analysis of the three anti-human P14 peptide monoclonal antibodies is shown in Table 1 below.

[0032] Table 1:

[0033] The specificity of the three rabbit monoclonal antibodies against human P14 peptide was detected by ELISA, and the steps are as follows: S1. Add 100 μL of coating solution containing human P14 peptide to each well of the microplate and incubate overnight at 4°C.

[0034] S2. Use a multichannel pipette to add 200 μL of washing buffer to each well and wash the plate three times. After each wash, treat the washing buffer by flipping the plate. After the last wash, invert the plate onto the lab bench with a thin sheet of paper and gently tap the plate to remove any residual liquid.

[0035] S3, add 200 μL of blocking solution to each well and incubate at 37°C for 2 hours.

[0036] S4. Clean the microplate using the method in step S2.

[0037] S4. Add 100 μL of antibody diluted with dilution buffer to each well according to a certain ratio. Start with 1 μg / ml antibody and dilute 3-fold, for a total of 8 gradients. Then incubate at 37°C for 1 hour.

[0038] S5. Clean the microplate using the method in step S2.

[0039] S6. Add 100 μL of horseradish peroxidase-conjugated goat anti-rabbit IgG secondary antibody diluted (1:5000) with dilution buffer to each well.

[0040] S7. Clean the microplate using the method in step S2.

[0041] S8, add 100L of ready-to-use TMB substrate to each well and incubate at 37°C for 5 minutes (or until the positive wells turn blue). S9, add 50 μL of 2M H2SO4 to each well to stop the reaction, and read the plate at 450 nm using an ELISA reader.

[0042] The test results are shown in Table 2 below: Table 2: ELISA results of three anti-human P14 peptide monoclonal antibodies

[0043] As shown in Table 2, all three anti-human P14 monoclonal antibodies could bind to the human P14 peptide at very low concentrations, with 5F6 binding slightly less strongly than 7F5 and 8E1, but significantly more strongly than NC. This indicates that the three anti-human P14 monoclonal antibodies have a high affinity for the human P14 peptide.

[0044] The ELISA results of the three anti-human P14 monoclonal antibodies in Table 2 were converted, with antibody concentration expressed in nM. The average of the two OD values ​​was taken (as shown in Table 3 below). ELISA binding curves were plotted (anti-human P14 monoclonal antibody 5F6, anti-human P14 monoclonal antibody 7F5, and anti-human P14 monoclonal antibody 8E1 are listed in order below). Figure 4-6 As shown in Table 3), the KD values ​​of the three monoclonal antibodies were obtained through unit point model analysis. Figure 4-6 It is known that the KD value of anti-human P14 monoclonal antibody 5F6 is 0.0150 nM, the KD value of anti-human P14 monoclonal antibody 7F5 is 0.0200 nM, and the KD value of anti-human P14 monoclonal antibody 8E1 is 0.0132 nM. Compared with commercially available ordinary monoclonal antibodies (KD is 1-10 nM), the affinity is increased by about 50-1000 times, which significantly improves the sensitivity of low-concentration target detection.

[0045] Table 3:

[0046] Example 2 Example 2 of the present invention provides a target cell, the construction method of which is as follows: S1. Construction of the expression vector: The nucleic acid sequences of the 8E1 heavy chain variable region (VH) and light chain variable region (VL) were synthesized by Nanjing GenScript Biotech Co., Ltd. The single-stranded variable region fragment (scFv, VH-Linker-VL, linker sequence (G4S)3) was amplified by PCR and linked to the transmembrane domain sequence of any membrane molecule (CD19 in this example). Subsequently, it was cloned into the multiple cloning site of the lentiviral vector pLVX-SFFV via homologous recombination, and an arbitrary fluorescent protein sequence (mCherry in this example) was linked to the transmembrane domain sequence using a flexible linker. Sanger sequencing confirmed the correctness of the inserted sequence.

[0047] S2, Lentiviral Packaging and Purification: HEK293T cells were co-transfected using a three-plasmid system: recombinant plasmid pLVX-αp14 scFv-CD19 TM-mCherry (10 μg), packaging plasmid psPAX2 (7.5 μg), and envelope plasmid pMD2.G (2.5 μg). PEI (polyethyleneimine) transfection reagent was used at a DNA:PEI ratio of 1:3. Supernatants containing viral particles were collected at 48 h and 72 h after transfection, filtered through a 0.45 μm PVDF membrane, and concentrated by ultracentrifugation (50,000 × g, 4 °C, 2 h).

[0048] S3, Cell Transfection and Screening (using K562 cell line [purchased from ATCC, USA] as an example): K562 cells were seeded in RPMI-1640 medium containing 10% FBS, and the cell density was adjusted to 5 × 10⁶ cells / year 24 hours before transfection. 5cells / mL; mix concentrated virus solution with polybrene (final concentration 8 μg / mL) to infect K562 cells at MOI=20, and centrifuge at 37℃ for infection (1000×g, 32℃, 90min).

[0049] S4, Expression Verification: 72 hours after infection, a small number of cells were taken and the positivity rate was detected by flow cytometry using an anti-G4S antibody conjugated with PE fluorescein (see results). Figure 7 The left image shows wild-type K562 cells with a positive rate of 1.09%, while the right image shows K562 cells transfected with scFv with a positive rate of 99.8%, indicating that the recombinant plasmid pLVX-αp14 scFv-CD19 TM-mCherry was successfully expressed in the K562 cell line.

[0050] S5, Functional Verification: The specific binding ability of scFv to the human P14 peptide antigen was verified using FITC-P14 conjugated with FITC (results are shown in [link to results]). Figure 8 The left image shows wild-type K562 cells with a positive rate of 2.30%, while the right image shows K562 cells transfected with scFv with a positive rate of 88.9%, indicating that αp14 scFv was successfully expressed on the cell membrane surface and could specifically bind to human P14 peptide.

[0051] This specific embodiment is merely an explanation of the present invention and is not intended to limit the invention. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they fall within the scope of the claims of the present invention.

Claims

1. A rabbit monoclonal antibody against human P14 polypeptide, characterized in that: The antibody includes a light chain variable region and a heavy chain variable region. The heavy chain variable region includes three complementarity-determining regions: HCDR1, HCDR2, and HCDR3. The light chain variable region includes three complementarity-determining regions: LCDR1, LCDR2, and LCDR3. The amino acid sequence of the anti-human P14 peptide rabbit monoclonal antibody includes: Group 1: The amino acid sequence of HCDR1 includes SEQ ID NO.1, the amino acid sequence of HCDR2 includes SEQ ID NO.2, the amino acid sequence of HCDR3 includes SEQ ID NO.3, the amino acid sequence of LCDR1 includes SEQ ID NO.4, the amino acid sequence of LCDR2 includes DAS, and the amino acid sequence of LCDR3 includes SEQ ID NO.5; Alternatively, the second group: the amino acid sequence of HCDR1 includes SEQ ID NO.6, the amino acid sequence of HCDR2 includes SEQ ID NO.7, the amino acid sequence of HCDR3 includes SEQ ID NO.8, the amino acid sequence of LCDR1 includes SEQ ID NO.9, the amino acid sequence of LCDR2 includes DAS, and the amino acid sequence of LCDR3 includes SEQ ID NO.10; Alternatively, the third group: the amino acid sequence of HCDR1 includes SEQ ID NO.11, the amino acid sequence of HCDR2 includes SEQ ID NO.12, the amino acid sequence of HCDR3 includes SEQ ID NO.13, the amino acid sequence of LCDR1 includes SEQ ID NO.14, the amino acid sequence of LCDR2 includes DTS, and the amino acid sequence of LCDR3 includes SEQ ID NO.

15.

2. The rabbit monoclonal antibody against human P14 polypeptide according to claim 1, characterized in that: The amino acid sequence of the rabbit monoclonal antibody against human P14 polypeptide includes: Group 1: includes a heavy chain variable region that is at least 90% homologous to the amino acid sequence of SEQ ID NO. 16 and a light chain variable region that is at least 90% homologous to the amino acid sequence of SEQ ID NO. 17; Alternatively, the second group includes a heavy chain variable region that is at least 90% homologous to the amino acid sequence of SEQ ID NO.18 and a light chain variable region that is at least 90% homologous to the amino acid sequence of SEQ ID NO.19; Alternatively, the third group includes a heavy chain variable region that is at least 90% homologous to the amino acid sequence of SEQ ID NO. 20 and a light chain variable region that is at least 90% homologous to the amino acid sequence of SEQ ID NO.

21.

3. An antigen-binding fragment of the rabbit monoclonal antibody against human P14 polypeptide according to any one of claims 1-2, characterized in that: Includes Fab fragments, Fab', F(ab')2 fragments, single-stranded variable fragments scFv, and scFv. At least one of the Fc fragment and the single-chain antibody ScAb.

4. A nucleic acid molecule encoding a rabbit monoclonal antibody against the anti-human P14 polypeptide as described in any one of claims 1-2 or an antigen-binding fragment as described in claim 3.

5. A recombinant expression vector comprising the nucleic acid molecule of claim 4.

6. A target cell, characterized in that: The host cells are obtained by transfecting host cells with the recombinant expression vector according to claim 5, wherein the host cells include one or more of human tumor cell lines, mouse tumor cell lines, immortalized human cell lines, and immortalized mouse cell lines.

7. A rabbit monoclonal antibody conjugate, characterized in that: It is prepared by reacting the rabbit monoclonal antibody containing the anti-human P14 polypeptide as described in any one of claims 1-2 and / or the antigen-binding fragment as described in claim 3 with the antigen and / or fluorescently labeled molecule.

8. The use of a rabbit monoclonal antibody against human P14 peptide according to any one of claims 1-2 or the antigen-binding fragment according to claim 3 in the detection of human P14 peptide.

9. The application of the rabbit monoclonal antibody against human P14 peptide according to any one of claims 1-2 or the antigen-binding fragment according to claim 3 in the detection of the positive rate of chimeric autoantibody receptor T cells developed based on human P14 peptide.

10. The application of the target cell of claim 6 in the in vitro activation, in vitro activation, proliferation and killing detection of chimeric autoantibody receptor T cells developed based on human P14 peptide.

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