A monoclonal antibody against APLN, its preparation method and application

Abstract

This invention discloses an APLN monoclonal antibody, its preparation method, and its applications. The APLN monoclonal antibody includes a light chain variable region and a heavy chain variable region. The amino acid sequence of the light chain variable region is shown in SEQ ID No:1, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID No:2. The APLN monoclonal antibody is obtained by immunizing mice with APLN protein, followed by single-cell B-cell sorting and single-cell PCR. Utilizing advanced single-cell B-cell cloning technology combined with single-cell PCR, a novel APLN monoclonal antibody has been developed. The obtained antibody sequence is the result of natural affinity maturation screening by the in vivo immune system, requiring no additional humanization or in vitro affinity maturation steps. It retains the natural high affinity and specificity, and can directly obtain all-natural antibodies with high affinity and high specificity.

Description

Technical Field

[0001] This invention relates to the fields of biotechnology and in vitro diagnostics, and more specifically, to an APLN monoclonal antibody, its preparation method, and its application. Background Technology

[0002] Liver cancer, especially hepatocellular carcinoma, is a major global health threat. A key reason for its high mortality rate is the difficulty in early diagnosis; most patients are diagnosed at an advanced stage, missing the optimal window for radical treatment. Currently, alpha-fetoprotein (AFP) is the widely used serological diagnostic biomarker for liver cancer. However, AFP has significant limitations in both sensitivity and specificity: approximately 30%-40% of liver cancer patients do not have elevated AFP levels, while some patients with benign diseases such as chronic hepatitis and cirrhosis may also have abnormally elevated AFP levels, leading to frequent false negatives and false positives. While imaging examinations (such as ultrasound, CT, and MRI) can detect lesions, they are difficult to differentiate between early-stage small liver cancers, atypical hyperplasia nodules, and early-stage liver cancer, and rely heavily on operator experience. Therefore, developing novel serological or histological diagnostic biomarkers and their corresponding testing reagents with higher sensitivity and specificity is crucial for achieving early detection, accurate subtyping, and individualized management of liver cancer.

[0003] Apolipoprotein (APLN) is an endogenous ligand of the APLN / APJ signaling pathway, and recent studies have revealed its multiple roles in tumorigenesis and development. In the field of liver cancer, numerous basic and clinical studies have confirmed that APLN is not only a functional molecule that promotes angiogenesis and tumor growth, but also a potentially valuable biomarker for diagnosis and prognosis. Studies have also found that APLN expression levels in liver cancer tissues are significantly higher than in adjacent normal liver tissues and benign liver lesions, and its expression is closely related to the malignancy of the tumor. Multiple clinical studies have shown that APLN levels in the peripheral blood of liver cancer patients are significantly higher than in healthy individuals and patients with chronic liver disease. Its serum concentration is positively correlated with key clinicopathological features such as tumor size, presence of portal vein tumor thrombus, and clinical stage, suggesting its potential as a non-invasive serum biomarker. Furthermore, both high tissue APLN expression and high serum APLN levels are significantly associated with an increased risk of postoperative recurrence, shortened disease-free survival, and reduced overall survival in liver cancer patients, demonstrating independent prognostic value.

[0004] While APLN shows promise as a diagnostic biomarker, its clinical translation depends on the development of stable, reliable, and highly specific detection tools. Currently, the APLN detection methods (mainly enzyme-linked immunosorbent assays) and their core reagents—APLN antibodies—used in the market and in research have significant shortcomings: (1) Poor performance of commercial antibodies: Most existing commercial anti-APLN polyclonal antibodies or early monoclonal antibodies generally have problems such as high cross-reactivity, large batch-to-batch differences, and insufficient affinity and specificity, which makes it difficult to meet the high standards of clinical diagnosis in terms of accuracy, stability and sensitivity of the test results.

[0005] (2) Bottlenecks in traditional antibody development technology: Although diagnostic monoclonal antibodies prepared using traditional hybridoma technology have solved the batch-to-batch consistency problem, the inherent immunogenicity risk of murine antibodies may cause interference in repeated testing, and the affinity optimization process is cumbersome. Conventional methods are difficult to efficiently obtain antibody pairs (capture antibody and detection antibody) with ultra-high affinity and specificity against the natural conformation APLN, which is the key to constructing high-performance "sandwich" ELISA kits.

[0006] In view of the above, this application is hereby submitted. Summary of the Invention

[0007] To address the aforementioned technical problems, this invention provides an APLN monoclonal antibody, its preparation method, and its application. Utilizing advanced single-B cell cloning technology combined with single-cell PCR technology, a novel APLN monoclonal antibody was developed. The obtained antibody sequence is the result of natural affinity maturation screening by the in vivo immune system, eliminating the need for additional humanization or in vitro affinity maturation steps. It retains the natural high affinity and specificity, allowing for the direct acquisition of all-natural antibodies with high affinity and high specificity.

[0008] This invention is achieved through the following technical solution: In a first aspect, the present invention discloses an APLN monoclonal antibody or a fragment thereof, the APLN monoclonal antibody comprising a light chain variable region and a heavy chain variable region, the amino acid sequence of the light chain variable region being shown in SEQ ID No:1, and the amino acid sequence of the heavy chain variable region being shown in SEQ ID No:2.

[0009] In one specific embodiment, the nucleotide sequence of the light chain variable region is shown in SEQ ID No:3.

[0010] In one specific embodiment, the nucleotide sequence of the heavy chain variable region is shown in SEQ ID No:4.

[0011] Secondly, the present invention provides a nucleic acid molecule encoding the APLN monoclonal antibody or a fragment thereof.

[0012] Thirdly, the present invention provides a carrier comprising the aforementioned nucleic acid molecule.

[0013] Fourthly, the present invention provides a method for preparing APLN monoclonal antibody or fragment thereof, which is obtained by immunizing mice with APLN protein and then using single B cell sorting technology and single cell PCR technology.

[0014] In a specific embodiment, the method for preparing APLN monoclonal antibody or a fragment thereof includes the following steps: The APLN gene was obtained, inserted into a vector plasmid, and amplified to obtain an expression plasmid. The expression plasmid was transferred into host cells, activated, induced, and purified to obtain the APLN protein. The obtained APLN protein was used as an antigen to immunize mice; After successful immunization, the mice were sacrificed and their spleens were removed. Plasma cells expressing APLN antibodies were then isolated using plasma cell sorting technology. Monoclonal B cells secreting antibodies targeting APLN were obtained from plasma cells using single B cell sorting technology. Antibodies were synthesized from sorted individual B cells using single-cell PCR technology.

[0015] Single-cell B-cell cloning technology is a revolutionary platform for next-generation antibody discovery. This technology can directly isolate antigen-specific single B cells from the spleen or peripheral blood of an immune host and directly clone the naturally paired antibody light and heavy chain variable region genes of these B cells using single-cell PCR. Compared with hybridoma or phage display technologies, this method has unique advantages: it retains natural high affinity and specificity: the obtained antibody sequences are the result of natural affinity maturation and selection by the in vivo immune system, eliminating the need for additional humanization or in vitro affinity maturation steps, and directly obtaining high-affinity, high-specificity all-natural antibodies. Through a reasonable immunization and screening strategy, multiple single-cell B-cell clones recognizing different epitopes of the APLN molecule can be simultaneously isolated from the same immunized animal, thereby rapidly obtaining naturally matched, high-performance paired antibody pairs.

[0016] Fifthly, the present invention provides the use of the APLN monoclonal antibody or a fragment thereof in the preparation of diagnostic or therapeutic drugs for cancer, wherein the cancer is a cancer associated with high expression of APLN.

[0017] In one specific implementation, the cancer is liver cancer, intestinal cancer, stomach cancer, pancreatic cancer, or bile duct cancer.

[0018] In a sixth aspect, the present invention provides a diagnostic reagent for cancer, comprising the APLN monoclonal antibody or a fragment thereof.

[0019] Compared with the prior art, the present invention has the following advantages and beneficial effects: 1. The present invention provides an APLN monoclonal antibody and its preparation method and application. By using advanced single B cell cloning technology combined with single cell PCR technology, a novel APLN monoclonal antibody was developed. The obtained antibody sequence is the result of natural affinity maturation screening by the in vivo immune system. No additional humanization or in vitro affinity maturation steps are required. It retains the natural high affinity and specificity, and can directly obtain all-natural antibodies with high affinity and high specificity. 2. The embodiment of the present invention provides an APLN monoclonal antibody and its preparation method and application. The DNA sequence and amino acid sequence of the heavy chain variable region (VH) and light chain variable region (VL) of the APLN monoclonal antibody are directly obtained by single-cell PCR technology, which ensures the specificity and targeting of the monoclonal antibody, thereby enabling it to recognize APLN antigen in serum or tissue samples of liver cancer patients with high specificity. 3. The APLN monoclonal antibody and its preparation method and application provided in the embodiments of the present invention utilize plasma cell separation technology and single B cell sorting technology to efficiently and faithfully separate single B cells that produce natural, high-affinity antibodies, perfectly preserving the natural pair selected in vivo; 4. The APLN monoclonal antibody, its preparation method, and its application provided in this embodiment of the invention have important value in disease diagnosis, especially in the early auxiliary diagnosis, prognostic assessment and efficacy monitoring of liver cancer, the development of immunoassay reagents, antibody drug design, and basic research. Attached Figure Description

[0020] To more clearly illustrate the technical solutions of the exemplary embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly described below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation of the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0021] Figure 1 The SDS-PAGE electrophoresis diagram of the antigen protein provided in the embodiments of the present invention (lane 1 is the protein marker from top to bottom: 180KDa, 130KDa, 95KDa, 70KDa, 55KDa, 43KDa, 33KDa, 25KDa, 17KDa, 10KDa; the other lanes are protein samples under different induction conditions). Figure 2 The antibody titer chart of immunized mouse serum provided in an embodiment of the present invention (the results show that the titer is still good at 1:500000). Figure 3 The results of CD138-positive cell sorting provided in this embodiment of the invention (the results show that the positive rate of B cells after enrichment exceeds 98%). Figure 4 The antigen coating and single B cell selection diagram provided in the embodiments of the present invention (the results show that the cells are evenly plated and picked up smoothly). Figure 5 This is a nucleic acid electrophoresis image of the antibody light and heavy chain sequence products amplified by PCR provided in an embodiment of the present invention; Figure 6 The SDS-PAGE electrophoresis diagram and SEC-HPLC diagram of the monoclonal antibody synthesis provided in the embodiments of the present invention; Figure 7 This is an ELISA image showing the binding of a monoclonal antibody to an antigen, provided in an embodiment of the present invention. Figure 8 The image shows the ELISA results of detecting APLN protein in the serum of liver cancer patients using the recombinant monoclonal antibody APLN provided in this embodiment of the invention. Detailed Implementation

[0022] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the embodiments. The illustrative embodiments and descriptions of this invention are only used to explain this invention and are not intended to limit this invention.

[0023] In the following description, numerous specific details are set forth in order to provide a thorough understanding of the invention. However, it will be apparent to those skilled in the art that these specific details are not necessary to practice the invention. In other embodiments, well-known materials or methods have not been specifically described in order to avoid obscuring the invention.

[0024] Throughout this specification, references to “an embodiment,” “an example,” or “an example” mean that a particular feature, structure, or characteristic described in connection with that embodiment or example is included in at least one embodiment of the invention. Therefore, the phrases “an embodiment,” “an example,” “an example,” or “an example” appearing in various places throughout the specification do not necessarily refer to the same embodiment or example. Furthermore, specific features, structures, or characteristics can be combined in one or more embodiments or examples in any suitable combination and / or sub-combination. The term “and / or” as used herein includes any and all combinations of one or more of the associated listed items.

[0025] The "range" disclosed in this application is defined by a lower limit and an upper limit. A given range is defined by selecting a lower limit and an upper limit, which define the boundaries of a particular range. Ranges defined in this way can include or exclude endpoints and can be arbitrarily combined; that is, any lower limit can be combined with any upper limit to form a range. For example, if ranges of 60–120 and 80–110 are listed for a specific parameter, it is understood that ranges of 60–110 and 80–120 are also expected. Furthermore, if minimum range values ​​of 1 and 2 are listed, and if maximum range values ​​of 3, 4, and 5 are listed, then the following ranges are all expected: 1–3, 1–4, 1–5, 2–3, 2–4, and 2–5. In this application, unless otherwise stated, the numerical range "a–b" represents a shortened representation of any combination of real numbers between a and b, where a and b are real numbers. For example, the numerical range "0~5" indicates that all real numbers between "0~5" have been listed in this article; "0~5" is simply a shortened representation of these numerical combinations. Furthermore, when a parameter is stated as an integer ≥2, it is equivalent to disclosing that the parameter is, for example, an integer such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, etc.

[0026] Unless otherwise specified, all steps in this application may be performed sequentially or randomly, preferably sequentially. For example, the method includes steps (a) and (b), indicating that the method may include steps (a) and (b) performed sequentially, or it may include steps (b) and (a) performed sequentially. For example, the method may also include step (c), indicating that step (c) may be added to the method in any order. For example, the method may include steps (a), (b), and (c), or it may include steps (a), (c), and (b), or it may include steps (c), (a), and (b), etc.

[0027] Example 1 This invention provides a method for preparing a monoclonal antibody targeting APLN, comprising the following steps: (1) First, a sequence encoding a 6×His tag was fused to the N-terminus of the full-length human APLN protein sequence, and the recombinant gene was obtained through gene synthesis and inserted into the vector plasmid G79168 / pT7-GST-6×His-APLN(human). Finally, positive clones containing the inserted fragment were screened and verified by sequencing to obtain expression plasmids containing the correct gene sequence, which were then expanded and cultured. The amplified expression plasmids were then extracted.

[0028] (2) Transform the expression plasmid G79168 into BL21(DE3) cells and activate the bacterial culture.

[0029] (3) Take 12 ml of activated bacterial culture into 1200 ml of LB medium, incubate at 37°C for 3 h, add IPTG, and induce overnight in a shaker at 30°C.

[0030] (4) After induction, the bacterial culture was centrifuged, broken, and the precipitate was dissolved and purified by nickel column. Impurities were washed away with buffer containing 6M urea, 20mM imidazole, and 50mM imidazole in sequence. Finally, the target protein was collected with buffer containing 6M urea, 100mM, 200mM, and 500mM imidazole in sequence.

[0031] (5) All samples were subjected to SDS-PAGE electrophoresis for detection. The detection results are as follows: Figure 1 As shown.

[0032] (6) The purified protein was used as an antigen to immunize three 6-week-old female BALB / c mice; The immunization protocol was as follows: Each mouse was subcutaneously injected with an emulsion mixture of 50 μg antigen and Freund's complete adjuvant (for primary immunization) or incomplete adjuvant (for booster immunization), at four injection sites per injection, for a total of four immunizations, with each injection spaced two weeks apart. One week after the last immunization, serum was collected from the mice via orbital blood collection.

[0033] (7) The titer of anti-APLN antibodies in serum was detected using enzyme-linked immunosorbent assay (ELISA). The specific steps are as follows: (7.1) Antigen coating: Dilute the antigen to 1 µg / ml with coating solution, add 100 µL / well to a polystyrene 96-well reaction plate, and incubate overnight at 4°C; (7.2) Washing: Discard the liquid in the hole the next day and wash 3 times with washing solution; (7.3) Blocking: Add 200 μL of blocking solution per well and let stand at room temperature for 1 hour; (7.4) Washing: Wash 3 times with detergent; (7.5) Add the sample to be tested (primary antibody): Add antiserum (take blood, incubate overnight at 4°C, centrifuge at 4000 r / min for 10 min to obtain supernatant), and serially dilute the serum at 1:5000, 1:10000, 1:20000, 1:50000, and 1:100000 (pre-immunization serum is used as a negative control), 100 µl per well, and incubate at room temperature for 1 h; (7.6) Washing: Wash 3 times with detergent; (7.7) Add enzyme-labeled anti-antibody: Add HRP-labeled anti-mouse IgG secondary antibody, 100µl / well, and incubate at 37℃ for 30min; (7.8) Washing: Wash 3 times with detergent; (7.9) Color development: Add 100µl of freshly prepared substrate solution per well and incubate at room temperature in the dark for 5–30 min; (7.10) Termination of reaction and colorimetric measurement: Add 50 µL / well of stop solution. The color turns yellow; measure the absorbance of each well at 450 nm using a microplate reader. Figure 2 As shown.

[0034] (8) After confirming successful immunization, select the mice with the highest immunization titer, sacrifice the mice, and aseptically remove the spleens. Prepare a single-cell suspension of the spleen by mechanical grinding. Subsequently, CD138+ plasma cell sorting magnetic beads are used to positively sort the CD138+ plasma cell population from the spleen cell suspension.

[0035] The specific steps are as follows: (8.1) Tissue disruption: Place the spleen in cold PBS (containing 2% FBS) and gently grind it into a single-cell suspension using a syringe plunger.

[0036] (8.2) Filter with a 70 μm nylon filter to remove tissue debris and cell clumps.

[0037] (8.3) Centrifugation: Centrifuge at 300 × g for 10 minutes and discard the supernatant.

[0038] (8.4) Resuspend the cells in cold, recommended buffer solution and adjust the concentration to 1 × 10⁻⁶. 8 Cells / mL, store on ice for later use.

[0039] (8.5) Plasma cells expressing antibodies were isolated from spleen cells by using CD138 positive cell sorting magnetic beads.

[0040] (9) The positive rate of CD138 in cells before and after sorting was determined by flow cytometry. The specific steps are as follows: Resuspend cells in 100 µl of cell staining buffer, add 1.25 µl of PE-labeled CD138 antibody, and incubate on ice in the dark for 15–20 minutes. Wash twice with at least 2 mL of cell staining buffer, centrifuge at 350 x g for 5 minutes, resuspend the cell pellet in 0.5 mL of cell staining buffer, and add 0.5 µl of 7-AAD activity staining solution to remove dead cells. Perform flow cytometry analysis. The proportion of CD138-positive spleen cells before and after sorting was statistically analyzed. The results are shown below. Figure 3 As shown.

[0041] (10) To obtain monoclonal B cells secreting antibodies targeting APLN from plasma cells, single-cell isolation was performed using Sartorius' CellCelector fully automated cell sorting system. Using the CellCelector fluorescence microscopy system, single B cells secreting specific antibodies were automatically identified and visualized, and directly sorted into 96-well PCR plates containing cell lysis buffer, and immediately used for subsequent reverse transcription.

[0042] The specific steps are as follows: (10.1) Nanoporous pre-wetting (10.2) Add 0.5 mL of APLN antigen (filtered for sterilization) at a concentration of 50 µg / mL and incubate overnight at 4°C.

[0043] (10.3) Use 2% BSA to seal the orifice plate.

[0044] (10.4) Prepare 1 mL of single-cell suspension per well, mixed with the following substances: 15,000 plasma cells and dye-bound secondary antibody (concentration 7.5 µg / mL).

[0045] (10.5) Incubate overnight at 37℃ and 5% CO2.

[0046] (10.6) Automated scanning and detection of secretory cells (Hits) were performed to confirm positive results using single-cell analysis. The results of selecting a single positive B cell are shown below. Figure 4 As shown.

[0047] (10.7) Hits were exported from the nanopores to a 96-well PCR plate for cDNA synthesis. Results before and after sorting are shown below. Figure 5 As shown.

[0048] (11) Synthesize single-cell cDNA using single-cell PCR technology The sorted individual B cells underwent mRNA reverse transcription and cDNA pre-amplification. The specific steps were as follows: (11.1) Lysis / hybridization reaction: Prepare lysis mixture: 10X lysis buffer: 1 µL; capture oligonucleotide: 1 µL; RNase inhibitor: 0.4 µL; nuclease-free water to bring the total reaction volume to 10 µL. Incubate at 72°C for 1 minute.

[0049] (11.2) Reverse transcription (RT) reaction: Add 10 µL of RT reaction mixture to the lysed sample: nuclease-free water: 4 µL; 4X cDNA synthesis premix: 5 µL; template switching oligonucleotide: 1 µL. Thermal cycling program: 50℃ for 30 minutes, 85℃ for 5 minutes. At this point, cDNA from a single B cell has been obtained.

[0050] (11.3) Pre-amplified cDNA: Add 80 µL of pre-amplification mixture: 29 µL of nuclease-free water; 50 µL of 2X pre-amplification mixture; 1 µL of pre-amplification primers. Thermal cycling program: Initial denaturation: 98℃ for 30 seconds; 21 cycles (denaturation / annealing / extension): 98℃ for 10 seconds → 65℃ for 10 seconds → 67℃ for 3 minutes; Final extension: 67℃ for 5 minutes.

[0051] (12) Using the pre-amplified products as templates, nested PCR was used to amplify the variable region genes of the antibody light and heavy chains, using a mixture of mouse IgG VH and IgG Vκ primers. All PCR reactions were performed using high-fidelity DNA polymerase to ensure amplification fidelity.

[0052] PCR conditions: IgH strand: First round (95℃ 3 min → 35 cycles: 95℃ 15 s, 56℃ 15 s, 72℃ 55 s); Second round annealing temperature 60℃. Finally, incubate at 72℃ for 5 minutes. Igκ strand: First round (95℃ 3 min → 35 cycles: 94℃ 15 s, 50℃ 15 s, 72℃ 55 s); Second round annealing temperature 45℃. Finally, incubate at 72℃ for 5 minutes.

[0053] (13) The PCR products were verified by agarose gel electrophoresis. The electrophoresis results are as follows: Figure 5 As shown. Products with an electrophoresis range of 350bp-450bp are considered correct. Correct product bands are purified by gel extraction and then subjected to Sanger sequencing.

[0054] Sequencing results: (1) The amino acid sequence of the V region of the APLN-ab light chain is shown in SEQ ID No:1: EIVLTQSPALMAASPGEKVTITTCSVSSSISSSNLHWYQQKSETSPKPWIYGTSNLASGVPVRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSYPLTFGAGTKLELK.

[0055] (2) The amino acid sequence of the V region of the APLN-ab heavy chain is shown in SEQ ID No:2: EVQLQESAAELARPGASVKMSCKASGYTFTSYTMHWVKQRPGQGLEWIGYINPSSGYTEYNQKFKDKTTLTADKSSSTAYMQLSSLTSEDSAVYYCARWGYGSSPWFAYWGQGTLVTVSA.

[0056] (3) The nucleotide sequence of the V region of the APLN-ab light chain is shown in SEQ ID No:3: GAAATTGTGCTGACCCAGAGCCCGGCGCTGATGGCGGCGAGCCCGGGCGAAAAAGTGACCATTACCTGCAGCGTGAGCAGCAGCATTAGCAGCAGCAACCTGCATTGGTATCAGCAGAAAAGCGAAACCAGCCCGAAACCGTGGATTTATGGCACCAGCAAC CTGGCGAGCGGCGTGCCGGTGCGCTTTAGCGGCAGCGGCAGCGGCACCAGCTATAGCCTGACATTAGCAGCATGGAAGCGGAAGATGCGGCGACCTATTATTGCCAGCAGTGGAGCAGCTATCCGCTGACCTTTGGCGCGGGCACCAAACTGGAACTGAAA.

[0057] (4) The nucleotide sequence of the V region of the APLN-ab heavy chain is shown in SEQ ID No:4: GAAGTGCAGCTGCAGGAAAGCGCGGCGGAACTGGCGCGCCCGGGCGCGAGCGTGAAAATGAGCTGCAAAGCGAGCGGCTATAACCTTTACCAGCTATACCATGCATTGGGTGAAACAGCGCCCGGGCCAGGGCCTGGAATGGATTGGCTATATTAACCCGAGCAGCGGCTATACCGAATAT AACCAGAAATTTAAAGATAAAACCACCCTGACCGCGGATAAAAGCAGCAGCACCGCGTATATGCAGCTGAGCAGCCTGACCAGCGAAGATAGCGCGGTGTATTATTGCGCGCGCTGGGGCTATGGCAGCAGCCCGTGGTTTTGCGTATTGGGGCCAGGGCACCCTGGTGACCGTGAGCGCG.

[0058] Example 2 This invention provides a method for preparing highly stable recombinant monoclonal antibodies, comprising the following steps: (1) Synthesizing recombinant monoclonal antibodies using a mammalian expression system Based on the sequencing results of Example 1, the recombinant chimeric antibody was expressed. The specific steps were as follows: (1.1) Insert the variable region sequence of the antibody into a human IgG antibody expression vector containing the constant region sequence of all human IgG1 antibodies except for the V region to construct a recombinant antibody expression vector.

[0059] (1.2) The paired light and heavy chain expression vectors were co-transfected into HEK293F cells for expression. Three days after transfection, the supernatant was collected and replaced with 25 ml of fresh DMEM medium supplemented with 1% Nutridoma-SP. The supernatant was collected again on the sixth day after transfection.

[0060] (1.3) The culture supernatant was centrifuged at 800×g for 10 minutes to remove cell debris and stored at 4°C. The recombinant chimeric antibody was purified by incubating with 25 µl Protein G beads overnight at 4°C using 25 ml of cell culture supernatant. After centrifugation, the supernatant was removed, the beads were resuspended in PBS, and transferred to a chromatographic column equilibrated with PBS. After two rounds of washing with 1 ml PBS, the antibody was eluted in 0.1 M glycine (pH 3.0).

[0061] (1.4) The purified antibody was subjected to reducing and non-reducing SDS-PAGE electrophoresis, and the results are as follows: Figure 6 As shown in the figure, the detection results indicate that the purified recombinant monoclonal antibody has high purity and its molecular weight is consistent with the prediction.

[0062] (1.5) The purified recombinant monoclonal antibody was analyzed by SEC-HPLC, and the results are as follows: Figure 6 As shown, the purified recombinant monoclonal antibody monomer content is all above 95%, indicating batch stability.

[0063] (2) Antibody affinity detection The binding affinity of the recombinant antibody to the antigen was determined using an indirect ELISA method. The recombinant His-APLN antigen was diluted to 1 μg / mL with coating buffer, and 100 μL / well was added to a 96-well plate for coating overnight at 4°C. The purified antibody was then serially diluted (starting from 20 nM and performing 3-fold serial dilutions) and added to the corresponding wells for incubation. The results are shown below. Figure 7 As shown, the OD values ​​of the blank and control groups were almost 0, while the added APLN recombinant monoclonal antibody had a stronger binding to the coated antigen, which was about 2.3 at 1:5000. The OD value showed a decreasing trend with increasing dilution factor, indicating high specificity and high sensitivity.

[0064] Example 3 This invention provides an application of the recombinant monoclonal antibody obtained in Example 2 in the detection of liver cancer, as detailed below: The ability of the recombinant APLN monoclonal antibody described in Example 2 to detect APLN protein in the serum of liver cancer patients was tested using ELISA technology. The results are as follows: Figure 8 As shown, the recombinant monoclonal antibody APLN can bind to the APLN protein in the serum of patients, and the concentration of APLN in the blood of liver cancer patients is higher than that in healthy individuals.

[0065] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above description is only a specific embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. An APLN monoclonal antibody or a fragment thereof, characterized in that, The APLN monoclonal antibody includes a light chain variable region and a heavy chain variable region. The amino acid sequence of the light chain variable region is shown in SEQ ID No:1, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID No:

2.

2. The APLN monoclonal antibody or a fragment thereof according to claim 1, characterized in that, The nucleotide sequence of the variable region of the light chain is shown in SEQ ID No:

3.

3. The APLN monoclonal antibody or a fragment thereof according to claim 1, characterized in that, The nucleotide sequence of the heavy chain variable region is shown in SEQ ID No:

4.

4. A nucleic acid molecule, characterized in that, Encodes the APLN monoclonal antibody or fragment thereof as described in any one of claims 1 to 3.

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

6. A method for preparing the APLN monoclonal antibody or a fragment thereof according to any one of claims 1 to 3, characterized in that, APLN protein was obtained by immunizing mice with mice and then sorting the mice using single B-cell sorting and single-cell PCR techniques.

7. The method for preparing the APLN monoclonal antibody or a fragment thereof according to claim 6, characterized in that, Includes the following steps: The APLN gene was obtained, inserted into a vector plasmid, and amplified to obtain an expression plasmid. The expression plasmid was transferred into host cells, activated, induced, and purified to obtain the APLN protein. The obtained APLN protein was used as an antigen to immunize mice; After successful immunization, the mice were sacrificed and their spleens were removed. Plasma cells expressing APLN antibodies were then isolated using plasma cell sorting technology. Monoclonal B cells secreting antibodies targeting APLN were obtained from plasma cells using single B cell sorting technology. Antibodies were synthesized from sorted individual B cells using single-cell PCR technology.

8. The use of any of the APLN monoclonal antibodies or fragments thereof as described in claims 1 to 3 in the preparation of diagnostic or therapeutic drugs for cancer, wherein the cancer is a cancer associated with high expression of APLN.

9. The application according to claim 8, characterized in that, The cancers mentioned are liver cancer, intestinal cancer, stomach cancer, pancreatic cancer, or bile duct cancer.

10. A diagnostic reagent for cancer, characterized in that, Includes the APLN monoclonal antibody or fragment thereof as described in any one of claims 1 to 3.

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