Monoclonal antibody for identifying proinsulin and application thereof

Abstract

The invention provides a monoclonal antibody for identifying proinsulin. A light chain CDR3 of the monoclonal antibody is shown as SEQ ID NO: 13, a light chain CDR1 of the monoclonal antibody is shown as SEQ ID NO: 14, a light chain CDR2 of the monoclonal antibody is shown as SEQ ID NO: 15, a heavy chain CDR3 of the monoclonal antibody is shown as SEQ ID NO: 16, a heavy chain CDR1 of the monoclonal antibody is shown as SEQ ID NO: 17, and a heavy chain CDR2 of the monoclonal antibody is shown as SEQ ID NO: 18. The monoclonal antibody is used for identifying human proinsulin. The monoclonal antibody can specifically recognize the specific epitope of the proinsulin, has the advantages of high recognition specificity, good affinity, high titer and the like, and has wide application value.

Description

[0001] This invention is a divisional application of the invention patent application filed on October 10, 2024, with application number 202411410572.7 and invention title "A monoclonal antibody for recognizing proinsulin and its application". Technical Field

[0002] This invention relates to the field of biotechnology, and more specifically to a monoclonal antibody that recognizes proinsulin and its applications. Background Technology

[0003] Proinsulin is an important precursor in the biosynthesis of insulin, possessing unique biological characteristics and clinical significance. Insulin biosynthesis begins with preproinsulin, a precursor in the cytoplasm. Newly synthesized preproinsulin contains 110 amino acids. Guided by its N-terminal signal peptide, it translocates across the membrane into the endoplasmic reticulum (ER), where it is cleaved by a signal peptidase to form another insulin precursor—proinsulin, composed of 86 amino acids. In the ER, proinsulin rapidly undergoes oxidative folding, forming three highly conserved disulfide bonds and its normal spatial conformation. This normally folded proinsulin is further transported from the ER to the Golgi apparatus for further processing and cleavage, forming insulin and C-peptide, which are stored in secretory granules.

[0004] Proinsulin consists of two parts: insulin and a C-peptide, exhibiting dual immunomodulatory activity. This means that proinsulin can bind to both insulin antibodies and C-peptide antibodies. Although proinsulin and insulin are similar in molecular structure, proinsulin's hypoglycemic activity is only 5%-10% of that of insulin. Under physiological conditions, only a very small amount of proinsulin is released into the bloodstream. However, in pathological conditions such as diabetes, pancreatic tumors, polycystic ovary syndrome, or chronic renal insufficiency, the body develops insulin resistance or synthesizes large amounts of insulin, leading to elevated levels of proinsulin in the blood. Previous research by the team developing this invention has also found that delayed proinsulin secretion can serve as an earlier and more sensitive clinical indicator for evaluating pancreatic dysfunction.

[0005] Currently, proinsulin detection reagents can be used to diagnose diseases such as diabetes, insulinoma, and insulin resistance syndrome, as well as to accurately assess the physiological function of pancreatic β cells. However, the methods for diagnosing abnormal proinsulin expression are not yet perfect, making it essential to develop a novel monoclonal antibody and detection reagent capable of recognizing specific epitopes of proinsulin. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention discloses a monoclonal antibody for recognizing proinsulin and its application. The monoclonal antibody has advantages such as good affinity and high potency, and exhibits strong specificity for recognizing proinsulin in biological samples.

[0007] To achieve the above technical objectives, in a first aspect, the present invention proposes a monoclonal antibody that recognizes proinsulin, wherein the amino acid sequence of the light chain CDR3 of the monoclonal antibody is shown in SEQ ID NO:7, SEQ ID NO:13 or SEQ ID NO:19; and the amino acid sequence of the heavy chain CDR3 of the monoclonal antibody is shown in SEQ ID NO:10, SEQ ID NO:16 or SEQ ID NO:22.

[0008] In a further example of the present invention, the amino acid sequence of the light chain CDR1 of the monoclonal antibody is shown in SEQ ID NO:8, SEQ ID NO:14 or SEQ ID NO:20; and / or, the amino acid sequence of the light chain CDR2 of the monoclonal antibody is shown in SEQ ID NO:9, SEQ ID NO:15 or SEQ ID NO:21.

[0009] In a further example of the present invention, the amino acid sequence of the heavy chain CDR1 of the monoclonal antibody is shown in SEQ ID NO:11, SEQ ID NO:17 or SEQ ID NO:23; and / or, the amino acid sequence of the heavy chain CDR2 of the monoclonal antibody is shown in SEQ ID NO:12, SEQ ID NO:18 or SEQ ID NO:24.

[0010] It should be noted that the partial mutations introduced into the hypervariable region of the monoclonal antibody described in this invention do not affect the binding of the antibody molecule to the antigen. In a further example of this invention, the amino acid sequence of the light chain variable region of the monoclonal antibody is as shown in SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:5; or an amino acid sequence of SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:5 with one or more amino acid substitutions and / or deletions and / or additions, and having the same function as the protein shown in SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:5; or an amino acid sequence with at least 90% homology to SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:5 (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, etc., but not limited to the exemplified values) and having the same function as the protein shown in SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:5.

[0011] Furthermore, the light chain amino acid sequence of the monoclonal antibody includes a light chain variable region and a constant region SEQ ID NO:35.

[0012] In a further example of the invention, the amino acid sequence of the heavy chain variable region of the monoclonal antibody is as shown in SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6; or an amino acid sequence of SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6 with one or more amino acid substitutions and / or deletions and / or additions, and having the same function as the protein shown in SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6; or an amino acid sequence with at least 90% homology to SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6 (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, etc., but not limited to the exemplified values) and having the same function as the protein shown in SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6.

[0013] Furthermore, the heavy chain amino acid sequence of the monoclonal antibody includes a heavy chain variable region and a constant region SEQ ID NO:36.

[0014] In a further example of the present invention, the monoclonal antibody is a whole antibody or the antigen-binding portion of a whole antibody; further, the whole antibody is of type IgG1; further, the antigen-binding portion is a Fab fragment, an F(ab')2 fragment, or a single-chain antibody.

[0015] In a further example of the invention, the amino acid sequence of proinsulin recognized by the monoclonal antibody is shown in SEQ ID NO:25, SEQ ID NO:26 or SEQ ID NO:27.

[0016] In a further example of the present invention, three monoclonal antibodies are provided, namely monoclonal antibody 1, monoclonal antibody 2 and monoclonal antibody 3, wherein the amino acid sequences of the light chain variable region and the heavy chain variable region of monoclonal antibody 1 are SEQ ID NO:1 and SEQ ID NO:2, respectively; the amino acid sequences of the light chain variable region and the heavy chain variable region of monoclonal antibody 2 are SEQ ID NO:3 and SEQ ID NO:4, respectively; and the amino acid sequences of the light chain variable region and the heavy chain variable region of monoclonal antibody 3 are SEQ ID NO:5 and SEQ ID NO:6, respectively.

[0017] Secondly, the present invention provides a nucleic acid molecule that encodes the aforementioned monoclonal antibody that recognizes proinsulin.

[0018] Furthermore, the nucleotide sequence encoding the light chain variable region of monoclonal antibody 1 is shown in SEQ ID NO:28, and the nucleotide sequence encoding the heavy chain variable region is shown in SEQ ID NO:29; the nucleotide sequence encoding the light chain variable region of monoclonal antibody 2 is shown in SEQ ID NO:30, and the nucleotide sequence encoding the heavy chain variable region is shown in SEQ ID NO:31; the nucleotide sequence encoding the light chain variable region of monoclonal antibody 3 is shown in SEQ ID NO:32, and the nucleotide sequence encoding the heavy chain variable region is shown in SEQ ID NO:33.

[0019] Thirdly, the present invention proposes the use of the above-mentioned monoclonal antibody that recognizes proinsulin in the detection of proinsulin in biological samples for non-diagnostic purposes.

[0020] The present invention does not limit the specific types of biological samples, such as human tissues, cells, supernatants, serum, plasma, etc. Those skilled in the art can use the monoclonal antibody of the present invention that identifies proinsulin to detect biological samples containing antigens as needed, without limiting the scope of protection of the present invention.

[0021] Fourthly, this invention proposes the application of the above-mentioned monoclonal antibody that recognizes proinsulin in the preparation of human proinsulin in vitro immunoassay reagents and kits; by specifically applying the above-mentioned monoclonal antibody that recognizes human proinsulin to a specific kit, the determination of proinsulin content in biological samples can be achieved.

[0022] Furthermore, the kit also includes any one or a combination of at least two of the following: an enzyme-labeled plate, a coating buffer, a substrate, a substrate chromogenic solution, or a reaction termination solution.

[0023] Furthermore, the pharmaceutical composition further includes a pharmaceutically acceptable carrier and / or diluent.

[0024] Furthermore, the present invention is not limited to the method for preparing hybridoma blast cells for producing the above-mentioned monoclonal antibodies, and those skilled in the art can choose accordingly. Further, the method for preparing hybridoma blast cells for producing the above-mentioned monoclonal antibodies may optionally include the following steps:

[0025] (1) Peptide synthesis: Peptides with amino acid sequences as shown in SEQ ID NO:25, SEQ ID NO:26 or SEQ ID NO:27 were synthesized using chemical synthesis methods and purified by HPLC.

[0026] (2) Antigen preparation: Using patented IEF (Immunogenicity Enhancement Factors), VLP (Virus-Like Particles) are combined with traditional KLH carrier proteins.

[0027] (3) Pre-immunization test: Five female BALB / c mice aged 8-12 weeks were selected. Blood was collected from the orbital cavity and serum was separated. The serum titer was detected by ELISA. The coating antigen was a polypeptide conjugate with BSA.

[0028] (4) Mouse immunization: Mice were immunized using a peptide-KLH conjugate as an immunogen. After the last immunization, blood was collected from mice to detect serum titer, using the same method as the pre-immunization test. Only mice with an antiserum titer greater than 10K were allowed to proceed to the sorting stage; otherwise, an additional immunization was administered.

[0029] (5) Antibody discovery: Hybridoma cell lines were obtained using standard hybridoma preparation procedures. The supernatant of the hybridoma cell lines was tested using a pre-immunoassay method, and the purified antibodies from the supernatant were used for retesting. The cell line with the highest ELISA titer was selected to obtain the hybridoma mother cells.

[0030] Furthermore, the present invention is not limited to the methods for producing the above-mentioned monoclonal antibodies, and those skilled in the art can choose accordingly. Further, the methods for preparing the monoclonal antibodies may be: (1) Hybridoma production: Cell production is carried out using a standard production process with serum-free culture medium, and antibodies are separated and purified using Protein A / G affinity column chromatography; (2) Ascites production: Antibodies are produced using a standard ascites production process, and antibodies are separated and purified using Protein A / G affinity column chromatography; (3) Recombinant production: Light and heavy chain nucleic acids of hybridoma cell lines are obtained through molecular biological operations, verified by agarose gel, constructed into an expression vector using restriction endonuclease and T4 ligase, and plasmids are amplified using a prokaryotic expression system. Cell production is carried out using transient transfection and construction of stable cell lines, and antibodies are separated and purified using Protein A / G affinity column chromatography.

[0031] The amino acid sequence involved in this invention includes:

[0032] (1) Monoclonal antibody 1

[0033] SEQ ID NO:1 (Light chain variable region):

[0034] DIVLTQSPLSLPVSLGDQASISCRSSQNIVHSNGKTYLEWYLQKPGQSPKLLIYNIYNRF SGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVPYTFGGGTKLEIK

[0035] SEQ ID NO:7 (Light chain CDR3):

[0036] FQGSHVPYT

[0037] SEQ ID NO:8 (Light chain CDR1):

[0038] RSSQNIVHSNGKTYLE

[0039] SEQ ID NO:9 (Light chain CDR2):

[0040] NIYNRFS

[0041] SEQ ID NO:2 (Heavy chain variable region):

[0042] QVQLQQSGAEVVKPGASVKMSCKASGYSFTSYNMHWIKQTPGQGLEWIGGIYPGNG ESSYNQKFKGKATMTADKSSNTAYMQFSSLTSEDSAVYYCARGDGFDYWGQGTTLTVSS

[0043] SEQ ID NO:10 (Heavy chain CDR3):

[0044] GDGFDY

[0045] SEQ ID NO:11 (Heavy chain CDR1):

[0046] SYNMH

[0047] SEQ ID NO:12 (Heavy chain CDR2):

[0048] GIYPGNGESSYNQKFKG

[0049] (2) Monoclonal antibody 2

[0050] SEQ ID NO:3 (Light chain variable region):

[0051] DIVLTQSPASLAVSLGQRATISCKASQSVDYNGDSYMNWYQQKPGQPPTLLIYVASKLE SGIPARFSGSGSGTDFTLNIHPVEEEDVATYYCQQTNEEPLTFGAGTKLELK

[0052] SEQ ID NO:13 (Light chain CDR3):

[0053] QQTNEEPLT

[0054] SEQ ID NO:14 (Light chain CDR1):

[0055] KASQSVDYNGDSYMN

[0056] SEQ ID NO:15 (Light chain CDR2):

[0057] VASKLES

[0058] SEQ ID NO:4 (Heavy chain variable region):

[0059] QVQLEQSGPELVKPGASVRISCKASKASGYTFTSYYVHWVRQRPGQGLEWIGWIHPGNINS KYNEKFKDKATLTADKSSTTAYMQLSSLTSEDSAVYFCSRSVDYWGQGTTLTVSS

[0060] SEQ ID NO:16 (Heavy chain CDR3):

[0061] SVDY

[0062] SEQ ID NO:17 (Heavy chain CDR1):

[0063] SYYVH

[0064] SEQ ID NO:18 (Heavy chain CDR2):

[0065] WIHPGNINSKYNEKFKD

[0066] (3) Monoclonal antibody 3

[0067] SEQ ID NO:5 (Light chain variable region):

[0068] DIVLTQSTALMAASPGEKVTITCSVSSSISSSYLHWYQQKSETSPKPWIYGTSNLASGVP VRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSNYPLTFGAGTKLELK

[0069] SEQ ID NO:19 (Light chain CDR3):

[0070] QQWSNYPLT

[0071] SEQ ID NO:20 (Light chain CDR1):

[0072] SVSSSISSSYLH

[0073] SEQ ID NO:21 (Light chain CDR2):

[0074] GTSNLAS

[0075] SEQ ID NO:6 (Heavy chain variable region):

[0076] QVQLEQSGGGLVQPKGSLKLSCAASGFNFNTNGMNWVRQAPGKGLEWVARIRSKSNNYATYYADSVKDRFTISRDDSQSMLYLQMNNLKTEDTAMYYCVRDGGYGAWFAYWGQGSLVTVSA

[0077] SEQ ID NO:22 (Heavy chain CDR3):

[0078] DGGYGAWFAY

[0079] SEQ ID NO:23 (Heavy chain CDR1):

[0080] TNGMN

[0081] SEQ ID NO:24 (Heavy chain CDR2):

[0082] RIRSKSNNYATYYADSVKD

[0083] Furthermore, the amino acid sequence of the light chain constant region is SEQ ID NO:34 (light chain constant region):

[0084] RADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTD QDSKDSTYSMSSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC

[0085] The amino acid sequence of the heavy chain constant region is SEQ ID NO:35 (heavy chain constant region):

[0086] ASTTPPSVYPLAPGSANSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHT AQTQPREEQFNSTFRSSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMNTNGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK

[0087] In addition, the amino acid sequences of SEQ ID NO:25, SEQ ID NO:26, or SEQ ID NO:27 in this invention are as follows:

[0088] SEQ ID NO:25:

[0089] PLALEGSLQKRGIV

[0090] SEQ ID NO:26:

[0091] PKTRREAEDLQVGQ

[0092] SEQ ID NO:27:

[0093] KSRREVEDPQVEQLEC

[0094] The nucleic acid molecular sequences involved in this invention include:

[0095] The nucleotide sequence encoding the light chain variable region of monoclonal antibody 1 is shown in SEQ ID NO:28, and the nucleotide sequence encoding the heavy chain variable region is shown in SEQ ID NO:29;

[0096] SEQ ID NO:28:

[0097] GACATTGTGCTCACCCAATCTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGCAGATCTAGTCAGAACATTGTACATAGTAATGGAAAGACCTATTTAGAGTGGTACCTGCAGAAACCAGGCCAGTCTCCAAAGCTCCTGATCTACAATATTTACAACCGGTTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAGCAGAGTGGAGGCTGAGGATCTGGGAGTTTATTACTGTTTTCAAGGTTCACATGTTCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAA

[0098] SEQ ID NO:29:

[0099] CAGGTCCAGCTGCAGCAGTCAGGGGCTGAGGTGGTGAAGCCTGGGGCCTCAGTGAAGATGTCCTGCAAGGCTTCTGGCTACTCATTTACCAGTTACAATATGCACTGGATAAAGCAGACACCTGGACAGGGCCTGGAATGGATTGGAGGTATTTATCCAGGAAATGGTGAAAGTTCCTACAATCAGAAGTTCAAAGGCAAGGCCACAATGACTGCAGACAAATCCTCCAACACAGCCTACATGCAGTTCAGCAGCCTGACATCTGAGGACTCTGCGGTCTATTATTGTGCAAGAGGGGACGGTTTTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA

[0100] The nucleotide sequence encoding the light chain variable region of monoclonal antibody 2 is shown in SEQ ID NO:30, and the nucleotide sequence encoding the heavy chain variable region is shown in SEQ ID NO:31:

[0101] SEQ ID NO:30:

[0102] GACATTTGTGCTCACCCAGTCTCCAGCCTCTTTGGCTGTGTCTCTAGGGCAGAGGGCCACCATCTCCTGCAAGGCCAGCCAAAGTGTTGATTATAATGGTGATAGTTATATGAACTGGTACCAACAGAAACCGGGACAGCCACCCACACTCCTCATCTATGTTGCAT CCAAGCTAGAATCTGGGATCCCAGCCAGGTTTAGTGGCAGTGGGTCTGGGACAGACTTCACCCTCAACATCCATCCTGTGGAGGAGGAGGATGTTGCAACCTATTACTGTCAGCAAACTAATGAGGAACCGCTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAA

[0103] SEQ ID NO:31:

[0104] CAAGTTCAGCTGGAGCAGTCTGGACCTGAGCTGGTGAAGCCTGGGGCTTCAGTGAGGATATCCTGCAAGGCTTCTGGCTACACCTTCACAAGCTACTATGTTCACTGGGTGAGGCAGAGGCCTGGACAGGGACTTGAGTGGATTGGATGGATTCATCCTGGAAATATTA ATTCTAAATACAATGAGAAATTCAAGGACAAGGCCACACTGACTGCAGACAAATCCTCCACCACAGCCTACATGCAGCTCAGCAGCCTGACCTCTGAGGACTCTGCGGTCTATTTCTGTTCAAGATCGGTTGACTACTGGGGCCAAGGGACCACTCTCACAGTCTCCTCA

[0105] The nucleotide sequence encoding the light chain variable region of monoclonal antibody 3 is shown in SEQ ID NO:32, and the nucleotide sequence encoding the heavy chain variable region is shown in SEQ ID NO:33.

[0106] SEQ ID NO:32:

[0107] GATATTGTGCTGACCCAGTCTACAGCACTCATGGCTGCATCTCCAGGGGAAGGTCACCATCACCTGCAGTGTCAGCTCAAGTATAAGTTCCAGCTACTTGCACTGGTACCAGCAGAAGTCAGAAACCTCCCCCAAACCCTGGATTTATGGCACATCCAAC CTGGCTTCTGGAGTCCCTGTTCGCTTCAGTGGCAGTGGATCTGGGACCTCTTATTCTCTCACAATCAGCAGCATGGAGGCTGAAGATGCTGCCACTTATTACTGTCAACAGTGGAGTAATTACCCACTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAA

[0108] SEQ ID NO:33:

[0109] CAAGTTCAGCTGGAGCAGTCTGGTGGAGGATTGGTGCAGCCTAAAGGGTCATTGAAACTCTCATGTGCAGCCTCTGGATTTAACTTCAATACCAATGGCATGAACTGGGTCCGCCAGGCCCCAGGAAAGGGTTTGGAGTGGGTTGCTCGCATAAGAAGTAAAAGTAATAATTATGCAACAT ATTATGCCGATTCAGTGAAAGACAGGTTCACCATCTCCAGAGATGATTCACAAAGCATGCTCTATCTGCAAATGAACAACTTGAAAACTGAGGACACAGCCATGTATTACTGTGTGAGAGACGGGGGTTACGGGGCCTGGTTTGCTTACTGGGGCCAAGGGTCTCTGGTCACTGTCTCTGCA

[0110] Compared with existing technologies, the monoclonal antibody of the present invention that recognizes proinsulin can specifically recognize specific antigenic epitopes in human / mouse proinsulin, with strong recognition specificity, good affinity and high titer, and has wide application value. Attached Figure Description

[0111] The accompanying drawings, which form part of this application, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0112] Figure 1The results of the immunoblotting experiment of the monoclonal antibody 1 protein of the present invention in Example 6 are shown; wherein, the left figure shows the results using the monoclonal antibody 1 of the present invention as the primary antibody recognition protein, and the right figure shows the results using the human insulin antibody as the primary antibody recognition protein; lane 1 represents the supernatant protein secreted by human insulinoma cells in the non-reduced state, lane 2 represents the supernatant protein secreted by human insulinoma cells in the reduced state, and lane M represents the marker (Thermo Fisher #26616);

[0113] Figure 2 The results of the immunoblotting experiment of the monoclonal antibody 2 protein of the present invention in Example 7 are shown; wherein, the left figure shows the results using the monoclonal antibody 2 of the present invention as the primary antibody recognition protein, and the right figure shows the results using the human insulin antibody as the primary antibody recognition protein; lane 1 represents the supernatant protein secreted by human insulinoma cells in the non-reduced state, lane 2 represents the supernatant protein secreted by human insulinoma cells in the reduced state, and lane M represents the marker (Thermo Fisher #26616);

[0114] Figure 3 Example 8 illustrates that the monoclonal antibody 3 of the present invention specifically recognizes murine proinsulin but not insulin. The left figure shows the results using the monoclonal antibody 3 of the present invention as the primary antibody recognition protein, and the right figure shows the results using a murine insulin antibody as the primary antibody recognition protein; lane 1 represents mouse islet cell protein; lane M represents marker (ThermoFisher#26616).

[0115] Note: The red arrows in the diagram represent the locations where proinsulin is detected, and the blue arrows represent the locations where insulin is detected. Detailed Implementation

[0116] To facilitate understanding of the present invention, a more comprehensive description will be provided below, along with preferred embodiments. However, it should be understood that these embodiments are merely for more detailed explanation and should not be construed as limiting the invention in any way, i.e., not intended to limit the scope of protection of the invention.

[0117] Unless otherwise defined, the technical terms used in the following embodiments have the same meanings as commonly understood by those skilled in the art to which this invention pertains. Unless otherwise specified, the experimental reagents used in the following embodiments are conventional biochemical reagents; and the experimental methods described are conventional methods.

[0118] Example 1: Preparation of hybridoma cell lines containing anti-proinsulin monoclonal antibodies

[0119] (1) Immunogen preparation: A polypeptide (amino acid sequence such as SEQ ID NO:25) is coupled to KLH (keyhole hemocyanin) carrier protein using conventional coupling methods to prepare an immunogen, and the polypeptide concentration is controlled to be ≥0.25mg / mL (thiol value ≥1.0);

[0120] Similarly, another polypeptide (amino acid sequence as shown in SEQ ID NO:25) was conjugated to the BSA carrier protein using conventional conjugation methods to prepare a screening antigen, with the polypeptide concentration controlled at ≥0.25 mg / mL (thiol value ≥1.0). Before immunization, the immunogen was mixed with Freund's complete adjuvant at a 1:1 ratio and thoroughly emulsified.

[0121] (2) Immunization: Five healthy 8-week-old BALB / c mice were selected as immunization subjects (the serum ELISA titer before immunization was tested, and it was best if the pre-immunization serum did not show a color reaction with the screening antigen, or the OD value of the undiluted serum ELISA was less than 2.1 times that of the PBS group).

[0122] The immunization regimen consisted of a second immunization three weeks after the initial immunization, followed by a third immunization two weeks after the second immunization, for a total immunization cycle of 7 weeks. Each mouse received 100 μL of immunization per dose. One week after the three immunizations, serum ELISA titers were measured (using screening antigen-coated plates). If the titer was greater than 256kJ, a single immunotherapy shock was administered, with cell fusion occurring 2-3 days after the shock. If the titer did not reach the target, a booster immunization was administered, followed by another shock once the titer reached the target.

[0123] (3) Fusion Screening: Mice to be fused were sacrificed using the carbon dioxide method, and spleens were aseptically collected. Red blood cells were lysed to prepare a spleen cell suspension, which was then counted. Thymus cells from 3-4 week old mice were aseptically collected and seeded onto 20-40 wells of 96-well plates using HAT medium, 100 μL per well. Myeloma cells in the logarithmic growth phase were selected, aseptically collected, and counted, ensuring cell viability was greater than 90%. Spleen cells and myeloma cells were mixed at a certain ratio (e.g., 3:1-5:1) and cell fusion was performed using electrofusion.

[0124] After fusion, the cell suspension was mixed thoroughly in HAT selective medium until the cell density was less than 15 cells / mL. The mixture was then aliquoted into 20-40 prepared 90-well plates, 100 μL per well (total 200 μL). After 1-2 weeks of cell growth, 100 μL of the supernatant from wells showing successful fusion was collected for ELISA testing (using a selection antigen coating). The wells with the highest ELISA values ​​were selected for subcloning. The goal was to ensure that subcloned cells were single cells per well (96 wells). Subcloning was further screened using the same method of ELISA testing the supernatant until the entire plate showed a positive ELISA result.

[0125] (4) Cell line selection: From the full-plate positive subclones, select 3-5 cell lines with the best growth status by microscopic examination. Incubate these cells statically in 50 mL of culture medium for 1-3 weeks. When cell viability is less than 40%, collect the supernatant. Purify the supernatant using Protein A / G affinity purification and label the antibody concentration using a BCA kit. Detect the EC50 values ​​of these 3-5 antibody lines using ELISA. The optimal cell line is selected as the final hybridoma cell line for the anti-proinsulin monoclonal antibody.

[0126] Example 2 Production of anti-proinsulin monoclonal antibody hybridoma cells

[0127] Using the culture component substitution method, the cell lines were acclimatized to a serum-free suspension culture state, at 10... 5 Inoculate gradually into 1L culture flasks (330mL of medium) at an inoculation density of 1 / mL and then incubate by shaking. When the sugar content of the medium is lower than 10 mmol / mL, add glucose solution to maintain the sugar content at 10-25 mmol / mL. Furthermore, when the inoculation density is >2×10⁻⁶, 6 When the cell count is 1 / mL, supplement with glutamine and other essential nutrients to maintain cell growth every two days. Collect the supernatant when the cell viability is less than 40%.

[0128] Place the prepared Protein A column on an AKTA instrument and equilibrate the column with 10 mM PBS (pH = 7.4 ± 0.05) at a flow rate of 10 mL / min. Collect the flow-through for a second column loading. Wash away impurities with glycine-HCl buffer (pH = 5.5) at a flow rate of 5 mL / min, with a washing volume of 2-5 times the column volume. After washing, equilibrate the column with equilibration buffer (10 mM PBS, pH = 7.4). Elute the antibody with glycine-HCl buffer (pH = 2.5) at a flow rate of 5 mL / min, maintaining an antibody concentration of 3-5 mg / mL, referencing the supernatant antibody concentration obtained from ELISA. Immediately after elution, add an equal volume of glycine-NaHCO3 buffer (pH = 9.5 ± 0.1) to neutralize the acidic environment.

[0129] The antibody buffer system was replaced with 10 mM PBS (pH = 7.4 ± 0.05) using an ultrafiltration centrifuge tube. The concentration was determined using a BCA kit and controlled at 2.0 mg / mL.

[0130] It should be noted that if the obtained antibodies are not used immediately, an appropriate amount of preservative (such as sodium azide) should be added and the antibodies stored in a refrigerator at -80°C.

[0131] Example 3: Recombinant Production of Anti-Insulin Proinsulin Monoclonal Antibody

[0132] The monoclonal cell line obtained in Example 1 was cultured to 75 cm⁻¹.2 Cells were cultured in carbon dioxide cell culture flasks until the bottom layer was 80% full. Cells were collected, resuspended in nuclease-free water containing RNase inhibitors, and then collected again by centrifugation. A portion of the cells were used for single-cell sequencing to obtain antibody sequence information.

[0133] Another part used a 5'RACE kit to extract total mRNA, reverse transcribed it into cDNA, and amplified the deoxyribonucleic acid sequence of the variable region of the antibody light and heavy chains.

[0134] Following the kit instructions, the pcDNA5.0 plasmid and the antibody light and heavy chains were digested with restriction endonucleases, respectively. The digestion products were recovered and purified using a nucleic acid recovery kit. After verification by dextran gel electrophoresis, the purified products were ligated using a T4 ligase kit to construct the antibody light and heavy chains into the plasmid, ensuring that the variable and constant regions of the light and heavy chains were intact on the plasmid.

[0135] The plasmid was transformed into E. coli (DH5α) competent cells using competent cells and heat shock method, the plasmid was amplified, and the plasmid was extracted using an endotoxin-free plasmid extraction kit.

[0136] The linearized light and heavy chain plasmids were simultaneously transformed into CHO cells using PEI transformation or electroporation.

[0137] Stable transfected cell lines can be screened using standard stable transfection cell line screening methods, or cell supernatant can be produced using transient transfection methods.

[0138] The production of antibodies from stable transgenic strains was carried out according to the method in Example 2.

[0139] The supernatant of cells transiently transfected for expression was purified using the antibody purification method described in Example 2.

[0140] In this embodiment, a specific monoclonal antibody 1 containing a partial sequence of anti-human insulin C-peptide-A chain linked to SEQ ID NO:25 (PLALEGSLQKRGIV) was prepared. This monoclonal antibody is of type IgG1 and the light chain is of type kappa.

[0141] Its light chain variable region sequence is SEQ ID NO:1. The amino acid sequences of the three hypervariable regions CDR1, CDR2, and CDR3 of the light chain variable region are shown in SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:7, respectively. Among them, CDR1 is located at positions 24-39 of the light chain variable region, CDR2 is located at positions 55-61 of the light chain variable region, and CDR3 is located at positions 94-102 of the light chain variable region. Its heavy chain variable region is SEQ ID NO:2. The amino acid sequences of the three hypervariable regions CDR1, CDR2, and CDR3 of the heavy chain variable region are shown in SEQ ID NO:11, SEQ ID NO:112, and SEQ ID NO:10, respectively. Among them, CDR1 is located at positions 31-35 of the heavy chain variable region, CDR2 is located at positions 50-66 of the heavy chain variable region, and CDR3 is located at positions 99-104 of the heavy chain variable region.

[0142] Its light chain variable region can be combined with SEQ ID NO:34 to form a complete light chain; its heavy chain variable region can be combined with SEQ ID NO:35 to form a complete heavy chain.

[0143] Example 4: Preparation of Proinsulin Monoclonal Antibody 2

[0144] Using the experimental methods described in Examples 1-3 above, a specific monoclonal antibody 2 containing a partial sequence SEQ ID NO:26 (PKTRREAEDLQVGQ) linked to the B-chain C peptide of human proinsulin can be obtained. This monoclonal antibody is of the IgG1 type and the light chain is of the kappa type.

[0145] Its light chain variable region sequence is SEQ ID NO:3. The amino acid sequences of the three hypervariable regions CDR1, CDR2, and CDR3 of the light chain variable region are shown in SEQ ID NO:14, SEQ ID NO:15, and SEQ ID NO:13, respectively. Among them, CDR1 is located at positions 24-38 of the light chain variable region, CDR2 is located at positions 54-60 of the light chain variable region, and CDR3 is located at positions 93-101 of the light chain variable region. Its heavy chain variable region is SEQ ID NO:4. The amino acid sequences of the three hypervariable regions CDR1, CDR2, and CDR3 of the heavy chain variable region are shown in SEQ ID NO:17, SEQ ID NO:18, and SEQ ID NO:16, respectively. Among them, CDR1 is located at positions 31-35 of the heavy chain variable region, CDR2 is located at positions 50-66 of the heavy chain variable region, and CDR3 is located at positions 99-102 of the heavy chain variable region.

[0146] Its light chain variable region can be combined with SEQ ID NO:34 to form a complete light chain; its heavy chain variable region can be combined with SEQ ID NO:35 to form a complete heavy chain.

[0147] The production of antibodies from stable transgenic strains was carried out according to the method in Example 2.

[0148] The supernatant of cells transiently transfected for expression was purified using the antibody purification method described in Example 2.

[0149] Example 5: Preparation of Proinsulin Monoclonal Antibody 3

[0150] Using the experimental methods described in Examples 1-3 above, a specific monoclonal antibody 3 containing a partial sequence SEQ ID NO:27 (KSRREVEDPQVEQLEC) linking the B-chain-C peptide of mouse proinsulin can be obtained. This monoclonal antibody is of the IgG1 type, and its light chain is of the kappa type.

[0151] Its light chain variable region sequence is SEQ ID NO:5. The amino acid sequences of the three hypervariable regions CDR1, CDR2, and CDR3 of the light chain variable region are shown in SEQ ID NO:20, SEQ ID NO:21, and SEQ ID NO:19, respectively. Among them, CDR1 is located at positions 24-35 of the light chain variable region, CDR2 is located at positions 51-57 of the light chain variable region, and CDR3 is located at positions 90-98 of the light chain variable region. Its heavy chain variable region is SEQ ID NO:6. The amino acid sequences of the three hypervariable regions CDR1, CDR2, and CDR3 of the heavy chain variable region are shown in SEQ ID NO:23, SEQ ID NO:24, and SEQ ID NO:22, respectively. Among them, CDR1 is located at positions 31-35 of the heavy chain variable region, CDR2 is located at positions 50-68 of the heavy chain variable region, and CDR3 is located at positions 101-110 of the heavy chain variable region.

[0152] Its light chain variable region can be combined with SEQ ID NO:34 to form a complete light chain; its heavy chain variable region can be combined with SEQ ID NO:35 to form a complete heavy chain.

[0153] The production of antibodies from stable transgenic strains was carried out according to the method in Example 2.

[0154] The supernatant of cells transiently transfected for expression was purified using the antibody purification method described in Example 2.

[0155] Example 6

[0156] Collect the supernatant from cultured immortalized human insulinoma cells. Take 60 μL of the supernatant and add 20 μL of 4×NPLDS SAMPLE BUF (Thermo Fisher, NP0007) to mix well. Boil at 95°C for 5 min to obtain the protein sample in the non-reduced state. Take 36 μL of the above sample and add 4 μL of 1M reducing agent DTT (Sigma, D9163) to mix well. Boil at 95°C for 5 min to obtain the protein sample in the reduced state.

[0157] The specific protein immunoblotting experiment was performed as described in the previous articles published by our team (e.g., Yang J, et al. PNAS; 2022; 119(45):e2204443119, etc.). 30 μL of the protein samples in both the non-reduced and reduced states were used for the protein immunoblotting experiment. The primary antibodies used were the monoclonal antibody 1 of this invention and the human insulin antibody, respectively. The results are as follows: Figure 1 As shown.

[0158] Figure 1 The left image shows the results using monoclonal antibody 1 prepared in Examples 1-3 of this invention as the primary antibody recognition protein, and the right image shows the results using human insulin antibody as the primary antibody recognition protein. Figure 1 A comparative analysis of the left and right images shows that the monoclonal antibody 1 of the present invention can specifically recognize proinsulin and only proinsulin, especially proinsulin in its reduced state. The human insulin antibody, as a primary antibody recognition protein, can recognize both proinsulin and insulin protein simultaneously. This result verifies that the monoclonal antibody 1 of the present invention does not cross-react with insulin protein during the recognition of proinsulin and has excellent specificity for recognizing proinsulin.

[0159] Example 7

[0160] Collect the supernatant from cultured immortalized human insulinoma cells. Take 60 μL of the supernatant and add 20 μL of 4×NPLDS SAMPLE BUF (Thermo Fisher, NP0007) to mix well. Boil at 95°C for 5 min to obtain the protein sample in the non-reduced state. Take 36 μL of the above sample and add 4 μL of 1M reducing agent DTT (Sigma, D9163) to mix well. Boil at 95°C for 5 min to obtain the protein sample in the reduced state.

[0161] The specific procedures for the protein immunoblotting experiment are as described in the previous articles published by our team (e.g., Yang J, et al. PNAS; 2022; 119(45):e2204443119, etc.). 30 μL of the protein samples in both the non-reduced and reduced states were taken for the protein immunoblotting experiment. The primary antibodies used were the monoclonal antibody 2 of this invention and the human insulin antibody, respectively. The results are as follows: Figure 2 As shown.

[0162] Figure 2 The left image shows the results using the monoclonal antibody 2 of this invention as the primary antibody recognition protein, and the right image shows the results using the human insulin antibody as the primary antibody recognition protein. Comparative analysis. Figure 2As shown in the left and right figures, the monoclonal antibody 2 of the present invention can specifically recognize proinsulin but not insulin protein, especially proinsulin in its reduced state. In contrast, the human insulin antibody, as a primary antibody recognition protein, can recognize both proinsulin and insulin protein simultaneously. This result confirms that the monoclonal antibody 2 of the present invention does not cross-react with insulin protein during the recognition of proinsulin and has excellent specificity for recognizing proinsulin.

[0163] Example 8

[0164] Sixty islets of pancreas from wild-type mice were extracted, and 30 μL of protein lysis buffer was added. The mixture was placed on ice for 20 min to lyse. After centrifugation at 13500 rpm and 4℃ for 15 min, 27 μL of supernatant was collected. 9 μL of 4×NP LDS SAMPLE BUF (Thermo Fisher, NP0007) and 4 μL of 1M reducing agent DTT (Sigma, D9163) were added and mixed. The mixture was then boiled at 95℃ for 5 min.

[0165] The specific procedures for islet extraction and protein immunoblotting are as described in previous articles published by our team (e.g., Yang J, et al. PNAS; 2022; 119(45):e2204443119, etc.). 30 μL of the above protein sample was used for protein immunoblotting, with the monoclonal antibody 3 of this invention and the mouse insulin antibody as the primary antibodies. Results are as follows: Figure 3 As shown.

[0166] Figure 3 The left figure shows the results using the monoclonal antibody 3 of the present invention as the primary antibody recognition protein, and the right figure shows the results using mouse-derived insulin antibody as the primary antibody recognition protein. The results show that the monoclonal antibody 3 of the present invention can specifically recognize proinsulin and does not recognize insulin protein, while the mouse-derived insulin antibody can recognize both proinsulin and insulin protein. This result verifies that the monoclonal antibody 3 of the present invention has no cross-reactivity with insulin protein and can specifically recognize proinsulin.

[0167] It should be noted that the above description, in conjunction with specific embodiments, provides a further detailed explanation of the present invention and should not be construed as limiting the specific implementation of the invention to these descriptions. For those skilled in the art, several simple modifications can be made without departing from the concept of the present invention, and all such modifications should be considered within the scope of protection of the present invention.

Claims

1. A monoclonal antibody which recognizes proinsulin, characterized in that, the light chain CDR3 of the monoclonal antibody is as set forth in SEQ ID NO: 13, the light chain CDR1 is as set forth in SEQ ID NO: 14, the light chain CDR2 is as set forth in SEQ ID NO: 15, the heavy chain CDR3 is as set forth in SEQ ID NO: 16, the heavy chain CDR1 is as set forth in SEQ ID NO: 17, the heavy chain CDR2 is as set forth in SEQ ID NO: 18; the monoclonal antibody is used for recognizing human proinsulin.

2. The monoclonal antibody that recognizes proinsulin according to claim 1, characterized in that, the amino acid sequence of the light chain variable region of the monoclonal antibody is as set forth in SEQ ID NO: 3, or an amino acid sequence that is derived from SEQ ID NO: 3 by substitution, deletion and / or addition of one or several amino acids and that has the same function as the protein set forth in SEQ ID NO: 3, or an amino acid sequence that has at least 90% homology with SEQ ID NO: 3 and that has the same function as the protein set forth in SEQ ID NO:

3.

3. The monoclonal antibody that recognizes proinsulin according to claim 1, characterized in that, the amino acid sequence of the heavy chain variable region of the monoclonal antibody is as set forth in SEQ ID NO: 4, or an amino acid sequence that is derived from SEQ ID NO: 4 by substitution, deletion and / or addition of one or several amino acids and that has the same function as the protein set forth in SEQ ID NO: 4, or an amino acid sequence that has at least 90% homology with SEQ ID NO: 4 and that has the same function as the protein set forth in SEQ ID NO:

4.

4. The monoclonal antibody that recognizes proinsulin according to claim 1, characterized in that, the monoclonal antibody is a whole antibody or an antigen-binding portion of a whole antibody.

5. The monoclonal antibody that recognizes proinsulin according to claim 4, characterized in that, the whole antibody is of the IgG1 type.

6. The monoclonal antibody that recognizes proinsulin according to claim 4, characterized in that, the antigen-binding portion is a Fab fragment, a F(ab')2 fragment or a single-chain antibody.

7. The monoclonal antibody that recognizes proinsulin according to claim 1, characterized in that, the amino acid sequence of the proinsulin recognized by the monoclonal antibody is as set forth in SEQ ID NO:

26.

8. A nucleic acid molecule, characterized in that, the nucleic acid molecule encodes the monoclonal antibody recognizing proinsulin according to any one of claims 1 to 7.

9. Use of the monoclonal antibody recognizing proinsulin according to any one of claims 1 to 7 for detecting proinsulin in a biological sample for non-diagnostic purposes.

10. Use of the monoclonal antibody recognizing proinsulin according to any one of claims 1 to 7 for the manufacture of an in vitro immunoassay reagent, kit for human proinsulin.

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