A monoclonal antibody for recognizing proinsulin and its application

Abstract

The present invention provides 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. The monoclonal antibody of the present invention can specifically recognize a specific antigenic epitope of proinsulin, exhibiting advantages such as strong recognition specificity, good affinity, and high potency, and has broad application value.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a monoclonal antibody for recognizing proinsulin and application thereof. Background Art

[0002] Proinsulin is a key precursor in insulin biosynthesis, possessing unique biological properties and clinical significance. Insulin biosynthesis begins with the cytoplasmic precursor, preproinsulin. Newly synthesized preproinsulin, comprising 110 amino acids, is guided by its amino-terminal signal peptide and translocated across the membrane into the endoplasmic reticulum (ER). Signal peptidase then cleaves it to form another insulin precursor, proinsulin, consisting of 86 amino acids. Within the ER, proinsulin rapidly undergoes oxidative folding, forming three pairs of disulfide bonds, a highly conserved evolutionary structure, and its normal spatial conformation. The properly folded proinsulin is then transported from the ER to the Golgi apparatus for further processing and cleavage, resulting in insulin and C-peptide, which are stored in secretory granules.

[0003] Proinsulin is composed of two parts, insulin and C-peptide, and has dual immune activity. This means that proinsulin can bind to both insulin antibodies and C-peptide antibodies. Although proinsulin and insulin are similar in molecular structure, its hypoglycemic activity is only 5%-10% of that of insulin. Under physiological conditions, only a very small amount of proinsulin is released into the blood. 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, and the level of proinsulin in the blood increases. Preliminary research by the research and development team of this invention also found that delayed proinsulin secretion can be used as an earlier and more sensitive clinical indicator for evaluating pancreatic islet dysfunction.

[0004] Currently, proinsulin detection reagents can be used to diagnose diseases such as diabetes, insulinoma, and insulin resistance syndrome, as well as accurately assess the physiological function of pancreatic beta cells. However, the methods for diagnosing abnormal proinsulin expression are still incomplete, and the development of novel monoclonal antibodies and detection reagents that can recognize specific proinsulin epitopes is highly desirable. Summary of the Invention

[0005] In view of the deficiencies in the prior art, the present invention discloses a monoclonal antibody for recognizing proinsulin and its application. The monoclonal antibody has the advantages of good affinity and high titer, and has strong specificity for recognizing proinsulin in biological samples.

[0006] In order to achieve the above technical objectives, in the first aspect, the present invention provides a monoclonal antibody that recognizes proinsulin, wherein the amino acid sequence of the light chain CDR3 of the monoclonal antibody is shown as SEQ ID NO:7, SEQ ID NO:13 or SEQ ID NO:19; the amino acid sequence of the heavy chain CDR3 of the monoclonal antibody is shown as SEQ ID NO:10, SEQ ID NO:16 or SEQ ID NO:22.

[0007] In a further example of the present invention, the amino acid sequence of the light chain CDR1 of the monoclonal antibody is shown as 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 as SEQ ID NO: 9, SEQ ID NO: 15 or SEQ ID NO: 21.

[0008] In a further example of the present invention, the amino acid sequence of the heavy chain CDR1 of the monoclonal antibody is shown as 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 as SEQ ID NO: 12, SEQ ID NO: 18 or SEQ ID NO: 24.

[0009] It should be noted that partial mutations introduced into the hypervariable region of the monoclonal antibody of the present invention do not affect the binding of the antibody molecule to the antigen. In a further example of the present 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, which has 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 that has at least 90% homology (such as 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, etc., but not limited to the exemplified values) to SEQ ID NO:1, SEQ ID NO:3 or SEQ ID NO:5 and has the same function as the protein shown in SEQ ID NO:1, SEQ ID NO:3 or SEQ ID NO:5.

[0010] Furthermore, the light chain amino acid sequence of the monoclonal antibody includes the light chain variable region and constant region SEQ ID NO: 35.

[0011] In a further example of the present 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 in which one or more amino acids are substituted and / or deleted and / or added to SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 6 and has 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 that has at least 90% homology (such as 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, etc., but not limited to the exemplified values) to SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 6 and has the same function as the protein shown in SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 6.

[0012] Furthermore, the heavy chain amino acid sequence of the monoclonal antibody includes the heavy chain variable region and constant region SEQ ID NO: 36.

[0013] In a further example of the present invention, the monoclonal antibody is a whole antibody or an antigen-binding portion of a whole antibody; further, the whole antibody is of IgG1 type; further, the antigen-binding portion is a Fab fragment, a F(ab')2 fragment or a single-chain antibody.

[0014] In a further example of the present invention, the amino acid sequence of proinsulin recognized by the monoclonal antibody is shown as SEQ ID NO: 25, SEQ ID NO: 26 or SEQ ID NO: 27.

[0015] 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.

[0016] In a second aspect, the present invention provides a nucleic acid molecule encoding the above-mentioned monoclonal antibody that recognizes proinsulin.

[0017] 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 of 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 of 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 of the heavy chain variable region is shown in SEQ ID NO: 33.

[0018] In a third aspect, the present invention proposes the use of the above-mentioned monoclonal antibody that recognizes proinsulin in detecting proinsulin in biological samples for non-diagnostic purposes.

[0019] The present invention is not limited to the specific types of the biological samples, such as human tissues, cells, supernatants, serum, plasma, etc. Those skilled in the art can use the monoclonal antibodies of the present invention that recognize proinsulin to detect biological samples containing antigens as needed, and this does not limit the scope of protection of the present invention.

[0020] Fourthly, the present invention proposes the use of the above-mentioned monoclonal antibody that recognizes proinsulin in the preparation of in vitro immunoassay reagents and kits for human proinsulin; by specifically applying the above-mentioned monoclonal antibody that recognizes human proinsulin to a specific kit, the proinsulin content in a biological sample can be determined.

[0021] Furthermore, the kit further comprises any one or a combination of at least two of an ELISA plate, a coating buffer, a substrate, a substrate color developing solution or a reaction termination solution.

[0022] Furthermore, the pharmaceutical composition also includes a pharmaceutically acceptable carrier and / or diluent.

[0023] In addition, the present invention is not limited to the method for preparing the hybridoma mother cell that produces the above-mentioned monoclonal antibody, and those skilled in the art can choose accordingly. Further, the method for preparing the hybridoma mother cell that produces the above-mentioned monoclonal antibody can optionally include the following steps:

[0024] (1) Peptide synthesis: A peptide having an amino acid sequence as shown in SEQ ID NO: 25, SEQ ID NO: 26 or SEQ ID NO: 27 was synthesized by chemical synthesis and purified by HPLC.

[0025] (2) Antigen preparation: Using patented IEF (Immunogenicity Enhancement Factors), VLP (Virus-Like Particles) is combined with traditional KLH carrier protein.

[0026] (3) Pre-immunization test: Five female BALB / c mice aged 8-12 weeks were selected, blood was collected from the eye sockets and the serum was separated. The serum titer was detected by ELISA, and the coating antigen was a conjugate of peptide and BSA.

[0027] (4) Mouse immunization: Peptide-KLH conjugates are used as immunogens for immunization. After the last injection, blood samples are collected from mice to test for serum titer. The testing method is the same as that for pre-immunization testing. Only mice with an antiserum titer greater than 10K can enter the sorting stage. Otherwise, an additional immunization is performed.

[0028] (5) Antibody discovery: Use conventional hybridoma preparation procedures to obtain hybridoma cell lines, use pre-immune detection methods to test the supernatant of the hybridoma cell lines, and use the supernatant purified antibodies for retesting. Select a cell line with the highest ELISA titer to obtain the hybridoma mother cell.

[0029] In addition, the present invention is not limited to the method for producing the above-mentioned monoclonal antibodies, and those skilled in the art can choose accordingly. Further, the preparation method of the monoclonal antibody can be selected as follows: (1) hybridoma production: cell production is carried out using the standard production process of serum-free medium, and the antibody is separated and purified using the Protein A / G affinity column chromatography method; (2) ascites production: antibody production is carried out using the standard production process of ascites, and the antibody is separated and purified using the Protein A / G affinity column chromatography method; (3) recombinant production: light and heavy chain nucleic acids of hybridoma cell lines are obtained through molecular biological operations, agarose gel verification, and constructed into expression vectors using restriction endonucleases and T4 ligases, and the plasmid is amplified using a prokaryotic expression system. Cell production is carried out using transient transfection and the construction of stable cell lines, and the antibody is separated and purified using the Protein A / G affinity column chromatography method.

[0030] The amino acid sequences involved in the present invention include:

[0031] (1) Monoclonal antibody 1

[0032] SEQ ID NO: 1 (light chain variable region):

[0033] DIVLTQSPLSLPVSLGDQASISCRSSQNIVHSNGKTYLEWYLQKPGQSPKLLIYNIYNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVPYTFGGGTKLEIK

[0034] SEQ ID NO:7 (light chain CDR3):

[0035] FQGSHVPYT

[0036] SEQ ID NO:8 (light chain CDR1):

[0037] RSSQNIVHSNGKTYLE

[0038] SEQ ID NO:9 (light chain CDR2):

[0039] NIYNRFS

[0040] SEQ ID NO: 2 (heavy chain variable region):

[0041] QVQLQQSGAEVVKPGASVKMSCKASGYSFTSYNMHWIKQTPGQGLEWIGGIYPGNGESSYNQKFKGKATMTADKSSNTAYMQFSSLTSEDSAVYYCARGDGFDYWGQGTTLTVSS

[0042] SEQ ID NO: 10 (heavy chain CDR3):

[0043] GDGFDY

[0044] SEQ ID NO: 11 (heavy chain CDR1):

[0045] SYNMH

[0046] SEQ ID NO: 12 (heavy chain CDR2):

[0047] GIYPGNGESSYNQKFKG

[0048] (2) Monoclonal antibody 2

[0049] SEQ ID NO: 3 (light chain variable region):

[0050] DIVLTQSPASLAVSLGQRATISCKASQSVDYNGDSYMNWYQQKPGQPPTLLIYVASKLESGIPARFSGSGSGTDFTLNIHPVEEEDVATYYCQQTNEEPLTFGAGTKLELK

[0051] SEQ ID NO: 13 (light chain CDR3):

[0052] QQTNEEPLT

[0053] SEQ ID NO: 14 (light chain CDR1):

[0054] KASQSVDYNGDSYMN

[0055] SEQ ID NO: 15 (light chain CDR2):

[0056] VASKLES

[0057] SEQ ID NO: 4 (heavy chain variable region):

[0058] QVQLEQSGPELVKPGASVRISCKASGYTFTSYYVHWVRQRPGQGLEWIGWIHPGNINSKYNEKFKDKATLTADKSSTTAYMQLSSLTSEDSAVYFCSRSVDYWGQGTTLTVSS

[0059] SEQ ID NO: 16 (heavy chain CDR3):

[0060] SVDY

[0061] SEQ ID NO: 17 (heavy chain CDR1):

[0062] SYYVH

[0063] SEQ ID NO: 18 (heavy chain CDR2):

[0064] WIHPGNINSKYNEKFKD

[0065] (3) Monoclonal antibody 3

[0066] SEQ ID NO: 5 (light chain variable region):

[0067] DIVLTQSTALMAASPGEKVTITCSVSSSISSSYLHWYQQKSETSPKPWIYGTSNLASGVPVRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSNYPLTFGAGTKLELK

[0068] SEQ ID NO: 19 (light chain CDR3):

[0069] QQWSNYPLT

[0070] SEQ ID NO: 20 (light chain CDR1):

[0071] SVSSSISSSYLH

[0072] SEQ ID NO: 21 (light chain CDR2):

[0073] GTSNLAS

[0074] SEQ ID NO: 6 (heavy chain variable region):

[0075] QVQLEQSGGGLVQPKGSLKLSCAASGFNFNTNGMNWVRQAPGKGLEWVARIRSKSNNYATYYADSVKDRFTISRDDSQSMLYLQMNNLKTEDTAMYYCVRDGGYGAWFAYWGQGSLVTVSA

[0076] SEQ ID NO: 22 (heavy chain CDR3):

[0077] DGGYGAWFAY

[0078] SEQ ID NO: 23 (heavy chain CDR1):

[0079] TNGMN

[0080] SEQ ID NO: 24 (heavy chain CDR2):

[0081] RIRSKSNNYATYYADSVKD

[0082] In addition, the amino acid sequence of the light chain constant region is SEQ ID NO: 34 (light chain constant region):

[0083] RADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC

[0084] The amino acid sequence of the heavy chain constant region is SEQ ID NO: 35 (heavy chain constant region):

[0085] ASTTPPSVYPLAPGSANSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHT AQTQPREEQFNSTFRSSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMNTNGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK

[0086] In addition, the amino acid sequences of SEQ ID NO: 25, SEQ ID NO: 26 or SEQ ID NO: 27 in the present invention are respectively:

[0087] SEQ ID NO: 25:

[0088] PLALEGSLQKRGIV

[0089] SEQ ID NO: 26:

[0090] PKTRREAEDLQVGQ

[0091] SEQ ID NO: 27:

[0092] KSRREVEDPQVEQLEC

[0093] The nucleic acid molecule sequences involved in the present invention include:

[0094] 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;

[0095] SEQ ID NO: 28:

[0096] GACATTGTGCTCACCCAATCTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGCAGATCTAGTCAGAACATTGTACATAGTAATGGAAAGACCTATTTAGAGTGGTACCTGCAGAAACCAGGCCAGTCTCCAAAGCTCCTGATCTACAATATTTACAACCGGTTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTC

[0097] AAGATCAGCAGAGTGGAGGCTGAGGATCTGGGAGTTTATTACTGTTTTCAAGGTTCAC

[0098] ATGTTCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAA

[0099] SEQ ID NO:29:

[0100] CAGGTCCAGCTGCAGCAGTCAGGGGCTGAGGTGGTGAAGCCTGGGGCCTCAGTGAAGATGTCCTGCAAGGCTTCTGGCTACTCATTTACCAGTTACAATATGCACTGGATAAAGCAGACACCTGGACAGGGCCTGGAATGGATTGGAGGTATTTATCCAGGAAATGGTGAAAGT

[0101] TCCTACAATCAGAAGTTCAAAGGCAAGGCCACAATGACTGCAGACAAATCCTCCAACACAGCCTACATGCAGTTCAGCAGCCTGACATCTGAGGACTCTGCGGTCTATTATTGTGCA

[0102] AGAGGGGACGGTTTTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA

[0103] The nucleotide sequence encoding the light chain variable region of monoclonal antibody 2 is shown as SEQ ID NO:30, and the nucleotide sequence of the heavy chain variable region is shown as SEQ ID NO:31:

[0104] SEQ ID NO:30:

[0105] GACATTTGTGCTCACCCAGTCTCCAGCCTCTTTGGCTGTGTCTCTAGGGCAGAGGGCCACCATCTCCTGCAAGGCCAGCCAAAGTGTTGATTATAATGGTGATAGTTATATGAACTGGTACCAACAGAAACCGGGACAGCCACCCACACTCCTCATCTATGTTGCAT CCAAGCTAGAATCTGGGATCCCAGCCAGGTTTAGTGGCAGTGGGTCTGGGACAGACTTCACCCTCAACATCCATCCTGTGGAGGAGGAGGATGTTGCAACCTATTACTGTCAGCAAACTAATGAGGAACCGCTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAA

[0106] SEQ ID NO:31:

[0107] CAAGTTCAGCTGGAGCAGTCTGGACCTGAGCTGGTGAAGCCTGGGGCTTCAGTGAGGATATCCTGCAAGGCTTCTGGCTACACCTTCACAAGCTACTATGTTCACTGGGTGAGGCAGAGGCCTGGACAGGGACTTGAGTGGATTGGATGGATTCATCCTGGAAATATTA ATTCTAAATACAATGAGAAATTCAAGGACAAGGCCACACTGACTGCAGACAAATCCTCCACCACAGCCTACATGCAGCTCAGCAGCCTGACCTCTGAGGACTCTGCGGTCTATTTCTGTTCAAGATCGGTTGACTACTGGGGCCAAGGGACCACTCTCACAGTCTCCTCA

[0108] 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:

[0109] SEQ ID NO: 32:

[0110] GATATTGTGCTGACCCAGTCTACAGCACTCATGGCTGCATCTCCAGGGGAAGGTCACCATCACCTGCAGTGTCAGCTCAAGTATAAGTTCCAGCTACTTGCACTGGTACCAGCAGAAGTCAGAAACCTCCCCCAAACCCTGGATTTATGGCACATCCAAC CTGGCTTCTGGAGTCCCTGTTCGCTTCAGTGGCAGTGGATCTGGGACCTCTTATTCTCTCACAATCAGCAGCATGGAGGCTGAAGATGCTGCCACTTATTACTGTCAACAGTGGAGTAATTACCCACTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAA

[0111] SEQ ID NO: 33:

[0112] CAAGTTCAGCTGGAGCAGTCTGGTGGAGGATTGGTGCAGCCTAAAGGGTCATTGAAACTCTCATGTGCAGCCTCTGGATTTAACTTCAATACCAATGGCATGAACTGGGTCCGCCAGGCCCCAGGAAAGGGTTTGGAGTGGGTTGCTCGCATAAGAAGTAAAAGTAATAATTATGCAACAT ATTATGCCGATTCAGTGAAAGACAGGTTCACCATCTCCAGAGATGATTCACAAAGCATGCTCTATCTGCAAATGAACAACTTGAAAACTGAGGACACAGCCATGTATTACTGTGTGAGAGACGGGGGTTACGGGGCCTGGTTTGCTTACTGGGGCCAAGGGTCTCTGGTCACTGTCTCTGCA

[0113] Compared with the prior art, the monoclonal antibody for recognizing proinsulin of the present invention can specifically recognize a specific antigen epitope in human / mouse proinsulin, has strong recognition specificity, good affinity, high potency, and has wide application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0114] The drawings in the specification, which constitute a part of this application, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0115] Figure 1The results of a protein immunoblotting experiment using monoclonal antibody 1 of the present invention in Example 6 are shown; the left figure shows the results using monoclonal antibody 1 of the present invention as the primary antibody to recognize the protein, and the right figure shows the results using a human insulin antibody as the primary antibody to recognize the protein; lane 1 represents the supernatant protein secreted by human insulinoma cells under a non-reduced state, lane 2 represents the supernatant protein secreted by human insulinoma cells under a reduced state, and lane M represents a marker (Thermo Fisher #26616);

[0116] Figure 2 The results of a protein immunoblotting experiment using monoclonal antibody 2 of the present invention in Example 7 are shown; the left figure shows the results using monoclonal antibody 2 of the present invention as the primary antibody to recognize the protein, and the right figure shows the results using a human insulin antibody as the primary antibody to recognize the protein; lane 1 represents the supernatant protein secreted by human insulinoma cells under a non-reduced state, lane 2 represents the supernatant protein secreted by human insulinoma cells under a reduced state, and lane M represents a marker (Thermo Fisher #26616);

[0117] Figure 3 This figure shows that monoclonal antibody 3 of the present invention in Example 8 specifically recognizes mouse proinsulin but not insulin. The left panel shows the results of using monoclonal antibody 3 of the present invention as the primary antibody to recognize the protein, while the right panel shows the results of using an antibody against mouse insulin as the primary antibody to recognize the protein. Lane 1 represents mouse islet cell protein; lane M represents a marker (ThermoFisher #26616).

[0118] Note: The red arrow in the figure indicates the location where proinsulin protein is detected, and the blue arrow indicates the location where insulin protein is detected. DETAILED DESCRIPTION

[0119] To facilitate understanding of the present invention, the present invention will be described in more detail below, with preferred embodiments of the present invention provided. However, it should be understood that these embodiments are merely for the purpose of further explanation and are not to be construed as limiting the present invention in any form, i.e., they are not intended to limit the scope of protection of the present invention.

[0120] Unless otherwise defined, the technical terms used in the following examples have the same meanings as commonly understood by those skilled in the art to which this invention belongs. The experimental reagents used in the following examples, unless otherwise specified, are conventional biochemical reagents; the experimental methods described, unless otherwise specified, are conventional methods.

[0121] Example 1 Preparation of Hybridoma Cell Lines Producing Anti-Proinsulin Monoclonal Antibodies

[0122] (1) Immunogen preparation: A polypeptide (amino acid sequence such as SEQ ID NO: 25) was coupled to a KLH (keyhole limpet hemocyanin) carrier protein using conventional coupling methods to prepare an immune antigen. The polypeptide concentration was controlled to be ≥ 0.25 mg / mL (thiol value ≥ 1.0);

[0123] Similarly, another peptide (amino acid sequence such as SEQ ID NO: 25) was conjugated to a BSA carrier protein using conventional conjugation methods to prepare a screening antigen, controlling the peptide concentration to ≥ 0.25 mg / mL (thiol value ≥ 1.0). Prior to immunization, the immunizing antigen was mixed with Freund's complete adjuvant in a 1:1 ratio and fully emulsified.

[0124] (2) Immunization: Five healthy 8-week-old BALB / c mice were selected as immunization subjects (the pre-immunization serum ELISA titer was tested before immunization. It was best if the pre-immunization serum did not have a color reaction with the screening antigen, or the OD value of the undiluted serum ELISA test was less than 2.1 times that of the PBS group).

[0125] The immunization schedule involves a second vaccination three weeks after the primary vaccination, followed by a third vaccination two weeks after the primary vaccination, for a total of seven weeks. Each mouse receives 100 μL of the vaccine per vaccination. Serum titers are measured by ELISA (using a screening antigen-coated plate) one week after the third vaccination. If the titer is greater than 256k, a boost is administered with the immunizing antigen. Cell fusion is achieved 2-3 days after the boost. If the titer is not reached, an additional vaccination is administered, and the boost is repeated once the titer is reached.

[0126] (3) Fusion screening: The mice to be fused were killed using the carbon dioxide method, the spleens were collected aseptically, the red blood cells were lysed, and the spleen cell suspension was prepared and counted. The thymus of 3-4 week old mice was collected aseptically, and the thymic cells were plated in 20-40 96-well plates using HAT medium, with 100 μL per well. Myeloma cells in the logarithmic growth phase were selected, collected aseptically, and counted to ensure that the cell viability was greater than 90%. The spleen cells and myeloma cells were mixed in a certain ratio (e.g., 3:1-5:1) and cell fusion was performed using the electrofusion method.

[0127] After fusion, place the cell suspension in HAT selective medium and mix well to a cell density of less than 15 cells / mL. Aliquot the suspension into 20-40 prepared 90-well plates, aliquoting 100 μL per well (200 μL total). After the cells have grown for 1-2 weeks, remove 100 μL of supernatant from wells that have successfully fused, as determined by microscopy, and perform ELISA analysis (coated with the screening antigen). Select the well with the highest ELISA value for subcloning. Try to minimize the number of subcloned cells per well. Screen subclones using the same ELISA method for supernatant analysis until the entire plate is positive.

[0128] (4) Cell Identification: From the entire plate of positive subclones, select 3-5 cell lines with the best growth status by microscopic examination. Use 50 mL culture system to culture for 1-3 weeks. When the cell viability is less than 40%, collect the supernatant, purify the supernatant using Protein A / G affinity purification, and calibrate the antibody concentration using a BCA kit. Measure the EC50 values ​​of these 3-5 antibody lines in the ELISA test. The best cell line is the final hybridoma cell line for anti-proinsulin monoclonal antibody.

[0129] Example 2 Production of anti-proinsulin monoclonal antibody hybridoma cells

[0130] The cell lines were acclimated to serum-free suspension culture using the culture component replacement method and cultured at 10 5 The inoculation density was gradually inoculated into 1L culture flasks (330mL culture medium) for shake flask culture. When the sugar content of the culture medium was lower than 10mmol / mL, glucose solution was added to control the sugar content of the culture medium at 10-25mmol / mL. In addition, when the density was greater than 2×10 6 When the cell viability is less than 40%, the supernatant is collected.

[0131] Place the prepared Protein A column on the AKTA instrument. 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 secondary column loading. Wash impurities with glycine-HCl buffer (pH 5.5) at a flow rate of 5 mL / min, using a wash 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. Adjust the antibody concentration to 3-5 mg / mL based on the antibody concentration in the supernatant determined by ELISA. Immediately after elution, add an equal volume of glycine-NaHCO₃ buffer (pH 9.5 ± 0.1) to neutralize the acidic environment.

[0132] Use an ultrafiltration centrifuge tube to replace the antibody buffer system to 10mM PBS (pH=7.4±0.05), use a BCA kit to standardize the concentration, and control the concentration at 2.0mg / mL.

[0133] Please note that if the antibody is not used immediately, it is necessary to add an appropriate amount of preservative (such as sodium azide) and store it in a -80℃ refrigerator.

[0134] Example 3 Recombinant production of anti-proinsulin monoclonal antibodies

[0135] The monoclonal cell line obtained in Example 1 was cultured to 75 cm2 The bottom of the CO2 cell culture flask was filled to 80% confluence, and the cells were harvested, resuspended in nuclease-free water containing RNase inhibitors, and centrifuged again to collect the cells. A portion of the cells was subjected to single-cell sequencing to obtain antibody sequence information.

[0136] The other part uses a 5'RACE kit to extract total mRNA, reverse transcribe it into cDNA, and amplify the deoxyribonucleotide sequences of the variable regions of the antibody light and heavy chains.

[0137] Following the kit instructions, the pcDNA5.0 plasmid and the antibody light and heavy chains were digested with restriction endonucleases. The digestion products were recovered and purified using a nucleic acid recovery kit. After verification of the purified products by dextran gel electrophoresis, they were ligated using a T4 ligase kit to construct the antibody light and heavy chains into the plasmids, ensuring that the variable and constant regions of the light and heavy chains were intact on the plasmids.

[0138] 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.

[0139] The linearized light and heavy chain plasmids were simultaneously transformed into CHO cells using PEI transformation or electroporation.

[0140] Stable transfected cells were screened by standard stable transfection cell line screening methods, or cell supernatants were produced using transient transfection methods.

[0141] The production of stable transgenic antibody was achieved according to the method in Example 2.

[0142] The antibody purification method in Example 2 was used to purify the supernatant of cells expressing transient transfection.

[0143] In this example, a specific monoclonal antibody 1 was prepared against the partial sequence of SEQ ID NO: 25 (PLALEGSLQKRGIV) linked to the human insulin C-peptide-A chain. The monoclonal antibody was of IgG1 type, and its light chain was of kappa type.

[0144] The sequence of its light chain variable region is SEQ ID NO:1, and 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, wherein 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; the sequence of its heavy chain variable region is SEQ ID NO:2, and 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, wherein 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.

[0145] 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.

[0146] Example 4 Preparation of Proinsulin Monoclonal Antibody 2

[0147] Using the experimental methods of Examples 1-3 above, a specific monoclonal antibody 2 against the partial sequence of SEQ ID NO: 26 (PKTRREAEDLQVGQ) of the human proinsulin B chain-C peptide junction was obtained. The monoclonal antibody is of IgG1 type, and its light chain is of kappa type.

[0148] The sequence of its light chain variable region is SEQ ID NO:3, and 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, wherein 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; the sequence of its heavy chain variable region is SEQ ID NO:4, and 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, wherein 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.

[0149] 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.

[0150] The production of stable transgenic antibody was achieved according to the method in Example 2.

[0151] The antibody purification method in Example 2 was used to purify the supernatant of cells expressing transient transfection.

[0152] Example 5 Preparation of proinsulin monoclonal antibody 3

[0153] Using the experimental methods of Examples 1-3 above, a specific monoclonal antibody 3 was obtained against the partial sequence of mouse proinsulin B chain-C peptide connection SEQ ID NO: 27 (KSRREVEDPQVEQLEC). The monoclonal antibody is of IgG1 type, and its light chain is of kappa type.

[0154] The sequence of its light chain variable region is SEQ ID NO:5, and 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, wherein 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; the sequence of its heavy chain variable region is SEQ ID NO:6, and 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, wherein 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.

[0155] 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.

[0156] The production of stable transgenic antibody was achieved according to the method in Example 2.

[0157] The antibody purification method in Example 2 was used to purify the supernatant of cells expressing transient transfection.

[0158] Example 6

[0159] The supernatant of the immortalized human insulinoma cells was collected, and 60 μL of the supernatant was added to 20 μL of 4× NPLDS SAMPLE BUF (Thermo Fisher, NP0007), mixed evenly, and boiled at 95°C for 5 min to prepare the protein sample under non-reduced state. 36 μL of the above sample was added to 4 μL of 1 M reducing agent DTT (Sigma, D9163), mixed evenly, and boiled at 95°C for 5 min to prepare the protein sample under reduced state.

[0160] The specific protein immunoblotting experimental procedures are similar to those previously published by the present invention team (e.g. Yang J, et al . PNAS ;2022;119 (45):e2204443119, etc.), 30 μL of the above non-reduced and reduced protein samples were taken for protein immunoblotting experiments, and the primary antibodies used were monoclonal antibody 1 of the present invention and human insulin antibody. The results are shown in Figure 4. Figure 1 shown.

[0161] Figure 1 The left figure shows the result of using the monoclonal antibody 1 prepared in Example 1-3 of the present invention as the primary antibody to recognize the protein, and the right figure shows the result of using the human insulin antibody as the primary antibody to recognize the protein. Figure 1 Comparative analysis of the middle left and right figures shows that monoclonal antibody 1 of the present invention can specifically recognize proinsulin protein and only recognize proinsulin protein, especially proinsulin protein in the reduced state, while human insulin antibody as a primary antibody recognition protein can simultaneously recognize proinsulin and insulin protein; this result verifies that monoclonal antibody 1 of the present invention has no cross-reaction with insulin protein during the process of recognizing proinsulin protein and has excellent specificity for recognizing proinsulin.

[0162] Example 7

[0163] The supernatant of the immortalized human insulinoma cells was collected, and 60 μL of the supernatant was added to 20 μL of 4× NPLDS SAMPLE BUF (Thermo Fisher, NP0007), mixed evenly, and boiled at 95°C for 5 min to prepare the protein sample under non-reduced state. 36 μL of the above sample was added to 4 μL of 1 M reducing agent DTT (Sigma, D9163), mixed evenly, and boiled at 95°C for 5 min to prepare the protein sample under reduced state.

[0164] The specific protein immunoblotting experimental procedures are similar to those previously published by the present invention team (e.g. Yang J, et al . PNAS ;2022;119 (45):e2204443119, etc.), 30 μL of the above non-reduced and reduced protein samples were taken for protein immunoblotting experiments, and the primary antibodies used were monoclonal antibody 2 of the present invention and human insulin antibody. The results are shown in Figure 2. Figure 2 shown.

[0165] Figure 2 The left figure shows the results of using the monoclonal antibody 2 of the present invention as the primary antibody to recognize the protein, and the right figure shows the results of using the human insulin antibody as the primary antibody to recognize the protein. Figure 2As can be seen from the left and right figures in the figure, the monoclonal antibody 2 of the present invention can specifically recognize proinsulin protein but not insulin protein, especially recognize proinsulin protein in a reduced state, while the human insulin antibody as a primary antibody recognition protein can simultaneously recognize proinsulin and insulin protein; this result confirms that the monoclonal antibody 2 of the present invention has no cross-reaction with insulin protein in the process of recognizing proinsulin protein and has excellent specificity for recognizing proinsulin.

[0166] Example 8

[0167] Sixty wild-type mouse islets were extracted, added with 30 μL of protein lysis buffer and lysed on ice for 20 min. After centrifugation at 13500 rpm and 4°C for 15 min, 27 μL of supernatant was collected, 9 μL of 4× NP LDS SAMPLE BUF (Thermo Fisher, NP0007) and 4 μL of 1 M reducing agent DTT (Sigma, D9163) were added, mixed, and boiled at 95°C for 5 min.

[0168] The specific islet extraction and protein immunoblotting experimental procedures are similar to those previously published by the present invention team (e.g. Yang J, et al . PNAS ;2022;119 (45):e2204443119, etc.), 30 μL of the above protein sample was taken for protein immunoblotting experiment, and the primary antibodies used were monoclonal antibody 3 of the present invention and mouse insulin antibody. The results are shown in Figure 3 shown.

[0169] Figure 3 The left figure shows the result of using the monoclonal antibody 3 of the present invention as the primary antibody to recognize the protein, and the right figure shows the result of using the mouse-derived insulin antibody as the primary antibody to recognize the protein; the results show that the monoclonal antibody 3 of the present invention can specifically recognize the proinsulin protein and does not recognize the insulin protein, while the mouse-derived insulin antibody can simultaneously recognize the proinsulin and insulin protein; this result verifies that the monoclonal antibody 3 of the present invention has no cross-reaction with the insulin protein and can specifically recognize the proinsulin protein.

[0170] It should be noted that the above is a further detailed description of the present invention in conjunction with specific embodiments, and the specific implementation of the present invention should not be considered to be limited to these descriptions. For those skilled in the art of the present invention, several simple improvements can be made without departing from the concept of the present invention, and all of these should be considered to fall within the scope of protection of the present invention.

Claims

1. A monoclonal antibody that recognizes proinsulin, characterized in that The light chain CDR3 of the monoclonal antibody is shown as SEQ ID NO: 7, the light chain CDR1 is shown as SEQ ID NO: 8, and the light chain CDR2 is shown as SEQ ID NO: 9; the heavy chain CDR3 is shown as SEQ ID NO: 10, the heavy chain CDR1 is shown as SEQ ID NO: 11, and the heavy chain CDR2 is shown as SEQ ID NO: 12; the monoclonal antibody is used to recognize human proinsulin.

2. The monoclonal antibody recognizing proinsulin according to claim 1, characterized in that The amino acid sequence of the light chain variable region of the monoclonal antibody is as shown in SEQ ID NO: 1, or an amino acid sequence obtained by substituting and / or deleting and / or adding one or more amino acids in SEQ ID NO: 1 and having the same function as the protein shown in SEQ ID NO: 1, or an amino acid sequence that has at least 90% homology with SEQ ID NO: 1 and has the same function as the protein shown in SEQ ID NO:

1.

3. The monoclonal antibody recognizing proinsulin according to claim 1, characterized in that The amino acid sequence of the heavy chain variable region of the monoclonal antibody is as shown in SEQ ID NO: 2, or an amino acid sequence in which one or more amino acids are substituted and / or deleted and / or added to SEQ ID NO: 2 and has the same function as the protein shown in SEQ ID NO: 2, or an amino acid sequence that has at least 90% homology with SEQ ID NO: 2 and has the same function as the protein shown in SEQ ID NO:

2.

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

6. The monoclonal antibody recognizing 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 recognizing proinsulin according to claim 1, characterized in that The amino acid sequence of proinsulin recognized by the monoclonal antibody is shown in SEQ ID NO:

25.

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 in 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 in the preparation of in vitro immunoassay reagents and kits for human proinsulin.

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