A-FABP monoclonal antibody 3H9 and preparation method and use thereof

Abstract

The present application relates to an anti-A-FABP monoclonal antibody 3H9 and a preparation method and application thereof, and specifically discloses an A-FABP monoclonal antibody 3H9 or an antigen binding fragment thereof, which has three heavy chain complementarity determining regions (CDRs) as shown in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, and has three light chain complementarity determining regions as shown in SEQ ID NO:10, SEQ ID NO:11 and SEQ ID NO:12. The present application provides a preparation method, identification and application of an anti-A-FABP monoclonal antibody, the monoclonal antibody has the advantages of high sensitivity and good specificity, and can be widely used for detecting A-FABP by different means such as Western blot, ELISA and flow cytometry, and provides a basis for studying the function of human A-FABP.

Description

Technical Field

[0001] This invention relates to the field of antibodies, specifically to an A-FABP monoclonal antibody 3H9, its preparation method, and its uses. Background Technology

[0002] Adipocyte-type fatty acid binding protein (A-FABP) is a member of the apolipoprotein family with a molecular weight of 14.6 kDa, primarily expressed in mature adipocytes and macrophages. The main function of A-FABP is as a carrier of free fatty acid molecules, regulating fat storage and breakdown in adipocytes; and regulating lipid accumulation and promoting the expression of various inflammatory factors, including MCP-1, TNF-α, IL-6, and IL-1β, in macrophages. A-FABP can be secreted into the extracellular and bloodstreams, promoting inflammatory responses and is closely related to the development of various metabolic diseases such as obesity, diabetes, lipid metabolism disorders, non-alcoholic steatohepatitis, and atherosclerosis. In animal experiments, bone marrow transplantation into A-FABP gene knockout mice has been shown to comprehensively improve atherosclerosis without metabolic side effects; simultaneously, mice with A-FABP gene mutations leading to reduced A-FABP expression levels have lower triglyceride levels, reducing the risk of cardiovascular disease and decreasing the incidence of obesity-related type 2 diabetes. Further research elucidated the molecular mechanism by which A-FABP protein upregulates the expression of multiple inflammatory factors in macrophages via the JNK / c-Jun / AP-1 signaling axis. It also confirmed that after ischemic stroke, the expression levels of A-FABP protein in both blood and brain tissue increase, promoting the expression of inflammatory factors and thus exacerbating post-stroke neuroinflammation. Therefore, A-FABP may serve as a potential therapeutic target for improving metabolic diseases and related cardiovascular and cerebrovascular diseases.

[0003] Given the crucial role of A-FABP protein in metabolic diseases and their cardiovascular complications, numerous pharmaceutical companies and research institutions worldwide have developed hundreds of A-FABP protein inhibitors, the vast majority of which are small molecule compounds. Although these inhibitors have demonstrated high activity and good specificity in in vivo experiments and shown promising therapeutic effects in various animal disease models, the low target specificity and numerous side effects, particularly cardiotoxicity, of small molecule chemical drugs mean that none have yet entered clinical trials. Harvard University developed a chimeric monoclonal antibody for rabbits and mice in 2015, but its affinity for A-FABP protein is very low, and it has not yet entered clinical trials.

[0004] As the most widely used tool in biological research, the status of antibody products is undeniable. However, the specificity and application scope of antibodies have long plagued the antibody industry and represent a significant crisis for the entire field of biological research. Poor quality antibody products directly lead to errors in experimental results and the inability to reproduce or replicate research findings. Research projects often stagnate due to problems caused by antibodies, resulting in staggering losses. According to 2015 statistics, in the United States alone, an average of $350 million is wasted annually on these ineffective antibodies. Globally, a staggering $800 million is wasted annually, accounting for 50% of total global research antibody expenditures. Summary of the Invention

[0005] This invention relates to an anti-A-FABP monoclonal antibody, its preparation method, and its uses. The anti-A-FABP monoclonal antibody, prepared using hybridoma technology, can be used in biological research experiments such as ELISA and Western blotting.

[0006] One aspect of the present invention provides an isolated anti-A-FABP antibody or its antigen-binding fragment having three heavy chain complementarity-determining regions (CDRs) as shown in SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4, and three light chain complementarity-determining regions as shown in SEQ ID NO:10, SEQ ID NO:11 and SEQ ID NO:12.

[0007] Another aspect of the present invention provides an isolated anti-A-FABP antibody or an antigen-binding fragment thereof having a heavy chain variable region as shown in SEQ ID No: 1 and a light chain variable region as shown in SEQ ID No: 9.

[0008] In another aspect, the present invention provides a nucleotide sequence characterized in that it encodes a monoclonal antibody against A-FABP as described above or an antigen-binding fragment thereof.

[0009] In the technical solution of this invention, the antibody is a monoclonal antibody.

[0010] In another aspect, the present invention provides a recombinant vector, characterized in that it comprises the aforementioned nucleotide sequence.

[0011] In another aspect, the present invention provides a host cell, characterized in that it comprises the aforementioned vector or vector group, preferably, the host cell is prokaryotic or eukaryotic, more preferably selected from yeast cells, mammalian cells or other cells suitable for preparing antibodies or their antigen-binding fragments.

[0012] In another aspect, the present invention provides a kit comprising an antibody or an antigen-binding fragment thereof as described above.

[0013] In another aspect, the present invention provides a detection reagent comprising an antibody or an antigen-binding fragment thereof as described above.

[0014] In another aspect, the present invention provides the use of the above-mentioned antibody or its antigen-binding fragment as a detection reagent, said reagent being used for: enzyme-linked immunosorbent assay (ELISA), Western blotting, flow cytometry (FACS), immunohistochemistry (IHC) detection, or immunoPCR.

[0015] In the aforementioned immunological assays, antibodies or their antigen-binding fragments can be used alone or linked by chemical bonds, electrostatic adsorption, or hydrophilic / hydrophobic adsorption. The conjugates include horseradish peroxidase (HRP), alkaline phosphatase (AP), biotin, fluorescein isothiocyanate (FITC), Cy3, Cy5, magnetic beads, and agarose, among other conjugates.

[0016] In the technical solution of the present invention, the detection reagent can be used for non-diagnostic therapeutic detection.

[0017] In another aspect, the present invention provides the use of the above-mentioned antibody or its antigen-binding fragment as a reagent for the in vitro isolation or purification of A-FABP.

[0018] In this invention, the above-mentioned antibody or its antigen-binding fragment is prepared by a hybridoma method.

[0019] In this invention, the nucleotide and amino acid sequences of the heavy and light chain variable regions of the antibody or its antigen-binding fragment are shown below.

[0020] Heavy chain variable region amino acid sequence:

[0021] VKLQESGAELVRSGASVKLSCTASGFNIKDYYMNWVKQRPEQGLEWIGWIDPENGDT EYAPKFQGKATMTADTSSNTAYLQLSSLTSEDTAVYYCNADYRYDGYAMDYWGQGTTVTV SS SEQ ID NO.1

[0022] The amino acid sequence of the heavy chain variable region CDR1 is: DYYMN SEQ ID NO.2

[0023] The amino acid sequence of the heavy chain variable region CDR2 is: WIDPENGDTEYAPKFQG SEQ ID NO.3

[0024] The amino acid sequence of the heavy chain variable region CDR3 is: DYRYDGYAMDY SEQ ID NO.4

[0025] The amino acid sequence of the heavy chain variable region FR1 is: VKLQESGAELVRSGASVKLSCTASGFNIK SEQ ID NO.5

[0026] The amino acid sequence of the heavy chain variable region FR2 is: WVKQRPEQGLEWIG SEQ ID NO.6

[0027] The amino acid sequence of the heavy chain variable region FR3 is: KATMTADSSNTAYLQLSSLTSEDTAVYYCNASEQ ID NO.7

[0028] The amino acid sequence of the heavy chain variable region FR4 is: WGQGTTVTVSS SEQ ID NO.8

[0029] Light chain variable region amino acid sequence:

[0030] DIQMTQTPLSLPVSLGDQASISCRSSQSIVHSNGNTYLDWYLQKPGQSPKLLIYKVSNR FSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVPYTFGGGTKLE SEQ ID NO.9

[0031] The amino acid sequence of the light chain variable region CDR1 is: RSSQSIVHSNGNTYLD SEQ ID NO.10

[0032] The amino acid sequence of the light chain variable region CDR2 is: KVSNRFS SEQ ID NO.11

[0033] The amino acid sequence of the CDR3 variable region of the light chain is: FQGSHVPYT SEQ ID NO.12

[0034] The amino acid sequence of the FR1 variable region of the light chain is: DIQMTQTPLSLPVSLGDQASISC SEQ ID NO.13

[0035] The amino acid sequence of the FR2 variable region of the light chain is: WYLQKPGQSPKLLIY SEQ ID NO.14

[0036] The amino acid sequence of the FR3 variable region of the light chain is: GVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCSEQ ID NO.15

[0037] The amino acid sequence of the FR4 variable region of the light chain is: FGGGTKLEI SEQ ID NO.16

[0038] The heavy chain variable region gene is 357 bp in length and encodes 119 amino acid residues. The nucleotide sequence of the heavy chain variable region is shown in SEQ ID NO:17, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:1, the amino acid sequence of heavy chain CDR1 is shown in SEQ ID NO:2, the amino acid sequence of heavy chain CDR2 is shown in SEQ ID NO:3, and the amino acid sequence of heavy chain CDR3 is shown in SEQ ID NO:4.

[0039] The full-length sequence of the light chain variable region gene is 333 bp, encoding 111 amino acid residues. The nucleotide sequence of the light chain variable region is shown in SEQ ID NO:18, the amino acid sequence of the light chain variable region is shown in SEQ ID NO:9, the amino acid sequence of light chain CDR 1 is shown in SEQ ID NO:10, the amino acid sequence of light chain CDR 2 is shown in SEQ ID NO:11, and the amino acid sequence of light chain CDR 3 is shown in SEQ ID NO:12.

[0040] Beneficial effects

[0041] This invention provides a method for preparing, identifying, and applying a monoclonal antibody against A-FABP. This monoclonal antibody exhibits advantages such as high sensitivity and specificity, and is suitable for various detection methods. The monoclonal antibody provided by this invention can be widely used in various techniques such as Western blot, ELISA, and flow cytometry to detect A-FABP, providing a foundation for studying the function of human A-FABP. Attached Figure Description

[0042] Figure 1 The results are from the specificity test of the A-FABP protein monoclonal antibody 3H9. Detailed Implementation

[0043] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below, but should not be construed as limiting the scope of the present invention.

[0044] Example 1: Mice were immunized with human A-FABP protein to screen for monoclonal antibodies against human A-FABP protein.

[0045] Purchased from Hong Kong Inno Diagnostics Co., Ltd.: Human A-FABP protein (2000μg, Cat. No. 41030), with a protein purity of >95%.

[0046] Female BALB / c mice (6 weeks old) were immunized. For the first immunization, Freund's complete adjuvant was used to emulsify the antigen, and it was administered subcutaneously at 6 points, with each mouse receiving 100 μg of antigen. Fourteen days later, a second immunization was performed, using Freund's incomplete adjuvant to emulsify the antigen, also administered subcutaneously at 6 points, with each mouse receiving 100 μg of antigen. Fourteen days later, a third immunization was performed, using the same method as the second immunization. Fourteen days later, a small amount of blood was collected from the mice by tail clipping for serum titer ELISA testing. Mice with the highest antibody titer (1:500,000) were selected for booster immunization, which did not require emulsification; instead, the antigen protein was injected intraperitoneally, with each mouse receiving 100 μg.

[0047] Three to five days after the fourth immunization, mice were sacrificed, and their spleen cells were fused with SP2 / 0 cells. Stable hybridoma cells were obtained by culturing in HAT medium. Hybridoma cells that secrete A-FABP antibodies were screened by ELISA. Subcloning was performed using limiting dilution to screen for monoclonal hybridoma cell line 3H9 that secretes A-FABP antibodies. The cells were then scaled up stepwise and cryopreserved in liquid nitrogen for preservation.

[0048] Preparation and purification of ascites antibodies: Female BALB / c mice (8 weeks old) were intraperitoneally injected with Freund's incomplete adjuvant, 0.5 ml per mouse. 3–5 days later, hybridoma cells 3H9 in the logarithmic growth phase were intraperitoneally injected, 5 × 10⁶ cells per mouse. 5 0.5 ml of cells were collected. Mice were sacrificed after 10 days to obtain ascites fluid. The ascites fluid was centrifuged at 5000 rpm and 4°C for 10 min to remove the precipitate. The ascites fluid was diluted with 10 volumes of 1×PBS solution, mixed well, and filtered through a 0.45 μm filter membrane. The ascites fluid was purified by affinity purification with Protein G (Protein G Sepharose 4 Fast Flow, GE Healthcare) to obtain purified A-FABP antibody.

[0049] Example 2: Specificity test of A-FABP protein monoclonal antibody 3H9

[0050] The following proteins were purchased from Inno Diagnostics (Hong Kong): Human A-FABP protein (200 μg, Cat. No. 41030) and Human E-FABP protein (100 μg, Cat. No. 41040); and Human H-FABP protein (100 μg, Cat. No. 12476-HNAE) from Beijing Yiqiao Shenzhou Company. The purity of all the above proteins was >95%.

[0051] The specificity of the monoclonal antibody 3H9 against A-FABP protein was further detected by ELISA using purified human A-FABP, H-FABP, and E-FABP as coating antigens. It was found that clone 3H9 specifically binds to A-FABP but not to H-FABP or E-FABP. See details... Figure 1 And Table 1.

[0052] Table 1. Specificity test of monoclonal antibody 3H9 against A-FABP protein

[0053]

[0054] Example: Detection of ascites titer of 3A-FABP protein monoclonal antibody 3H9.

[0055] Ascites fluid was prepared by intraperitoneal injection of 3H9 hybridoma cells into BALB / c mice. Ten days after intraperitoneal injection of the clone, the ascites fluid was collected and purified. The antibody titer (coating antigen: A-FABP 1 μg / mL) was detected by ELISA, revealing that the affinity-purified 3H9 antibody titer reached 1 / 200,000. See Table 2 for details.

[0056] Table 2. Ascites titer test of A-FABP protein monoclonal antibody 3H9

[0057] 1 / 1,000 1 / 2,000 1 / 20,000 1 / 200,000 FT NC 3H9 NaN 3.619 0.531 0.055 -0.008 0.018

[0058] Example 4: Detection of the titer of the A-FABP protein monoclonal antibody 3H9

[0059] Further detection was performed using ELISA with different concentrations of A-FABP protein (coating antigen: A-FABP).

[0060] The sensitivity of monoclonal antibody 3H9 was tested at concentrations of 1 μg / ml, 0.5 μg / ml, 0.05 μg / ml, 0.005 μg / ml, and 0.0005 μg / ml. Antibody 3H9 was found to be highly sensitive to low concentrations of A-FABP protein. See Table 3 for details.

[0061] Table 3. Antibody titer of A-FABP protein monoclonal antibody 3H9

[0062] 1 μg / mL 0.5 μg / mL 0.05 μg / mL 0.005 μg / mL 0.0005 μg / mL NC 3H9 0.509 0.243 0.006 0.013 0.062 0.05

[0063] Example 5: Sequencing of the 3H9 gene of the A-FABP protein monoclonal antibody.

[0064] After immunization, fusion and monoclonalization, antibody purification, and identification of antibody specificity and titer, total RNA was extracted from 3H9 monoclonal antibody cell lines and reverse transcribed into cDNA. Then, using the cDNA as a template, the variable regions of the heavy and light chains of the antibody were amplified by PCR. Total RNA was extracted from the 3H9 monoclonal antibody cell lines using the Invitrogen TRIzol reagent kit (15596-026) according to its instructions. Then, using the Takara 5'RACE FULL kit (D315), the total RNA was reverse transcribed into first-strand cDNA using the random primers in the kit. PCR amplification was then performed on the heavy chain using primers designed for the constant region and adapter primers provided in the kit, and on the light chain using primers designed for the constant region and adapter primers provided in the kit.

[0065] The PCR fragments were recovered using an agarose gel extraction kit, and after TA cloning, single clones were selected for PCR identification. Samples from correctly identified strains were sent to Invitrogen for sequencing. The final determined nucleotide sequence of the heavy chain variable region was SEQ ID NO:1, the nucleotide sequence of the light chain variable region was SEQ ID NO:2, the amino acid sequence of the heavy chain variable region was SEQ ID NO:3, and the amino acid sequence of the light chain variable region was SEQ ID NO:4 (see Table 4).

[0066] Table 4. Specific sequences of the variable regions of the heavy chain and light chain of the 3H9 antibody.

[0067] The amino acid sequence of the variable region of the 3H9 antibody heavy chain is as follows (SEQ ID NO:1):

[0068] VKLQESGAELVRSGASVKLSCTASGFNIKDYYMNWVKQRPEQGLEWIGWIDPENGDT EYAPKFQGKATMTADTSSNTAYLQLSSLTSEDTAVYYCNADYRYDGYAMDYWGQGTTVTV SS Wherein, the amino acid sequence of the heavy chain variable region CDR1 is: DYYMN SEQ ID NO.2

[0069] The amino acid sequence of the heavy chain variable region CDR2 is: WIDPENGDTEYAPKFQG SEQ ID NO.3

[0070] The amino acid sequence of the heavy chain variable region CDR3 is: DYRYDGYAMDY SEQ ID NO.4

[0071] The amino acid sequence of the heavy chain variable region FR1 is: VKLQESGAELVRSGASVKLSCTASGFNIK SEQ ID NO.5

[0072] The amino acid sequence of the heavy chain variable region FR2 is: WVKQRPEQGLEWIG SEQ ID NO.6

[0073] The amino acid sequence of the heavy chain variable region FR3 is: KATMTADSSNTAYLQLSSLTSEDTAVYYCNASEQ ID NO.7

[0074] The amino acid sequence of the heavy chain variable region FR4 is: WGQGTTVTVSS SEQ ID NO.8

[0075] The amino acid sequence of the variable region of the light chain of the 3H9 antibody is as follows (SEQ ID NO:9):

[0076] DIQMTQTPLSLPVSLGDQASISCRSSQSIVHSNGNTYLDWYLQKPGQSPKLLIYKVSNR FSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVPYTFGGGTKLEI

[0077] The amino acid sequence of the light chain variable region CDR1 is: RSSQSIVHSNGNTYLD SEQ ID NO.10

[0078] The amino acid sequence of the light chain variable region CDR2 is: KVSNRFS SEQ ID NO.11

[0079] The amino acid sequence of the CDR3 variable region of the light chain is: FQGSHVPYT SEQ ID NO.12

[0080] The amino acid sequence of the FR1 variable region of the light chain is: DIQMTQTPLSLPVSLGDQASISC SEQ ID NO.13

[0081] The amino acid sequence of the FR2 variable region of the light chain is: WYLQKPGQSPKLLIY SEQ ID NO.14

[0082] The amino acid sequence of the FR3 variable region of the light chain is: GVPDRFSGSGSGTDFTLKISRVEAEDLGVYYC

[0083] SEQ ID NO.15

[0084] The amino acid sequence of the FR4 variable region of the light chain is: FGGGTKLEI SEQ ID NO.16

[0085] The nucleotide sequence of the variable region of the 3H9 antibody heavy chain is as follows (SEQ ID NO:17):

[0086] GTCAAACTGCAGGAGTCTGGGGCAGAGCTTGTGAGGTCAGGGGCCTCAGTCAAGTTGT

[0087] CCTGCACAGCTTCTGGCTTCAACATTAAAGACTACTATATGAACTGGGTGAAGCAGAGG

[0088] CCTGAACAGGGCCTGGAGTGGATTGGATGGATTGATCCTGAGAATGGTGATACTGAATAT

[0089] GCCCCGAAGTTCCAGGGCAAGGCCACTATGACTGCAGACACATCCTCCAACACAGCCTA

[0090] CCTGCAGCTCAGCAGCCTGACATCTGAGGACACTGCCGTCTATTACTGTAATGCCGACTA

[0091] TAGGTACGACGGGTATGCTATGGACTACTGGGGCCAAGGGACCACGGTCACCGTCTCCT

[0092] CA

[0093] The nucleotide sequence of the light chain variable region of the 3H9 antibody is as follows (SEQ ID NO:18):

[0094] GACATTCAGATGACCCAGACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGCAGATCTAGTCAGAGCATTGTACATAGTAATGGAAACACCTATTTAGACTGGTACCTGCAGAAACCAGGCCAGTCTCCAAAGCTCCTGATCTACAAAGTTTCCAACCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAGCAGAGTGGAGGCTGAGGATCTGGGAGTTTATTACTGCTTTCAAGGTTCACATGTTCCGTACACGTTCGGAGGGGGGACCAAGCTGGAGATC。

Claims

1. An isolated monoclonal antibody or antigen-binding fragment thereof against A-FABP, characterized in that: which has three heavy chain complementarity determining regions as shown in SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4, and has three light chain complementarity determining regions as shown in SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12; wherein the heavy chain complementarity determining region CDR1 is as shown in SEQ ID NO: 2; the heavy chain complementarity determining region CDR2 is as shown in SEQ ID NO: 3; the heavy chain complementarity determining region CDR3 is as shown in SEQ ID NO: 4; the light chain complementarity determining region CDR1 is as shown in SEQ ID NO: 10; the light chain complementarity determining region CDR2 is as shown in SEQ ID NO: 11; the light chain complementarity determining region CDR3 is as shown in SEQ ID NO:

12.

2. An isolated monoclonal antibody or antigen-binding fragment thereof against A-FABP, characterized in that: which has a heavy chain variable region as shown in SEQ ID No: 1, and a light chain variable region as shown in SEQ ID No:

9.

3. A nucleic acid molecule, characterized in that: which encodes the monoclonal antibody or antigen-binding fragment thereof as claimed in claim 1 or 2.

4. A recombinant vector, characterized by: comprising the nucleic acid molecule as claimed in claim 3.

5. A host cell, characterized by: comprising the recombinant vector as claimed in claim 4.

6. The host cell of claim 5, characterized in that: The host cell is prokaryotic or eukaryotic.

7. The host cell of claim 5, characterized by: The host cell is selected from a yeast cell, a mammalian cell or other cell suitable for producing an antibody or antigen-binding fragment thereof.

8. A kit for detecting A-FABP, characterized by: The kit comprises the monoclonal antibody or antigen-binding fragment thereof as claimed in claim 1 or 2.

9. A test reagent for detecting A-FABP, characterized by: The detection reagent comprises the monoclonal antibody or antigen-binding fragment thereof as claimed in claim 1 or 2.

10. The detection reagent of claim 9, characterized in that: The detection reagent is used in enzyme-linked immunosorbent assay, immunoblotting, flow cytometry, immunohistochemical detection or immunoprecipitation.

11. Use of the monoclonal antibody or antigen-binding fragment thereof as claimed in claim 1 or 2 for the preparation of a reagent for detecting A-FABP.

12. Use according to claim 11, characterized in that, The reagent is used in a reagent for enzyme-linked immunosorbent assay, immunoblotting, flow cytometry, immunohistochemical detection or immunoprecipitation.

13. Use of the monoclonal antibody or antigen-binding fragment thereof as claimed in claim 1 or 2 for the preparation of a reagent for in vitro separation or purification of A-FABP.

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