A-FABP monoclonal antibody 3H9 as well as preparation method and application thereof

Abstract

The invention relates to an anti-A-FABP monoclonal antibody 3H9 as well as a preparation method and application thereof, and particularly discloses an A-FABP monoclonal antibody 3H9 or an antigen binding fragment thereof, the A-FABP monoclonal antibody 3H9 or the antigen binding fragment thereof has three heavy chain complementary determining regions (CDR) as shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, and has three light chain complementary determining regions as shown in SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12. The invention provides a preparation method, identification and application of the monoclonal antibody for resisting A-FABP, the monoclonal antibody has the advantages of high sensitivity, good specificity and the like, can be widely used for detecting A-FABP by different means such as Western blot, ELISA (enzyme-linked immuno sorbent assay), flowCytometry and the like, and provides a basis for researching functions of human A-FABP.

Description

Technical Field

[0001] The present invention relates to the field of antibodies, and in particular to an A-FABP monoclonal antibody 3H9 and a preparation method and use thereof. Background Art

[0002] Adipocyte-type fatty acid binding protein (A-FABP) is a member of the apolipoprotein family with a molecular weight of 14.6KD. It is mainly expressed in mature adipocytes and macrophages. The main function of A-FABP protein is to act as a carrier of free fatty acid molecules, regulating the storage and decomposition of fat in adipocytes; regulating lipid accumulation and promoting the expression of various inflammatory factors in macrophages, including MCP-1, TNF-α, IL-6, IL-1β, etc. A-FABP protein can be secreted outside cells and into the blood, promoting inflammatory response, and is closely related to the occurrence and development of various metabolic diseases such as obesity, diabetes, lipid metabolism disorders, non-alcoholic fatty liver disease, and atherosclerosis. In animal experiments, transplantation of A-FABP gene knockout mouse bone marrow has been shown to comprehensively improve atherosclerosis without metabolic side effects; at the same time, mice with reduced A-FABP expression levels due to A-FABP gene mutations have lower triglyceride levels, reduced the risk of cardiovascular disease, and reduced obesity-induced type 2 diabetes. Related studies have also clarified the molecular mechanism by which A-FABP protein upregulates the expression of multiple inflammatory factors in macrophages through the JNK / c-Jun / AP-1 signaling axis, and confirmed that after ischemic stroke, the expression levels of A-FABP protein in the blood and brain tissue increase, promoting the expression of inflammatory factors, thereby exacerbating post-stroke neuroinflammation. Therefore, A-FABP can be used as a potential therapeutic target for improving metabolic diseases and related cardiovascular and cerebrovascular diseases.

[0003] In view of the important role of A-FABP protein in metabolic diseases and their cardiovascular and cerebrovascular complications, many pharmaceutical companies and research institutions in the world have developed hundreds of A-FABP protein inhibitors, most of which are small molecule compounds. Although these inhibitors have shown high activity and good specificity in in vivo experiments and good therapeutic effects in various animal disease models, due to the low in vivo target specificity and many side effects of chemical small molecule drugs, especially cardiac toxicity, no A-FABP protein small molecule inhibitor has entered the clinical trial stage. Harvard University in the United States developed a rabbit and mouse chimeric monoclonal antibody 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 unquestionable. However, the specificity and application range of antibodies have long plagued the antibody industry and are also a great crisis in the entire biological research field. The poor quality of antibody products will directly lead to errors in experimental results and the inability to repeat and reproduce research results. The progress of research projects often stagnates due to problems caused by antibodies, and the losses caused are also staggering. According to statistics in 2015, an average of $350 million is wasted on these ineffective antibodies in the United States alone each year. And worldwide, $800 million is wasted each year, accounting for 50% of the total global scientific research antibody expenditure. Summary of the invention

[0005] The present invention relates to an anti-A-FABP monoclonal antibody and a preparation method and use thereof. The anti-A-FABP monoclonal antibody is prepared by hybridoma technology and can be used in biological research experiments such as ELISA and immunoblotting.

[0006] One aspect of the present invention provides an isolated anti-A-FABP antibody or an antigen-binding fragment thereof, which has 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, 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.

[0008] In yet another aspect, the present invention provides a nucleotide sequence, characterized in that it encodes the aforementioned anti-A-FABP monoclonal antibody or antigen-binding fragment thereof.

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

[0010] In yet 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, and more preferably is selected from yeast cells, mammalian cells or other cells suitable for preparing antibodies or antigen-binding fragments thereof.

[0012] In yet another aspect, the present invention provides a kit, comprising the aforementioned antibody or antigen-binding fragment thereof.

[0013] In yet another aspect, the present invention provides a detection reagent, which comprises the aforementioned antibody or antigen-binding fragment thereof.

[0014] In another aspect, the present invention provides the use of the above-mentioned antibody or its antigen-binding fragment as a detection reagent, wherein the reagent is used for the following purposes: enzyme-linked immunosorbent assay (ELISA), immunoblotting (Western Blot), flow cytometry (FACS), immunohistochemistry (IHC) detection or immune PCR.

[0015] In the above-mentioned immunological detection, the antibody or its antigen-binding fragment can be connected alone or with conjugates such as horseradish peroxidase (HRP), alkaline phosphatase (AP), biotin, fluorescein isothiocyanate (FITC), Cy3, Cy5, magnetic beads and agarose by chemical bond coupling, electrostatic adsorption or hydrophilic adsorption.

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

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

[0018] In the present invention, the above-mentioned antibodies or antigen-binding fragments thereof are prepared by the hybridoma method.

[0019] In the present invention, the nucleotide and amino acid sequences of the heavy chain and light chain variable regions of the antibody or antigen-binding fragment thereof are as follows.

[0020] Heavy chain variable region amino acid sequence:

[0021] VKLQESGAELVRSGASVKLSCTASGFNIKDYYMNWVKQRPEQGLEWIGWIDPENGDT EYAPKFQGKATMTADTSSNTAYLQLSSLTSEDTAVYYCNADYRYDGYAMDYWGQGTTVTV SS SEQ ID NO.1

[0022] Among them, 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: KATMTADTSSNTAYLQLSSLTSEDTAVYYCNASEQ 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] Among them, 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 light chain variable region CDR3 is: FQGSHVPYT SEQ ID NO.12

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

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

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

[0037] The amino acid sequence of the light chain variable region FR4 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 heavy chain variable region nucleotide sequence is shown in SEQ ID NO: 17, the heavy chain variable region amino acid sequence is shown in SEQ ID NO: 1, the heavy chain CDR1 amino acid sequence is shown in SEQ ID No: 2, the heavy chain CDR 2 amino acid sequence is shown in SEQ ID No: 3, and the heavy chain CDR3 amino acid sequence is shown in SEQ ID No: 4.

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

[0040] Beneficial Effects

[0041] The present invention provides a method for preparing, identifying and applying a monoclonal antibody against A-FABP, which has the advantages of high sensitivity and good specificity and is suitable for different detection methods. The monoclonal antibody provided by the present invention can be widely used in different methods such as Western blot, ELISA, flowCytometry, etc. to detect A-FABP, providing a basis for studying the function of human A-FABP. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 This is the specificity test result of A-FABP protein monoclonal antibody 3H9. DETAILED DESCRIPTION

[0043] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and understandable, the specific implementation modes of the present invention are described in detail below, but it should not be understood as limiting the applicable scope of the present invention.

[0044] Example 1 Immunizing mice with human A-FABP protein to screen for human A-FABP protein monoclonal antibodies

[0045] The human A-FABP protein (2000 μg, Cat. No. 41030) was purchased from Hong Kong Inno Diagnostics Co., Ltd. The protein purity was >95%.

[0046] Female BALB / c mice (6 weeks old) were immunized. Freund's complete adjuvant was used for the first immunization to emulsify the antigen, and the antigen was injected at 6 points subcutaneously. The amount of antigen injected to each mouse was 100ug. After 14 days, the second immunization was performed, and the antigen was emulsified with Freund's incomplete adjuvant and injected at 6 points subcutaneously. The amount of antigen injected to each mouse was 100ug. After 14 days, the third immunization was performed, and the method was the same as the second immunization. After 14 days, the tails of the mice were cut to collect a small amount of blood for serum titer ELISA detection, and the mice with the highest antibody titer (1:500000) were selected for booster immunization without emulsification. The antigen protein was injected intraperitoneally, and 100μg was injected to each mouse.

[0047] 3-5 days after the fourth immunization, the mice were killed and their spleen cells were fused with SP2 / 0 cells, and stable hybridoma cells were obtained by culturing in HAT medium. Hybridoma cells that can secrete A-FABP antibodies were screened by ELISA, and subcloned by limiting dilution to screen the monoclonal hybridoma cell line 3H9 that can secrete A-FABP antibodies, which was expanded step by step and frozen in liquid nitrogen for seed 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. After 3 to 5 days, hybridoma cells 3H9 in the logarithmic growth phase were intraperitoneally injected, 5×10 5 cells (0.5 ml). After 10 days, the mice were killed and ascites was obtained. The solution was centrifuged at 5000 rpm and 4°C for 10 min to remove the precipitate. The ascites was diluted with 10 times the volume of 1× PBS solution, mixed and filtered through a 0.45 μm filter membrane. The ascites was affinity purified by Protein G (Protein G Sepharose4Fast Flow, GE Healthcare) to obtain purified A-FABP antibody.

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

[0050] Human A-FABP protein (200 μg, Cat. No. 41030) and human E-FABP protein (100 μg, Cat. No. 41040) were purchased from Hong Kong Inno Diagnostics Co., Ltd.; human H-FABP protein (100 μg, Cat. No. 12476-HNAE) was purchased from Beijing Sino Biological Co., Ltd. The purity of the above proteins was > 95%.

[0051] Purified human A-FABP, H-FABP, and E-FABP were used as coating antigens and the specificity of the A-FABP protein monoclonal antibody 3H9 was further tested by ELISA. It was found that clone 3H9 specifically bound to A-FABP, but not to H-FABP or E-FABP. Figure 1 And Table 1.

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

[0053]

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

[0055] 3H9 hybridoma cells were injected intraperitoneally into BALB / c mice to prepare ascites. Ten days after intraperitoneal injection of clones, ascites were collected and purified. Antibody titer was detected by ELISA (coating antigen: A-FABP 1 μg / mL), and it was found that the affinity-purified antibody 3H9 had a titer of 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 4A-FABP protein monoclonal antibody 3H9 antibody titer detection

[0059] ELISA was used to detect the A-FABP protein with different concentrations (coating antigen: A-FABP

[0060] The sensitivity of monoclonal antibody 3H9 was tested by ELISA (1 μg / ml, 0.5 μg / ml, 0.05 μg / ml, 0.005 μg / ml, 0.0005 μg / ml). It was found that antibody 3H9 was highly sensitive to low concentration A-FABP protein. See Table 3 for details.

[0061] Table 3. Antibody titer test 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 5A-FABP protein monoclonal antibody 3H9 gene sequencing

[0064] After immunization, fusion and monoclonalization, antibody purification, and antibody specificity and titer identification, 3H9 monoclonal antibody cell line was selected for total RNA extraction and reverse transcription into cDNA, and then the heavy chain variable region and light chain variable region of the antibody were amplified by PCR using cDNA as a template. Invitrogen's TRIzol reagent kit (15596-026) was used to extract total RNA from 3H9 monoclonal antibody cell line according to its instructions, followed by Takara's 5'RACE FULL kit (D315), using total RNA as a template and random primers in the kit for reverse transcription into first-strand cDNA, and then the heavy chain was PCR amplified using constant region designed primers and adapter primers in the kit, and the light chain was PCR amplified using constant region designed primers and adapter primers in the kit.

[0065] The PCR fragments were recovered by agarose gel recovery kit for TA cloning, and then single clones were selected for PCR identification. Some samples were selected from the identified correct strains and sent to Invitrogen for sequencing. The heavy chain variable region nucleotide sequence was finally determined to be SEQ ID NO: 1, the light chain variable region nucleotide sequence was SEQ ID NO: 2, the heavy chain variable region amino acid sequence was SEQ ID NO: 3, and the light chain variable region amino acid sequence was SEQ ID NO: 4, as shown in Table 4.

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

[0067] The amino acid sequence of the heavy chain variable region of the 3H9 antibody 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: KATMTADTSSNTAYLQLSSLTSEDTAVYYCNASEQ 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 light chain variable region of the 3H9 antibody is as follows (SEQ ID NO: 9):

[0076] DIQMTQTPLSLPVSLGDQASISCRSSQSIVHSNGNTYLDWYLQKPGQSPKLLIYKVSNR FSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVPYTFGGGTKLEI

[0077] Among them, 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 light chain variable region CDR3 is: FQGSHVPYT SEQ ID NO.12

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

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

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

[0083] SEQ ID NO.15

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

[0085] The nucleotide sequence of the heavy chain variable region of the 3H9 antibody 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 anti-A-FABP monoclonal antibody or antigen-binding fragment thereof, characterized in that: It 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 complementary determining region CDR1 is shown in SEQ ID NO:2; The heavy chain complementary determining region CDR2 is shown in SEQ ID NO: 3; The heavy chain complementary determining region CDR3 is shown in SEQ ID NO:4; The light chain complementary determining region CDR1 is shown in SEQ ID NO: 10; The light chain complementary determining region CDR2 is shown in SEQ ID NO: 11; The light chain complementary determining region CDR3 is shown in SEQ ID NO:

12.

2. An isolated anti-A-FABP monoclonal antibody or antigen-binding fragment thereof, characterized in that: It 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 nucleotide, characterized in that: It encodes the monoclonal antibody or antigen-binding fragment thereof as claimed in claim 1 or 2.

4. A recombinant vector, characterized in that: Comprising the nucleotide sequence of claim 3.

5. A host cell, characterized in that: Comprising the recombinant vector according to claim 4; Preferably, the host cell is prokaryotic or eukaryotic; Preferably, the host cell is selected from yeast cells, mammalian cells or other cells suitable for producing antibodies or antigen-binding fragments thereof.

6. A kit for detecting A-FABP, characterized in that: The kit comprises the monoclonal antibody or antigen-binding fragment thereof according to claim 1 or 2.

7. A detection reagent for detecting A-FABP, characterized in that: The detection reagent comprises the monoclonal antibody or antigen-binding fragment thereof according to claim 1 or 2; Preferably, the detection reagent is used for enzyme-linked immunosorbent assay, immunoblotting, flow cytometry, immunohistochemistry or immuno-PCR.

8. Use of the monoclonal antibody or antigen-binding fragment thereof according to claim 1 or 2 in the preparation of a reagent for detecting A-FABP.

9. The use according to claim 8, characterized in that The reagent is used for the following purposes: enzyme-linked immunosorbent assay, immunoblotting, flow cytometry, immunohistochemistry or immuno-PCR.

10. Use of the monoclonal antibody or antigen-binding fragment thereof according to claim 1 or 2 in preparing a reagent for separating or purifying A-FABP in vitro.

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