Monoclonal antibody 34C1 for resisting vitamin B12 and application of monoclonal antibody 34C1

Abstract

The invention provides a monoclonal antibody 34C1 for resisting vitamin B12. The sequence of a heavy chain CDR1 of the monoclonal antibody 34C1 is as shown in SEQ ID NO: 1, the sequence of CDR2 of the monoclonal antibody 34C1 is as shown in SEQ ID NO: 2, and the sequence of CDR3 of the monoclonal antibody 34C1 is as shown in SEQ ID NO: 3; the sequence of the light chain CDR1 is shown as SEQ ID NO: 4, the sequence of the CDR2 is shown as SEQ ID NO: 5, and the sequence of the CDR3 is shown as SEQ ID NO: 6. The antibody can specifically recognize VB12, and can be developed into an immunodetection reagent based on a competition law principle or a sandwich law principle.

Description

Technical Field

[0001] This invention relates to a monoclonal antibody 34C1 against vitamin B12 and its applications, belonging to the field of antibody technology. Background Technology

[0002] Vitamin B12 (VB12), also known as cobalamin, is an essential micronutrient for maintaining normal human metabolism and function. As a cofactor of methyltransferases, it participates in the synthesis of methionine, thymine, and other amino acids, as well as the transfer and storage of folic acid within cells, and is a crucial coenzyme in DNA synthesis.

[0003] A deficiency in vitamin B12 can impair the body's utilization of folic acid, and is associated with early pregnancy loss, recurrent miscarriage, and may also lead to megaloblastic anemia, neuropathy (such as peripheral nerve damage, cognitive impairment, and depression), coronary heart disease, and metabolic abnormalities. Since vitamin B12 is absorbed in the ileum via intrinsic factor, malabsorption (such as atrophic gastritis or intestinal diseases), drug interference (such as proton pump inhibitors), or insufficient dietary intake (vegetarians) can all cause vitamin B12 deficiency. Detecting vitamin B12 levels is crucial for early diagnosis and intervention, making vitamin B12 testing clinically significant.

[0004] Currently, the main methods for detecting VB12 include microbiological methods, radioimmunoassay (RIA), chemiluminescent immunoassay (CLIA), enzyme-linked immunosorbent assay (ELISA), and liquid chromatography-mass spectrometry (LC-MS / MS).

[0005] The principle of the microbiological method is to quantify VB12 by utilizing the growth dependence of specific microorganisms (such as Lactobacillus rosimanis). Its advantages include low cost and the ability to detect extremely low concentrations of VB12. Disadvantages include long processing time (24-48 hours), cumbersome operation, low accuracy, poor repeatability, inability to distinguish between VB12 and folic acid, and susceptibility to the effects of antibiotics or sample pretreatment.

[0006] Radioimmunoassay (RIA) works by using radiolabeled vitamin B12 to competitively bind intrinsic factor or transcobalamin proteins with vitamin B12 in the sample. Its advantages include high sensitivity and suitability for batch detection. Disadvantages include the risk of radioactive contamination and poor reagent stability, and it is gradually being replaced by other methods.

[0007] Chemiluminescent immunoassay (CLIA) works on the principle of antigen-antibody reaction combined with chemiluminescent signal detection. Its advantages include high automation, fast detection speed (1-2 hours), and excellent sensitivity and specificity. Its disadvantage is that it may be affected by factors such as rheumatoid factor.

[0008] The principle of enzyme-linked immunosorbent assay (ELISA) is that enzyme-labeled antibodies bind to vitamin B12, resulting in colorimetric quantification. Its advantages include the absence of complex equipment, making it suitable for small to medium-sized laboratories. Disadvantages include lower sensitivity, susceptibility to cross-reactions (such as with vitamin B12 analogs), poor specificity, and significant deviations in results between different kits.

[0009] The principle of liquid chromatography-mass spectrometry (LC-MS / MS) is to separate and quantify VB12 and its metabolites in a sample. Its advantages include high specificity, the ability to distinguish between active and inactive forms. Disadvantages include expensive equipment, complex operation, the need for specialized technicians, low throughput, and limited adoption.

[0010] In summary, chemiluminescence and immunofluorescence methods based on the principle of immune binding have certain advantages in terms of labor and instrument costs, sensitivity, and detection throughput. However, there is a lack of monoclonal antibodies with high affinity and high specificity for VB12 in current technologies. Summary of the Invention This invention provides a monoclonal antibody 34C1 against vitamin B12 and its application, which can effectively solve the above-mentioned problems.

[0011] A monoclonal antibody 34C1 against vitamin B12 has the following sequences: heavy chain CDR1 as shown in SEQ ID NO:1, CDR2 as shown in SEQ ID NO:2, and CDR3 as shown in SEQ ID NO:3; and light chain CDR1 as shown in SEQ ID NO:4, CDR2 as shown in SEQ ID NO:5, and CDR3 as shown in SEQ ID NO:6. In some embodiments, the monoclonal antibody 34C1 against vitamin B12 has the heavy chain variable region sequence shown in SEQ ID NO:7 and the light chain variable region sequence shown in SEQ ID NO:8.

[0012] In some embodiments, the anti-vitamin B12 monoclonal antibody 34C1 has the heavy chain shown in SEQ ID NO:9 and the light chain shown in SEQ ID NO:10.

[0013] A reagent for detecting vitamin B12, comprising the aforementioned anti-vitamin B12 monoclonal antibody 34C1.

[0014] A kit for detecting vitamin B12, comprising the aforementioned anti-vitamin B12 monoclonal antibody 34C1.

[0015] A method for detecting vitamin B12, using the aforementioned anti-vitamin B12 monoclonal antibody 34C1 for vitamin B12 detection.

[0016] In some embodiments, the detection method is a competition method or a sandwich method.

[0017] The beneficial effects of this invention are: The monoclonal antibody 34C1 provided by this invention possesses high specificity, enabling it to accurately recognize and bind to vitamin B12 (VB12). Based on this characteristic, the monoclonal antibody 34C1 has broad application potential in the field of immunoassay, particularly suitable for developing immunoassay reagents based on competitive or sandwich assay principles. By utilizing the specific binding of the 34C1 antibody to VB12, highly sensitive and specific detection methods can be designed, thus playing an important role in clinical diagnosis, food safety testing, and biomedical research. Detailed Implementation

[0018] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Therefore, the following detailed description of the embodiments of the present invention is not intended to limit the scope of the claimed invention, but merely to represent selected embodiments of the present invention.

[0019] Example 1 1. Preparation of monoclonal antibody 34C1 The reagents or kits involved in this application and their sources are as follows: Freund's Adjuvant, Complete (Catalog No. 77140, Thermo Fisher); Freund's Adjuvant, Incomplete (Catalog No. 77145, Thermo Fisher); HAT Media Supplement (50×) (Catalog No.: 21060017, Thermo Fisher); HT Media Supplement (50×) (Catalog No. H0111067030, Thermo Fisher); PEG (Catalog No. P7181, Sigma-Aldrich); RPMI 1640 (Catalog No. L210KJ, Shanghai Yuanpei Biotechnology); Fetal Bovine Serum (FBS) (C04001-500, Shanghai Xiaopeng Biotechnology); DMEM (Catalog No. L310KJ, Shanghai Yuanpei Biotechnology); Penicillin-St reptomycin (catalog number 15140122, Gibco); HRP-labeled goat anti-mouse antibody (catalog number D110087, Shanghai Sangon Biotech); Protein A Resin (catalog number SA023010, Changzhou Tiandi Renhe Biotechnology Co., Ltd.); VB12 antigen (catalog number: VB12401, Boyue Biotechnology).

[0020] 1.1 Mouse Immunization After dissolving the VB12 antigen, emulsify it evenly with an equal volume of Freund's complete adjuvant. Take 6-8 week old SPF-grade Balb / c mice (Fuzhou Wu's Animal Experiment Center) and inject 200 μg / mouse subcutaneously at multiple sites. Two weeks later, emulsify the antigen with Freund's incomplete adjuvant and inject 100 μg / mouse subcutaneously at multiple sites. Administer two booster immunizations. Three days before fusion, administer a shock immunization via intraperitoneal injection.

[0021] 1.2 Preparation of feeder cells BALB / c mouse peritoneal macrophages were used as feeder cells. One day before fusion, BALB / c mice were euthanized by cervical retraction, immersed in 75% alcohol, and under aseptic conditions in a laminar flow hood, the abdominal skin was cut open with scissors to expose the peritoneum. 5 mL of RPMI 1640 basal culture medium containing 1% penicillin-streptomycin was injected intraperitoneally using a syringe. The cells were repeatedly rinsed, and the rinsing solution was collected. The cells were centrifuged at 1000 rpm for 5 minutes, and the pellet was resuspended in RPMI 1640 complete culture medium containing 1% HAT. The cell concentration was adjusted to 1 × 10⁻⁶ cells / mL. 5 Add 150 μL / well to a 96-well plate and incubate overnight at 37°C with 5% CO2.

[0022] 1.3 Preparation of immune spleen cells Three days after the last immunization of mice, the spleen was removed under aseptic conditions, placed in a petri dish, rinsed once with RPMI 1640 basal culture medium, and then ground and filtered on a nylon mesh in a small beaker to prepare a cell suspension. The suspension was centrifuged, the supernatant was discarded, and the cells were resuspended in RPMI 1640 basal culture medium. This process was repeated three times, and the cells were counted.

[0023] 1.4 Cell Fusion (1) Take 40 mL of HAT culture medium, 15 mL of DMEM serum-free culture medium and 1 mL of 50% PEG (M12000) and place them in a 37°C water bath for preheating; (2) Take mouse myeloma cells Sp2 / 0 (2-5 × 10⁷ cells) and the above-mentioned immune spleen cells (10⁸ cells) suspensions respectively, add them to a 50 mL centrifuge tube, mix well, and add DMEM serum-free culture medium to a final volume of 40 mL. Centrifuge for 10 minutes, discard the supernatant, and mix well; (3) Place the centrifuge tube in pre-warmed water at 37°C, take 0.7 mL of pre-warmed 50% PEG solution, and let it stand for 90 seconds. Immediately add 15 mL of pre-warmed serum-free culture medium at 37°C; (4) Add DMEM serum-free culture medium to 40 mL, centrifuge for 10 minutes, and discard the supernatant. Add 40 mL of HAT culture medium containing 15%–20% fetal bovine serum. Mix well with a pipette and add 2 drops to each of the four wells of a 96-well cell culture plate containing feeder cells. Incubate at 37°C and 7% CO2.

[0024] 1.5 Selection and Culture of Hybridoma Cells The cells were cultured in the aforementioned HAT medium on days 1, 3, 5, and 7 after fusion. The cells that survived were hybridoma cells, while the non-hybridoma cells died, thus selecting the true hybrid cells.

[0025] 1.6 Detection of specific antibodies and cloning of hybridoma cells The supernatant from each culture well was collected, and an indirect ELISA was used to detect the presence of VB12-BSA, a small molecule that specifically recognizes BSA, in the culture medium. Wells that did not recognize BSA, with an OD490 value greater than 2 for VB12-BSA and less than 0.2 for BSA, were considered positive hybridoma cell lines. The day before cloning, feeder cells were prepared and plated according to step 3. The selected positive hybridoma cells were mixed by pipetting, and the cells in each well were diluted with HT medium to one cell per well. The cells were incubated at 37°C and 5% CO2 for 7-10 days. Antibody detection was performed when visible clones appeared. Wells with only a single clone were marked under an inverted microscope. The 34C1 monoclonal antibody hybridoma cell line was obtained through preliminary screening.

[0026] 1.7 Antibody sequencing Cloned 34C1 antibody cells were sent to Shanghai Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The amino acid sequence of the monoclonal antibody 34C1 was obtained as follows: The heavy chain CDR1 sequence is: GFSLSSYT (SEQ ID NO:1) The heavy chain CDR2 sequence is: IYASGST (SEQ ID NO:2) The heavy chain CDR3 sequence is: ARGGYGIYGYGTYFNL (SEQ ID NO:3) The light chain CDR1 sequence is: SGNIEDYY (SEQ ID NO:4) The light chain CDR2 sequence is: NDDQ (SEQ ID NO:5) The light chain CDR3 sequence is: LSSDSSANPV (SEQ ID NO:6) The heavy chain variable region sequence is as follows: QEQLKESGGRLVTPGTPLTLTCTVSGFSLSSYTMGWVRQAPGKGLEYIGIIYASGSTYYAAWARGRFTISKTSTTVDLKMTSLTTEDTATYFCARGGYGIYGYGTYFNLWGQGTLVTVSS (SEQ ID NO:7).

[0027] The light chain variable region sequence is as follows: QFVLTQPQSVSGSLGQTVSISCNRDSGNIEDYYVHWYQQHPGKAPTTVIYNDDQRPSGVPDRFSGSIDSTSNSASLTITGLLAEDEADYYCLSSDSSANPVFGGGTQLTVT (SEQ ID NO: 8).

[0028] The constant region sequence of this monoclonal antibody can be from any species, but is preferably the constant region of the mouse κ light chain and the constant region of the IgG1 heavy chain.

[0029] The heavy chain sequence of this monoclonal antibody is as follows: QEQLKESGGRLVTPGTPLTLTCTVSGFSLSSYTMGWVRQAPGKGLEYIGIIYASGSTYYAAWARGRFTISKTSTTVDLKMTSLTTEDTATYFCARGGYGIYGYGTYFNL WGQGTLVTVSSPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSPRPSETVTCNVAHPASSTKVDKKIVPRDCGC KPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTIS KTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMNTNGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK (SEQ ID NO:9). The light chain sequence is: QFVLTQPQSVSGSLGQTVSISCNRDSGNIEDYYVHWYQQHPGKAPTTVIYNDDQRPSGVPDRFSGSIDSTSNSASLTITGLLAEDEADYYCLSSDSSANPV FGGGTQLTVTSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:10).

[0030] 1.8 Sequence Synthesis and Expression The 34C1 sequence of the aforementioned monoclonal antibody was synthesized by Shanghai Sangon Biotech (Shanghai) Co., Ltd., and inserted into the PTT5 vector. Following the Thermo Fisher FreeStyle™ 293 Expression System User Manual, the vector containing the recombinant protein sequence was transfected into HEK293-F cells, cultured, and the cell culture supernatant was collected.

[0031] The above operations are well known to those skilled in the art, and other plasmid construction, cell transfection, and culture methods in the public domain can also be used to obtain the above single-chain antibodies.

[0032] 1.9 Separation and Purification Cell supernatant from HEK293-F cells expressing protein A was fed into a protein A affinity chromatography column equilibrated with 20 mM phosphate buffer (pH 7.4) at a linear flow rate of 200–400 cm⁻¹ / h for enrichment of the target protein. After loading, elution was performed with 20 mM acetate buffer (pH 3.5), and the eluent was collected. Collect the avidin affinity chromatography elution fraction, concentrate it using a 30kD ultrafiltration tube and replace the buffer, and store the recombinant protein in 20mM phosphate buffer containing 100mM sodium chloride at pH 7.4.

[0033] The above operations are well known to those skilled in the art, and other purification and separation methods in the public domain can also be used to obtain the above recombinant protein.

[0034] 2. Competitive ELISA method for detecting the specificity and affinity of monoclonal antibody 34C1 Using VB12-BSA as the coating antigen, monoclonal antibody 34C1 as the primary antibody, and HRP-labeled goat anti-mouse IgG as the secondary antibody, a competitive ELISA assay was performed. Small molecule VB12 was serially diluted 5-fold with PBS and co-incubated with monoclonal antibody 34C1 for 1 hour. The incubated complex was then added to the test wells and incubated for another hour. Subsequently, the mixture was incubated with HRP-labeled goat anti-mouse IgG for color development, and the results were read. The results are shown in the table below. Table 1. ELISA detection of monoclonal antibody 34C1 activity

[0035] From the experimental data detailed in Table 1, we can clearly observe a significant trend: as the amount of small molecule VB12 co-incubated with monoclonal antibody 34C1 gradually increases, the OD450 optical density reading obtained by ELISA detection shows a continuous decrease. This phenomenon clearly indicates that in the formed complex, monoclonal antibody 34C1 has the ability to specifically recognize small molecule VB12 and VB12-BSA. Specifically, when small molecule VB12 and VB12-BSA are present simultaneously in the reaction system, they compete with each other for the specific recognition site of monoclonal antibody 34C1. Based on this finding, we can infer that monoclonal antibody 34C1 has potential application value in the field of immunoassay, especially suitable for developing immunoassay reagents based on the principles of competitive assays and / or sandwich assays, thereby providing a powerful tool for the diagnosis and detection of related diseases.

[0036] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the invention by those skilled in the art. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the invention should be included within the scope of protection of the invention.

Claims

1. A monoclonal antibody 34C1 against vitamin B12, characterized in that, The heavy chain CDR1 sequence is shown in SEQ ID NO:1, the CDR2 sequence is shown in SEQ ID NO:2, and the CDR3 sequence is shown in SEQ ID NO:3; the light chain CDR1 sequence is shown in SEQ ID NO:4, the CDR2 sequence is shown in SEQ ID NO:5, and the CDR3 sequence is shown in SEQ ID NO:

6.

2. The monoclonal antibody 34C1 against vitamin B12 according to claim 1, characterized in that, The sequence of its heavy chain variable region is shown in SEQ ID NO:7, and the sequence of its light chain variable region is shown in SEQ ID NO:

8.

3. The monoclonal antibody 34C1 against vitamin B12 according to claim 1, characterized in that, Its heavy chain is shown in SEQ ID NO:9, and its light chain is shown in SEQ ID NO:

10.

4. A reagent for detecting vitamin B12, characterized in that, Including the anti-vitamin B12 monoclonal antibody 34C1 as described in any one of claims 1 to 3.

5. A kit for detecting vitamin B12, characterized in that, Including the anti-vitamin B12 monoclonal antibody 34C1 as described in any one of claims 1 to 3.

6. A method for detecting vitamin B12, characterized in that, Vitamin B12 was detected using the monoclonal antibody 34C1 against vitamin B12 as described in any one of claims 1 to 3.

7. The method for detecting vitamin B12 according to claim 6, characterized in that, The detection method is either the competition method or the sandwich method.

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