A folate sandwich method antibody mAb12 or antigen-binding fragment thereof, and preparation method and application thereof
By developing the folic acid sandwich antibody mAb12 with excellent specificity and affinity, the problems of low sensitivity and insufficient specificity in existing folic acid detection technologies have been solved, achieving efficient and accurate folic acid detection, which is suitable for the diagnosis and treatment of a variety of diseases.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- ORIGENE WUXI BIOTECHNOLOGY CO LTD
- Filing Date
- 2026-01-24
- Publication Date
- 2026-05-29
Smart Images
Figure CN122103353A_ABST
Abstract
Description
Technical Field
[0001] This application relates to the field of immunoassay technology, specifically to a folic acid sandwich antibody mAb12 or its antigen-binding fragment, its preparation method, and its application. Background Technology
[0002] Folic acid (vitamin B9) is a water-soluble B vitamin that acts as a key coenzyme in cell metabolism and proliferation, and participates in the synthesis of nucleic acids and amino acids. It is an essential nutrient for maintaining normal human growth, development, and physiological functions. Recent studies have further revealed that the clinical role of folic acid has transcended the traditional scope of nutritional support. It has demonstrated significant value in preventing neural tube defects in fetuses, reducing the risk of cardiovascular and cerebrovascular diseases, assisting in tumor prevention and treatment, and delaying the progression of neurodegenerative diseases, thus becoming a highly regarded micronutrient in the field of precision medicine. Folic acid deficiency can lead to various clinical problems such as megaloblastic anemia, decreased immune function, and fetal developmental abnormalities; however, excessive supplementation also carries corresponding potential risks. Therefore, establishing an accurate and efficient method for folic acid detection is of significant practical importance for guiding clinical nutritional intervention strategies and achieving early screening and dynamic monitoring of related diseases.
[0003] Currently, folic acid testing mainly falls into two categories: one is the gold standard method based on mass spectrometry, which boasts high accuracy and specificity, but relies on expensive instruments, complex sample pretreatment, and specialized personnel, making it difficult to meet the needs of large-scale, time-sensitive clinical testing; the other is immunological testing methods, which are simple, rapid, and easily automated, and have become the routine choice for clinical laboratories. However, due to the small molecular weight and weak immunogenicity of folic acid, traditional immunoassays often employ competitive methods. These methods have inherent limitations: generally low sensitivity, specificity easily affected by sample matrix interference, insufficient stability of test results, complex process development, and difficulty in achieving precise quantification. This limits their application in low-concentration sample testing and high-end clinical diagnostics.
[0004] Therefore, existing immunoassay techniques still have limitations in meeting the clinical demand for highly sensitive and specific quantitative analysis of folic acid. To overcome this bottleneck, it is necessary to overcome the inherent obstacle of detecting small molecules using sandwich assays and develop anti-folate monoclonal antibodies with high affinity and high specificity, thus providing a crucial foundation for constructing a dual-antibody sandwich assay system. Summary of the Invention
[0005] This application provides folic acid sandwich antibody mAb12 or its antigen-binding fragment with excellent specificity and affinity, and covers its preparation method and application. This antibody or its fragment can enable rapid detection of folic acid in double-antibody sandwich ELISA or chemiluminescent immunoassay kits, which is of significant value for the diagnosis and treatment of birth defects, cardiovascular diseases, tumors, and neurodegenerative diseases.
[0006] In a first aspect, this application provides a folic acid sandwich antibody mAb12 or its antigen-binding fragment, comprising a light chain variable region VL and a heavy chain variable region VH. The light chain variable region VL includes LCDR1, LCDR2, and LCDR3. The amino acid sequence of LCDR1 is the sequence shown in SEQ ID NO.1. The amino acid sequence of LCDR2 is STS (SEQ ID NO.2). The amino acid sequence of LCDR3 is the sequence shown in SEQ ID NO.3; The heavy chain variable region VH includes HCDR1, HCDR2, and HCDR3. The amino acid sequence of HCDR1 is shown in SEQ ID NO.4. The amino acid sequence of HCDR2 is shown in SEQ ID NO.5. The amino acid sequence of HCDR3 is the sequence shown in SEQ ID NO.6; Furthermore, the folic acid sandwich antibody mAb12 or its antigen-binding fragment can specifically bind to folic acid.
[0007] Further, the light chain variable region VL comprises an amino acid sequence having more than 98% homology to the amino acid sequence shown in SEQ ID NO.7, wherein any one or more amino acids have been substituted, deleted, and / or added, and / or terminally modified; the heavy chain variable region VH comprises an amino acid sequence having more than 98% homology to the amino acid sequence shown in SEQ ID NO.8, wherein any one or more amino acids have been substituted, deleted, and / or added, and / or terminally modified.
[0008] Furthermore, amino acid sequences with 98%, 98.5%, 99%, or 99.5% identity.
[0009] Furthermore, the light chain variable region VL includes the amino acid sequence shown in SEQ ID NO.7, and the heavy chain variable region VH includes the amino acid sequence shown in SEQ ID NO.8.
[0010] Specifically, the light chain variable region VL has a full length of 105 amino acids, with the number of amino acids in the four domains of its FR being 26, 17, 36, and 10, respectively, and the number of amino acids in the three domains of its LCDR being 5, 3, and 8, respectively. The regions of LCDR1, LCDR2, and LCDR3 are 27aa-31aa, 49aa-51aa, and 88aa-95aa, respectively, and their amino acid sequences are: SSVNY (SEQ ID NO.1), STS (SEQ ID NO.2), and HQWSSYRT (SEQ ID NO.3), respectively.
[0011] Specifically, the heavy chain variable region VH has a total length of 121 amino acids, with the number of amino acids in the four domains of its FR being 24, 17, 36, and 11, respectively, and the number of amino acids in the three domains of HCDR being 8, 8, and 17, respectively. HCDR1, HCDR2, and HCDR3 are 25aa-32aa, 50aa-57aa, and 94aa-110aa, respectively, and their amino acid sequences are GYTFADYG (SEQ ID NO.4), INTYSGEP (SEQ ID NO.5), and AREALYYRYPYFYTMDY (SEQ ID NO.6), respectively.
[0012] Furthermore, the antigen-binding fragment is one of F(ab')2, Fab', Fab, Fv, scFv, dsFv, and bispecific antibodies.
[0013] Secondly, this application also provides a hybridoma cell line that secretes folic acid sandwich antibody mAb12, the hybridoma cell line having the accession number CGMCC No. 46776, which was deposited at the China General Microbiological Culture Collection Center (CGMCC) on January 14, 2026.
[0014] Thirdly, this application also provides a biomaterial selected from polynucleotides, carriers, or cells. The polynucleotide encodes the folic acid sandwich antibody mAb12 or its antigen-binding fragment as described in the first aspect; The vector carries the polynucleotide; The cell carries the polynucleotide, or contains the carrier, or is capable of expressing the folic acid sandwich antibody mAb12 or its antigen-binding fragment as described in the first aspect.
[0015] Furthermore, the cells are eukaryotic cells, preferably mammalian cells, more preferably 293 cells or CHO cells.
[0016] Fourthly, this application also provides a method for preparing the folic acid sandwich antibody mAb12 or its antigen-binding fragment as described in the first aspect, wherein the preparation method is selected from any of the following: 1) Obtained from the hybridoma cell line mAb12 described in the second aspect; 2) Cultivating the cells described in the third aspect, wherein the cells are prepared by transforming the cells with a polynucleotide encoding a folic acid sandwich antibody mAb12 or an antigen-binding fragment thereof, the polynucleotide including a heavy chain expression plasmid and a light chain expression plasmid, and the transformation includes co-transforming the heavy chain expression plasmid and the light chain expression plasmid into the cells.
[0017] Fifthly, this application also provides the application of the folic acid sandwich antibody mAb12 or its antigen-binding fragment as described in the first aspect, or the biological material as described in the third aspect, wherein the application is selected from one or more of the following: 1) Folic acid testing for purposes other than disease diagnosis and treatment; 2) Prepare immunoassay products for detecting folic acid; 3) Used for purifying folic acid; 4) Prepare products for purifying folic acid.
[0018] In this application, the application is for non-diagnostic and therapeutic purposes, and can be used for folic acid level screening in health checkups, pre-pregnancy / pregnancy care, and health management of the elderly; it can be used in clinical trials or consumer tracking to objectively assess changes in human folic acid levels after ingestion of folic acid-fortified foods or supplements; it can be used in cell experiments and animal models to quantitatively detect folic acid levels in biological samples (cell lysates, tissue homogenates, serum, etc.) to study the mechanism of action of folic acid in physiological and biochemical processes such as metabolism, proliferation, and gene expression; it can also be used to study the absorption, distribution, metabolism, and excretion kinetics of folic acid in humans or model organisms.
[0019] Sixthly, this application also provides a folic acid immunoassay product, said immunoassay product comprising the folic acid sandwich antibody mAb12 or its antigen-binding fragment as described in the first aspect or the biological material as described in the third aspect.
[0020] Furthermore, the folic acid immunoassay product is a reagent or kit for detection using enzyme-linked immunosorbent assay (ELISA), Western blot, immunohistochemical staining (IHC), flow cytometry (FCM), immunoprecipitation, and magnetic microparticle chemiluminescence immunoassay.
[0021] Preservation Information Taxonomic nomenclature: Hybridoma cell line Accession number: CGMCC No. 46776 Deposit date: January 14, 2026 Preservation institution: China General Microbiological Culture Collection Center (CGMCC); Address of the depository: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.
[0022] Compared with the prior art, this application provides a folic acid-specific antibody mAb12 or its antigen-binding fragment, which exhibits both high specificity and high affinity when binding folic acid, with an affinity constant (Ka) reaching 6 × 10⁻⁶. 9 L / mol. The antibody is suitable for preparing immunoassay kits for folic acid detection, especially showing excellent performance in double-antibody sandwich ELISA and chemiluminescence detection systems. Experiments have demonstrated that the detection sensitivity of the folic acid-specific antibody mAb12 based on the double-antibody sandwich chemiluminescence method for folic acid standard antigen is less than 0.1 ng / ml; analysis of clinical samples using a magnetochemiluminescence platform showed good correlation with the reference method (R method) within a concentration range of 0–20 ng / mL. This antibody and related detection methods have significant clinical application prospects in the auxiliary diagnosis and treatment monitoring of birth defect screening, cardiovascular diseases, tumors, and neurodegenerative diseases. Attached Figure Description
[0023] Figure 1 This is an electrophoresis diagram of the full-length amplification products of the mAb12 heavy and light chains, where M is the DNA molecular weight marker.
[0024] Figure 2 The mAb12 sandwich ELISA method was used to detect cross proteins and target antigen samples. The vertical axis represents the detected OD value.
[0025] Figure 3 The standard curve was used to detect folic acid standard antigen using mAb12 magnetic microparticle chemiluminescence immunoassay. The x-axis represents the concentration of folic acid standard antigen (ng / mL), and the y-axis represents the detected luminescence value. The R-value of the standard curve is... 2 =0.9905, linear detection range 0-20ng / mL, the sample concentration calculation formula is derived as: y=63648x-42983.
[0026] Figure 4 The standard curve was used to detect folic acid in clinical samples using mAb12 magnetic microparticle chemiluminescence immunoassay. The x-axis represents the concentration of free folic acid in the clinical samples (ng / mL), and the y-axis represents the detected luminescence value. The R-value of the standard curve is... 2 =0.9637, linear detection range 0-20ng / mL, the sample concentration calculation formula is derived as: y=43425x-23794. Detailed Implementation
[0027] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions, conditions described in a laboratory manual, or conditions recommended by the manufacturer.
[0028] Example 1: Preparation and screening of anti-folate monoclonal antibodies secreted by hybridoma cell lines
[0029] 1) Preparation of immunogens The folic acid complex was prepared with an immunogen purity of over 90%, meeting the purity requirements for preparing monoclonal antibodies.
[0030] 2) Animal immunization The folic acid complex prepared above was emulsified with complete Freund's adjuvant and immunized 6-8 week old BALB / c mice subcutaneously or intraperitoneally at a dose of 80 μg / mouse. A second immunization was performed two weeks later with incomplete Freund's adjuvant emulsification at a dose of 60 μg / mouse. Tail blood was collected after both immunizations and serum titers were determined using a serially diluted ELISA method. Based on the titer results, a booster immunization was determined, and mice with the highest antibody titers were selected for cell fusion.
[0031] 3) Cell fusion and clone screening Spleen cells from immunized mice were fused with mouse myeloma cells SP2 / 0 at a ratio of 10:1 using 50% PEG 4000. After centrifugation, the cells were resuspended in HAT selective medium and seeded into 96-well microplates containing feeder cells. The plates were incubated at 37°C with 5% CO2. On days 4 and 7 post-fusion, half of the HAT medium was replaced, and after 10 days, HT medium was used. The growth of hybridoma cells was observed. When the clones reached 1 / 4 to 1 / 3 of the well bottom area, the culture supernatant was collected, and antibody detection was performed using ELISA to screen for positive clones. The hybridoma cells from the positive wells were cloned using limiting dilution until the antibody positivity rate of the cloned cells reached 100%. High-secretion-specific cell lines (i.e., ELISA titers of 1:10) were selected. 4 The above positive clones were used to obtain the hybridoma cell line mAb12, which secretes anti-folate complex monoclonal antibody.
[0032] 4) Preparation and purification of monoclonal antibodies Hybridoma cell line mAb12 was cultured in serum-free medium. After approximately one week, when the cell supernatant volume reached the target volume and the cell death rate reached 60%-70%, the cell suspension was collected, the supernatant was collected by centrifugation, and antibody purification was performed using affinity chromatography. The appropriate column material was selected based on the antibody subtype for purification. The concentration of the purified monoclonal antibody was determined using the BCA method, and then aliquoted, frozen, and stored to obtain the monoclonal antibody mAb12.
[0033] Example 2: Identification of monoclonal antibodies using the folic acid sandwich method
[0034] 1) Specificity identification of monoclonal antibodies Indirect ELSA was used for detection. The ELISA plate was coated with cross-linked protein and folic acid complex antigen at a concentration of 1 μg / mL and incubated overnight at 4°C. The plate was then blocked with PBST containing 1% BSA. 4 The purified monoclonal antibody was diluted 10 times and reacted at 37°C for 50 min. The plate was washed 3 times with PBST, HRP-goat anti-mouse IgG secondary antibody was added, and the plate was reacted at 37°C for 50 min. The plate was washed 5 times with PBST, TMB was added for color development for 10 min, stop solution was added, and the A450 was measured by microplate reader.
[0035] Figure 2 The results showed that the cross protein reacted negatively with the monoclonal antibody mAb12, with OD450 values less than 0.1; the folic acid complex reacted positively with the mAb12 antibody, with OD values much higher than those of the cross protein, indicating that the folic acid sandwich monoclonal antibody of this application specifically recognizes the folic acid complex.
[0036] 2) Determination of the affinity constant of monoclonal antibodies Affinity constant (Ka) was determined using a non-competitive ELISA method.
[0037] Coating: Dilute the antigen with carbonate buffer to concentrations of 1, 0.5, 0.1, and 0.05 μg / mL, add 100 μL / well to a 96-well microplate for coating, and incubate at 4°C for 24 h.
[0038] Blocking: Wash the plate 4 times with PBST, add BSA solution at 200 μL / well, and incubate at 37°C for 2 h.
[0039] Add monoclonal antibody: Wash the plate 4 times with PBST, serially dilute the monoclonal antibody starting at 100 μg / mL with carbonate buffer, add 100 μL to each well, and incubate at 37°C for 2 h.
[0040] Add enzyme-labeled secondary antibody: Wash the plate 4 times with PBST, add 100 μL of HRP enzyme-labeled goat anti-mouse Ig secondary antibody diluted 1:10000 to each well, and incubate at 37℃ for 30 min.
[0041] Color development and termination: Wash the plate 4 times with PBST, add 100 μL of substrate color development solution to each well, and react at 37℃ in the dark for 15 min; add 50 μL of 1.0 mol / L H2SO4 stop solution to each well to terminate the reaction.
[0042] Detection: The absorbance value at a wavelength of 450 nm (A450nm) was measured.
[0043] Plotting the logarithm of antibody concentration on the x-axis and OD value on the y-axis, an S-shaped curve was obtained. The calculated affinity constant Ka for the folic acid sandwich monoclonal antibody mAb12 was 6 × 10⁻⁶. 9 L / mol.
[0044] 3) Sandwich antibody pairing To select the optimal combination of coating and detection antibodies, folic acid-binding protein was coated onto an ELISA plate and incubated overnight at 4°C. The next day, the plate was removed, washed once with PBST, blocked with 1% BSA solution at 37°C for 2 hours, and washed three times with PBST. 100 μl of folic acid (20 ng / mL) was added to each well, and the plate was incubated at 37°C for 1 hour. After incubation, the plate was removed, washed three times with PBST, and HRP-labeled monoclonal antibody mAb12 was added as the detection antibody, incubated at 37°C for 1 hour. The plate was washed five times with PBST, TMB substrate was added, and the plate was incubated at 37°C for 10 minutes. After incubation, stop solution was added, and the OD450 reading was measured using an ELISA reader. Based on the OD values of the samples and the background value of the negative control, the most ideal antibody pair was selected. The pairing screening results are shown in Table 1.
[0045] Table 1. Results of antibody pairing assay
[0046] Therefore, the antibody mAb12 involved in this application is optimal for sandwich assay.
[0047] Example 3: Analysis of the gene and amino acid sequence of the variable region of a monoclonal antibody Using the recombinant plasmid of the antibody as a DNA template, sequencing primers for the light chain variable region and heavy chain variable region were designed based on the vector sequences at the 5' ends of the light and heavy chains on the template. Sequencing was performed using an ABI 3730 sequencer. The nucleotide sequences of the light and heavy chain variable regions of the monoclonal antibody were obtained by sequencing.
[0048] Using the internet and the IMGT / V-QUEST analysis software at http: / / www.imgt.org, the nucleotide sequences of the light chain variable region and the heavy chain variable region were analyzed. The amino acid sequence of the light chain variable region of monoclonal antibody mAb12 is shown in SEQ ID NO.7, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.8.
[0049] The VL is 105 amino acids in length. The number of amino acids in the four domains of its FR are 26, 17, 36 and 10, respectively. The number of amino acids in the three domains of LCDR are 5, 3 and 8, respectively. The regions of LCDR1, LCDR2 and LCDR3 are 27aa-31aa, 49aa-51aa and 88aa-95aa, respectively. Their amino acid sequences are: SSVNY (SEQ ID NO.1), STS (SEQ ID NO.2) and HQWSSYRT (SEQ ID NO.3), respectively.
[0050] The VH is 121 amino acids long. The number of amino acids in the four domains of its FR are 24, 17, 36 and 11, respectively. The number of amino acids in the three domains of HCDR are 8, 8 and 17, respectively. HCDR1, HCDR2 and HCDR3 are 25aa-32aa, 50aa-57aa and 94aa-110aa, respectively. Their amino acid sequences are GYTFADYG (SEQ ID NO.4), INTYSGEP (SEQ ID NO.5) and AREALYYRYPYFYTMDY (SEQ ID NO.6), respectively.
[0051] Example 4: Folic acid sandwich monoclonal antibody used for magnetic microparticle chemiluminescent immunoassay. 1. Detection Principle and Method A magnetic microparticle chemiluminescence immunoassay technique based on the double-antibody sandwich method was employed. Biotin-labeled folic acid-binding protein (FAB) was immobilized with SA magnetic beads, and ALP was conjugated to a folic acid sandwich monoclonal antibody. Simultaneously, folic acid, ALP substrate, and corresponding buffer components were placed on a Cosmetic fully automated magnetic microparticle chemiluminescence analyzer, and the instrument program was set for detection. A positive result was interpreted as a signal-to-noise ratio (SNR) greater than 2.0. The magnitude of the luminescence value reflects the amount of bound enzyme-labeled antibody and is directly proportional to the folic acid concentration in the sample. A standard curve was plotted based on the measured luminescence values of the standards, as shown below. Figure 3 The folic acid concentration in the sample to be tested can be obtained from the standard curve.
[0052] 2. Composition of the magnetic microparticle chemiluminescence detection kit for folic acid detection
[0053] 1) SA magnetic beads bound to biotin-folic acid binding protein: Take 50 μL of magnetic beads into a 0.5 mL centrifuge tube, place it on a magnetic rack, and remove the supernatant after 1 min; wash the magnetic beads 3 times with 0.5 mL of antibody dilution buffer; add a certain amount of biotin-labeled folic acid binding protein and mix by rotating at room temperature for 60 min; after magnetic separation, resuspend in magnetic preservation buffer at a working concentration of 0.5 mg / mL.
[0054] 2) mAb12 conjugation with ALP: First, the 2-IT antibody mAb12 is reduced; then, an ALP-SMCC intermediate is formed; finally, ALP-SMCC is conjugated with the reducing antibody. After conjugation, the ALP is diluted to the working concentration using ALP storage buffer.
[0055] 3) Washing buffer: is standard pH 7.4 PBST containing 0.05% Proclin 300, prepared as a 20-fold concentrate.
[0056] 4) Chemiluminescent colorimetric solution: purchased from Aivid Biotechnology.
[0057] 5) Sample diluent: PBST containing 1% BSA and 0.05% Proclin 300, filtered for sterilization.
[0058] 6) Standard: Folic acid (small molecule), diluted to 5 μg / mL with PBS containing 1% BSA, 5% sucrose, 10% glycerol and 0.05% Proclin 300, filtered for sterilization and aseptically dispensed.
[0059] 3. Testing of folic acid in clinical samples Clinical samples with different folic acid concentrations were processed. Using folic acid-binding protein as the coating antibody and mAb12 antibody as the detection antibody, the above-described detection method was used to detect clinical samples of different concentrations. The results are shown below. Figure 4 .
[0060] Based on the results, the monoclonal antibody described in this application, used in a magnetic microparticle chemiluminescent immunoassay reagent, showed good correlation with clinical results within a sample range of 0-20 ng / mL.
[0061] In summary, when the folic acid sandwich monoclonal antibody mAb12 of this application is applied to immunoassay kits prepared by double-antibody sandwich ELISA or chemiluminescence methods, the detection sensitivity of folic acid standard antigen by the double-antibody sandwich chemiluminescence platform is less than 0.1 ng / mL. When detecting clinical samples by magnetochemiluminescence, the clinical composite rate is >0.96 in the sample range of 0-20 ng / mL, which is significantly higher than that of traditional competitive detection methods. Moreover, the process is simple and breaks through the limitations of traditional competitive methods.
[0062] This specific embodiment is merely an explanation of this application and is not intended to limit it. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they fall within the scope of the claims of this application.
Claims
1. A folic acid sandwich antibody mAb12 or its antigen-binding fragment, characterized in that, Includes the light chain variable region VL and the heavy chain variable region VH. The light chain variable region VL includes LCDR1, LCDR2, and LCDR3. The amino acid sequence of LCDR1 is the sequence shown in SEQ ID NO.
1. The amino acid sequence of LCDR2 is STS. The amino acid sequence of LCDR3 is the sequence shown in SEQ ID NO.3; The heavy chain variable region VH includes HCDR1, HCDR2, and HCDR3. The amino acid sequence of HCDR1 is shown in SEQ ID NO.
4. The amino acid sequence of HCDR2 is shown in SEQ ID NO.
5. The amino acid sequence of HCDR3 is the sequence shown in SEQ ID NO.6; Furthermore, the folic acid sandwich antibody mAb12 or its antigen-binding fragment can specifically bind to folic acid.
2. The folic acid sandwich antibody mAb12 or its antigen-binding fragment according to claim 1, characterized in that, The light chain variable region VL comprises an amino acid sequence having more than 98% homology to the amino acid sequence shown in SEQ ID NO. 7, obtained by substitution, deletion, and / or addition of one or more amino acids and / or terminal modification of any one or more amino acids; the heavy chain variable region VH comprises an amino acid sequence having more than 98% homology to the amino acid sequence shown in SEQ ID NO. 8, obtained by substitution, deletion, and / or addition of one or more amino acids and / or terminal modification of any one or more amino acids.
3. The folic acid sandwich antibody mAb12 or its antigen-binding fragment according to claim 2, characterized in that, The light chain variable region VL includes the amino acid sequence shown in SEQ ID NO.7, and the heavy chain variable region VH includes the amino acid sequence shown in SEQ ID NO.
8.
4. A hybridoma cell line that secretes folic acid sandwich antibody mAb12, characterized in that, The hybridoma cell line has the accession number CGMCC No. 46776 and was deposited at the China General Microbiological Culture Collection Center (CGMCC) on January 14, 2026.
5. A biomaterial, characterized in that, The biomaterial is selected from polynucleotides, carriers, or cells. The polynucleotide encodes the folic acid sandwich antibody mAb12 or its antigen-binding fragment as described in any one of claims 1-3; The vector carries the polynucleotide; The cell carries the polynucleotide, or contains the carrier, or is capable of expressing the folic acid sandwich antibody mAb12 or its antigen-binding fragment as described in any one of claims 1-3.
6. The method for preparing folic acid sandwich antibody mAb12 or its antigen-binding fragment according to any one of claims 1-3, characterized in that, The preparation method is selected from any of the following: 1) Obtained by secretion from the hybridoma cell line mAb12 as described in claim 4; 2) Cultivating the cells as described in claim 5, wherein the cells are prepared by transforming the cells with a polynucleotide encoding a folic acid sandwich antibody mAb12 or an antigen-binding fragment thereof, the polynucleotide including a heavy chain expression plasmid and a light chain expression plasmid, and the transformation includes co-transforming the heavy chain expression plasmid and the light chain expression plasmid into the cells.
7. The application of the folic acid sandwich antibody mAb12 or its antigen-binding fragment according to any one of claims 1-3, or the biomaterial according to claim 5, characterized in that, The application is selected from one or more of the following: 1) Folic acid testing for purposes other than disease diagnosis and treatment; 2) Prepare immunoassay products for detecting folic acid; 3) Used for purifying folic acid; 4) Prepare products for purifying folic acid.
8. A folic acid immunoassay product, characterized in that, The immunoassay product comprises the folic acid sandwich antibody mAb12 or its antigen-binding fragment as described in any one of claims 1-4, or the biological material as described in claim 5.
9. The folic acid immunoassay product according to claim 8, characterized in that, The folic acid immunoassay products are reagents or kits for detection using enzyme-linked immunosorbent assay (ELISA), Western blot, immunohistochemical staining (IHC), flow cytometry (FCM), immunoprecipitation, and magnetic microparticle chemiluminescence immunoassay.