Folic acid sandwich method antibody mAb15 or antigen binding fragment thereof, and preparation method and application thereof

Abstract

The invention belongs to the technical field of immunodetection, and discloses a folic acid sandwich method antibody mAb15 or an antigen binding fragment thereof as well as a preparation method and application of the folic acid sandwich method antibody mAb15 or the antigen binding fragment thereof. The folic acid sandwich antibody mAb15 or the antigen binding fragment thereof comprises a light chain variable region VL and a heavy chain variable region VH, the light chain variable region VL comprises complementary determining regions LCDR1, LCDR2 and LCDR3, and the heavy chain variable region VH comprises complementary determining regions HCDR1, HCDR2 and HCDR3. When the folic acid sandwich method antibody mAb15 is used for detecting a folic acid standard antigen, the detection sensitivity is lower than 0.15 ng / mL, a clinical sample is detected through a magnetochemiluminescence method, the correlation with R clinical comparison is good in the sample range of 0-20 ng / mL, and the folic acid sandwich method antibody mAb15 has important significance in the field of diagnosis and treatment of birth defects, cardiovascular diseases, tumors and neurodegenerative diseases.

Description

Technical Field

[0001] This application relates to the field of immunoassay technology, specifically to a folic acid sandwich antibody mAb15 or its antigen-binding fragment, its preparation method, and its application. Background Technology

[0002] Folic acid, chemically known as vitamin B9, is a water-soluble B vitamin, originally extracted from spinach leaves and named after them. This substance is a collective term for a class of structurally similar compounds whose molecules consist of a pteridine ring, para-aminobenzoic acid, and one or more L-glutamic acids linked by conjugation. Its biologically active form is tetrahydrofolate (THF). Folic acid plays a crucial role in maintaining cell proliferation, promoting growth and development, and ensuring overall health, especially in the biosynthesis of proteins and DNA. It has clear nutritional and clinical value in preventing neural tube defects in newborns, cardiovascular diseases, malignant tumors, and neurodegenerative diseases, and has become an important essential micronutrient. Abnormal folic acid levels can trigger various pathological changes: deficiency can lead to megaloblastic anemia, decreased immune function, and fetal malformations; excessive intake also poses potential risks. Therefore, establishing accurate and reliable folic acid detection methods is of great significance for clinical diagnosis and health assessment.

[0003] Currently, mass spectrometry is the accepted reference method for clinical folic acid testing. However, this method relies on complex sample pretreatment procedures, is time-consuming, and requires high automation, making it difficult to widely implement in routine testing. Immunological assays, due to their simplicity and speed, have become a widely used alternative in clinical practice. However, because folic acid is a small molecule and lacks sufficient immunogenic epitopes, existing assays are mostly based on competitive immunoassay principles. These methods generally suffer from limitations such as low sensitivity, insufficient specificity, susceptibility to false positives, and complex processing procedures.

[0004] Therefore, developing anti-folate monoclonal antibodies with high affinity and high specificity provides a key reagent for the double-antibody sandwich method of folic acid detection, which can significantly improve the sensitivity and specificity of detection, simplify the operation process, and promote the development of folic acid clinical testing towards a more efficient and reliable direction. Summary of the Invention

[0005] This application provides folic acid sandwich antibody mAb15 or its antigen-binding fragment, which has high specificity and affinity, and can be used to successfully achieve rapid detection of folic acid in immunoassay kits prepared by double antibody sandwich ELISA or chemiluminescence method. This has important significance in the diagnosis and treatment of birth defects, cardiovascular diseases, tumors and neurodegenerative diseases.

[0006] In one aspect, this application provides a folic acid sandwich antibody mAb15 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 or a variant with one or two amino acid substitutions, deletions, or additions compared to the sequence shown in SEQ ID NO.1. The amino acid sequence of LCDR2 is RAS (SEQ ID NO.2) or a variant having an amino acid substitution, deletion, or addition compared to RAS. The amino acid sequence of LCDR3 is the sequence shown in SEQ ID NO.3 or a variant with one or two amino acid substitutions, deletions, or additions compared to 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 the sequence shown in SEQ ID NO.4 or a variant with one or two amino acid substitutions, deletions, or additions compared to the sequence shown in SEQ ID NO.4. The amino acid sequence of HCDR2 is the sequence shown in SEQ ID NO.5 or a variant with one or two amino acid substitutions, deletions, or additions compared to the sequence shown in SEQ ID NO.5. The amino acid sequence of HCDR3 is the sequence shown in SEQ ID NO.6 or a variant with one or two amino acid substitutions, deletions or additions compared to the sequence shown in SEQ ID NO.6; Furthermore, the folic acid sandwich antibody mAb15 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 95% 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 95% 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 95%, 96%, 97%, 98%, or 99% 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] Furthermore, the antigen-binding fragment is one of F(ab')2, Fab', Fab, Fv, scFv, dsFv, and bispecific antibodies.

[0011] Secondly, this application also provides a biomaterial selected from polynucleotides, carriers, or cells. The polynucleotide encodes the folic acid sandwich antibody mAb15 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 mAb15 or its antigen-binding fragment as described in the first aspect.

[0012] Thirdly, this application also provides a method for preparing the folic acid sandwich antibody mAb15 or its antigen-binding fragment as described in the first aspect, comprising culturing cells as described in the second aspect, wherein the cells are prepared by transforming the cells with a polynucleotide encoding a folic acid sandwich antibody mAb15 or its antigen-binding fragment, the polynucleotide comprising a heavy chain expression plasmid and a light chain expression plasmid, and the transformation comprising co-transforming the heavy chain expression plasmid and the light chain expression plasmid into the cells.

[0013] Fourthly, this application also provides the application of the folic acid sandwich antibody mAb15 or its antigen-binding fragment as described in the first aspect, or the biological material as described in the second 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.

[0014] In this application, the application is not for diagnostic or therapeutic purposes. Specifically, it can be used to assess the folic acid nutritional status of specific regions and populations (such as pregnant women and the elderly) to provide data support for public health policies; it can be used for antibody detection to rapidly screen engineered strains or cell lines that produce high levels of folic acid or its intermediate metabolites (such as dihydrofolate and tetrahydrofolate); it can also be used in microbial fermentation monitoring to monitor the concentration of folic acid in the fermentation broth in real time and optimize fermentation conditions during the production of folic acid using probiotics or yeast; it can also be used to study the metabolic, transport, and regulatory mechanisms of folic acid in organisms, as well as its interactions with other nutrients or biomolecules.

[0015] Fifthly, this application also provides a folic acid immunoassay product, which comprises the folic acid sandwich antibody mAb15 or its antigen-binding fragment as described in the first aspect, or the biological material as described in the second aspect.

[0016] 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), and immunoprecipitation.

[0017] Compared to existing technologies, the folic acid sandwich antibody mAb15 or its antigen-binding fragment of this application can bind to folic acid with high specificity and high affinity, with an affinity constant Ka reaching 1×10⁻⁶. 9 L / mol. The folic acid sandwich antibody mAb15 of this application can also be used to prepare various immunoassay kits for folic acid detection, especially for use in immunoassay kits prepared by double antibody sandwich ELISA or chemiluminescence methods. The double antibody sandwich chemiluminescence platform for detecting folic acid standard antigens has a detection sensitivity of less than 0.15 ng / mL, while the magnetochemiluminescence method for detecting clinical samples shows good correlation with clinical results in the 1-20 ng / mL sample range, demonstrating significant potential for diagnosis and treatment of birth defects, cardiovascular diseases, tumors, and neurodegenerative diseases. Attached Figure Description

[0018] Figure 1 This is an electrophoresis diagram of the full-length amplified heavy and light chains of mAb15, where M is the DNA molecular weight marker.

[0019] Figure 2 The mAb15 sandwich ELISA method was used to detect cross proteins and target antigen samples. The vertical axis represents the detected OD value.

[0020] Figure 3 The standard curve was used to detect folic acid standard antigen using mAb15 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 =0.9996, linear detection range 0-20 ng / mL, derive the sample concentration calculation formula: y=4021.1x 2 +20867x+11904.

[0021] Figure 4 The standard curve was used to detect folic acid in clinical samples using mAb15 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=0.9626, linear detection range 0-20ng / mL, the formula for calculating sample concentration is derived as: y=113507x-193330. Detailed Implementation

[0022] 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.

[0023] Example 1: Preparation of rabbit monoclonal antibody using the folic acid sandwich method

[0024] 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.

[0025] 2) Animal immunization The folic acid complex prepared above was emulsified with complete Freund's adjuvant at a 1:1 volume ratio and administered subcutaneously to approximately 2 kg New Zealand white rabbits at a dose of 800 μg / rabbit. A second immunization was performed two weeks later, emulsified with incomplete Freund's adjuvant at a 1:1 volume ratio, at a dose of 400 μg / rabbit. Tail blood was collected after both immunizations and serum titers were determined using a serially diluted ELISA method. The OD450 at an ELISA titer of 128000 was considered to be greater than 1.0. Based on the results, it was determined whether to collect PBMCs or continue immunization. Rabbits with the highest antibody titers were selected for PBMC collection.

[0026] 3) PBMC isolation, specific B cell sorting, and clonal recombination The rabbit was placed supine on the operating table. The fur around the heart was trimmed, and the skin was disinfected with alcohol. The area with the most prominent heartbeat was selected and punctured with a 50mL syringe. Blood flowed into the syringe immediately after the needle entered the heart. The needle was quickly withdrawn after obtaining the required amount of blood. The whole blood in the syringe was transferred into a sterile 50mL tube and mixed with an equal amount of PBS. The mixture was then slowly added dropwise to the lymphocyte separation medium. The mixture was centrifuged at 400×g for 30 minutes at room temperature. After centrifugation, the liquid surface separated into four layers from top to bottom: a yellow plasma layer, a white thin film layer (i.e., a mononuclear cell layer), a separation medium layer, and a red blood cell layer. The mononuclear cell layer was carefully aspirated and washed with PBS to remove platelets and lymphocyte separation medium, thus obtaining rabbit PBMCs.

[0027] Antigen-specific B cells were further sorted from rabbit PBMCs and cultured. Positive clones were selected from the B cell supernatant using antigen-coated ELISA plates. Cells from positive clones were collected, lysed, and RNA was extracted and reverse transcribed into cDNA. The full-length light and heavy chain sequences of naturally paired rabbit monoclonal antibodies were amplified from the cDNA of the corresponding positive clones. Rabbit monoclonal antibody expression vectors were constructed using clonal recombination methods, and the sequences were confirmed by sequencing. The results of the amplified full-length PCR products are shown below. Figure 1 .

[0028] 4) Preparation and purification of monoclonal antibodies To obtain multiple rabbit monoclonal antibodies that recognize folic acid molecules, the heavy and light chain genes of rabbit monoclonal antibodies were loaded into an expression vector. The plasmid was transfected into KEK293 cells, and after 120-144 hours, the culture supernatant contained recombinant rabbit monoclonal antibodies recognizing folic acid molecules. The cell suspension was collected, the supernatant was obtained by centrifugation, and the antibody was purified by affinity chromatography. The concentration of the purified monoclonal antibody was determined by the BCA method, and then aliquoted, lyophilized, and named rabbit monoclonal antibody mAb15.

[0029] Example 2: Identification of rabbit monoclonal antibodies using the folic acid sandwich method 1) Specificity identification of rabbit 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 rabbit 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-rabbit 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.

[0030] Figure 2 The results showed that the cross protein reacted negatively with the rabbit monoclonal antibody mAb15, with OD450 values ​​less than 0.1; the folic acid complex reacted positively with the mAb15 antibody, with OD values ​​much higher than those of the cross protein, indicating that the folic acid sandwich method rabbit monoclonal antibody of this application specifically recognizes the folic acid complex.

[0031] 2) Determination of affinity constant of rabbit monoclonal antibody Affinity constant (Ka) was determined using a non-competitive ELISA method.

[0032] 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.

[0033] Blocking: Wash the plate 4 times with PBST, add BSA solution at 200 μL / well, and incubate at 37°C for 2 h.

[0034] Add monoclonal antibody: Wash the plate 4 times with PBST, serially dilute the rabbit monoclonal antibody with carbonate buffer starting at 100 μg / mL, add 100 μL to each well, and incubate at 37°C for 2 h.

[0035] Add enzyme-labeled secondary antibody: Wash the plate 4 times with PBST, add 100 μL of HRP enzyme-labeled goat anti-rabbit Ig secondary antibody diluted 1:10000 to each well, and incubate at 37℃ for 30 min.

[0036] 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.

[0037] Detection: The absorbance value at a wavelength of 450 nm (A450nm) was measured.

[0038] Plotting the logarithm of antibody concentration on the x-axis and OD value on the y-axis, an S-shaped curve was constructed. The calculated affinity constant Ka for the folic acid sandwich monoclonal antibody mAb15 was 1 × 10⁻⁶. 9 L / mol.

[0039] 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 rabbit monoclonal antibody mAb15 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.

[0040] Table 1. Results of antibody pairing assay

[0041] Therefore, the antibody mAb15 involved in this application is optimal for sandwich assay.

[0042] 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 rabbit monoclonal antibody were obtained by sequencing.

[0043] 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 sequenced and analyzed. The amino acid sequence of the light chain variable region of rabbit monoclonal antibody mAb15 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.

[0044] The VL is 110 amino acids in length. The number of amino acids in the four domains of its FR are 26, 16, 36 and 11, respectively. The number of amino acids in the three domains of LCDR are 6, 3 and 12, respectively. The regions of LCDR1, LCDR2 and LCDR3 are 27aa-32aa, 49aa-51aa and 88aa-99aa, respectively. Their amino acid sequences are ESINNY (SEQ ID NO.1), RAS (SEQ ID NO.2) and QQGYSGSNVNNV (SEQ ID NO.3), respectively.

[0045] The VH is 121 amino acids long. The number of amino acids in the four domains of its FR are 25, 17, 38 and 11, respectively. The number of amino acids in the three domains of HCDR are 8, 7 and 15, respectively. HCDR1, HCDR2 and HCDR3 are 26aa-33aa, 51aa-57aa and 96aa-110aa, respectively. Their amino acid sequences are GFSLTSYD (SEQ ID NO.4), IWSSGST (SEQ ID NO.5) and ARDQGYGDRSYSFNL (SEQ ID NO.6), respectively.

[0046] Example 4: Folic acid sandwich method rabbit monoclonal antibody used for magnetic microparticle chemiluminescent immunoassay.

[0047] 1. Detection Principle and Method

[0048] A magnetic microparticle chemiluminescence immunoassay technique based on the double-antibody sandwich method was employed. Biotin-labeled folic acid-binding protein was immobilized with SA magnetic beads, and ALP was conjugated to a rabbit monoclonal antibody using the folic acid sandwich method. Simultaneously, folic acid, ALP substrate, and corresponding buffer components were placed on a Cosma 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.

[0049] 2. Composition of the magnetic microparticle chemiluminescence detection kit for folic acid detection

[0050] 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 at room temperature for 60 min by rotation; after magnetic separation, resuspend in magnetic preservation buffer at a working concentration of 0.5 mg / mL.

[0051] 2) mAb15 conjugation with ALP: First, the 2-IT antibody mAb15 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.

[0052] 3) Washing buffer: is standard pH 7.4 PBST containing 0.05% Proclin 300, prepared as a 20-fold concentrate.

[0053] 4) Chemiluminescent colorimetric solution: purchased from Aivid Biotechnology.

[0054] 5) Sample diluent: PBST containing 1% BSA and 0.05% Proclin 300, filtered for sterilization.

[0055] 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.

[0056] 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 mAb15 antibody as the detection antibody, the above detection method was used to detect clinical samples of different concentrations. The results are shown below. Figure 4 .

[0057] Based on the results, the rabbit 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.

[0058] In summary, when the folic acid sandwich rabbit monoclonal antibody mAb15 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 on the double-antibody sandwich chemiluminescence platform is less than 0.15 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.

[0059] 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 mAb15 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 or a variant with one or two amino acid substitutions, deletions, or additions compared to the sequence shown in SEQ ID NO.

1. The amino acid sequence of LCDR2 is RAS or a variant that has an amino acid substitution, deletion, or addition compared to RAS. The amino acid sequence of LCDR3 is the sequence shown in SEQ ID NO.3 or a variant with one or two amino acid substitutions, deletions, or additions compared to 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 the sequence shown in SEQ ID NO.4 or a variant with one or two amino acid substitutions, deletions, or additions compared to the sequence shown in SEQ ID NO.

4. The amino acid sequence of HCDR2 is the sequence shown in SEQ ID NO.5 or a variant with one or two amino acid substitutions, deletions, or additions compared to the sequence shown in SEQ ID NO.

5. The amino acid sequence of HCDR3 is the sequence shown in SEQ ID NO.6 or a variant with one or two amino acid substitutions, deletions or additions compared to the sequence shown in SEQ ID NO.6; Furthermore, the folic acid sandwich antibody mAb15 or its antigen-binding fragment can specifically bind to folic acid.

2. The folic acid sandwich antibody mAb15 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 95% 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 95% 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 mAb15 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. The folic acid sandwich antibody mAb15 or its antigen-binding fragment according to claim 1, characterized in that, The antigen-binding fragment is one of F(ab')2, Fab', Fab, Fv, scFv, dsFv, and bispecific antibodies.

5. A biomaterial, characterized in that, The biomaterial is selected from polynucleotides, carriers, or cells. The polynucleotide encodes the folic acid sandwich antibody mAb15 or its antigen-binding fragment as described in any one of claims 1-4; The vector carries the polynucleotide; The cell carries the polynucleotide, or contains the carrier, or is capable of expressing the folic acid sandwich antibody mAb15 or its antigen-binding fragment as described in any one of claims 1-4.

6. The method for preparing folic acid sandwich antibody mAb15 or its antigen-binding fragment according to any one of claims 1-4, characterized in that, The method includes culturing cells as described in claim 5, wherein the cells are prepared by transforming cells with a polynucleotide encoding a folic acid sandwich antibody mAb15 or an antigen-binding fragment thereof, the polynucleotide comprising a heavy chain expression plasmid and a light chain expression plasmid, and the transformation comprising 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 mAb15 or its antigen-binding fragment according to any one of claims 1-4, 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 mAb15 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), and immunoprecipitation.

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