A folate sandwich method antibody mAb16 or antigen-binding fragment thereof, and preparation method and application thereof

Abstract

The application belongs to the technical field of immune detection, and discloses a folate sandwich method antibody mAb16 or an antigen binding fragment thereof, and a preparation method and application thereof. The folate sandwich method antibody mAb16 or the antigen binding fragment thereof comprises a light chain variable region and a heavy chain variable region, the light chain variable region comprises complementarity determining regions LCDR1, LCDR2 and LCDR3, and the heavy chain variable region comprises complementarity determining regions HCDR1, HCDR2 and HCDR3. The folate sandwich method antibody mAb16 is used for detecting folate standard antigens, the detection sensitivity is lower than 0.2 ng / ml, clinical samples are detected by a magnetic chemiluminescence method, in a range of 0-20 ng / ml samples, and a good correlation is obtained by comparison with R clinical samples, and the folate sandwich method antibody mAb16 has important significance in the fields 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 mAb16 or its antigen-binding fragment, its preparation method, and its application. Background Technology

[0002] Folic acid is a water-soluble B vitamin, chemically known as vitamin B9, named after its initial extraction from spinach leaves. Folic acid is a collective term for a class of compounds with similar chemical structures, composed of pteridine, p-para-aminobenzoic acid, and one or more L-glutamic acids conjugated together. Its biologically active form is tetrahydrofolic acid (THF). The molecular formula of folic acid is C1. 19 H 19 Folic acid (N7O6) is increasingly recognized for its importance in the diet, especially its role in preventing birth defects, cardiovascular diseases, tumors, and neurodegenerative diseases, making it a crucial micronutrient. Folic acid plays a vital role in cell proliferation, promoting human growth and development, and maintaining human health, ensuring the synthesis of proteins and DNA. Folic acid deficiency leads to megaloblastic anemia (fatigue, pallor, difficulty breathing), weakened immunity, and neural tube defects in the fetus; folic acid excess also carries risks. Therefore, folic acid testing has significant clinical diagnostic value.

[0003] Currently, mass spectrometry is considered the "gold standard" for folic acid testing. However, mass spectrometry requires complex and time-consuming sample pretreatment procedures, and clinical applications demand high levels of automation, making large-scale application difficult. Therefore, immunoassay is commonly used in clinical practice. Immunoassay is simple to operate and rapid, making it a feasible alternative to small molecule mass spectrometry. Because small molecules lack antigenic epitopes, competitive immunoassays are commonly used in clinical practice. Competitive immunoassays suffer from low sensitivity, poor specificity, a high risk of false positives, and complex processes.

[0004] Therefore, there is an urgent need to develop highly specific and sensitive monoclonal antibodies for the folic acid sandwich method to achieve folic acid detection using the double antibody sandwich method. Summary of the Invention

[0005] This application provides folic acid sandwich antibody mAb16 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] On one hand, this application provides a folic acid sandwich antibody mAb16 or its antigen-binding fragment, comprising a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises complementarity-determining regions LCDR1, LCDR2 and LCDR3, wherein the amino acid sequence of LCDR1 is ENINNY (SEQ ID NO.1), the amino acid sequence of LCDR2 is RGS, and the amino acid sequence of LCDR3 is QQGVSGSDVDNA (SEQ ID NO.3).

[0007] In some embodiments, the heavy chain variable region includes complementarity-determining regions HCDR1, HCDR2, and HCDR3, wherein the amino acid sequence of HCDR1 is GFSLSSYD (SEQ ID NO.4), the amino acid sequence of HCDR2 is IWSNDNT (SEQ ID NO.5), and the amino acid sequence of HCDR3 is ARDQGYGDRSYSFNL (SEQ ID NO.6).

[0008] In some embodiments, the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO.7 or an amino acid sequence having at least 95% identity with the amino acid sequence shown in SEQ ID NO.7; specifically, the VL is 111 amino acids in length, the number of amino acids in the four domains of its FR are 26, 17, 36 and 11 respectively, the number of amino acids in the three domains of LCDR are 6, 3 and 12 respectively, and the regions of LCDR1, LCDR2 and LCDR3 are 27aa-32aa, 50aa-52aa and 89aa-100aa respectively, and their amino acid sequences are: ENINNY (SEQ ID NO.1), RGS (SEQ ID NO.2), QQGVSGSDVDNA (SEQ ID NO.3).

[0009] In some embodiments, the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO. 8 or an amino acid sequence having at least 95% identity with the amino acid sequence shown in SEQ ID NO. 8. Specifically, the VH is 123 amino acids in length, with the number of amino acids in the four domains of its FR being 25, 17, 40, and 11, the number of amino acids in the three domains of HCDR being 8, 7, and 15, and the amino acid sequences of HCDR1, HCDR2, and HCDR3 being 26aa-33aa, 51aa-57aa, and 98aa-112aa, respectively, and their amino acid sequences being GFSLSSYD (SEQ ID NO. 4), IWSNDNT (SEQ ID NO. 5), and ARDQGYGDRSYSFNL (SEQ ID NO. 6), respectively.

[0010] In some embodiments, the antigen-binding fragment is one of F(ab')2, Fab', Fab, Fv, scFv, dsFv, bispecific antibody, and antibody minimum recognition unit; preferably, the remaining sequence of the antibody is derived from one or more species including rabbit, mouse, rat, guinea pig, hamster, ferret, cat, dog, goat, sheep, cow, pig, horse, monkey, and human.

[0011] On the other hand, this application also provides biological materials comprising polynucleotides, carriers, or cells, wherein the polynucleotides encode the folic acid sandwich antibody mAb16 or its antigen-binding fragment; the carrier carries the polynucleotide; the cells carry the polynucleotide, or contain the carrier, or are capable of expressing the folic acid sandwich antibody mAb16 or its antigen-binding fragment.

[0012] On the other hand, this application also provides a method for preparing the folic acid sandwich antibody mAb16 or its antigen-binding fragment, including culturing the cells; optionally, the cells are prepared by transforming the cells with a polynucleotide encoding a polynucleotide including the folic acid sandwich antibody mAb16 or its antigen-binding fragment, the polynucleotide including a heavy chain expression plasmid and a light chain expression plasmid, the transformation including co-transforming the heavy chain expression plasmid and the light chain expression plasmid into the cells.

[0013] In some embodiments, the cells are eukaryotic cells, preferably mammalian cells, more preferably 293 cells or CHO cells.

[0014] On the other hand, this application also provides the use of folic acid sandwich antibody mAb16 or its antigen-binding fragment or the biological material described herein in any of the following:

[0015] 1) Folic acid testing for non-diagnostic and non-therapeutic purposes;

[0016] 2) Prepare products for the detection of folic acid;

[0017] 3) Used for purifying folic acid;

[0018] 4) Prepare products for purifying folic acid.

[0019] On the other hand, this application also provides a detection reagent or detection kit containing the folic acid sandwich antibody mAb16 or its antigen-binding fragment, or the biological material.

[0020] In some embodiments, the detection kit is a double-antibody sandwich ELISA or an immunoassay kit prepared by chemiluminescence.

[0021] Compared to existing technologies, the folic acid sandwich antibody mAb16 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 8.5 × 10⁻⁶. 8 L / mol. The folic acid sandwich antibody mAb16 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 0.2 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 importance in the diagnosis and treatment of birth defects, cardiovascular diseases, tumors, and neurodegenerative diseases. Attached Figure Description

[0022] Figure 1 This is an electrophoresis diagram of the full-length amplification products of mAb16 heavy and light chains, where M is the DNA molecular weight marker.

[0023] Figure 2 The mAb16 sandwich ELISA method was used to detect cross proteins and target antigen samples. The vertical axis represents the detected OD value.

[0024] Figure 3 The standard curve was used for the chemiluminescence assay of mAb16 magnetic microparticles to detect folic acid standard antigen. 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 =0.9976, linear detection range 0-20ng / mL, the sample concentration calculation formula is derived as: y = 32805x + 8307.7.

[0025] Figure 4 The standard curve was used to detect folic acid in clinical samples using mAb16 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 =0.9374, linear detection range 0-20ng / mL, the sample concentration calculation formula is derived as: y = 15840x - 12652. Detailed Implementation

[0026] 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.

[0027] Example 1: Preparation of rabbit monoclonal antibody using the folic acid sandwich method

[0028] 1) Preparation of immunogens

[0029] 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

[0031] 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 128,000 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.

[0032] 3) PBMC isolation, specific B cell sorting, and clonal recombination

[0033] 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 volume 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.

[0034] 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 .

[0035] 4) Preparation and purification of monoclonal antibodies

[0036] 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 mAb16.

[0037] Example 2: Identification of rabbit monoclonal antibodies using the folic acid sandwich method

[0038] 1) Specificity identification of rabbit monoclonal antibodies

[0039] 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.

[0040] Figure 2 The results showed that the cross protein reacted negatively with the rabbit monoclonal antibody mAb16, with OD450 values ​​less than 0.1; the folic acid complex reacted positively with the mAb16 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.

[0041] 2) Determination of affinity constant of rabbit monoclonal antibody

[0042] Affinity constant (Ka) was determined using a non-competitive ELISA method.

[0043] 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 and coat accordingly. Incubate at 4°C for 24 h.

[0044] Blocking: Wash the plate 4 times with PBST, add BSA solution at 200 μL / well, and incubate at 37°C for 2 h;

[0045] 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;

[0046] 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 times to each well, and incubate at 37℃ for 30 min;

[0047] 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;

[0048] Detection: The absorbance value at a wavelength of 450 nm (A450nm) was measured.

[0049] An S-shaped curve was plotted with the logarithm of antibody concentration on the x-axis and OD value on the y-axis. The calculated affinity constant Ka for the folic acid sandwich monoclonal antibody mAb16 was 8.5 × 10⁻⁶. 8 L / mol.

[0050] 3) Sandwich antibody pairing

[0051] 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 mAb16 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.

[0052] Table 1. Results of antibody pairing assay

[0053]

[0054] Therefore, the antibody mAb16 involved in this application is optimal for sandwich assay.

[0055] Example 3: Analysis of the variable region gene and amino acid sequence of the 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 through sequencing.

[0056] 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 mAb16 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.

[0057] The VL is 111 amino acids long. The number of amino acids in the four domains of its FR are 26, 17, 36 and 11, respectively. The number of amino acids in the three domains of 7LCDR are 6, 3 and 12, respectively. The regions of LCDR1, LCDR2 and LCDR3 are 27aa-32aa, 50aa-52aa and 89aa-100aa, respectively. Their amino acid sequences are: ENINNY (SEQ ID NO.1), RGS (SEQ ID NO.2) and QQGVSGSDVDNA (SEQ ID NO.3).

[0058] The VH is 123 amino acids in length. The number of amino acids in the four domains of its FR are 25, 17, 40 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 98aa-112aa, respectively. Their amino acid sequences are GFSLSSYD (SEQ ID NO.4), IWSNDNT (SEQ ID NO.5) and ARDQGYGDRSYSFNL (SEQ ID NO.6), respectively.

[0059] Example 4: Folic acid sandwich method rabbit monoclonal antibody used for magnetic microparticle chemiluminescent immunoassay.

[0060] 1. Detection Principle and Method

[0061] 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.

[0062] 2. Composition of the magnetic particle chemiluminescence detection kit for folic acid detection

[0063] 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; after magnetic separation, resuspend in magnetic preservation buffer at a working concentration of 0.5 mg / ml.

[0064] 2) mAb16 conjugation with ALP: First, the 2-IT antibody mAb16 is reduced; then, the 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 preservation buffer.

[0065] 3) Washing buffer: is standard pH 7.4 PBST containing 0.05% Proclin 300, prepared as a 20-fold concentrate.

[0066] 4) Chemiluminescent colorimetric solution: purchased from Aivid Biotechnology.

[0067] 5) Sample diluent: PBST containing 1% BSA and 0.05% Proclin 300, filtered for sterilization.

[0068] 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 as diluent, filtered for sterilization and aseptically dispensed.

[0069] 3. Testing of folic acid in clinical samples

[0070] Clinical samples with different folic acid concentrations were processed. Using folic acid-binding protein as the coating antibody and mAb16 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 .

[0071] 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 1-80 pmol / L.

[0072] In summary, when the folic acid sandwich rabbit monoclonal antibody mAb16 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 0.2 ng / ml. When detecting clinical samples by magnetochemiluminescence, the clinical composite rate is >0.95 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.

[0073] 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 mAb16 or its antigen-binding fragment, characterized in that, It includes a light chain variable region and a heavy chain variable region. The light chain variable region includes complementarity-determining regions LCDR1, LCDR2, and LCDR3. The amino acid sequence of LCDR1 is shown in SEQ ID NO.1, the amino acid sequence of LCDR2 is RGS, and the amino acid sequence of LCDR3 is shown in SEQ ID NO.

3. The heavy chain variable region includes complementarity-determining regions 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, and the amino acid sequence of HCDR3 is shown in SEQ ID NO.

6.

2. The folic acid sandwich antibody mAb16 or its antigen-binding fragment according to claim 1, characterized in that, The light chain variable region includes an amino acid sequence that has at least 95% identity with the amino acid sequence shown in SEQ ID NO:

7.

3. The folic acid sandwich antibody mAb16 or its antigen-binding fragment according to claim 2, characterized in that, The light chain variable region includes an amino acid sequence as shown in SEQ ID NO.

7.

4. The folic acid sandwich antibody mAb16 or its antigen-binding fragment according to claim 1, characterized in that, The heavy chain variable region includes an amino acid sequence that has at least 95% identity with the amino acid sequence shown in SEQ ID NO:

8.

5. The folic acid sandwich antibody mAb16 or its antigen-binding fragment according to claim 4, characterized in that, The heavy chain variable region includes an amino acid sequence as shown in SEQ ID NO:

8.

6. The folic acid sandwich antibody mAb16 or its antigen-binding fragment according to any one of claims 1-5, characterized in that, The antigen-binding fragment is selected from any one of F(ab')2, Fab', Fab, Fv, scFv and dsFv.

7. A biomaterial, characterized in that, The biomaterial includes a polynucleotide, a carrier, or a cell, wherein the polynucleotide encodes the folic acid sandwich antibody mAb16 or its antigen-binding fragment as described in any one of claims 1-6; the carrier carries the polynucleotide; the cell carries the polynucleotide, or contains the carrier, or is capable of expressing the folic acid sandwich antibody mAb16 or its antigen-binding fragment as described in any one of claims 1-6.

8. The method for preparing folic acid sandwich antibody mAb16 or its antigen-binding fragment according to any one of claims 1-6, characterized in that, Includes culturing the cells as described in claim 7; The cells are prepared by converting cells with a polynucleotide encoding a folic acid sandwich antibody mAb16 or its antigen-binding fragment thereof. The polynucleotide includes a heavy chain expression plasmid and a light chain expression plasmid. The conversion includes co-converting the heavy chain expression plasmid and the light chain expression plasmid into the cells.

9. The preparation method according to claim 8, characterized in that, The cells in question are eukaryotic cells.

10. The preparation method according to claim 9, characterized in that, The cells in question are mammalian cells.

11. The preparation method according to claim 10, characterized in that, The cells are either 293 cells or CHO cells.

12. The use of the folic acid sandwich antibody mAb16 or its antigen-binding fragment according to any one of claims 1-6, or the biomaterial according to claim 7, in any of the following: 1) Folic acid testing for non-diagnostic and non-therapeutic purposes; 2) Prepare products for folic acid detection; 3) Used for purifying folic acid; 4) Prepare products for purifying folic acid.

13. A detection reagent or detection kit, characterized in that, The detection reagent or kit comprises the folic acid sandwich antibody mAb16 or its antigen-binding fragment as described in any one of claims 1-6, or the biological material as described in claim 7.

14. The detection reagent or detection kit according to claim 13, characterized in that, The detection kit is an immunoassay kit prepared by double antibody sandwich ELISA or chemiluminescence method.

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