Anti-phytase monoclonal antibody or antigen-binding fragment thereof, kit and application thereof

Abstract

The invention provides an anti-phytase monoclonal antibody or an antigen-binding fragment thereof, a kit and an application thereof. The invention adopts a flow cytometer to sort single B cells and successfully screens out anti-phytase specific antibodies. The recombinant antibodies obtained by screening are functionally verified using an ELISA method to verify that they have a specific binding ability with a phytase antigen. These recombinant antibodies have high activity, good stability, and strong specificity, and can be used as reference standards for qualitative detection of phytase positivity.

Description

Technical Field

[0001] The patent of the present invention relates to an anti-phytase monoclonal antibody or its antigen binding fragment, a kit and its application, and belongs to the field of bioengineering technology. Background Art

[0002] The antibody molecules screened naturally by traditional hybridomas have advantages, but the platform efficiency is low. The single-cell photoconductive system is expensive. Protein display technologies such as phage display, yeast display, and mammalian cell display avoid the shortcomings of instability and repeated subcloning of fusion hybridomas, and are widely used in the screening and preparation of recombinant antibodies, providing a broad space for the development of monoclonal antibodies. However, in most display systems, the constructed antibody library is a random combination of heavy chain and light chain genes, so it is difficult to screen antibodies with high specificity of the original pairing of heavy and light chains.

[0003] The currently widely used method for separating single B cells is flow cytometry fluorescence sorting (FACS), which can separate antigen-specific B cells. This is a widely used technology based on flow cytometry, which detects specific molecules on the surface of B cells by fluorescently labeling proteins that recognize specific molecules to screen out single B cells. Its principle can also be considered as the specific binding of antigens and antibodies. This method retains the natural pairing of light and heavy chain variable regions, has the advantages of good genetic diversity, high efficiency, and a small number of cells required. It is one of the newest and most efficient antibody screening methods.

[0004] Phytase is an enzyme that can catalyze the hydrolysis of phytic acid (also known as inositol hexaphosphate). It is an extracellular enzyme that is widely present in nature and is found in animals, plants, and microorganisms. Phytase has been found in many plant tissues (such as grains, beans, and vegetables), especially in germinating seeds and pollen. In addition, there are many types of microorganisms that produce phytase in nature, such as bacteria, molds, and fungi. Phytase can hydrolyze phosphate residues from phytic acid, thereby destroying the strong affinity of phytic acid for mineral elements, so phytase can increase the nutritional value of mineral elements. However, there are relatively few publicly available detection antibodies for phytase. Summary of the invention

[0005] The purpose of the present invention is to provide an anti-phytase monoclonal antibody or an antigen-binding fragment thereof, which has high affinity to phytase.

[0006] The technical solution adopted by the present invention is:

[0007] An anti-phytase monoclonal antibody or an antigen-binding fragment thereof, comprising the protein described in a1 or a2 below:

[0008] a1: The amino acid sequence of the heavy chain CDR1 of the monoclonal antibody is shown in SEQ ID NO.1, the amino acid sequence of CDR2 is shown in SEQ ID NO.2, and the amino acid sequence of CDR3 is shown in SEQ ID NO.3; the amino acid sequence of the light chain CDR1 is shown in SEQ ID NO.4, the amino acid sequence of CDR2 is shown in SEQ ID NO.5, and the amino acid sequence of CDR3 is shown in SEQ ID NO.6;

[0009] a2: The amino acid sequence of the heavy chain CDR1 of the monoclonal antibody is shown in SEQ ID NO.7, the amino acid sequence of CDR2 is shown in SEQ ID NO.8, and the amino acid sequence of CDR3 is shown in SEQ ID NO.9; the amino acid sequence of the light chain CDR1 is shown in SEQ ID NO.10, the amino acid sequence of CDR2 is shown in SEQ ID NO.11, and the amino acid sequence of CDR3 is shown in SEQ ID NO.12.

[0010] Preferably, it comprises the protein described in b1 or b2 below:

[0011] b1: The amino acid sequence of the heavy chain variable region of the monoclonal antibody includes the sequence shown in SEQ ID NO.13; or an amino acid sequence having at least 80%, 85%, 90% or 95% sequence identity with SEQ ID NO.13 and having the same or similar affinity as phytase; the amino acid sequence of the light chain variable region of the monoclonal antibody includes the sequence shown in SEQ ID NO.14; or an amino acid sequence having at least 80%, 85%, 90% or 95% sequence identity with SEQ ID NO.14 and having the same or similar affinity as phytase;

[0012] b2: The amino acid sequence of the heavy chain variable region of the monoclonal antibody includes the sequence shown in SEQ ID NO.15; or an amino acid sequence that has at least 80%, 85%, 90% or 95% sequence identity with SEQ ID NO.15 and has the same or similar affinity as phytase, and the amino acid sequence of the light chain variable region of the monoclonal antibody includes the sequence shown in SEQ ID NO.16; or an amino acid sequence that has at least 80%, 85%, 90% or 95% sequence identity with SEQ ID NO.16 and has the same or similar affinity as phytase.

[0013] In the present invention, the amino acid sequence of the heavy chain variable region of the monoclonal antibody in b1 includes the sequence shown in SEQ ID NO.13, which includes the highly variable region (CDRs) and the framework region of the heavy chain variable region, wherein the framework region may have at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity with the corresponding framework region in the sequence shown in SEQ ID NO.13. The framework region of the light chain variable region of the monoclonal antibody in b1 may have at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity with the corresponding framework region in the sequence shown in SEQ ID NO.14. The framework region of the heavy chain variable region of the monoclonal antibody in b2 may have at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity with the corresponding framework region in the sequence shown in SEQ ID NO.15. The framework region of the light chain variable region of the monoclonal antibody in b2 may have at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or more sequence identity with the corresponding framework region in the sequence shown in SEQ ID NO.16.

[0014] Preferably, the monoclonal antibody or antigen-binding fragment thereof further comprises a constant region.

[0015] Preferably, the constant region comprises a heavy chain constant region and a light chain constant region; the constant region is selected from the constant region of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD.

[0016] Preferably, the antibody is derived from cattle, horses, pigs, sheep, goats, rats, mice, rabbits, chickens, ducks or humans, more preferably mice.

[0017] The coding gene of the above-mentioned anti-phytase monoclonal antibody or its antigen-binding fragment.

[0018] The expression vector or host bacteria of the above-mentioned anti-phytase monoclonal antibody or its antigen-binding fragment.

[0019] The use of the above anti-phytase monoclonal antibody or its antigen-binding fragment in detecting the phytase content. The content here is not limited to absolute content, but also includes relative content, and the situation of reflecting the presence of phytase (such as positive reaction).

[0020] The invention also discloses an antibody conjugate, which comprises the above-mentioned monoclonal antibody or antigen-binding fragment thereof.

[0021] Preferably, the monoclonal antibody or its antigen-binding fragment is coupled to biotin or a label. The label is selected from fluorescent dyes, enzymes, radioisotopes, chemiluminescent agents or nanoparticle labels, etc. More preferably, the monoclonal antibody or its antigen-binding fragment is coupled to biotin.

[0022] The invention also discloses a kit containing the anti-phytase monoclonal antibody or its antigen binding fragment or antibody conjugate.

[0023] Preferably, the kit is an immunofluorescence detection kit, an immunohistochemistry detection kit, a flow cytometry detection kit, a protein immunoblotting detection kit or an ELISA detection kit.

[0024] The present invention also discloses a method for detecting the content of phytase in a sample, wherein the monoclonal antibody described in a1 or b1 is used as a coating antibody, the monoclonal antibody described in a2 or b2 is used as a detection antibody, and the ELISA method is used for detection. In some embodiments of the present invention, the monoclonal antibody described in b1 is used as a coating antibody, the monoclonal antibody described in b2 is used as a detection antibody, and the ELISA method is used for detection. In some embodiments of the present invention, when the monoclonal antibody described in b2 is used as a detection antibody, the monoclonal antibody described in b2 is coupled to biotin.

[0025] As used herein, identity refers to the identity of an amino acid sequence or a nucleotide sequence. Percent sequence identity can be calculated by any method known in the art, for example using the BLOSUM62 matrix, with reference to Henikoff et al. NAS, 89 (22):10915-10919 (1992) The method described in .

[0026] In the present invention, as used herein, the terms "CDR", "CDRs" or "complementarity determining region" refer to the highly variable regions of the heavy and light chains of immunoglobulins, and refer to regions containing one or more or even all of the major amino acid residues that play a role in the binding of an antibody or antigen-binding fragment to an antigen or epitope it recognizes. In a specific embodiment of the present disclosure, CDRs refer to the highly variable regions of the heavy and light chains of an antibody. In the present invention, the "FR" or "framework region" region includes the heavy chain framework region and the light chain framework region, and refers to the region other than CDR in the heavy chain variable region and the light chain variable region of an antibody; wherein the heavy chain framework region can be further subdivided into adjacent regions separated by CDRs, including HFR1, HFR2, HFR3 and HFR4 framework regions; the light chain framework region can be further subdivided into adjacent regions separated by CDRs, including LFR1, LFR2, LFR3 and LFR4 framework regions. In the present invention, the heavy chain variable region of an anti-phytase monoclonal antibody includes a highly variable region and a framework region; and its light chain variable region includes a highly variable region and a framework region.

[0027] The present invention uses phytase antigen to perform multiple phytase immunization on experimental mice, and performs serum titer test after immunization; and separates the spleen tissue of mice with high titer, performs single B cell sorting and culture; extracts RNA from positive clones and reversely transcribes them into cDNA, and amplifies antibody VH and VL gene fragments therein. The VH and VL gene fragments amplified in vitro are cloned into an expression vector containing mouse IgG constant region for recombinant expression. The recombinant antibodies are functionally verified by ELISA method, and finally mouse monoclonal antibodies with good specificity and strong affinity are screened out. The recombinant antibodies obtained by screening are functionally verified by ELISA method, and the recombinant antibodies are verified to have specific binding ability with phytase antigen. These recombinant antibodies have high activity, good stability, and strong specificity, and can be used as reference standards for qualitative detection of phytase positive. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is the standard curve of sandwich Elisa for detecting phytase. DETAILED DESCRIPTION

[0029] The specific implementation modes of the present invention are further described below in conjunction with the accompanying drawings, but the description of the embodiments does not impose any limitation on the protection scope of the present invention.

[0030] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by technicians in the technical field to which the present invention belongs. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0031] Unless otherwise specified, the materials and instruments used in the following examples can be obtained from conventional commercial channels.

[0032] The screening process of the anti-phytase high-affinity monoclonal antibody described in the present invention is as follows:

[0033] (1) Using naturally extracted phytase (Hubei Changyao Biological, cas37288-11-2) as an antigen, experimental Balb / c mice were immunized with phytase multiple times, and serum titer was tested after immunization. The spleen tissue of immunized Balb / c mice with high serum titer was aseptically separated, and spleen cell suspension was obtained after red lysis. Through B cell staining and flow sorting, 1045 single B cell clones were isolated and cultured (11×95 wells, 1 well per 96-well plate as a control), and positive clones that specifically recognized the antigen were screened out by ELISA after 13-14 days of culture.

[0034] (2) Use the PrimeScript™ II 1st Strand cDNA Synthesis Kit (Takara, catalog number 6210A) to reverse transcribe the RNA of the single B cell positive clone into cDNA, and amplify the antibody VH and VL gene fragments. The VH and VL gene fragments amplified in vitro are cloned into an expression vector containing the mouse IgG constant region through cloning technology for recombinant expression.

[0035] (3) The expressed recombinant antibody was functionally verified using the ELISA method to verify its specific binding ability with the phytase antigen. Finally, monoclonal antibodies with good specificity and strong affinity were screened out.

[0036] Example 1 Preparation of phytase mouse monoclonal antibody

[0037] 1. Animal immunization

[0038] Balb / c mice were selected as immunized animals. For the first immunization, 50, 100 or 200 μg of phytase was emulsified with an equal volume of complete Freund's adjuvant and injected subcutaneously into Balb / c mice. Seven days later, the second immunization was performed, and 50, 100 or 200 μg of phytase was emulsified with an equal volume of incomplete Freund's adjuvant and injected subcutaneously into Balb / c mice. Seven days later, the third immunization was performed, and 50, 100 or 200 μg of phytase was emulsified with an equal volume of incomplete Freund's adjuvant and injected subcutaneously into Balb / c mice. Seven days later, the fourth immunization was performed, and 50, 100 or 200 μg of phytase was emulsified with an equal volume of incomplete Freund's adjuvant and injected subcutaneously into Balb / c mice. After four immunizations, the serum titer was determined by ELISA, and the results are shown in Table 1.

[0039] Table 1 Elisa detection of phytase titer in mice

[0040]

[0041] 2. B lymphocyte acquisition

[0042] Take one immune mouse with high serum titer, separate spleen tissue aseptically, treat with red blood cell lysis buffer and centrifuge, wash cells once with sterile 1640 culture medium, and count suspended cells.

[0043] 3. Feeder cell laying

[0044] Resuscitated feeder cells (NIH / 3T3 cells overexpressing CD40L, treated with 5ug / mL mitomycin for 4hrs) were plated in 96-well plates at 1.4E4 / well in 100μL culture medium. The culture medium was IMDM+12.5%FBS+50ng / mL mIL-21 (mouse interleukin 21)+50ng / mL mIL-2 (mouse interleukin 2)+2%PS.

[0045] 4. B lymphocyte sorting and culture

[0046] 4.1 Splenocytes were resuspended in PBS + 0.5% BSA, and memory B cells were sorted using the Memory B Cell Isolation Kit (Miltenyi Biotec, 130-095-838). Memory B cells were then incubated with 10ug / mL phytase-Biotin coupled to PE Streptavidin (1:4) and LIVE / DEAD™ Fixable Purple Dead Cell Staining Kit (Invitrogen, L34955) (1:500) at 4°C for 20min. Resuspend in 10 times the volume of PBS + 0.5% BSA and centrifuge at 1600rpm for 5min at room temperature.

[0047] 4.2 Resuspend cells in IMDM + 17.5% FBS, select lymphocytes using FSC-A / SSC-A, and remove adherent cells using FSC-W / FSC-A. Select APC-positive, PE-positive and 405-negative B cells.

[0048] 4.3 By flow cytometry, one B cell was added to each well of a 96-well plate containing 100 μL of feeder cell culture medium and cultured at 37°C, 5% CO2 for 14 days. During this period, the culture medium was replenished once, 100 μL / well. The culture medium was IMDM + 12.5% ​​FBS + 50 ng / mL mIL-21 + 50 ng / mL mIL-2 + 1% PS.

[0049] 5. Identification of B lymphocytes

[0050] ELISA was performed on the supernatant of cells cultured on day 14. ELISA plates were coated with 100 μL (1 μg / mL) of phytase antigen per well and incubated at 4°C overnight.

[0051] Discard the liquid in the ELISA plate and wash each well three times with 300 μL of PBST. Add 300 μL of 1% BSA (dissolved in PBST) blocking solution to each well and block at room temperature for 2 hours. Discard the liquid in the ELISA plate and wash each well once with 300 μL of PBST. Add 100 μL of the twice-diluted supernatant to be tested to each well and incubate at 37°C for 1 hour. Discard the liquid in the ELISA plate and wash each well three times with 300 μL of PBST. Add 100 μL of secondary antibody diluted in blocking solution (Peroxidase AffiniPure Goat Anti-Mouse IgG(H+L), 33201ES60, brand: Yeasen) to each well and incubate at room temperature for 1 hour. Discard the liquid in the ELISA plate and wash each well three times with 300 μL of PBST. Add 100 μL TMB to each well, incubate at 37°C for 5-10 minutes, and then add 50 μL 1 M H2SO4 to each well to terminate the reaction. Use a microplate reader to read the values ​​at 450 nm-630 nm, and obtain 306 monoclonal clones with OD values ​​> 0.4. Select 2 clones with OD values ​​> 2.2 for antibody gene sequence retrieval.

[0052] 6. Amplification and sequencing analysis of variable regions of positive cloned antibodies

[0053] 6.1 The two B cells identified as positive were collected, and RNA was extracted using the PureLink RNA Mini Kit (invitrogen, 12183018A) and then reverse transcribed into cDNA. PCR amplification was performed using amplification primers, and the amplified product was subcloned into an expression vector containing the mouse IgG constant region for sequencing analysis. The heavy chain and light chain sequences of the antibody are shown in Table 2-3. After 4 days of transient expression of the recombinant antibody using Expi293, the supernatant was collected for Elisa detection.

[0054] Table 2 CDR sequences of heavy and light chains of antibodies

[0055]

[0056] Table 3 Antibody numbers and their corresponding heavy / light chain variable region sequences

[0057]

[0058] Example 2 Functional verification of 293 transient expression supernatant

[0059] Coat the ELISA plate, add 100 μL (1 μg / mL) of phytase antigen to each well, and incubate overnight at 4°C. Discard the liquid in the ELISA plate, and wash each well with 300 μL of PBST. Add 300 μL of 1% BSA (dissolved in PBS) blocking solution to each well and block at room temperature for 2 hours. Discard the liquid in the ELISA plate, and wash each well three times with 300 μL of PBST. Add 100 μL of the gradient diluted recombinant antibody 2B10 or 3B9 transient transfection supernatant to each well and incubate at room temperature for 1 hour. Discard the liquid in the ELISA plate, and wash each well three times with 300 μL of PBST. Add 100 μL of secondary antibody (peroxidase-labeled goat anti-mouse IgG (H+L) Peroxidase AffiniPure Goat Anti-Mouse IgG (H+L), 33201ES60, brand: Yeasen) diluted in blocking solution to each well and incubate at room temperature for 1 hour. Discard the liquid in the ELISA plate and wash each well three times with 300 μL of PBST. Add 100 μL of TMB to each well, incubate at 37°C for 5-10 minutes, and then add 50 μL of 1 M H2SO4 to each well to terminate the reaction. Use an ELISA reader to read the values ​​at 450 nm-630 nm, and the test results are shown in Table 4. As can be seen from Table 4, the screened anti-phytase monoclonal antibody expression supernatant has a strong specific binding ability with phytase. At present, there are relatively few patented antibodies for phytase, and the present invention fills the gap in this field. Use Protein G (rProteinG Seplife 4FF, SEPLIFE, A4083105) to routinely purify the above-mentioned 2B10 or 3B9 transient supernatant for subsequent experiments.

[0060] Table 4 Phytase titer test results

[0061]

[0062] Example 3 Sandwich Elisa for Detection of Phytase

[0063] Coat the ELISA plate, add 100 μL (4 μg / mL) of phytase-coated antibody 2B10 to each well, and incubate overnight at 4°C. Discard the liquid in the ELISA plate, and wash each well three times with 300 μL of PBST. Add 300 μL of 1% BSA (dissolved in PBS) blocking solution to each well and block at room temperature for 2 hours. Discard the liquid in the ELISA plate, and wash each well three times with 300 μL of PBST. Add 100 μL of antigen diluted in blocking solution to each well, and incubate at room temperature for 1 hour. Discard the liquid in the ELISA plate, and wash each well three times with 300 μL of PBST. Add 100 μL of detection antibody 3B9-Biotin (pre-biotin labeled) diluted in blocking solution, 40 ng / mL, to each well, and incubate at room temperature for 1 hour. Wash each well three times with 300 μL of PBST. Incubate with secondary antibody (horseradish peroxidase-labeled streptavidin B110053-0100, brand: Biotech) diluted in blocking solution at room temperature for 40 minutes. Discard the liquid in the ELISA plate and wash each well three times with 300 μL of PBST. Add 100 μL of TMB to each well, incubate at 37°C for 5-10 minutes, and then add 50 μL of 1 M H2SO4 to each well to terminate the reaction. Use an ELISA reader to read the values ​​at 450 nm-630 nm. The test results are shown in Table 5.

[0064] Table 5 Results of sandwich Elisa testing of phytase standards

[0065]

[0066] STD (standard curve) detection starts at 8000pg / ml and dilutes 11 samples in half (duplicate wells), see Table 5. As shown in Table 5, sandwich Elisa can accurately detect phytase at a minimum of 31pg / mL, and the linear range of the standard curve is 31.25pg / mL~1ng / mL ( Figure 1 ).

[0067] In summary, the monoclonal antibody screened in the present invention has a strong specific binding ability with the phytase antigen, and the obtained recombinant antibody has high activity, good stability, and strong specificity, and can be used as a reference standard for qualitative detection of phytase positivity. It can be subsequently developed into an Elisa detection kit.

[0068] The applicant declares that the present invention illustrates the detailed method of the present invention through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned detailed method, that is, it does not mean that the present invention must rely on the above-mentioned detailed method to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of various raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. A kit, characterized in that: Containing the anti-phytase monoclonal antibody or its antigen-binding fragment shown in a1, and the anti-phytase monoclonal antibody or its antigen-binding fragment shown in a2; a1: the amino acid sequence of the heavy chain CDR1 of the anti-phytase monoclonal antibody or the antigen-binding fragment thereof is shown in SEQ ID NO.1, the amino acid sequence of CDR2 is shown in SEQ ID NO.2, and the amino acid sequence of CDR3 is shown in SEQ ID NO.3; the amino acid sequence of the light chain CDR1 is shown in SEQ ID NO.4, the amino acid sequence of CDR2 is shown in SEQ ID NO.5, and the amino acid sequence of CDR3 is shown in SEQ ID NO.6; a2: The amino acid sequence of the heavy chain CDR1 of the anti-phytase monoclonal antibody or its antigen-binding fragment is shown in SEQ ID NO.7, the amino acid sequence of CDR2 is shown in SEQ ID NO.8, and the amino acid sequence of CDR3 is shown in SEQ ID NO.9; the amino acid sequence of the light chain CDR1 is shown in SEQ ID NO.10, the amino acid sequence of CDR2 is shown in SEQ ID NO.11, and the amino acid sequence of CDR3 is shown in SEQ ID NO.

12.

2. The kit according to claim 1, characterized in that: The amino acid sequence of the heavy chain variable region of the anti-phytase monoclonal antibody or antigen-binding fragment thereof shown in a1 includes the sequence shown in SEQ ID NO.13, or an amino acid sequence having at least 80%, 85%, 90% or 95% sequence identity with SEQ ID NO.13 and having the same or similar affinity as phytase; the amino acid sequence of the light chain variable region includes the sequence shown in SEQ ID NO.14, or an amino acid sequence having at least 80%, 85%, 90% or 95% sequence identity with SEQ ID NO.14 and having the same or similar affinity as phytase; The amino acid sequence of the heavy chain variable region of the anti-phytase monoclonal antibody or antigen-binding fragment thereof shown in a2 includes the sequence shown in SEQ ID NO.15, or an amino acid sequence that has at least 80%, 85%, 90% or 95% sequence identity with SEQ ID NO.15 and has the same or similar affinity as phytase; the amino acid sequence of the light chain variable region includes the sequence shown in SEQ ID NO.16, or an amino acid sequence that has at least 80%, 85%, 90% or 95% sequence identity with SEQ ID NO.16 and has the same or similar affinity as phytase.

3. The kit according to claim 1 or 2, characterized in that: The anti-phytase monoclonal antibody or antigen-binding fragment thereof shown in a1 or a2 further includes a constant region.

4. The kit according to claim 3, characterized in that: The constant region includes a heavy chain constant region and a light chain constant region; the constant region is selected from the constant region of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD.

5. A kit, characterized in that: Containing two antibody conjugates, wherein one antibody conjugate contains the anti-phytase monoclonal antibody or its antigen-binding fragment shown in a1, and the other antibody conjugate contains the anti-phytase monoclonal antibody or its antigen-binding fragment shown in a2; a1: the amino acid sequence of the heavy chain CDR1 of the anti-phytase monoclonal antibody or the antigen-binding fragment thereof is shown in SEQ ID NO.1, the amino acid sequence of CDR2 is shown in SEQ ID NO.2, and the amino acid sequence of CDR3 is shown in SEQ ID NO.3; the amino acid sequence of the light chain CDR1 is shown in SEQ ID NO.4, the amino acid sequence of CDR2 is shown in SEQ ID NO.5, and the amino acid sequence of CDR3 is shown in SEQ ID NO.6; a2: The amino acid sequence of the heavy chain CDR1 of the anti-phytase monoclonal antibody or its antigen-binding fragment is shown in SEQ ID NO.7, the amino acid sequence of CDR2 is shown in SEQ ID NO.8, and the amino acid sequence of CDR3 is shown in SEQ ID NO.9; the amino acid sequence of the light chain CDR1 is shown in SEQ ID NO.10, the amino acid sequence of CDR2 is shown in SEQ ID NO.11, and the amino acid sequence of CDR3 is shown in SEQ ID NO.

12.

6. The kit according to claim 5, characterized in that: The amino acid sequence of the heavy chain variable region of the anti-phytase monoclonal antibody or antigen-binding fragment thereof shown in a1 includes the sequence shown in SEQ ID NO.13, or an amino acid sequence having at least 80%, 85%, 90% or 95% sequence identity with SEQ ID NO.13 and having the same or similar affinity as phytase; the amino acid sequence of the light chain variable region includes the sequence shown in SEQ ID NO.14, or an amino acid sequence having at least 80%, 85%, 90% or 95% sequence identity with SEQ ID NO.14 and having the same or similar affinity as phytase; The amino acid sequence of the heavy chain variable region of the anti-phytase monoclonal antibody or antigen-binding fragment thereof shown in a2 includes the sequence shown in SEQ ID NO.15, or an amino acid sequence that has at least 80%, 85%, 90% or 95% sequence identity with SEQ ID NO.15 and has the same or similar affinity as phytase; the amino acid sequence of the light chain variable region includes the sequence shown in SEQ ID NO.16, or an amino acid sequence that has at least 80%, 85%, 90% or 95% sequence identity with SEQ ID NO.16 and has the same or similar affinity as phytase.

7. The kit according to claim 5 or 6, characterized in that: The anti-phytase monoclonal antibody or antigen-binding fragment thereof shown in a1 or a2 further includes a constant region.

8. The kit according to claim 7, characterized in that: The constant region includes a heavy chain constant region and a light chain constant region; the constant region is selected from the constant region of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD.

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