A hybridoma cell line and an antibody for detecting rituximab and its biosimilars, and their applications

Abstract

The present invention relates to a hybridoma cell line and an antibody for detecting rituximab and its biosimilars, and their applications, belonging to the technical field of biological detection. The hybridoma cell line provided by the present invention is a combination of hybridoma cell lines, including hybridoma cell line RTX-9C02 and hybridoma cell line RTX-5B12, both of which can secrete monoclonal antibodies that specifically bind to rituximab. The produced monoclonal antibodies, RTX-9C02 antibody and RTX-5B12 antibody, can be jointly used to detect the blood drug concentration of rituximab and its biosimilars, achieving the effect of stably, accurately and highly specifically detecting rituximab and its biosimilars.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological detection, and particularly relates to a hybridoma cell line and an antibody for detecting rituximab and its biosimilars, and their applications. Background Art

[0002] Rituximab (trade name MabThera, developed by Roche), is a human-mouse chimeric monoclonal antibody that acts on human CD20. Since CD20 is mainly expressed on the surface of B lymphocytes, rituximab can be used to treat diseases caused by excessive B lymphocytes, including lymphoma, chronic lymphocytic leukemia, transplant rejection, and certain autoimmune diseases. To ensure the effectiveness and safety of rituximab, monitoring of its blood drug concentration is crucial. Among them, the rituximab drug that has already bound to CD20 will be cleared more rapidly, so the correlation between the blood drug concentration of free rituximab and the drug efficacy is more closely related.

[0003] However, in current domestic clinical practice, the monitoring of rituximab blood drug concentration is rarely carried out, which is related to the cumbersome detection method of rituximab blood drug concentration and people's lack of awareness of blood drug concentration monitoring. The main detection and analysis methods of rituximab are ELISA and LC-MS methods. However, the LC-MS method has a long analysis time and complex pretreatment operations, which are difficult to meet the requirements of high efficiency in clinical testing, and usually can only measure the total rituximab drug concentration. The ELSIA method has low automation and long testing time, and cannot meet the clinical requirements of high throughput, rapid and accurate detection. For example, the prior art CN 111239273 A discloses a scheme for measuring rituximab by mass spectrometry, which requires multiple steps such as long-term overnight enzymatic digestion. CN 112661816 A uses a fusion protein related to the CD20 sequence to capture rituximab, and then uses anti-human IgG for detection. This method is affected by a large amount of endogenous irrelevant IgG, and the cross-reaction of this artificial antigen with other CD20 monoclonal antibodies has not been evaluated.

[0004] Therefore, there is currently a lack of a free blood drug concentration detection method for rituximab and its biosimilars with high specificity, simplicity, rapidity, and high automation. Summary of the Invention

[0005] In view of this, the purpose of the present invention is to provide a hybridoma cell line and an antibody for detecting rituximab and its biosimilars, and their applications. By secreting a monoclonal antibody that specifically binds to rituximab from the hybridoma cell line, and then using the secreted specific monoclonal antibody to detect rituximab and its biosimilars, the blood drug concentration of rituximab and its biosimilars can be detected rapidly, accurately, precisely, and with high specificity by using the fully automated chemiluminescence method.

[0006] The present invention provides a hybridoma cell line combination that secretes monoclonal antibodies against rituximab, and the hybridoma cell line combination includes hybridoma cell line RTX-9C02 and hybridoma cell line RTX-5B12;

[0007] The hybridoma cell line RTX-9C02 is deposited in the General Microbiology Center of the China Committee for Culture Collection of Microorganisms, and the deposit number is CGMCC No. 46301;

[0008] The hybridoma cell line RTX-5B12 is deposited in the General Microbiology Center of the China Committee for Culture Collection of Microorganisms, and the deposit number is CGMCC No. 46302.

[0009] The present invention provides the application of the above-mentioned hybridoma cell line combination in the preparation of monoclonal antibodies that specifically bind to rituximab and its biosimilars.

[0010] The present invention provides a monoclonal antibody combination that specifically binds to rituximab and its biosimilars. The monoclonal antibody combination is secreted by the above-mentioned hybridoma cell line combination, and the monoclonal antibody combination includes RTX-9C02 antibody and RTX-5B12 antibody.

[0011] The present invention provides the application of the above-mentioned hybridoma cell line combination or the above-mentioned monoclonal antibody combination in the preparation of products for detecting rituximab and its biosimilars.

[0012] Preferably, the product includes a kit.

[0013] Preferably, the detection of rituximab and its biosimilars is for detecting the blood drug concentration of rituximab and its biosimilars.

[0014] The present invention provides a kit for detecting rituximab and its biosimilars, and the kit includes the above-mentioned monoclonal antibody combination.

[0015] Preferably, the RTX-5B12 antibody is a capture antibody, and the RTX-9C02 antibody is a detection antibody.

[0016] Preferably, the kit further includes magnetic microparticles and alkaline phosphatase.

[0017] Preferably, the mass ratio of the magnetic microparticles to the capture antibody is 50∶(0.2 - 2), and the mass ratio of the alkaline phosphatase to the detection antibody is 1∶(0.2 - 1).

[0018] Advantages of the present invention:

[0019] The hybridoma cell line combination provided by the present invention can secrete monoclonal antibodies that specifically bind to rituximab and its biosimilars. When the secreted monoclonal antibodies are used in combination, based on immunological principles and the fully automated chemiluminescence method, the blood drug concentration of rituximab and its biosimilars can be detected, achieving a detection effect that is fast, accurate, stable, and highly specific.

[0020] Biological preservation information

[0021] The hybridoma cell line RTX-9C02, classified and named as hybridoma cell line Mus musculus ; It was deposited at the General Microbiology Center of the China National Committee for Culture Collection of Microorganisms on December 17, 2024; The deposit address is: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, and the deposit number is: CGMCC No. 46301.

[0022] The hybridoma cell line RTX-5B12, classified and named as hybridoma cell line Mus musculus ; It was deposited at the General Microbiology Center of the China National Committee for Culture Collection of Microorganisms on December 17, 2024; The deposit address is: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, and the deposit number is: CGMCC No. 46302. Brief description of the drawings

[0023] Figure 1 It is a SDS-PAGE identification result diagram of each raw material and product in the preparation process of the rituximab (Fab)2 fragment immunogen.

[0024] Figure 2 It is a standard curve for detecting the blood drug concentration of rituximab by the magnetic particle luminescence method. Detailed implementation manners

[0025] The present invention provides a hybridoma cell line combination that secretes monoclonal antibodies against rituximab. The hybridoma cell line combination includes the hybridoma cell line RTX-9C02 and the hybridoma cell line RTX-5B12;

[0026] The hybridoma cell line RTX-9C02 is deposited at the General Microbiology Center of the China National Committee for Culture Collection of Microorganisms, and the deposit number is CGMCC No. 46301;

[0027] The hybridoma cell line RTX-5B12 is deposited at the General Microbiology Center of the China National Committee for Culture Collection of Microorganisms, and the deposit number is CGMCC No. 46302.

[0028] The hybridoma cell line provided by the present invention is obtained by immunizing an animal with the (Fab)2 fragment of rituximab, then fusing the spleen cells of the animal with myeloma cells, and further screening the hybridoma cells obtained by fusion with rituximab.

[0029] The present invention provides the application of the described hybridoma cell line combination in the preparation of monoclonal antibodies that specifically bind to rituximab and its biosimilars.

[0030] The present invention provides a monoclonal antibody combination that specifically binds to rituximab and its biosimilars. The monoclonal antibody combination is secreted by the described hybridoma cell line combination. The monoclonal antibody combination includes the RTX-9C02 antibody and the RTX-5B12 antibody. The RTX-9C02 antibody is secreted by the RTX-9C02 hybridoma cell line, and the RTX-5B12 antibody is secreted by the RTX-5B12 hybridoma cell line.

[0031] In the present invention, the steps for the hybridoma cell line to secrete monoclonal antibodies include: injecting the hybridoma cell line into the abdominal cavity of a mouse, then collecting the obtained ascites, and purifying to obtain the monoclonal antibody.

[0032] The present invention provides the application of the described hybridoma cell line combination or the described monoclonal antibody combination in the preparation of products for detecting rituximab and its biosimilars.

[0033] In the present invention, the product preferably includes a kit, and the kit preferably includes an ELISA kit. Preferably, the detection of rituximab and its biosimilars is for detecting the blood drug concentration of rituximab and its biosimilars. The detection sample is preferably a serum sample or a plasma sample.

[0034] The hybridoma cell line combination described in the present invention can secrete monoclonal antibodies that specifically bind to rituximab and its biosimilars. By jointly applying the secreted monoclonal antibodies to detect the blood drug concentration of rituximab and its biosimilars, a detection effect that is fast, accurate, stable, and highly specific can be achieved.

[0035] The present invention provides a kit for detecting rituximab and its biosimilars. The kit includes the described monoclonal antibody combination. Preferably, in the kit, the RTX-5B12 antibody is a capture antibody, and the RTX-9C02 antibody is a detection antibody. The kit preferably further includes magnetic microparticles and alkaline phosphatase.

[0036] In the kit of the present invention, preferably, the capture antibody and magnetic particles are made into a magnetic particle working solution. The mass ratio of the magnetic particles to the capture antibody is preferably 50:(0.2 - 2), more preferably 50:(0.5 - 1.5), and further preferably 50:1. The mass concentration of the magnetic particles in the magnetic particle working solution is preferably 0.2 - 0.6 mg / mL, more preferably 0.3 - 0.5 mg / mL, and further preferably 0.4 mg / mL.

[0037] In the kit of the present invention, preferably, the alkaline phosphatase and the detection antibody are made into an enzyme-labeled working solution. The mass ratio of the alkaline phosphatase to the detection antibody is preferably 1:(0.2 - 1), more preferably 1:(0.3 - 0.8), and further preferably 1:0.5. The concentration of the alkaline phosphatase in the enzyme-labeled working solution is preferably 0.1 - 2 μg / mL, more preferably 0.5 - 1.5 μg / mL, and further preferably 1 μg / mL. When preparing the enzyme-labeled working solution, preferably, a coupling agent can also be added. The coupling agent is preferably glutaraldehyde.

[0038] When using the kit of the present invention for detection, preferably, the sample to be tested is diluted 20 times and then reacted with the magnetic particle working solution. After the reaction, it is washed, then the enzyme-labeled working solution is added for reaction. After the reaction, it is washed again, and then the alkaline phosphatase substrate is added for a color reaction. The luminescence value is statistically analyzed to obtain the concentration of rituximab or its biosimilar in the sample to be tested. The volume ratio of the diluted sample to be tested, the magnetic particle working solution, and the enzyme-labeled working solution is preferably (2 - 100):(10 - 150):(10 - 150), more preferably (5 - 50):(20 - 100):(20 - 100), and further preferably 10:50:50. The present invention does not have special limitations on the specific measurement steps and parameters during the detection process, and conventional measurement steps and parameters in the art can be used.

[0039] The technical solutions provided by the present invention will be described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0040] In the following embodiments, unless otherwise specified, all are conventional methods.

[0041] In the following embodiments, the materials, reagents, etc. used, unless otherwise specified, can all be obtained from commercial channels.

[0042] Example 1

[0043] Preparation of the immunogen of rituximab (Fab) 2 fragment

[0044] 1. Dilute 20 mg of rituximab drug with 20 mM PBS buffer (pH = 7.4) to 5 mg / mL to obtain 4 mL of the dilution.

[0045] 2. Add 2 mg of Ide-Z protease (Beijing Deaoping Biology) to the diluent and incubate at 37 °C for 30 min to obtain an incubation product;

[0046] 3. Take the supernatant of the incubation product, first adsorb Ide-Z protease through a Ni-TED affinity column to obtain the Ni column purified flow-through liquid, then adsorb the unreacted full-length antibody and the cleaved Fc fragment through a ProteinA column, and collect the flow-through liquid to obtain "Ni column purified flow-through through ProteinA flow-through", which is the immunogen of rituximab (Fab)2 fragment; elute the adsorbed substance on the ProteinA column to obtain "Ni column purified flow-through through ProteinA eluate".

[0047] 4. Determine the concentration of "Ni column purified flow-through through ProteinA flow-through" by OD280, and the result is 3.2 mg / mL, and store it frozen at -20 °C.

[0048] 5. Perform SDS-PAGE identification on Ide-Z protease, rituximab (denoted as rituximab drug), supernatant of the incubation product (denoted as cleavage system mixture (before purification)), Ni column purified flow-through liquid (denoted as cleavage system mixture (Ni column purified flow-through)), Ni column purified flow-through through ProteinA flow-through, and Ni column purified flow-through through ProteinA eluate. The results are as Figure 1 shown. The full-length rituximab is completely cleaved, and the immunogen of rituximab (Fab)2 fragment is obtained after adsorbing impurity fragments through the Ni-TED affinity column and ProteinA column.

[0049] Example 2

[0050] The preparation of hybridoma cells and idiotypic antibodies (antibodies that specifically bind to rituximab and its biosimilars) against the (Fab)2 region of rituximab is prepared by using the hybridoma cell fusion technology. The steps are as follows:

[0051] 1. Preparation of hybridoma cells

[0052] Dilute the rituximab (Fab)2 fragment immunogen prepared in Example 1 to 1 mg / mL with PBS buffer, add an equal volume of Freund's complete adjuvant, and emulsify completely. The mice are immunized for the first time at a dose of 0.1 mg / animal; after an interval of 4 weeks, take 1 mg of the rituximab (Fab)2 fragment immunogen prepared in Example 1 and mix it with an equal volume of Freund's incomplete adjuvant, emulsify it, and the mice are immunized for the second time at a dose of 0.1 mg / animal;

[0053] 3 days later, fuse the spleen cells of the second immunization with Sp2 / 0 cells (mouse myeloma cells), distribute them into a 96-well cell culture plate, and place the fused cells in CO2 Cultured in an incubator, at 6 days, half of the culture medium was replaced with HT medium (400 mL of ordinary DMEM medium + 100 mL of fetal bovine serum + 10 mL of glutamine solution + 6.5 mL of penicillin-streptomycin mixture + 5 mL of HT). At 14 days, the supernatant of the fused cells in different wells was aspirated for clone screening. Specifically, a 96-well ELISA plate was coated with 2 μg / mL of rituximab drug. After taking 100 μL of the above-mentioned fused cell supernatant and reacting for 1 h, the ELISA plate was washed 5 times with PBS. Then, 100 μL of goat anti-mouse IgG-Fc-HRP (horseradish peroxidase) secondary antibody was added and reacted for 1 h. The ELISA plate was washed 5 times with PBS, 100 μL of TMB (tetramethylbenzidine) substrate solution was added and reacted for 5 min, and the OD value was read with an enzyme-linked immunosorbent assay (ELISA) reader for titer determination. Clones with positive titer detection were further subjected to competitive screening.

[0054] For competitive screening, a 96-well ELISA plate was coated with 2 μg / mL of rituximab and then 50 μL of 1 μg / mL CD20 protein and 50 μL of the fused cell supernatant were added. After reacting for 1 h, the ELISA plate was washed 5 times with PBS. Then, 100 μL of goat anti-mouse IgG-Fc-HRP secondary antibody was added and reacted for 1 h. The ELISA plate was washed 5 times with PBS, 100 μL of TMB substrate solution was added and reacted for 5 min, and the OD value was read with an ELISA reader. Clones with strong positive (high OD) in cell titer determination and weak positive or negative (low OD) in competitive screening were selected. Finally, 5 hybridoma cells secreting idiotypic antibodies against the (Fab)2 region of rituximab were screened, as shown in Table 1.

[0055]

[0056] 2. Preparation of idiotypic antibodies against the (Fab)2 region of rituximab

[0057] The 5 hybridoma cells in Table 1 above were separately injected into the peritoneal cavity of 10-week-old Kunming mice that had been intraperitoneally injected with 0.1 mL of liquid paraffin 10 days in advance. After 24 days, the ascites of each Kunming mouse was collected. The ascites was diluted 10 times with PBS buffer and then purified through a Protein A affinity column equilibrated with PBS buffer. Then, it was dialyzed into PBS buffer using a 7KD dialysis bag to obtain 5 idiotypic antibodies against the (Fab)2 region of rituximab (RTX-5B12 antibody, RTX-9C02 antibody, RTX-1E23 antibody, RTX-22A1 antibody, RTX-9H1 antibody), which were stored frozen at -20 °C.

[0058] Example 3

[0059] Screening of paired antibodies

[0060] Using the checkerboard method, the ELISA 96-well plate was coated with 2 μg / mL of the idiotypic antibody against the anti-rituximab (Fab) 2 region. Rituximab drug at 0 or 20 ng / mL was added, and after reacting for 30 min, it was washed 3 times with PBST (phosphate buffered saline - Tween 20). After adding 2 μg / mL of the idiotypic antibody against the anti-rituximab (Fab) 2 region - HRPP (horseradish peroxidase) conjugate and reacting for 30 min, it was washed 3 times with PBST. Then, TMB chromogenic solution was added, and the results were read using an enzyme-linked immunosorbent assay reader. One paired result is shown in Table 2.

[0061]

[0062] The results showed that RTX-5B12 / RTX-9C02 had a good low-value signal-to-noise ratio. Therefore, this pair was subsequently used to establish a method for detecting the free rituximab blood drug concentration.

[0063] Example 4

[0064] Establishment of a method for detecting the free rituximab blood drug concentration by chemiluminescence immunoassay with magnetic microparticles

[0065] 1. Detection method

[0066] (1) 50 mg of Dynal beads M280 Tosyl magnetic beads were diluted in 2 mL of 50 mM pH 8.0 borate (BB) buffer. 1 mg of the RTX-5B12 antibody obtained in Example 2 was added, and after mixing evenly, it was reacted with shaking at 37 °C for 8 h.

[0067] The supernatant was removed by magnetic absorption, and TBST (50 mM Tris, 0.9 wt% NaCl, 0.1 wt% TW20, pH 7.4) was added and reacted at 37 °C for 12 h.

[0068] The supernatant was removed by magnetic absorption, and TBST was added and diluted to 0.4 mg / mL, named magnetic microparticle working solution, and stored at 2 - 8 °C for later use.

[0069] (2) 1 mg of alkaline phosphatase (ALP) was dissolved in 1 mL of PBS buffer. 0.5 mg of the RTX-9C02 antibody obtained in Example 2 was added and mixed evenly. 10 μL of 500 mg / mL glutaraldehyde solution was added, and it was mixed evenly at room temperature for 2 h to obtain a conjugate solution. The conjugate solution was dialyzed with PBS buffer and diluted to 1 μg / mL with a mixed buffer (50 mM MES, 0.9% NaCl, 5 mg / mL BSA, 1 mM MgCl 2 , pH 6.7), named enzyme-labeled working solution.

[0070] (3) Reaction procedure: After the test sample is automatically diluted 20 times with PBS buffer, 10 μL is taken and reacted with 50 μL of the magnetic particle working solution for 5 minutes, then washed. Then, 50 μL of the enzyme-labeled working solution is added, incubated at 37 °C for 5 minutes, washed, and AMPPD luminescent solution is added for color development, and the luminescence value is counted. The total detection time is 5 minutes.

[0071] 2. Methodological verification

[0072] (1) Standard curve drawing: Calibrators of rituximab at different concentrations (0, 0.5, 5, 10, 20, 50, 100 μg / mL) are prepared using human serum matrix that has not been treated with rituximab as the test samples. The corresponding luminescence values are measured using the method in step 1. (3) of this example, and a standard curve is formulated. The results are shown in Table 3 and Figure 2 as follows.

[0073]

[0074] From Figure 2 and Table 3, it can be seen that the calibration curve obtained by the magnetic particle luminescence method is: four-parameter equation: Y = (A - D) / [1 + (X / C)^B] + D; where: A = 9888.8964239; B = 1.0488411; C = 78558.6687904; D = 364047351.389; correlation coefficient R 2 : 0.999.

[0075] (2) Precision detection: Rituximab is diluted using human serum matrix that has not been treated with rituximab to prepare rituximab solutions at two concentrations, high (50 μg / mL) and low (10 μg / mL). According to the method in step 1. (3) of this example, the luminescence values of the rituximab solutions at high and low concentrations are detected. In one experiment, each concentration point is measured 10 times (numbered 1 to 10 in sequence), and CV is calculated according to SD / Mean. The results are shown in Table 4.

[0076]

[0077] From Table 4, it can be seen that the magnetic particle luminescence method reagent for measuring rituximab blood drug concentration prepared with the RTX-5B12 / RTX-9C02 antibody pair has high precision. And the detected values account for 93% - 104.6% of the actual values, indicating high recovery and good detection accuracy.

[0078] (3) Cross - reaction detection: Different amounts of cross - test substances (cross - reactants) were added to rituximab solution samples with concentrations of 10 μg / mL and 50 μg / mL respectively for cross - reaction. The obtained cross - reaction samples were used as test samples. According to the method in item (3) of step 1 of this example, the rituximab concentration in the cross - reaction samples was measured, and the cross - reaction rate was further calculated. The cross - reaction rate was calculated according to the following formula: Cross - reaction rate = (measured concentration - theoretical concentration) / concentration of cross - reactant × 100%;

[0079] Among them, the measured concentration is the rituximab concentration in the detected cross - reaction sample; the theoretical concentration is the concentration of the rituximab solution sample, that is, 10 μg / mL or 50 μg / mL; the concentration of the cross - reactant is the concentration of the cross - reactant in the cross - reaction sample.

[0080] The names, concentrations of cross - reactants, concentrations of rituximab, and test results in the cross - reaction samples are shown in Table 5:

[0081]

[0082] From the results in Table 5, it can be concluded that the reagent for determining rituximab blood drug concentration by the magnetic particle luminescence method prepared in the present invention has no cross - reaction with a variety of endogenous proteins and possible concomitant medications, and has high specificity.

[0083] Example 5

[0084] Determination of biosimilars of rituximab

[0085] The biosimilars of rituximab, Hanlikang and Dabo Hua, were prepared into 10 μg / mL and 50 μg / mL with human serum matrix not treated with rituximab and used as test samples. They were measured by the method in item (3) of step 1 of Example 4, and the results are shown in Table 6.

[0086]

[0087] The results show that the deviation between the measured concentrations of the 2 biosimilars and the theoretically prepared concentrations < ± 10%, indicating that the determination method established with the monoclonal antibody of the present invention can be used to determine a variety of rituximab biosimilars.

[0088] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A hybridoma cell line combination secreting a monoclonal antibody against rituximab, characterized in that: The hybridoma cell line combination includes hybridoma cell line RTX-9C02 and hybridoma cell line RTX-5B12; The hybridoma cell line RTX-9C02 is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with the deposit number of CGMCC No.46301; The hybridoma cell line RTX-5B12 is deposited in the General Microbiology Center of China National Microbiological Culture Collection Administration, with the deposit number being CGMCC No.46302.

2. Use of the hybridoma cell line combination according to claim 1 in the preparation of monoclonal antibodies that specifically bind to rituximab and its biosimilars; the biosimilars are Hanlicon or Dabohua.

3. A monoclonal antibody combination that specifically binds to rituximab and its biosimilars, characterized in that: The monoclonal antibody combination is secreted and produced by the hybridoma cell line combination according to claim 1, and the monoclonal antibody combination includes RTX-9C02 antibody and RTX-5B12 antibody; the biosimilar drug is Hanlicon or Dabohua.

4. Use of the hybridoma cell line combination according to claim 1 or the monoclonal antibody combination according to claim 3 in the preparation and detection of rituximab and its biosimilar products; the biosimilar is Hanlicon or Dabohua.

5. The use according to claim 4, characterized in that: The products include kits.

6. The use according to any one of claims 4 to 5, characterized in that: The detection of rituximab and its biosimilars is to detect the blood concentration of rituximab and its biosimilars.

7. A kit for detecting rituximab and its biosimilars, characterized in that: The kit comprises the monoclonal antibody combination as described in claim 3; the biosimilar drug is Hanlicon or Dabohua.

8. The kit according to claim 7, characterized in that The RTX-5B12 antibody is a capture antibody, and the RTX-9C02 antibody is a detection antibody.

9. The kit according to claim 8, characterized in that The kit also includes magnetic particles and alkaline phosphatase.

10. The kit according to claim 9, characterized in that The mass ratio of the magnetic particles to the capture antibody is 50:(0.2-2), and the mass ratio of the alkaline phosphatase to the detection antibody is 1:(0.2-1).

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