A detection kit for urinary kallidin residual protein and a preparation method and application thereof

Abstract

The application discloses a kind of ecallantide residual protein detection kit and its preparation method and application, the detection kit includes capture antibody and detection antibody, the capture antibody includes anti-ecallantide residual protein polyclonal antibody, the detection antibody includes biotin-labelled anti-ecallantide residual protein polyclonal antibody, the anti-ecallantide residual protein polyclonal antibody is obtained after multiple immunization of animal with ecallantide residual protein as immunogen, and the ecallantide residual protein is further purified after removing ecallantide using anti-ecallantide monoclonal antibody affinity column adsorption from human prekallikrein crude preparation.The application fills the detection blank of urinary protein drug residual protein, is strong in specificity, antibody coverage reaches more than 70%, method limit of quantification is about 4ng / ml, for the detection limit of ecallantide residual protein, high detection sensitivity, without expensive instrument, simple operation.

Description

Technical Field

[0001] This invention belongs to the field of biomedical detection technology, specifically relating to a detection kit for residual urokinase protein, its preparation method, and its application. Background Technology

[0002] Urokinase is a protein drug whose raw material is human urine. During its production, human urine undergoes multiple steps, including concentration and chromatography, to prepare various urokinase drugs. According to biopharmaceutical requirements, impurity studies, such as residual protein detection, are necessary. Urokinase is a protein drug extracted and purified from human urine. As a commonly used clinical protein drug, its production process may leave residual host proteins and process-related protein impurities (such as proteins shed from purification packing materials and residual protein fragments from enzymatic hydrolysis). These residual proteins not only reduce the purity and efficacy of Urokinase but may also trigger immune responses (such as antibody production and allergic reactions), affecting the safety and effectiveness of the drug. Based on the requirements of the Chinese Pharmacopoeia regarding the limits of residual proteins in biopharmaceuticals, sensitive and specific detection methods (such as enzyme-linked immunosorbent assay, high-performance liquid chromatography, etc.) need to be established to quantitatively or qualitatively analyze residual proteins in the finished Urokinase product, ensuring that its content meets drug quality standards and guaranteeing the safety and stability of clinical use.

[0003] Currently, techniques commonly used for detecting residual proteins in urokinase include ELISA, SDS-PAGE, HPLC, and mass spectrometry. HPLC offers high resolution and repeatability, allowing for the quantification of specific components. However, for extremely complex mixtures, HPLC struggles to comprehensively quantify the total residual amount and suffers from high instrument costs and stringent operator skill requirements. SDS-PAGE can visually distinguish contaminating protein bands and determine their molecular weight, making it suitable for identifying residual protein types, but it only provides semi-quantitative analysis with limited precision. Liquid chromatography-tandem mass spectrometry (LC-MS / MS) is primarily used to identify specific host protein residues in samples (providing a protein list), for process weakness analysis and purification process optimization. By using isotopically labeled standard peptides (such as AQUA peptide), it enables precise absolute quantification of key residual or indicator proteins. However, LC-MS / MS instruments are expensive, operation is complex, data analysis requires specialized expertise, and costs are high. Therefore, it is typically not used as a routine release testing method but rather for process characterization, validation, and troubleshooting. ELISA has extremely high sensitivity (down to ng / mL or even pg / mL level), high throughput, and relatively standardized operation, making it suitable for release testing and process monitoring. However, one of the most challenging aspects of the ELISA method is that antibody quality is crucial, and it is essential to ensure broad coverage and specificity of residual proteins.

[0004] Since both urokinase and urokinase residue protein originate from urine and have complex compositions, the detection and removal of residue protein is crucial throughout the entire process. Complete urokinase residue protein is difficult to obtain, and there is currently no ELISA method or kit for detecting urokinase residue protein in existing technologies. At present, there is no commercially available detection kit for residue protein in urokinase active pharmaceutical ingredient that is easy to operate, suitable for routine quality control, and provides accurate quantification. Summary of the Invention

[0005] Purpose of the invention: The first objective of this invention is to provide a detection kit for qualitative and / or quantitative determination of residual urokinase protein that is highly sensitive, specific, accurate, precise, and easy to operate.

[0006] A second objective of the present invention is to provide a method for preparing the aforementioned detection kit.

[0007] A third objective of this invention is to provide the application of the aforementioned detection kit in the qualitative and / or quantitative determination of residual urokinase protein.

[0008] Technical Solution: To solve the above-mentioned technical problems, the present invention provides a detection kit for residual urokinase protein. The detection kit includes a capture antibody and a detection antibody. The capture antibody includes a polyclonal antibody against residual urokinase protein, and the detection antibody includes a biotin-labeled polyclonal antibody against residual urokinase protein. The polyclonal antibody against residual urokinase protein is obtained by immunizing animals multiple times with residual urokinase protein as an immunogen. The residual urokinase protein is obtained by further purifying crude human urinary kininogenase by adsorption and removal of urokinase using an anti-urokinase monoclonal antibody affinity column.

[0009] Preferably, the further purification includes endotoxin removal from the crude human urinary kininogenase product after urokinase removal. Endotoxin removal can be performed using conventional methods for removing endotoxins from proteins, such as a protein endotoxin removal kit. More preferably, the endotoxin content in the urokinase-residual protein is controlled to <500 EU / mg through endotoxin removal.

[0010] In this invention, the preparation process of the polyclonal antibody against urokinase residual protein includes: eluting the animal antiserum obtained by repeatedly immunizing the animal with urokinase residual protein as an immunogen to obtain affinity-purified antibody.

[0011] The preparation process of the polyclonal antibody against urokinase residual protein further includes: further binding the affinity-purified antibody with urokinase to obtain the polyclonal antibody after reverse screening.

[0012] In this invention, the anti-urokinase monoclonal antibody affinity column is obtained by coupling an anti-urokinase monoclonal antibody to an affinity column, wherein the anti-urokinase monoclonal antibody comprises one or more selected from CELL-2, CELL-5, and CELL-6. The amino acid sequence information of CELL-2, CELL-5, and CELL-6 can be found in Chinese patent application publication number CN120887993A.

[0013] The concentration of the anti-Urokinase residual protein polyclonal antibody is 0.8–1.2 µg / ml, for example, 0.8 µg / ml, 0.9 µg / ml, 1 µg / ml, 1.1 µg / ml, 1.2 µg / ml, or any value between the two aforementioned values; the concentration of the biotin-labeled anti-Urokinase residual protein polyclonal antibody is 0.15–1.2 µg / ml, for example, 0.15 µg / ml, 0.3 µg / ml, 0.45 µg / ml, 0.6 µg / ml, 0.75 µg / ml, 0.9 µg / ml, 1.05 µg / ml, 1.2 µg / ml, or any value between the two aforementioned values. The detection kit further includes one or more of streptavidin, coating buffer, chromogenic solution, washing solution, and stop solution. The streptavidin is preferably luciferase-labeled streptavidin, and the luciferase is selected from one or more of horseradish peroxidase, alkaline phosphatase, and phosphodiesterase, more preferably horseradish peroxidase; correspondingly, the chromogenic solution contains a luciferase substrate, for example, selected from one or more of chromogenic peroxidase substrate, chromogenic alkaline phosphatase substrate, and chromogenic phosphodiesterase substrate, more preferably chromogenic peroxidase substrate.

[0014] In this invention, the buffer solution of the detection kit can be an appropriate immunoassay buffer. Preferably, the buffer solution used with the capture antibody is PBS buffer, the buffer solution used with the detection antibody is PBST buffer containing 0.4-0.6 wt% bovine serum albumin, and the buffer solution used with streptavidin is PBST buffer containing 0.4-0.6 wt% bovine serum albumin.

[0015] In this invention, the washing solution and the stop solution can be appropriate immunoassay reagents. Preferably, the washing solution is a PBS buffer containing 0.04-0.06 wt% Tween 20, and the stop solution is a sulfuric acid solution with a volume content of 10-12%.

[0016] The preparation method of the urokinase residual protein detection kit of the present invention includes the following steps:

[0017] (1) Preparation of residual urokinase protein: urokinase is obtained by removing urokinase from crude human urinary kininogenase by adsorption using an anti-urokinase monoclonal antibody affinity column and then further purified; preferably, the further purification includes the removal of bacterial endotoxins;

[0018] (2) Preparation of capture antibody against polyclonal antibody against residual urokinase protein: Animal antiserum obtained by immunizing animals multiple times with residual urokinase protein as an immunogen is bound to residual urokinase protein and eluted to obtain affinity purified antibody; preferably, the affinity purified antibody is further bound to urokinase to obtain polyclonal antibody after reverse screening.

[0019] (3) Preparation of biotin-labeled polyclonal antibody against residual protein of ursocrine: The polyclonal antibody against residual protein of ursocrine prepared in step (2) is biotin-labeled.

[0020] The preparation steps of the anti-Urokinase monoclonal antibody affinity column in step (1) are as follows: the anti-Urokinase monoclonal antibody is mixed and dissolved with NaHCO3+NaCl buffer, and then mixed and coupled with affinity filler (the conditions for the mixing and coupling include a temperature of 2-6℃ and a time of 3-5h). Then, it is washed with NaHCO3+NaCl buffer (mainly used to remove unbound proteins) to obtain the coupled filler. The coupled filler is mixed with glycine+NaHCO3+NaCl buffer and stirred (the conditions for the stirring reaction include a temperature of 2-6℃ and a time of 1-2h). Then, it is washed several times with NaAc-HAc+NaCl buffer and Tris-HCl+NaCl buffer in sequence.

[0021] In this invention, the affinity filler can be a cyanide-activated agarose gel, such as CNBr Focurose 4FF.

[0022] In this invention, to enhance the affinity of the anti-urokinase monoclonal antibody affinity column and improve the removal efficiency of urokinase, the pH of the NaHCO3+NaCl buffer is preferably 8.0-8.5. More preferably, to further improve the removal efficiency of urokinase from crude human urinary kininogenase, the pH of the NaHCO3+NaCl buffer is 8.3, and the concentration of NaHCO3 in the NaHCO3+NaCl buffer is 0.08-0.12M and the concentration of NaCl is 0.4-0.6M.

[0023] Before and after the anti-Urokinase monoclonal antibody was bound to the affinity filler, the protein peak area was observed by connecting the instrument. The results showed that when the pH of the NaHCO3+NaCl buffer was 8.3, the peak area of ​​the unbound protein (anti-Urokinase monoclonal antibody) was the smallest, which means that the binding efficiency of the anti-Urokinase monoclonal antibody to the affinity filler was the highest.

[0024] Preferably, the pH of the glycine + NaHCO3 + NaCl buffer solution is 8.0-8.5. More preferably, the concentration of glycine in the glycine + NaHCO3 + NaCl buffer solution is 0.08-0.12M, the concentration of NaHCO3 is 0.08-0.12M, and the concentration of NaCl is 0.4-0.6M. The pH of the NaAc-HAc + NaCl buffer solution is 3.5-4.5. More preferably, the concentration of NaAc-HAc in the NaAc-HAc + NaCl buffer solution is 0.08-0.12M, and the concentration of NaCl is 0.4-0.6M. The pH of the Tris-HCl + NaCl buffer solution is 7.5-8.5. More preferably, the concentration of Tris-HCl in the Tris-HCl + NaCl buffer solution is 0.08-0.12M, and the concentration of NaCl is 0.4-0.6M.

[0025] Preferably, to more effectively remove urokinase from crude human urinary kininogenase, the adsorption removal process in step (1) includes: regenerating the anti-urokinase monoclonal antibody affinity column with NaAc-HAc+NaCl buffer, equilibrating and rinsing with PB+NaCl buffer, repeatedly loading the crude human urinary kininogenase onto the antibody affinity column to remove urokinase, using PB+NaCl buffer as the permeation buffer, collecting the permeation eluent, and then concentrating it by ultrafiltration to obtain crude urokinase residual protein. More preferably, the crude urokinase residual protein is further purified by endotoxin removal to obtain urokinase residual protein.

[0026] Preferably, the pH of the PB+NaCl buffer solution is 7.8-8.2, wherein the concentration of PB (dihydrogen phosphate and / or dihydrogen phosphate) is 0.08-0.12M and the concentration of NaCl is 0.15-0.25M.

[0027] The crude human urinary kininogenase product of this invention can be prepared by methods known in the art or commercially available. Preferably, the preparation process of the crude human urinary kininogenase product includes: adsorbing urinary proteins from urine using an adsorbent, collecting the adsorbent after adsorption, eluting the adsorbent, loading the eluted urinary protein solution onto a metal chelate affinity chromatography column equilibrated with a buffer, eluting with NaAc-HAc buffer, collecting the eluent, adding solid ammonium sulfate and diatomaceous earth sequentially to the eluent, allowing it to stand, filtering, and collecting the precipitate. More preferably, the equilibration buffer of the metal chelate affinity chromatography column is 0.01-0.2M phosphate buffer, with a NaCl concentration of 0-2M and a pH of 6.0-9.0, and the metal ion chelated by the metal chelate affinity chromatography column is Cu. 2+ Zn 2+ Ni 2+Fe 3+ Any one of them.

[0028] In this invention, the adsorbent for adsorbing urine can be a macroporous anion exchange resin, and the urine protein solution can be eluted with NaCl solution.

[0029] To improve the efficiency of preparing polyclonal antibodies against urokinase residue, preferably, the process of multiple immunizations of animals in step (2) includes: using multiple rabbits (e.g., 8-12 rabbits), each rabbit receiving an initial immunization injection of 4-6 mg of urokinase residue and a booster immunization injection of 2-3 mg of urokinase residue; during the first injection, 0.8-1.2 mL of complete Freund's adjuvant is mixed with each 1 mL of urokinase residue, and during each subsequent injection, 0.8-1.2 mL of incomplete Freund's adjuvant is mixed with each 1 mL of urokinase residue, using multiple injection sites, for a total of 4-6 immunizations.

[0030] To improve the preparation efficiency and purity of polyclonal antibodies against urokinase residues, preferably, the process of eluting the animal antiserum from the urokinase residues includes: mixing and coupling the urokinase residues with an agarose gel to prepare the corresponding antigen affinity purification agarose gel, and equilibrating it with PBS buffer; diluting the animal antiserum with PBS buffer, mixing it with the prepared antigen affinity purification agarose gel, and incubating it; after the antigen and antibody are fully bound, equilibrating the affinity column with PBS buffer, washing away unbound impurities, adding glycine elution buffer after the PBS buffer has drained, connecting the instrument to display peak changes, and collecting the antibody-containing eluent; dialyzing the eluent with PBS buffer overnight, and then sterilizing and filtering it to obtain the affinity purified antibody. In this process, the pH of the PBS buffer is preferably 7.2-7.6, the volume ratio of animal antiserum to PBS buffer during dilution is preferably 1:0.9-1.1, and the incubation conditions include a temperature of 2-6℃ and a time of 2-4 hours.

[0031] In this invention, to improve the purity of the polyclonal antibody against urokinase residual protein, preferably, the preparation process of the polyclonal antibody against urokinase residual protein further includes: further conjugating the affinity-purified antibody with urokinase to obtain the polyclonal antibody after reverse screening. This is achieved by coupling urokinase with agarose gel to prepare a target protein affinity-purified agarose gel, and then incubating the affinity-purified antibody with this gel (the incubation conditions include a temperature of 2-6°C and a time of 2-4 hours) until the antigen and antibody are fully bound, effectively removing the urokinase target antibody from the affinity-purified antibody, and obtaining the polyclonal antibody after reverse screening as the urokinase residual protein polyclonal antibody, thus making the polyclonal antibody more specific for urokinase residual protein. The urokinase can be obtained using urokinase raw material or urokinase standard, prepared by a disclosed method or obtained commercially.

[0032] In this invention, biotin labeling of the anti-Urokinase residual protein polyclonal antibody can be performed by conventionally mixing biotin with the residual protein polyclonal antibody.

[0033] This invention also includes the application of the aforementioned detection kit in the qualitative and / or quantitative determination of residual urokinase protein. The sample to be measured can be any type of urokinase sample, such as urokinase active pharmaceutical ingredient, urokinase components involved in protein separation and purification processes, etc.

[0034] This invention, after obtaining crude human urinary kininogenase, innovatively establishes an anti-Urokinase monoclonal antibody affinity column to adsorb and remove the target protein Urokinase from the crude human urinary kininogenase, preparing high-purity Urokinase residual protein. This protein is then used as an immunogen to repeatedly immunize animals to obtain animal antiserum. The animal antiserum is then purified by antigen-antibody affinity of Urokinase residual protein to obtain affinity-purified antibodies that react with Urokinase residual protein (antigen). Further, target protein antigen-residual protein polyclonal antibody affinity purification is performed to obtain specific antibodies that do not react with the target protein (Urokinase), resulting in highly specific and high-coverage anti-Urokinase residual protein polyclonal antibodies, which are then used to prepare a detection kit.

[0035] Compared with existing technologies, this invention has the following significant advantages: This invention is developed from the source, using an anti-urokinase monoclonal antibody affinity column to remove urokinase (target protein) from crude human urinary kininogenase to prepare urokinase residual protein. The urokinase residual protein is then used as an immunogen to immunize animals to prepare residual protein polyclonal antibodies. A portion of the residual protein polyclonal antibodies are selected as capture antibodies to capture the antigen in the test sample; another portion is selected as reporter antibodies for detecting and / or quantifying urokinase residual protein. Based on this, an enzyme-linked immunosorbent assay (ELISA) for detecting residual protein in human urinary total protein is developed, filling the gap in the detection of drug residual proteins in urinary sources. The detection kit provided by this invention has high specificity for detecting urokinase residual protein (antibody coverage reaches over 70%), a method quantification limit of approximately 4 ng / ml, a low detection limit for urokinase residual protein, high detection sensitivity, requires no expensive instruments, and is easy to operate. Attached Figure Description

[0036] Figure 1 The concentration and absorbance (OD) of the residual protein standard of urokinase in Example 2. 450 The fitted standard curve is given, where x represents the concentration of the residual protein standard of urokinase, and y represents the absorbance value OD. 450 ;

[0037] Figure 2 The concentration and absorbance (OD) of the residual protein standard of urokinase in Example 3. 450The fitted standard curve is given, where x represents the concentration of the residual protein standard of urokinase, and y represents the absorbance value OD. 450 . Detailed Implementation

[0038] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0039] To further illustrate the present invention, the following detailed description, in conjunction with the accompanying drawings and embodiments, provides a detection kit for quantitative determination of residual urokinase protein, its preparation method, and its application. However, these descriptions should not be construed as limiting the scope of protection of the present invention.

[0040] Example 1: Preparation and purification of polyclonal antibodies against residual urokinase protein

[0041] 1. Preparation of Urokinase Residual Protein (Immunogen)

[0042] 1.1 Synthesis of Anti-KLK1 (Urokinase) Antibody Affinity Filler

[0043] 10 mg of anti-KLK1 (Urokinase) antibody (the antibody being CELL-5, published in Chinese patent application CN120887993A, entitled "An Anti-Human Tissue Kallikrein 1 Monoclonal Antibody and Its Application") was dissolved in 30 ml of 0.1 M NaHCO3 + 0.5 M NaCl buffer at pH 8, pH 8.3, and pH 8.5, respectively. Then, each solution was mixed and coupled with 10 ml of antibody affinity packing material CNBr Focurose 4FF (manufacturer: Huiyan Biotechnology, catalog number: HQ030301500M). The mixture was stirred slowly at 100 rpm using a magnetic stirrer at 4°C. After 4 hours of reaction, the antibody affinity packing material reaction solution was removed by filtration, and the unbound proteins were washed with 0.1 M NaHCO3 + 0.5 M NaCl buffer at the corresponding pH values. Finally, the packing material was placed in a 0.1M glycine + 0.1M NaHCO3 + 0.5M NaCl buffer solution at pH 8.3 and reacted slowly with stirring at 4°C. After reacting for 1.5 h, the packing material was washed four times sequentially with 0.1M NaAc-HAc + 0.5M NaCl buffer solution (pH 4.0) and 0.1M Tris-HCl + 0.5M NaCl buffer solution (pH 8.0) to obtain three different anti-KLK1 (Urokinase) antibody affinity packing materials, which were then placed in 20% ethanol for later use.

[0044] To determine the binding efficiency between the antibody and the packing material, peak areas were observed using a high-performance liquid chromatograph (HPLC). After treatment with NaHCO3+NaCl buffer solutions at pH 8.0, pH 8.3, and pH 8.5, the binding efficiencies of the anti-Urokinase monoclonal antibody and the packing material were 90%, 99.5%, and 95%, respectively. Therefore, under the NaHCO3+NaCl buffer solution condition at pH 8.3, the peak area of ​​the unbound protein (antibody) was the smallest, indicating that the anti-Urokinase monoclonal antibody had the highest binding efficiency with the affinity packing material.

[0045] The formula for calculating the binding efficiency between the antibody and the filler is as follows:

[0046] Antibody-filler binding efficiency = (initial peak area - peak area of ​​unbound protein after 4 hours) / initial peak area * 100%

[0047] 1.2 Removal of urokinase from crude human urinary kininogenase

[0048] Take 8 ml of the anti-KLK1 (Urokinase) antibody affinity packing prepared in 1.1, regenerate it with 0.05M NaAc-HAc + 1M NaCl buffer (pH=4.0), and equilibrate and wash it with 0.1M PB + 0.2M NaCl buffer (pH=8.0). Repeatedly load 15g of crude human urinary kallikrein (KN crude product, prepared according to Example 1 of Chinese patent application CN102660525A, entitled "A Method for Preparing Crude Human Urinary Kallikrein") through the antibody affinity column to remove Urokinase. Use 0.1M PB + 0.2M NaCl buffer (pH=8.0) as the breakthrough eluent, collect a total of 770ml of breakthrough eluent, and then concentrate it through a 3KD ultrafiltration tube (manufacturer: Merck Millipore, batch number: 0000340557) to obtain 143ml of crude Urokinase residual protein.

[0049] 1.3 Removal of endotoxins

[0050] Purchase a commercially available protein endotoxin removal kit (manufacturer: Beyotime, product number: C0268S). Refer to the instructions in the kit to remove endotoxins from the crude urokinase residual protein obtained in step 1.2. Under the premise of minimal protein loss, control the bacterial endotoxin level to <500 EU / mg to obtain urokinase residual protein (urokinase residual protein content is 7.34 mg / ml).

[0051] 2. Preparation of polyclonal antibodies

[0052] 2.1 Animal Immunization

[0053] Ten New Zealand white rabbits, aged three months and weighing 2.3-2.5 kg, were immunized with the residual urokinase protein obtained in step 1.3. The initial immunization dose was 5 mg / rabbit, and the booster dose was 2.5 mg / rabbit. For the first injection, 1 ml of immunogen (urokinase protein with a concentration of 7.34 mg / ml obtained in step 1.3 diluted to 5 mg / ml) was mixed with 1 ml of complete Freund's adjuvant (manufacturer: Sigma, catalog number: F5881) and injected subcutaneously at multiple sites on the inner thigh. The next injection was given every 14 days, with 1 ml of immunogen (urokinase protein with a concentration of 7.34 mg / ml obtained in step 1.3 diluted to 2.5 mg / ml) mixed with 1 ml of incomplete Freund's adjuvant (manufacturer: Sigma, catalog number: F5506) and injected subcutaneously at multiple sites on the inner thigh. After five immunizations, the antiserum titer was determined using an indirect ELISA method. The residual protein of urokinase was immobilized on an enzyme-linked immunosorbent assay (ELISA) plate. The antiserum from the fifth immunization was added as the primary antibody to specifically bind to the antigen. Goat anti-rabbit HRP (manufacturer: Nanjing Huading Biotechnology Co., Ltd., batch number: HDPA080124) was then added as the secondary antibody to specifically bind to the primary antibody. Finally, the substrate TMB (manufacturer: Nanjing Zhongding Biotechnology Co., Ltd., catalog number: 80010) was added to react with the secondary antibody for color development, allowing the antiserum titer to be determined.

[0054] The results showed that the antiserum indirect ELISA titer against residual urokinase protein reached 3280.5K-fold dilution after five immunizations, with a titer of 1:3280500.

[0055] 2.2 Polyclonal antibody purification

[0056] 2.2.1 Polyclonal antibody affinity purification

[0057] Take 10 mg of immunogen (Urokinase residual protein prepared according to the method in step 1.3) and mix it with 10 ml of agarose gel (brand: Zhejiang Yishengke Biotechnology Co., Ltd., catalog number: HC1130702) to prepare the corresponding antigen affinity purification agarose gel, and equilibrate it with 100 ml of PBS (0.01 M, pH=7.4).

[0058] Take 400 ml of antiserum from the fifth immunization of rabbits in step 2.1, dilute it with 400 ml of PBS at a 1:1 volume ratio, mix it with 10 ml of antigen affinity purification agarose gel prepared in step 2.2.1, and transfer it to a centrifuge tube. Incubate at 4°C for 3 hours. After the antigen and antibody have fully bound, wash the affinity column with 100 ml of PBS to remove unbound proteins. After the PBS has drained, add glycine elution buffer (50 ml, 0.1 M, pH 2.5), connect the instrument to observe the peak change, and collect 42 ml of antibody-containing eluent. The eluent was dialyzed with 9000 ml PBS (pH=7.4) overnight at 4°C, and then sterilized by filtration (0.22 μm filter membrane). Finally, antibody identification was performed to obtain affinity-purified antibody. The results showed that 400 ml of affinity-purified antiserum yielded 33.6 mg of affinity-purified antibody with a titer of 1:9841500 (detection method is the same as the intermediate indirect ELISA method in 2.1).

[0059] 2.2.2 Target protein reverse screening for polyclonal antibodies

[0060] Take 5 mg of urokinase (the preparation method is described in Example 1 of Chinese patent application CN119144590A, entitled "A method for preparing human urinary kininogenase with reduced bacterial endotoxin") and mix it with 5 ml of agarose gel (Zhejiang Yishengke Biotechnology Co., Ltd., catalog number: HC1130702) to prepare target protein affinity purification agarose gel, and equilibrate it with 100 ml of PBS (pH=7.4).

[0061] The affinity-purified antibody obtained in step 2.2.1 was mixed with the target protein affinity-purified agarose gel prepared in step 2.2.2 and transferred to a centrifuge tube. The mixture was incubated at 4°C for 3 hours. After sufficient antigen-antibody binding, 30 ml of the eluent was collected. This eluent contained the specific residual protein polyclonal antibody (reverse-screened polyclonal antibody, 8.1 mg, concentration 0.27 mg / ml) after removing the urokinase target antibody. 30 ml of glycine elution buffer (0.1 M, pH 2.5) was added for elution. The instrument was connected to display the peak change. The resulting 14 ml of eluent contained the urokinase target antibody (6.44 mg), which was used for sample pretreatment in subsequent polyclonal antibody coverage detection.

[0062] 2.3 Polyclonal antibody-labeled biotin

[0063] Take 3 mg of the polyclonal antibody obtained in step 2.2.2 after reverse screening and biotin label it using Thermo Biotin (batch number: XG348276). The specific steps are as follows: Weigh 1 mg of biotin (Thermo, batch number: XG348276) and dissolve it in 180 μl of pure water to make a solvent of 5.5 mg / ml. Slowly add 41 μl of biotin to 3 mg of the polyclonal antibody obtained in step 2.2.2 and rotate the reaction at room temperature in the dark for 1 h. After the reaction, dialyze the above solution with more than 200 times the volume (about 10 mL) of PBS at 4°C overnight to obtain biotin-labeled polyclonal antibody. After dialyzing, add 10 μl of antibacterial agent ProClin300 (Suzhou Jingmao Biotechnology Co., Ltd., catalog number: GB01003) to each 1 ml of biotin-labeled polyclonal antibody, aliquot and store at -20°C. After labeling, the titer was determined using an indirect ELISA method. The residual protein of urokinase obtained in step 1.3 was immobilized on an ELISA plate as an antigen. 100 μl of the biotin-labeled polyclonal antibody was added to each well as the primary antibody to specifically bind to the antigen. Then, 100 μl of SA-HRP solution (prepared by diluting SA-HRP stock solution 10,000 times; the manufacturer of SA-HRP stock solution is Diamond, catalog number B110053, concentration 500 U / ml) was added as the secondary antibody to specifically bind to the primary antibody. Finally, the substrate TMB was added to react with the secondary antibody for color development, and the titer of the biotin-labeled polyclonal antibody could be determined.

[0064] The results showed that the titer of the biotin-labeled polyclonal antibody was 1:138240 dilution.

[0065] 2.4 Polyclonal antibody coverage detection

[0066] 2.4.1 Pretreatment of Urokinase Residual Protein Samples

[0067] Take 6 mg of urokinase target antibody (obtained in step 2.2.2) and mix it with 6 ml of agarose gel (Zhejiang Yishengke Biotechnology Co., Ltd., catalog number: HC1130702) to prepare target antibody affinity purification agarose gel, and equilibrate it with 100 ml of PBS (pH=7.4); add 35 mg of urokinase residual protein (prepared using the same method as in step 1.3) and mix it with 6 ml of target antibody affinity purification agarose gel, and incubate at room temperature by rotation for 1 h. After the antigen and antibody are fully bound, collect the effluent, and try to ensure that urokinase (target protein) in the urokinase residual protein is fully adsorbed, thus obtaining the sample without AAE treatment (pre-AAE sample).

[0068] Take 3 mg of polyclonal antibody after reverse screening (prepared according to the same method as in step 2.2.2) and mix it with 3 ml of agarose gel (Zhejiang Yishengke Biotechnology Co., Ltd., catalog number: HC1130702) to prepare the corresponding antibody affinity purification agarose gel. Mix it with the pre-AAE sample and incubate at room temperature for 3 h. After the antigen and antibody are fully bound, wash away the unbound protein with 1000 ml of PBS (pH=7.4). Then add 5 ml of glycine elution buffer (manufacturer: Shanghai Yuanye Biotechnology Co., Ltd., batch number: J12IS217225), incubate for 5 min and collect the elution buffer to obtain the AAE-treated sample (post-AAE sample). This step is called antibody affinity extraction (AAE).

[0069] 2.4.2 Coverage Detection

[0070] The procedure in 2.4.1 was repeated three times. The obtained pre-AAE and post-AAE samples were concentrated using a 3KD ultrafiltration tube (brand: Cytiva, catalog number: 28932218). Mass spectrometry analysis was then performed by Nanjing Zhongding Biotechnology Co., Ltd. The results showed that the pre-AAE sample contained 414 proteins, the post-AAE sample contained 407 proteins, and there were 251 common proteins. The coverage was calculated using the following formula:

[0071] Coverage = Number of proteins in post-AAE samples / (Number of proteins in pre-AAE samples + Number of proteins in post-AAE samples - Number of common proteins);

[0072] The polyclonal antibody coverage rate calculated using this formula is 71%, which meets industry requirements.

[0073] Example 2: Development of a polyclonal antibody pair for the detection of residual proteins in urokinase and its method.

[0074] 1. The checkerboard method was used to determine the detection antibody and the working concentration of SA-HRP.

[0075] Using the polyclonal antibody pair obtained in Example 1 (the polyclonal antibody prepared according to the same method as step 2.2.2 in Example 1 and the biotin-labeled polyclonal antibody prepared according to the same method as step 2.3 in Example 1), a checkerboard orthogonal method experiment was performed according to the double antibody sandwich ELISA operation principle to screen the optimal working concentration of the detection antibody (biotin-labeled polyclonal antibody) and horseradish peroxidase-labeled streptavidin (SA-HRP).

[0076] 1.1 Solution Preparation

[0077] PBS buffer: Weigh 8g NaCl, 0.2g KCl, 3.63g Na2HPO4·12H2O, and 0.24g KH2PO4, dissolve in water, adjust the pH to 7.4, and bring the volume to 1L. Store at room temperature for 1 month.

[0078] PBST buffer: Measure 100 ml of PBS buffer and mix with 50 μl of Tween 20.

[0079] PBST solution containing 0.5% BSA (diluent): Weigh approximately 0.5g of BSA, dissolve it in 100ml of PBST buffer, mix well, and store in a refrigerator (2~8℃). Shelf life is 7 days.

[0080] Antibody coating solution: Dilute the polyclonal antibody after reverse screening prepared according to the same method as step 2.2.2 in Example 1 with PBS buffer to 1.0 μg / ml, and use immediately.

[0081] Standard (antigen) solution: The residual protein of urokinase prepared according to step 1.3 in Example 1 was diluted with PBST solution containing 0.5% BSA to 128 ng / ml, 16 ng / ml and 2 ng / ml respectively;

[0082] Zero-well solution: 0.5% BSA in PBST solution (diluent);

[0083] Antibody solution for detection: Dilute the biotin-labeled polyclonal antibody obtained in step 2.3 with PBST solution containing 0.5% BSA to concentrations of 1.2 μg / ml, 0.6 μg / ml, 0.3 μg / ml, and 0.15 μg / ml, respectively, and prepare fresh for each use;

[0084] Preparation of horseradish peroxidase-labeled streptavidin solution (SA-HRP solution): dilute SA-HRP stock solution (manufacturer: Diamond, catalog number: B110053, concentration: 500U / ml) with 0.5% BSA in PBST solution to 10,000 times and 20,000 times respectively.

[0085] Colorimetric solution: TMB single-component colorimetric solution (manufacturer: Sangon Biotech, product number: E661007);

[0086] Washing solution: Washing solution (20×) (manufacturer: Sangon Biotech, product number: E661005), dilute at a ratio of 1:20 and mix well;

[0087] Termination solution: Manufacturer: Sangon Biotech, Product No.: E661006;

[0088] 1.2 Operating Procedures

[0089] (1) Coating: Add 100 μl of the prepared coating antibody solution with a concentration of 1.0 μg / ml to the wells of the plate, and seal the plate with sealing film. Coat overnight at 2~8℃.

[0090] (2) Washing the plate: Discard the liquid inside the plate, add 300µl of washing solution to each well 3 times, each time for more than 30 seconds. After the last wash, pat the remaining washing solution dry on absorbent paper.

[0091] (3) Sealing: Add 200 μl of 0.5% BSA in PBST solution to each well, seal the plate with a sealing membrane, and incubate at 37±1℃ for 1 h.

[0092] (4) Washing the plate: Discard the liquid inside the plate, add 300µl of washing solution to each well once, and pat the remaining washing solution dry on absorbent paper.

[0093] (5) Adding samples: According to the experimental layout, add 100 μl of zero-well solution and standard (antigen) solutions of different concentrations (128 ng / ml, 16 ng / ml, 2 ng / ml) to the corresponding wells. Each sample is replicated. Seal the plate with the sealing film and incubate at 37℃±1℃ for 1 h.

[0094] (6) Washing the plate: Discard the liquid inside the plate, add 300 μl of washing solution to each well 3 times, each time for more than 30 seconds. After the last wash, pat the remaining washing solution dry on absorbent paper.

[0095] (7) Add detection solution: Add 100µl of biotin-labeled polyclonal antibody solution of different concentrations (1.2μg / ml, 0.6μg / ml, 0.3μg / ml and 0.15μg / ml) to each well and incubate at 37±1℃ for 1h.

[0096] (8) Washing the plate: Discard the liquid inside the plate, add 300µl of washing solution to each well 3 times, each time for more than 30 seconds. After the last wash, pat the remaining washing solution dry on absorbent paper.

[0097] (9) Add SA-HRP solution: Add 100µL of peroxidase-labeled streptomycin solution (SA-HRP solution) (0.05U / ml or 0.025U / ml) to each well and incubate at 37±1℃ for 1h.

[0098] (10) Washing the plate: Discard the liquid inside the plate, add 300µl of washing solution to each well 4 times, each time for more than 30 seconds. After the last wash, pat the remaining washing solution dry on absorbent paper.

[0099] (11) Color development: Add 100µl of color development solution to each well and incubate at 37±1℃ for 15±2min.

[0100] (12) Termination: Add 100µl of termination solution to each well to terminate the reaction.

[0101] (13) Detection reading: The OD value of each well is read by the microplate reader and the wavelength is measured at 450 nm (measured within 10 min after the stop solution is added).

[0102] OD 450 The test results are shown in Table 1. Based on the results in Table 1, the S / N ratio was calculated. S / N is the ratio of the absorbance value (S) of the positive well (or sample well) to the absorbance value (N) of the negative control well (or background well). In this embodiment, it is the ratio of the absorbance of the standard wells with antigen concentrations of 2, 16, and 128 ng / ml to the absorbance of the zero well. The results are shown in Table 2.

[0103] Table 1

[0104]

[0105] Table 2

[0106]

[0107] Analysis of the data in Table 2 shows that the optimal S / N ratio can be obtained under the experimental conditions of a coating antibody concentration of 1.0 µg / ml, a detection antibody concentration of 0.3 µg / ml, and SA-HRP diluted 20,000 times (i.e., a concentration of 0.025 U / ml). Further method development can be conducted under these experimental conditions.

[0108] 2. Application of polyclonal antibody pairs in the development and validation of methods for the urokinase residual protein detection kit

[0109] 2.1 Solution Preparation

[0110] PBS buffer: Weigh 8g NaCl, 0.2g KCl, 3.63g Na2HPO4·12H2O, and 0.24g KH2PO4, dissolve in water, adjust the pH to 7.4, and bring the volume to 1L. Store at room temperature for 1 month.

[0111] PBST buffer: Measure 100 ml of PBS buffer and mix with 50 μl of Tween 20.

[0112] PBST solution containing 0.5% BSA (diluent): Weigh approximately 0.5g of BSA, dissolve in 100ml of PBST buffer, mix well, and store in a refrigerator (2~8℃). Shelf life is 7 days.

[0113] Antibody coating solution: Dilute the polyclonal antibody obtained after reverse screening according to step 2.2.2 in Example 1 with PBS buffer to 1.0 μg / ml, and use immediately.

[0114] Antibody solution for detection: Dilute the biotin-labeled polyclonal antibody obtained in step 2.3 of Example 1 to a concentration of 0.3 μg / ml with 0.5% BSA in PBST solution, and use immediately.

[0115] Preparation of horseradish peroxidase-labeled streptavidin solution (SA-HRP solution): Dilute the SA-HRP stock solution (manufacturer: Diamond, catalog number: B110053, concentration: 500 U / ml) with 0.5% BSA in PBST solution to a concentration of 0.025 U / ml (20,000 times) and use immediately.

[0116] Colorimetric solution: TMB single-component colorimetric solution (manufacturer: Sangon Biotech, product number: E661007);

[0117] Preparation of washing solution: Washing solution (20×) (manufacturer: Sangon Biotech, product number: E661005), dilute at a ratio of 1:20 and mix well;

[0118] Termination solution: Manufacturer: Sangon Biotech, Product No.: E661006;

[0119] Preparation of standards: The preparation process of protein calibration standards is shown in Table 3, and the preparation process of quality control standards is shown in Table 4.

[0120] In Table 3, P1: The residual protein of urokinase was prepared according to the method of Example 1 and prepared into a 1,000,000 ng / ml residual protein solution of urokinase;

[0121] P2: Prepare a 20000 ng / ml residual protein solution of urokinase from P1;

[0122] P3: Prepare a 400 ng / ml residual protein solution of urokinase from P2;

[0123] STD1 (Standard 1): Prepared from P3 as a 256 ng / ml residual protein solution of urokinase;

[0124] STD2 (Standard 2): Prepared from STD1 as a 128 ng / ml residual protein solution of urokinase;

[0125] STD3 (Standard 2): Prepare a 64 ng / ml residual protein solution of urokinase from STD2;

[0126] STD4 (Standard 2): Prepared from STD3 as a 32 ng / ml solution of residual protein in urokinase;

[0127] STD5 (Standard 2): Prepared from STD4 as a 16 ng / ml residual protein solution of urokinase;

[0128] STD6 (Standard 2): Prepared from STD5 to an 8 ng / ml solution of residual protein in urokinase;

[0129] STD7 (Standard 2): Prepared from STD6 as a 4 ng / ml solution of residual protein in urokinase;

[0130] STD0 (Standard 2): Diluent (PBST solution containing 0.5% BSA).

[0131] In Table 4, P4: The residual protein of urokinase was prepared according to the method of Example 1 and prepared into a 1,000,000 ng / ml residual protein solution of urokinase;

[0132] P5: Prepared from P4 as a 20000 ng / ml residual protein solution of urokinase;

[0133] P6: Prepared from P5 as a 400 ng / ml residual protein solution of urokinase;

[0134] ULOQ (Upper Limit of Quantitation): Prepared from P6 as a 256 ng / ml residual protein solution of Urokinase;

[0135] HQC (High Quality Control): Prepared by ULOQ as a 192 ng / ml residual protein solution of Urokinase;

[0136] MQC (Medium Quality Control): Prepared by HQC as a 100 ng / ml solution of residual protein in urokinase;

[0137] LQC (Low Quality Control): A 10 ng / ml residual protein solution of ursodeoxylin prepared from MQC;

[0138] LLOQ (Lower Limit of Quantitation): Prepared by LQC as a 4 ng / ml residual protein solution of urokinase.

[0139] Sensitivity verification samples: 20 zero-well samples, i.e., dilution solution (PBST solution containing 0.5% BSA).

[0140] Table 3

[0141]

[0142] Table 4

[0143]

[0144] 2.2 Operating Procedures

[0145] (1) Coating: Add 100 μl of the prepared coating antibody solution with a concentration of 1.0 μg / ml to the wells of the plate, and seal the plate with sealing film. Coat overnight at 2~8℃.

[0146] (2) Washing the plate: Discard the liquid inside the plate, add 300µl of washing solution to each well 3 times, each time for more than 30 seconds. After the last wash, pat the remaining washing solution dry on absorbent paper.

[0147] (3) Blocking: Add 200 μl of PBST solution containing 0.5% BSA to each well, seal the plate with a sealing membrane, and incubate at 37±1℃ for 1 h.

[0148] (4) Washing the plate: Discard the liquid inside the plate, add 300µl of washing solution to each well once, and pat the remaining washing solution dry on absorbent paper.

[0149] (5) Adding samples: According to the experimental layout, add 100 μl of the standard, quality control and sensitivity verification samples as described above to the corresponding wells. Each sample is duplicated. Seal the plate with a sealing film and incubate at 37℃±1℃ for 1 h.

[0150] (6) Washing the plate: Discard the liquid inside the plate, add 300 μl of washing solution to each well 3 times, each time for more than 30 seconds. After the last wash, pat the remaining washing solution dry on absorbent paper.

[0151] (7) Add detection solution: Add 100µl of prepared biotin-labeled polyclonal antibody solution with a concentration of 0.3μg / ml to each well and incubate at 37±1℃ for 1h.

[0152] (8) Washing the plate: Discard the liquid inside the plate, add 300µl of washing solution to each well 3 times, each time for more than 30 seconds. After the last wash, pat the remaining washing solution dry on absorbent paper.

[0153] (9) Add SA-HRP solution: Add 100µL of peroxidase-labeled streptomycin solution (SA-HRP solution, concentration of 0.025U / ml) to each well and incubate at 37±1℃ for 1h.

[0154] (10) Washing the plate: Discard the liquid inside the plate, add 300µl of washing solution to each well 4 times, each time for more than 30 seconds. After the last wash, pat the remaining washing solution dry on absorbent paper.

[0155] (11) Color development: Add 100µl of color development solution to each well and incubate at 37±1℃ for 15±2min.

[0156] (12) Termination: Add 100 µl of termination solution to each well to terminate the reaction.

[0157] (13) Detection reading: The OD value of each well is read by the microplate reader and the wavelength is measured at 450 nm (measured within 10 min after the stop solution is added).

[0158] 2.3 Standard Curve and Results of Sensitivity and Accuracy Verification Experiments

[0159] With theoretical concentration as the x-axis and the difference between the measured OD value and the mean OD value of the blank replicate well as the y-axis, a standard curve is fitted using a four-parameter regression model.

[0160] Four-parameter regression model: y = (AD) / [1 + (x / C)] B ]+D, where A is the estimated value of the asymptote below the curve, D is the estimated value of the asymptote above the curve, B is the slope of the curve, and C is the concentration corresponding to half of the maximum binding. The OD values ​​of the measured standard, quality control, and sensitivity verification samples are... 450 The values ​​are shown in Tables 5-1 and 5-2. The formula for the standard curve is:

[0161]

[0162] Standard curve see Figure 1 The corresponding sensitivity sample test results are shown in Table 6, and the corresponding quality control sample test results are shown in Table 7.

[0163] Table 5-1

[0164]

[0165] Table 5-2

[0166]

[0167] Table 6

[0168]

[0169] Table 7

[0170]

[0171] As can be seen from the above, the polyclonal antibody has a good linear correlation with the standard curve, the accuracy deviation of the quality control is ≤10%, the method quantitation limit is 4 ng / ml, the method sensitivity is 0.0231 ng / ml, the standard curve is stable, the data is accurate, and it can be used for sample detection.

[0172] Example 3: Application of the Urokinase Residual Protein Detection Kit in the Tracking and Detection of Urokinase Raw Material

[0173] The kit solution prepared in 2.1 of Example 2 was used to detect residual proteins in multiple batches of Urokinase active pharmaceutical ingredient (the preparation method is described in Example 1 of Chinese patent application CN119144590A, entitled "A method for preparing human urinary kininogenase to reduce bacterial endotoxins").

[0174] Three batches of urokinase active pharmaceutical ingredient were diluted with PBST solution containing 0.5% BSA to a protein concentration of approximately 0.125 mg / ml, resulting in Sample-1, Sample-2, and Sample-3.

[0175] The preparation and operation steps of other solutions in the kit are the same as in Example 2. Each sample was run in duplicate. The detection results are shown in Tables 8 and 9, and the formula for the standard curve is as follows:

[0176]

[0177] Standard curve see Figure 2 .

[0178] Table 8

[0179]

[0180] Table 9

[0181]

[0182] As shown in Tables 8 and 9, the residual protein content in three batches of Urokinase raw materials was detected using the above-mentioned kit, and the CV was <5% for all of them. This indicates that the polyclonal antibody of the present invention has good reproducibility when used in the enzyme-linked immunosorbent assay kit to detect the residual protein content in Urokinase raw materials.

Claims

1. A kit for detecting residual urokinase protein, characterized in that, The detection kit includes a capture antibody and a detection antibody. The capture antibody includes a polyclonal antibody against residual urokinase protein, and the detection antibody includes a biotin-labeled polyclonal antibody against residual urokinase protein. The polyclonal antibody against residual urokinase protein is obtained by immunizing animals multiple times with residual urokinase protein as an immunogen. The residual protein of urokinase is obtained by further purifying the crude human urinary kininogenase product after removing urokinase by adsorption using an anti-urokinase monoclonal antibody affinity column; the anti-urokinase monoclonal antibody affinity column is obtained by coupling an anti-urokinase monoclonal antibody to an affinity column, and the anti-urokinase monoclonal antibody is CELL-5; The preparation process of the polyclonal antibody against urokinase residue protein includes: immunizing animals multiple times with urokinase residue protein as an immunogen to obtain animal antiserum, binding and eluting with urokinase residue protein to obtain affinity-purified antibody; further binding the affinity-purified antibody with urokinase to obtain polyclonal antibody after reverse screening. The process of multiple immunizations in animals includes: using multiple rabbits, each rabbit receiving an initial immunization injection of 4-6 mg of residual urokinase protein and a booster immunization injection of 2-3 mg of residual urokinase protein; during the first injection, 0.8-1.2 mL of complete Freund's adjuvant is mixed with each 1 mL of residual urokinase protein, and during each subsequent injection, 0.8-1.2 mL of incomplete Freund's adjuvant is mixed with each 1 mL of residual urokinase protein, using multiple injection sites, for a total of 4-6 immunizations.

2. The detection kit for residual urokinase protein according to claim 1, characterized in that, The concentration of the anti-Urokinase residual protein polyclonal antibody is 0.8~1.2µg / ml, and the concentration of the biotin-labeled anti-Urokinase residual protein polyclonal antibody is 0.15~1.2µg / ml; and / or, the detection kit further includes one or more of streptavidin, coating buffer, chromogenic solution, washing solution and stop solution.

3. The method for preparing the detection kit for residual urokinase protein according to any one of claims 1 to 2, characterized in that, Includes the following steps: (1) Preparation of residual urokinase protein: urokinase was removed from the crude human urinary kininogenase product by adsorption using an anti-urokinase monoclonal antibody affinity column and then further purified. (2) Preparation of capture antibody against polyclonal antibody against residual urokinase protein: Animal antiserum obtained by immunizing animals multiple times with residual urokinase protein as an immunogen was used to bind and elute the residual urokinase protein to obtain affinity purified antibody. (3) Preparation of biotin-labeled polyclonal antibody against residual urokinase protein: The polyclonal antibody against residual urokinase protein prepared in step (2) was biotin-labeled; The steps for preparing the anti-Urokinase monoclonal antibody affinity column in step (1) are as follows: the anti-Urokinase monoclonal antibody is mixed and dissolved with NaHCO3+NaCl buffer, then mixed and coupled with affinity packing material, and then washed with NaHCO3+NaCl buffer to obtain the coupled packing material; the coupled packing material is mixed with glycine+NaHCO3+NaCl buffer and stirred for reaction, and then washed several times with NaAc-HAc+NaCl buffer and Tris-HCl+NaCl buffer in sequence; the concentration of NaHCO3 in the NaHCO3+NaCl buffer is 0.08-0.12M, the concentration of NaCl is 0.4-0.6M, and the pH is 8.0-8.

5.

4. The preparation method according to claim 3, characterized in that, The adsorption removal process described in step (1) includes: regenerating the anti-Urokinase monoclonal antibody affinity column with NaAc-HAc+NaCl buffer, equilibrating and rinsing with PB+NaCl buffer, repeatedly loading the crude human urinary kininogenase product through the antibody affinity column to remove Urokinase, using PB+NaCl buffer as the permeation solution, collecting the permeation eluent, and then concentrating it by ultrafiltration.

5. The preparation method according to claim 3, characterized in that, The further purification process described in step (1) includes the removal of bacterial endotoxins.

6. The preparation method according to claim 3, characterized in that, The preparation process of the crude human urinary kininogenase in step (1) includes: after adsorbing urinary protein in urine with an adsorbent, collecting the adsorbent after adsorbing urinary protein, eluting it in a concentrated manner, loading the eluted urinary protein solution onto a metal chelate affinity chromatography column that has been equilibrated with a balancing solution, eluting with NaAc-HAc buffer and collecting the eluent, adding solid ammonium sulfate and diatomaceous earth to the eluent in sequence, letting it stand, filtering and collecting the precipitate.

7. The preparation method according to claim 3, characterized in that, The glycine + NaHCO3 + NaCl buffer solution has a glycine concentration of 0.08-0.12M, a NaHCO3 concentration of 0.08-0.12M, a NaCl concentration of 0.4-0.6M, and a pH of 8.0-8.

5.

8. The preparation method according to claim 6, characterized in that, The equilibration solution of the metal chelate affinity chromatography column is 0.01-0.2M phosphate buffer, NaCl concentration of 0-2M, pH 6.0-9.0, and the metal ion chelated by the metal chelate affinity chromatography column is Cu. 2+ Zn 2+ Ni 2+ Fe 3+ Any one of them.

9. The use of the detection kit according to claim 1 or 2, or the detection kit prepared by the preparation method according to any one of claims 3 to 8, in the qualitative and / or quantitative determination of residual urokinase protein.

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