Method for determining kininogenase and used kit

Abstract

The invention relates to a method for determining kininogenase and a used kit. On one hand, the invention relates to a kit for determining the content of kininogenase in human plasma, the kit comprises a test solution for testing, an antigen standard substance, a capture antibody, an enzyme-labeled antibody and a test method description file which are separately packaged by containers, and the test solution comprises PBS, PBST, a washing solution, a confining solution, a sample diluent, a stop solution, a coating buffer solution, a color developing solution A and a color developing solution B. The invention also relates to a method for determining the content of kininogenase in human plasma by using the kit. According to the kit and the method provided by the invention, the amount of endogenous kininogenase or exogenous kininogenase in a human body can be accurately and effectively measured, and a beneficial tool is provided for diagnosis and treatment of clinical diseases.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology and relates to a method for measuring kininogenase in a biological sample, particularly human plasma, and a kit for use in the method. The kininogenase is preferably human tissue kallikrein 1 (also known as pancreatic / renal kallikrein, human kallikrein 1, human tissue kallikrein-1, hK1, KLK1, kininogenase, or kininogenase). Background Art

[0002] As early as 1909, Abelous et al. first reported that intravenous injection of human urine could induce a transient decrease in dog blood pressure, thus identifying the presence of a hypotensive substance in urine. Kraut et al. subsequently discovered high concentrations of this substance in the pancreas and named it "kallikrein." Kallikreins are divided into two major categories: tissue kallikrein and plasma kallikrein.

[0003] Human tissue kallikreins are serine proteases located on human chromosomes 19q13.13-19q13.14. Different functions are associated with the different serine proteases they encode. Human tissue kallikreins form a large multigene family consisting of 15 members. Only one enzyme in both human and animal tissue kallikreins effectively releases the bioactive kinin, TK, from kininogen.

[0004] Research has found that the tissue kallikrein-kinin / bradykinin system (KKS), also known as the kinin system, is widely present in multiple systems in animals, with a particularly dense distribution within the cardiovascular system. This kinin system can interact with the renin-angiotensin system (RAS) to exert a range of biological activities. The tissue kallikrein-kinin / bradykinin system has a wide range of biological activities and is closely connected and cross-talks with the coagulation system, the renin-angiotensin system, and various vasoactive factor systems. Together, they maintain the normal physiological functions of multiple organs in the human body and participate in various complex pathophysiological processes. It regulates the cardiovascular, renal, and nervous systems, as well as glucose metabolism, relaxes blood vessels, and participates in inflammatory responses, pain stimulation, and shock reactions.

[0005] TK, a key enzyme in the human KKS system, can release bradykinin and kinin-like substances by cleaving low-molecular-weight kininogens. These substances then bind to bradykinin receptor 2 (B2) on endothelial smooth muscle cells in the vascular wall and elsewhere, activating signaling pathways such as nitric oxide-cyclic adenosine monophosphate (NO-cAMP) and prostacyclin-cyclic adenosine monophosphate (Prostacyclin-cAMP). This triggers a series of biologically active effects, including vasodilation, smooth muscle contraction and relaxation, inhibition of apoptosis, inflammation, proliferation, hypertrophy, and fibrosis, and promotion of angiogenesis and neurogenesis. Because B2 receptors are widely present in tissues and cells throughout the human body, such as smooth muscle cells, neurons, renal tubular epithelial cells, and cardiomyocytes, TK has a wide range of biological effects.

[0006] Human tissue kallikreins belong to a subgroup of secreted serine proteases within the S1 family of clan SA [Borgono CA, Michael IP, Diamandis EP. Human tissue kallikreins: physiologic roles and applications in cancer. Mol Cancer Res 2004;2:257-80]. Until recently, there has been considerable interest in the three so-called classic kallikreins: KLK1, KLK2, and KLK3. Of all the known tissue kallikreins, only human tissue kallikrein 1, or KLK1, can effectively hydrolyze low molecular weight kininogen (LMWK), releasing active kinins that regulate cardiovascular and renal function.

[0007] KLK1 has been shown to lower blood pressure, reduce renal and cardiac hypertrophy and fibrosis, and play a role in cardiac remodeling, alleviating kidney damage, reducing the incidence of cerebral infarction, and minimizing the risk of neurological damage. Furthermore, exogenous administration has demonstrated the role of KLK1 in preventing stroke, cardiovascular and cerebrovascular diseases, and kidney diseases. In recent years, further research has demonstrated that KLK1 levels have important clinical significance in the development and progression of cardiovascular and cerebrovascular diseases and may serve as a predictor of stroke incidence. KLK1 levels can predict stroke incidence and five-year event-free survival, allowing patients to take preventive measures early, thereby reducing the risk of stroke. Furthermore, KLK1 levels can be used to diagnose early-stage diabetic nephropathy earlier than urinary microalbumin excretion. In summary, KLK1 levels have important predictive value for cardiovascular and cerebrovascular diseases and diabetic kidney disease. Therefore, accurate measurement of KLK1 levels is of great significance in both clinical and scientific research. Summary of the Invention

[0008] The present application aims to provide a method for determining kallikrein. Based on the determination method, the present application aims to provide a kit for determining kallikrein. It has been unexpectedly found that the amount of human kallikrein, especially KLK1, can be effectively determined by using the method of the present application, and the present application is based on such a finding.

[0009] In the first aspect of the present application, a kit for determining the content of kallikrein, especially KLK1, in human plasma is provided, which comprises test solution, antigen standard, capture antibody (or coating antibody), enzyme-labeled antibody (or detection antibody), and test method instruction, which are respectively packaged in containers. The test solution comprises PBS with pH=7.4, PBST, washing solution, blocking solution, sample diluent, termination solution, coating buffer, color developing solution A, and color developing solution B.

[0010] According to the kit of the first aspect of the present application, the PBS is prepared as follows: 0.2 g of KH2PO4, 1.44 g of Na2HPO4, 8 g of NaCl, and 0.2 g of KCl are dissolved in water, and the pH is adjusted to 7.4 with hydrochloric acid or sodium hydroxide, and then water is added to 1 L.

[0011] According to the kit of the first aspect of the present application, the PBST is prepared as follows: 1 L of PBS and 0.5 mL of Tween-20 are mixed to obtain a PBS containing 0.05% Tween, which can also be recorded as 0.05% PBST.

[0012] According to the kit of the first aspect of the present application, the washing solution is prepared as follows: 1 L of PBS and 2 mL of Tween-20 are mixed to obtain a PBS containing 0.2% Tween, which can also be recorded as 0.2% PBST.

[0013] According to the kit of the first aspect of the present application, the blocking solution is prepared as follows: 2 g of BSA and 100 mL of PBST are mixed to obtain a PBS containing 2% BSA and 0.05% Tween.

[0014] According to the kit of the first aspect of the present application, the sample diluent is prepared as follows: 1 mL of the blocking solution and 19 mL of 0.05% PBST are mixed to obtain a 0.05% PBST containing 0.1% BSA with pH 7.4, which is used for diluting the antigen standard and the test sample. The test sample is a plasma sample.

[0015] According to the kit of the first aspect of the present application, the termination solution is prepared as follows: 21.7 mL of 98% concentrated sulfuric acid is added dropwise to 178.3 mL of distilled water, and then mixed to obtain the termination solution.

[0016] According to the kit of the first aspect of the present application, the coating buffer is prepared by dissolving 0.159 g of Na2CO3 and 0.293 g of NaHCO3 in water and diluting to 100 mL, which is a 0.05 M carbonate buffer at pH 9.6.

[0017] According to the kit of the first aspect of the present application, the color developing solution A is prepared by dissolving 13.6 g of sodium acetate, 1.6 g of citric acid, 0.3 mL of 30% hydrogen peroxide in water, and adding water to 500 mL; it can also be referred to as substrate A.

[0018] According to the kit of the first aspect of the present application, the color developing solution B is prepared by dissolving 0.2 g of disodium edetate, 0.95 g of citric acid, 50 mL of glycerol, 0.15 g of TMB hydrochloride in water, and adding water to 500 mL; it can also be referred to as substrate B.

[0019] According to the kit of the first aspect of the present application, the antigen standard is recombinant human kallikrein; further, the recombinant human kallikrein is prepared by the following method: Cloning the KLK1 cDNA into the multiple cloning site of the pCMV6-XL5 vector between the cytomegalovirus promoter and the polyadenylation signal to control transcription of the cDNA encoding pro-human KLK1; Transfecting the human KLK1 cDNA in pCMV6-XL5 into a CHO cell line using the FreeStyle™ MAX CHO expression system; After transfection and expression of recombinant human KLK1, collecting the culture supernatant of the cell culture, filtering, concentrating, reacting with trypsin to activate the recombinant human KLK1, and then inactivating the trypsin with soybean trypsin inhibitor; Eluting the activated recombinant human kallikrein on an OCTYL SEPHAROSE® column, purifying it with a benzamidine affinity column, purifying it with a DEAE column, and replacing the resulting active KLK1 component into PBS buffer to obtain purified recombinant human KLK1 as an antigen standard.

[0020] According to the kit of the first aspect of the present application, the capture antibody and enzyme-labeled antibody are prepared by the following method: Intraperitoneally injecting 6-8 week old Balb / c mice with antigen recombinant human KLK1 (e.g. 80 μg / time) and adjuvant (e.g. phytane, e.g. 0.5 mL / time), boosting every 2 weeks after the initial immunization, a total of 3 times; 3 days before the last immunization, intravenously injecting the antigen (half the amount of antigen), promoting the migration of plasma cells to the spleen, and taking the mouse serum 3 days after the last immunization, detecting the antibody titer by ELISA, and selecting the titer ≥1:104 Spleen cells were extracted from the animals; The immunized mice were sacrificed, the spleen was removed aseptically and placed in RPMI1640 medium containing 10% fetal bovine serum, the spleen was ground with a syringe core and filtered through a 200-mesh cell sieve to prepare a single-cell suspension, which was centrifuged and washed with medium, and the viable cells were counted; The mouse myeloma cells SP2 / 0 were cultured in RPMI1640 medium containing 10% FBS to maintain the logarithmic growth phase; The cells were mixed at a ratio of spleen cells:myeloma cells = 5-10:1, the supernatant was discarded, 50% PEG4000 (pH 7.2) preheated to 37°C was slowly added to the cell pellet, then preheated RPMI1640 medium was added to terminate the reaction and washed with medium to remove residual PEG, and the fused cells were obtained; The fused cells were cultured in HAT medium, half-volume replacement was performed at regular intervals to transition to complete medium, the enzyme-labeled plate was coated with the target antigen, the cell supernatant was added, after incubation, the HRP-labeled secondary antibody was added, and the OD value was measured after color development, the wells with OD values significantly higher than the negative control were selected, the positive well cells were diluted to 1 / well, and were plated on the enzyme-labeled plate, and the process was repeated 2-3 times until the positive rate of monoclonal cells was 100%; The same mice were injected intraperitoneally with norhopane (e.g., 0.5 mL), 7 days later, hybridoma cells were injected, and 10-14 days later, the antibody-containing ascites were collected, the ascites were purified using a Protein A affinity chromatography column, and seven antibodies labeled as MM01, MM02, MM03, MM04, MM05, MM06, and MM07 were obtained, which were used as capture antibodies or coating antibodies; The seven capture antibodies were HRP-labeled using a horseradish peroxidase-labeled antibody protein kit, and seven antibodies were obtained, labeled as MM01 / HRP, MM02 / HRP, MM03 / HRP, MM04 / HRP, MM05 / HRP, MM06 / HRP, and MM07 / HRP, which were used as enzyme-labeled antibodies or detection antibodies; The seven capture antibodies and the seven enzyme-labeled antibodies were paired two by two, and sandwich method verification was performed, the capture antibody concentration was 2 μg / mL, the enzyme-labeled antibody concentration was 0.5 μg / mL, the antigen standard, recombinant human kininogenase, was set at 0 ng / mL (i.e., blank), 1 ng / mL, and 10 ng / mL, respectively, and the absorbance of each well was read at 450 nm wavelength, the results were provided by subtracting the blank well value (i.e., OD450-K) from the standard 1 ng / mL and 10 ng / mL well absorbance, and the combination of OD450 value <0.1 for the blank pair group and OD450-K response value >4.0 for the 1 ng / mL and 10 ng / mL concentration standard groups was selected as the capture antibody and enzyme-labeled antibody.

[0021] The kit according to the first aspect of the present invention may optionally include an ELISA plate, which may also be provided separately from the kit. The ELISA plate is preferably a 96-well plate, preferably made of polystyrene, but polyvinyl chloride may also be used. In one embodiment, the kit may optionally include an anticoagulant tube for collecting blood and preparing test plasma. In one embodiment, the anticoagulant tube is prepared as follows: an aqueous solution containing 50 mg / mL sodium fluoride, 100 mg / mL sodium oxalate, and 10 mg / mL choline chloride is prepared using purified water, and the pH is adjusted to 6.5 using hydrochloric acid to prepare the anticoagulant solution. The anticoagulant solution is added to an empty blood collection plastic tube, and the ratio of the labeled volume of the tube to the volume of the anticoagulant solution is 20:1, thereby obtaining the anticoagulant tube.

[0022] According to the kit of the first aspect of the present invention, the test method description document records an operating method for determining the content of kininogenase in human plasma using the kit, comprising the following steps: 1) Coating: Dilute the capture antibody to a working concentration of 2 μg / mL in coating buffer, add 100 μL per well of the ELISA plate, and incubate at 4°C overnight. 2) Washing the plate: Wash the ELISA plate with washing solution; 3) Blocking: Add 200 μL of blocking solution to each well and incubate at room temperature for 1 hour; 4) Washing the plate: Wash the ELISA plate with washing solution; 5) Set up blank wells, wells with different dilutions of antigen recombinant human kininogenase standard, and wells with plasma test sample, add 100 μL to each well, and incubate at room temperature for 2 hours; 6) Washing the plate: Wash the ELISA plate with washing solution; 7) Add 100 μL of detection antibody diluted to 0.5 μg / mL in sample diluent to each well and incubate at room temperature for 1 hour; 8) Washing the plate: Wash the ELISA plate with washing solution; 9) Mix chromogenic solution A and chromogenic solution B at a volume ratio of 1:1, add 100 μL / well to the ELISA plate, and incubate at room temperature in the dark for 15 minutes; 10) Add 50 μL of stop solution to each well and measure the OD value of each well at 450 nm within 15 minutes; 11) Using the concentration of the antigen recombinant human kininogenase standard as the X-axis and the OD value as the Y-axis, a four-parameter logistic curve was fitted to obtain a standard curve; 12) Substitute the OD value measured in the test sample well into the standard curve and combine it with the dilution factor of the test sample to calculate the concentration of kininogenase in the test sample.

[0023] According to the kit of the first aspect of the present invention, in step 5), the plasma test sample is plasma appropriately diluted with a sample diluent so that the OD value falls within the range of the standard curve.

[0024] According to the kit of the first aspect of the present invention, plasma is obtained by the following method: collecting venous blood into an anticoagulant tube, mixing it with an anticoagulant solution, and immediately separating the plasma by high-speed centrifugation. The plasma is then frozen and stored at -22 to -18°C until testing. In one embodiment, the anticoagulant tube is prepared by preparing an aqueous solution containing 50 mg / mL sodium fluoride, 100 mg / mL sodium oxalate, and 10 mg / mL choline chloride using purified water, adjusting the pH to 6.5 with hydrochloric acid, and adding the anticoagulant solution to an empty plastic blood collection tube, such that the ratio of the labeled volume of the tube to the volume of the anticoagulant solution is 20:1, thereby obtaining the anticoagulant tube.

[0025] Furthermore, the second aspect of the present invention provides a method for determining the content of kininogenase in human plasma, which uses the test solution, antigen standard, capture antibody (or coating antibody), and enzyme-labeled antibody (or detection antibody) in the kit described in any one of the first aspects of the present invention to measure the collected and optionally diluted plasma of the subject, comprising the following steps: 1) Coating: Dilute the capture antibody to a working concentration of 2 μg / mL in coating buffer, add 100 μL per well of the ELISA plate, and incubate at 4°C overnight. 2) Washing the plate: Wash the ELISA plate with washing solution; 3) Blocking: Add 200 μL of blocking solution to each well and incubate at room temperature for 1 hour; 4) Washing the plate: Wash the ELISA plate with washing solution; 5) Set up blank wells, wells with different dilutions of antigen recombinant human kininogenase standard, and wells with plasma test sample, add 100 μL to each well, and incubate at room temperature for 2 hours; 6) Washing the plate: Wash the ELISA plate with washing solution; 7) Add 100 μL of detection antibody diluted to 0.5 μg / mL in sample diluent to each well and incubate at room temperature for 1 hour; 8) Washing the plate: Wash the ELISA plate with washing solution; 9) Mix chromogenic solution A and chromogenic solution B at a volume ratio of 1:1, add 100 μL / well to the ELISA plate, and incubate at room temperature in the dark for 15 minutes; 10) Add 50 μL of stop solution to each well and measure the OD value of each well at 450 nm within 15 minutes; 11) Using the concentration of the antigen recombinant human kininogenase standard as the X-axis and the OD value as the Y-axis, a four-parameter logistic curve was fitted to obtain a standard curve; 12) Substitute the OD value measured in the test sample well into the standard curve and combine it with the dilution factor of the test sample to calculate the concentration of kininogenase in the test sample.

[0026] The method of the present application can be used to determine the content of endogenous or exogenous kallikrein (KLK1) in human plasma. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 : Four-parameter Logistic fitted curve.

[0028] Figure 2 : Linear regression curve. DETAILED DESCRIPTION

[0029] The present application can be further described by the following examples, however, the scope of the present application is not limited to the following examples. Those skilled in the art will appreciate that various modifications and changes can be made to the present application without departing from the spirit and scope thereof. The present application generally and / or specifically describes the materials used in the experiments and the experimental methods. Although many of the materials and methods used to achieve the present application are known in the art, the present application is described as much as possible. The following examples further illustrate the present application, but do not limit the present application.

[0030] Example 1: Preparation of recombinant human kininogenase The recombinant human kallikrein (also known as human tissue kallikrein-1 or KLK1 or hKLK1 or human tissue kallikrein-1) prepared in this example can be used as an antigen standard for the detection kit of the present application, which can be used to detect kallikrein (human tissue kallikrein-1 or KLK1) in human plasma.

[0031] Recombinant human kininogenase can be prepared using methods known in the art, for example, as described in WO2013173923A1 or US20170340559A1. Specifically, a cDNA encoding a pro-human KLK1 with a 262 amino acid residue sequence can be purchased from OriGene Technologies, Inc. (Rockville, MD, US; https: / / www.origene.com / catalog / cdna-clones / expression-plasmids / sc122623-kallikrein-1-klk1-nm-002257-human-untagged-clone). The KLK1 cDNA, product number SC122623, is a human cDNA open reading frame clone cloned into the multiple cloning site of OriGene's pCMV6-XL5 vector, located between the cytomegalovirus (CMV) promoter and the polyadenylation signal to control transcription of the cDNA encoding pre-human KLK1. This KLK1 clone was sequenced and translated using translation software, revealing its identity to sequence 2 of US20170340559A1.

[0032] The FreeStyle MAX system is a complete system for transient protein production in mammals that combines Gibco FreeStyle medium, FreeStyle MAX reagents, and FreeStyle CHO-S cells or FreeStyle 293 cells. The FreeStyle™ MAX CHO Expression System (Gibco™, Thermofisher, Cat. No. K900020; https: / / www.thermofisher.cn / order / catalog / product / K900020) was used to transfect the human KLK1 cDNA in pCMV6-XL5 into the CHO cell line. This kit allows for transient transfection of the vector into Chinese hamster ovary (CHO) cells. The transfected cells are grown in 5 L of culture medium and express the protein in serum-free medium. CHO cells are grown in suspension and transiently transfected with the KLK1 vector using the lipid reagent provided in the kit according to the expression system instructions.

[0033] Recombinant human KLK1 was expressed and purified essentially according to the method described by Hsieng S. Lu (see Hsieng S. Lu, et al. Purification and Characterization of Human Tissue Prokallikrein and Kallikrein Isoforms Expressed in Chinese Hamster Ovary Cells, Protein Expression and Purification (1996), 8, 227-237). Briefly, after transfection and allowing sufficient time for expression of recombinant human KLK1, the culture supernatant of 5 liters of cell culture was collected by centrifugation and then filtered through a 0.2 μm filter. The clarified supernatant was concentrated and reacted with trypsin to activate recombinant human KLK1. Because transient transfection was performed using a cDNA encoding pro-human KLK1, recombinant human KLK1 secreted from CHO cells was in the form of an inactive proprotein. Therefore, assaying KLK1 activity in the cell culture supernatant required an activation step using trypsin. Specifically, trypsin was activated at a final concentration of 10 nM at room temperature for 2.5 hours, and then inactivated using soybean trypsin inhibitor (Gibco™, ThermoFisher, Cat. No. 17075029).

[0034] After activation of recombinant human kininogenase (recombinant human KLK1), ammonium sulfate was added to the supernatant and loaded onto an OCTYL SEPHAROSE® column (Octyl Sepharose™ 4 Fast Flow, cytivalifesciences). The octyl column elution pool of active KLK1 was further purified by a benzamidine affinity column. The active fractions on the benzamidine column were then mixed, buffer exchanged into DEAE equilibration buffer, and then purified using a DEAE column. The active KLK1 fractions from DEAE were combined and the buffer was exchanged into PBS buffer to obtain purified (HPLC purity 99.1%) recombinant human KLK1, which was divided into glass bottles and stored at -20°C as an antigen recombinant human kininogenase (KLK1) standard.

[0035] Example 2: Preparation of antibodies This example describes a method for preparing monoclonal antibodies against recombinant human kininogenase.

[0036] 1. Antigen preparation and animal immunization 1. Antigen preparation The present example uses recombinant human kallikrein as an antigen. Of course, the skilled person will know that purified human kallikrein from natural sources can also be used as an antigen.

[0037] 2. Immunization of animals 1) 6-8 week old Balb / c mice (uniform genetic background, high efficiency of fusion of splenocytes with SP2 / 0 myeloma cells; SCXK (E) 2022-0030, Vivotecnia) were selected, and before immunization, they were tested for the absence of specific pathogens (SPF level).

[0038] 2) Immunization route: Intraperitoneal injection of antigen recombinant human KLK1 (80 μg / time) + adjuvant phytane (0.5 mL / time), with a booster every 2 weeks after the first immunization, for a total of 3 times; 3 days before the last immunization, intravenous injection of antigen (half the amount of antigen), to promote the migration of plasma cells to the spleen.

[0039] 3) Titer detection: 3 days after the last immunization, the mouse serum was taken, and the antibody titer was detected by ELISA, and animals with a titer of ≥ 1:10 4 were selected for splenocyte extraction. The above immunization routes are conventional methods in the art.

[0040] 2. Cell Preparation and Fusion 1. Obtaining splenocytes (aseptic operation) The immunized mouse was sacrificed, the abdomen was disinfected with 75% ethanol, and the spleen was aseptically removed and placed in RPMI1640 medium (Thermo Fisher Scientific Inc.) containing 10% fetal bovine serum (FBS); The spleen was ground with a syringe core, filtered through a 200-mesh cell sieve to make a single-cell suspension, centrifuged (1500 rpm x 5 min) and washed twice with RPMI1640 medium, and the viable cells were counted (trypan blue staining, viability > 95%) and the concentration was adjusted to 1-2 x 10 6 cells / mL.

[0041] 2. Myeloma cell culture Mouse myeloma cells SP2 / 0 (mouse origin, HGPRT - , do not secrete antibodies) were cultured in RPM11640 medium containing 10% FBS, maintained in the logarithmic growth phase (cell doubling time 18-24 h), and subcultured no more than 20 passages. The viable cells were counted after washing twice with serum-free RPMI1640 and resuspending in RPMI1640 medium, and the concentration was adjusted to 1-2 x 10 6 cells / mL (ensure viability ≥ 95%).

[0042] 3. PEG-induced fusion Cell ratio: spleen cells: myeloma cells = 5-10:1 (the optimal ratio is 6:1 in this example). Calculate the required cell volume (in this example, take 6 mL of spleen cells + 1 mL of myeloma cells), mix them in a 15 mL centrifuge tube, centrifuge at 1500 rpm for 5 minutes, discard the supernatant, and gently tap the bottom of the tube to evenly pellet the cells. Fusion: Slowly add 1 mL of 50% PEG4000 (pH 7.2), preheated to 37°C, to the mixed cell pellet over 90 seconds while gently shaking the centrifuge tube. After 90 seconds, immediately add 10 mL of preheated RPMI1640 medium (containing Hepes buffer) to terminate the reaction. Centrifuge the terminated fused cells at 800-1000 rpm for 5 minutes, discard the supernatant, and resuspend the cell pellet in 5 mL of RPMI1640 medium containing 10% FBS.

[0043] Note that prolonged PEG exposure can lead to cytotoxicity, so strict timing is required. After fusion, the cells should be washed twice with incomplete culture medium to remove residual PEG.

[0044] 3. HAT screening and positive clone identification The procedures are performed using general procedures well known in the art.

[0045] 1. Principle of Selective Culture HAT medium components: hypoxanthine (H), aminopterin (A), thymidine (T); Screening mechanism: Aminopterin inhibits the de novo DNA synthesis pathway of cells. Myeloma cells die due to the lack of HGPRT and are unable to synthesize DNA through the salvage pathway. Spleen cells (HGPRT + ) can synthesize DNA but cannot proliferate on its own; hybridoma cells have both HGPRT and unlimited proliferation capabilities, so they survive.

[0046] 2. Cloning and Screening Process Plating culture: 1×10 5 Plate cells / well in a 96-well microtiter plate (HAT medium, ClonaCell™, Cat. No. 03831), place in a 37°C, 5% CO2 incubator, and change the medium by half every 3 days (the order of medium change is: HAT → HT → complete medium). Positive clone screening: After 7-10 days of culture, the culture supernatant was detected by indirect ELISA; Coating: Target antigen (1-10 μg / mL) is coated on the ELISA plate at 4°C overnight; Detection: Add cell supernatant, incubate, add HRP-labeled secondary antibody, measure OD value after color development, and screen wells with OD value significantly higher than negative control; Limiting dilution cloning: Dilute the cells in the positive wells to 1 / well and plate them in a 96-well plate (containing feeder cells and mouse peritoneal macrophages to provide growth factors). Repeat 2-3 times until the monoclonal positive rate reaches 100%.

[0047] IV. Hybridoma Cell Expansion, Preservation, and Antibody Production 1. Large-scale cultivation In vitro culture: Transfer to a 24-well plate → culture flask → bioreactor, culture in serum-free medium, and collect the supernatant (antibody concentration approximately 1-10 μg / mL); Induction of ascites in vivo: 0.5 mL of pristane was injected into the peritoneal cavity of mice of the same strain for pretreatment, and 1×10 6 hybridoma cells and collect ascites after 10-14 days (antibody concentration can reach 1-10 mg / mL).

[0048] 2. Cell Cryopreservation Cryopreservation conditions: Take cells in logarithmic growth phase, add freezing solution (90% FBS + 10% DMSO), and press 1×10 6 The volume / mL was divided into cryopreservation tubes and the temperature was programmed to cool (4°C for 30 min → -20°C for 30 min → -80°C overnight → liquid nitrogen storage); 3. Antibody Purification and Identification The culture medium and ascites were purified using Protein A affinity chromatography column (Protein A 4FF Chromatography Column, Yisheng) to obtain antibodies.

[0049] Using the above steps, seven antibodies were prepared, labeled as MM01, MM02, MM03, MM04, MM05, MM06, and MM07, which are generally referred to as capture antibodies or coating antibodies or other similar names in the art.

[0050] 5. Antibody Pairing The seven antibodies MM01 to MM07 prepared above were labeled with horseradish peroxidase (HRP) using the antibody protein kit (Cat. No. SYLD032) according to the instructions as follows: 1 mg of HRP was dissolved in 250 μl of water and then added with 50 μl of oxidation solution. The mixture was reacted in a magnetic stirrer in the dark at room temperature for 2 min, with a pH of 6.0-6.5, to obtain solution a; 1 mg of antibody was added to 100 μl of labeling buffer, with a volume ratio of antibody to buffer of 10 / 1 and a pH of 8.5, to obtain solution b; Liquid B was slowly added to liquid B while shaking. After mixing, the pH was adjusted to 7.0-7.5. The reaction was continued in a dark and room temperature shaking incubator for 1.5 h. After the reaction was completed, 10% glycerol was added and mixed, and the pH was adjusted to about 7.0. Seven antibodies labeled with HRP were obtained, which were respectively designated as MM01 / HRP, MM02 / HRP, MM03 / HRP, MM04 / HRP, MM05 / HRP, MM06 / HRP, and MM07 / HRP. They are generally referred to as enzyme-labeled antibodies, detection antibodies, or other similar names in the art.

[0051] The seven capture antibodies obtained above were paired with seven HRP-labeled enzyme-labeled antibodies in pairs for sandwich validation (see Example 3 of the present invention). The concentration of the capture antibodies was set to 2 μg / mL, the concentration of the HRP-labeled enzyme-labeled antibodies was set to 0.5 μg / mL, and the concentration of the antigen standard, recombinant human kininogenase, was set to 0 (i.e., blank), 1 ng / mL, and 10 ng / mL, respectively. The absorbance of each well was read at a wavelength of 450 nm. The absorbance of the positive wells with the 1 ng / mL and 10 ng / mL standards was subtracted from the blank well value (i.e., OD450-K) to provide the result.

[0052] The test results of the sandwich method verification are shown in the following table: , Continued table: .

[0053] Through the above pairing, a combination was selected in which the OD450 value of the blank well was as small as possible (preferably <0.1) and the OD450-K response value of the two concentrations of positive wells was as high as possible (preferably >3.5, more preferably >4.0). Finally, MM04 was selected as the coating antibody (concentration of 2 μg / mL) and MM07-HRP was selected as the detection antibody (concentration of 0.5 μg / mL).

[0054] Example 3: Kit and method for detecting kininogenase This example provides a kit for detecting kininogenase and a method for detecting kininogenase. The kit includes the pre-prepared or freshly prepared solutions described in "1. Preparation of Solutions for the Kit," the antibodies and antigens described in "2. Antibodies and Antigens for Experimental Use," and optionally the anticoagulant tube described in Example 4.

[0055] 1. Preparation of solutions used in the kit PBS : Take 0.2g of KH2PO4, 1.44g of Na2HPO4, 8g of NaCl, and 0.2g of KCl, add appropriate amount of water to dissolve, adjust the pH to 7.4 with hydrochloric acid or sodium hydroxide, and then add water to 1L to obtain (the pH of the obtained PBS is 7.4).

[0056] PBST : Mix 1 L of PBS and 0.5 mL of Tween-20 to obtain (PBS containing 0.05% Tween).

[0057] detergent : Mix 1 L of PBS and 2 mL of Tween-20 to obtain (PBS containing 0.2% Tween).

[0058] Blocking solution : Mix 2 g of BSA and 100 mL of PBST (PBS containing 2% BSA and 0.05% Tween).

[0059] Sample diluent : Mix 1 mL of blocking solution and 19 mL of 0.05% PBST to obtain (0.05% PBST containing 0.1% BSA, pH 7.4), which is used to dilute antigen standards and test samples.

[0060] Stop solution: Add 21.7 mL of 98% concentrated sulfuric acid dropwise to 178.3 mL of distilled water and mix well to obtain the product.

[0061] Coating buffer: Dissolve 0.159 g of Na2CO3 and 0.293 g of NaHCO3 in water and dilute to 100 mL to obtain 0.05 M carbonate buffer solution (pH 9.6).

[0062] Color development solution A: Take 13.6g of sodium acetate, 1.6g of citric acid, and 0.3mL of 30% hydrogen peroxide, add appropriate amount of water to dissolve, and add water to 500mL to obtain (also known as substrate A).

[0063] Color development solution B: Dissolve 0.2 g of disodium edetate, 0.95 g of citric acid, 50 mL of glycerol, and 0.15 g of TMB hydrochloride in appropriate amount of water, and add water to 500 mL to obtain (also known as substrate B).

[0064] ELISA plate: The ELISA plate used in the experiment is a 96-well polystyrene plate (the brand used in the experiment is Yisheng, product number 83650ES03). Polyvinyl chloride plates can also be used.

[0065] In the above-mentioned color developing solution B, TMB used is 3,3',5,5'-tetramethylbenzidine. If TMB is used instead of its hydrochloride, it can be dissolved in an appropriate amount of DMSO and then added to water.

[0066] 2. Antibodies and antigens for experiments: Capture antibody: capture antibody MM04 obtained in Example 2, with a working concentration of 2 μg / mL; Enzyme-labeled antibody: the enzyme-labeled antibody MM07 / HRP obtained in Example 2, with a working concentration of 0.5 μg / mL; Antigen: The recombinant human kininogenase obtained in Example 1 was used as an antigen to prepare standards of different concentrations in order to prepare a standard curve.

[0067] 3. Operation steps This procedure should be provided in the form of a paper document in the kit, or it can be provided in electronic form or other forms. All reagents used should be returned to room temperature before use.

[0068] 1) Coating: Dilute the capture antibody MM04 in coating buffer to the required working concentration (working concentration after dilution is 2 μg / mL), add 100 μL to each well, and incubate at 4°C overnight; 2) Washing: Add 300 μL of washing solution to each well and wash for 5 minutes. Repeat this three times, shaking thoroughly each time and patting dry on clean absorbent paper. 3) Blocking: Add 200 μL of blocking solution to each well and incubate at room temperature for 1 hour; 4) Washing: Add 300 μL of washing solution to each well and wash for 5 minutes. Repeat this three times, shaking thoroughly each time and patting dry on clean absorbent paper. 5) Set up blank wells and (antigen recombinant human kininogenase) standard wells (or other test sample wells). Add the blank well to well 1, and add different dilution concentrations of standard to wells 2 to 8, adding 100 μL to each well. Perform a triplicate well operation (the average OD value of the three wells is used as the blank, standard, or test sample value). Incubate at room temperature for 2 hours. 6) Washing: Add 300 μL of washing solution to each well and wash for 5 minutes. Repeat this three times, shaking thoroughly each time and patting dry on clean absorbent paper. 7) Dilute the detection antibody MM07 / HRP (i.e., enzyme-labeled antibody, working concentration after dilution: 0.5 μg / mL) with sample diluent to the required working concentration, add 100 μL / well to each well, and incubate at room temperature for 1 hour; 8) Washing: add 300 μL washing solution to each well, wash for 5 min, repeat for 3 times, and dry on clean paper after each washing; 9) Mix color developing solution A and B at a volume ratio of 1:1 (the mixed solution should be protected from light), add 100 μL / well, and incubate at room temperature for 15 min in the dark; 10) Add 50 μL stop solution to each well, and measure the OD value of each well at 450 nm within 15 min; 11) Take the concentration of the (antigen recombinant human kallikrein) standard as the X axis, and the OD value as the Y axis, and perform four-parameter Logistic curve fitting (the standard with a concentration of 0 can be regarded as a negative control or blank, and the blank can be deducted when fitting, and the OD value of the blank point should be ≤0.1), to obtain a standard curve; 12) According to the above steps, set the wells for adding the test sample (plasma) in step 5), and appropriately dilute the test sample plasma with a sample diluent if necessary, so that the OD value falls within the interval of the standard curve, and the kallikrein concentration in the test sample diluent obtained by the standard curve is multiplied by the dilution factor of the test sample to obtain the kallikrein concentration in the test sample.

[0069] 4. Standard curve Using steps 1) to 11) of the above "3, operation steps", the standard curves of the two methods can be obtained by four-parameter Logistic curve fitting and linear regression, respectively, as follows: .

[0070] The four-parameter Logistic curve fitting is shown in Figure 1 , and the linear regression curve is shown in Figure 2 . The X axis is the concentration of the antigen recombinant human kallikrein standard, and the Y axis is the absorbance measured at 450 nm. As can be seen from the results in the figure, the curves obtained by the two methods have excellent linear characteristics in the concentration range of 12.5-800 pg / mL of the antigen recombinant human kallikrein standard. In addition, the above standard curve results show that the equation obtained by four-parameter Logistic curve fitting is more optimal, and therefore, in the context of the present application, if not otherwise specified, the four-parameter Logistic curve fitting equation is used when calculating the kallikrein content using the standard curve.

[0071] Example 4: Detection of Kininogenase Content in Human Body Using Kit It is well known that there is an endogenous, natural kallikrein in the human body, such as human plasma, which is human tissue kallikrein 1 or KLK1 or hKLK1. This part of kallikrein is the background amount in the body, and the basic concentration of different individuals may have slight differences. The same individual may also have differences in different body states (such as healthy state and suffering from acute ischemic disease). In addition, people give patients exogenous KLK1 for the purpose of treating some diseases, such as the KLK1 described in WO2013173923A1, which is used for injection of type 1 diabetes or type 2 diabetes treated by drugs, and for example, Scott E. K. et al. reported the use of recombinant KLK1 to treat acute ischemic stroke and prevent recurrence (Scott E. Kasner, et al, Recombinant Human Tissue Kallikrein-1 for Treating Acute Ischemic Stroke and Preventing Recurrence, Stroke. 2025; 56: 745-753). Therefore, it is of great significance to determine the endogenous KLK1 content of healthy people or patients with diseases to evaluate the health status of the human body, and it is also of great significance to determine the change of KLK1 content in the body before and after the patient is administered with KLK1 for the purpose of monitoring the drug level in the process of treating diseases such as the above-mentioned diabetes or acute ischemic stroke.

[0072] This example uses the kit and its detection method provided in Example 3 to determine the kallikrein content in the plasma of 7 volunteers (5 men and 2 women) of the research team of the applicant, and the specific method steps are as follows: (a) Preparation of anticoagulant tube: prepare an aqueous solution containing sodium fluoride 50 mg / mL, sodium oxalate 100 mg / mL, and choline chloride 10 mg / mL (adjust pH to 6.5 with hydrochloric acid) with purified water as anticoagulant, and add the anticoagulant to the empty blood collection plastic tube. The ratio of the labeled volume of the tube containing blood to the volume of the anticoagulant is 20:1, and the anticoagulant tube is obtained (a blood collection anticoagulant tube with a labeled volume of 2 mL or 5 mL or other capacity as needed is obtained, and if not specified, a 2 mL tube is used. The anticoagulant tube can be configured in the kit described in Example 3); (b) Blood sample collection: each volunteer collects 2 mL of venous blood in the morning on an empty stomach and mixes it with the anticoagulant. Blood is collected every 2 days, a total of 7 times (blood collection period of 18 days, respectively, on day 0, day 3, every 6 days, …, day 18), and the plasma is separated by high-speed centrifugation within 5 minutes after blood collection. The plasma is stored at -22~-18°C and is ready for testing; (c) Take the above plasma, use the method of Example 3, and determine the KLK1 content (ng / mL) in the plasma using the "four-parameter Logistic curve fitting" equation according to steps 1) to step 12) in "3. Operation steps", and the results are as follows: the KLK1 content (ng / mL) of subject volunteer 1 (male, 52y, 69kg) measured 7 times was 262.4, 247.5, 303.2, 277.4, 259.7, 232.8, 313.7, and the mean ± sd = 271.0 ± 29.2; the KLK1 content (ng / mL) of subject volunteer 2 (male, 41y, 73kg) measured 7 times was 234.7, 198.3, 209.5, 253.4, 251.3, 248.3, 273.5, and the mean ± sd = 238.4 ± 26.4; the KLK1 content (ng / mL) of subject volunteer 3 (male, 24y, 63kg) measured 7 times was 313.5, 283.7, 327.2, 293.3, 263.8, 284.8, 334.8, and the mean ± sd = 300.2 ± 25.8; the KLK1 content (ng / mL) of subject volunteer 4 (female, 28y, 51kg) measured 7 times was 283.4, 225.8, 242.2, 292.7, 238.6, 247.4, 278.4, and the mean ± sd = 258.4 ± 25.9; the KLK1 content (ng / mL) of subject volunteer 5 (female, 26y, 55kg) measured 7 times was 313.2, 287.6, 302.5, 279.4, 228.8, 304.7, 268.4, and the mean ± sd = 283.5 ± 28.7; the KLK1 content (ng / mL) of subject volunteer 6 (male, 26y, 76kg) measured 7 times was 224.6, 186.3, 253.5, 231.8, 274.2, 246.3, 193.8, and the mean ± sd = 230.1 ± 31.7; the KLK1 content (ng / mL) of subject volunteer 7 (male, 28y, 66kg) measured 7 times was 286.4, 264.8, 340.2, 294.6, 273.6, 268.4, 286.3, and the mean ± sd = 287.8 ± 25.5; the KLK1 content (ng / mL) of the 7 volunteers measured was 186.3-340.2 ng / mL, and the mean ± sd = 267.0 ± 35.6.

[0073] The above determination results are comparable to the KLK1 content (ng / mL) in the plasma of normal people described in the literature, for example, CN101672851, and it should be noted that these results are the KLK1 concentration or content in the original plasma after dilution conversion, and in the context of this paper, the units of these results are ng / mL.

[0074] The above 49 blood samples were mixed in equal volume ratios to prepare mixed plasma, and the KLK1 content therein was measured at the same time. The result was 274.2 ng / mL, which was basically consistent with the above results.

[0075] (d) Referring to steps (a)-(c) above, except that choline chloride was not added when preparing the anticoagulant tube in step (a), seven blood samples were collected from volunteers 1 and 2 in parallel with step (b). The plasma obtained by the same method was processed and assayed according to step (c). The KLK1 content was 215.4±26.8 and 177.7±29.7, respectively, which was significantly lower. (e) Referring to steps (a)-(c) above, except that equimolar amounts of potassium fluoride and potassium oxalate were used instead of sodium fluoride and sodium oxalate when preparing the anticoagulant tube in step (a). Seven blood samples were collected from volunteers 3 and 6 in parallel with step (b). The plasma obtained by the same method was processed and assayed according to step (c). The KLK1 content was 258.1±30.2 and 185.6±27.5, respectively, which was significantly lower. (f) Referring to steps (a) to (c) above, in step (b), 1.0 µg of the recombinant human kininogenase standard obtained in Example 1 was additionally added to seven 2 mL aliquots of whole blood collected from Volunteer 2, Volunteer 3, Volunteer 4, and Volunteer 5 (equivalent to an additional standard concentration of 500 ng / mL in the whole blood). The plasma obtained by the same treatment was assayed according to step (c). The KLK1 content results were 747.6 ± 28.3, 788.4 ± 24.4, 767.8 ± 30.3, and 775.2 ± 33.2, respectively, which were generally consistent with the background value and the additional amount added. (g) Referring to step (d) above, in step (b), 1.0 μg of the recombinant human kininogenase standard obtained in Example 1 was additionally added to seven 2 mL aliquots of whole blood collected from volunteers 2 and 3 (equivalent to a concentration of 500 ng / mL of the additional standard added to the whole blood). The plasma obtained by the same treatment was then assayed according to step (c). The KLK1 content results were 621.2 ± 32.1 and 671.8 ± 27.4, respectively, which were significantly low. (h) Referring to step (e) above, in step (b), 1.0 μg of the recombinant human kininogenase standard obtained in Example 1 was additionally added to seven 2 mL aliquots of whole blood collected from volunteers 6 and 7 (equivalent to a concentration of 500 ng / mL of the additional standard added to the whole blood). The plasma obtained by the same treatment was then assayed according to step (c). The KLK1 content results were 638.5 ± 29.6 and 674.8 ± 30.7, respectively, which were significantly low. These unexpected findings suggest that adding trace amounts of choline chloride to the anticoagulant solution and simultaneously avoiding potassium salts can make the assay more accurate.

[0076] The kit and method provided by the application can accurately and effectively determine the amount of endogenous or exogenous kininogenase in human body, and provide a beneficial tool for clinical disease diagnosis and treatment.

[0077] The application is described by the above examples, but the application is not limited to the above detailed methods, that is, the application does not mean that the application must rely on the above detailed methods to be implemented. It should be understood by those skilled in the art that any improvement of the application, equivalent replacement of each raw material of the product of the application, addition of auxiliary ingredients, selection of specific methods, etc. fall within the protection scope and disclosure scope of the application.

Claims

1. A kit for determining the content of kininogenase in human plasma, comprising a test solution, an antigen standard, a capture antibody, an enzyme-labeled antibody, and test method instructions, each packaged in a container. The test solution comprises: PBS, PBST, washing solution, blocking solution, sample diluent, stop solution, coating buffer, chromogen A, chromogen B.

2. The kit according to claim 1, wherein The PBS is prepared as follows: 0.2 g of KH2PO4, 1.44 g of Na2HPO4, 8 g of NaCl, and 0.2 g of KCl are dissolved in an appropriate amount of water, the pH is adjusted to 7.4 with hydrochloric acid or sodium hydroxide, and then water is added to 1 L. The PBST is prepared as follows: 1 L of PBS and 0.5 mL of Tween-20 are mixed. The washing solution is prepared as follows: 1 L of PBS and 2 mL of Tween-20 are mixed. And / or, the blocking solution is prepared as follows: 2 g of BSA and 100 mL of PBST are mixed.

3. The kit according to claim 1, wherein The sample diluent was prepared as follows: 1 mL of blocking solution and 19 mL of 0.05% PBST is mixed evenly to obtain; the stop solution is prepared as follows: 21.7 mL of 98% concentrated sulfuric acid is added dropwise to 178.3 mL of distilled water and mixed evenly to obtain; the coating buffer is prepared as follows: 0.159 g of Na2CO3 and 0.293 g of NaHCO3 are dissolved in water and diluted to 100 mL to obtain; the color developing solution A is prepared as follows: 13.6 g of sodium acetate, 1.6 g of citric acid, and 0.3 mL of 30% hydrogen peroxide are dissolved in appropriate amount of water and water is added to 500 mL to obtain; and / or, the color developing solution B is prepared as follows: 0.2 g of disodium edetate, 0.95 g of citric acid, 50 mL of glycerol, and 0.15 g of TMB hydrochloride are dissolved in appropriate amount of water and water is added to 500 mL to obtain.

4. The kit according to claim 1, wherein the antigen standard is recombinant human kininogenase, which is prepared by the following method: The KLK1 cDNA was cloned into the multiple cloning site of the pCMV6-XL5 vector, located between the cytomegalovirus promoter and the polyadenylation signal, to control the transcription of the cDNA encoding the progenitor KLK1; Human KLK1 cDNA in pCMV6-XL5 was transfected into CHO cell lines using the FreeStyle™ MAX CHO Expression System. After transfection and expression of recombinant human KLK1, the culture supernatant of the cell culture was collected, filtered, concentrated, reacted with trypsin to activate recombinant human KLK1, and then the trypsin was inactivated with soybean trypsin inhibitor; The activated recombinant human kininogenase was eluted on an OCTYL SEPHAROSE® column, purified using a benzamidine affinity column, and then purified using a DEAE column. The resulting active KLK1 fraction was exchanged into PBS buffer to obtain purified recombinant human KLK1 as an antigen standard.

5. The capture antibody and enzyme-labeled antibody of the kit according to claim 1 are prepared by the following method: 6-8 week old Balb / c mice were intraperitoneally injected with antigen recombinant human KLK1 (e.g., 80 μg / time) and adjuvant (e.g., pristane, e.g., 0.5 mL / time). After the initial immunization, the mice were boosted every 2 weeks for a total of 3 times. Three days before the last immunization, the antigen (the amount of antigen was halved) was injected intravenously to promote the migration of plasma cells to the spleen. Three days after the last immunization, the mouse serum was collected and the antibody titer was detected by ELISA. The titer ≥1:10 was selected. 4 Splenocyte extraction was performed on the animals; Immunized mice were sacrificed, and the spleens were aseptically removed and placed in RPMI1640 medium containing 10% fetal bovine serum. The spleens were ground with a syringe plunger, filtered, and prepared into a single-cell suspension. After centrifugation, the suspensions were washed with medium, and the viable cells were counted. Mouse myeloma SP2 / 0 cells were cultured in RPMI1640 medium containing 10% FBS and maintained in the logarithmic growth phase; Mix the cells at a ratio of 5-10:1 between spleen cells and myeloma cells, discard the supernatant, and slowly add 50% PEG4000 preheated to 37°C dropwise to the cell pellet. Then, add preheated RPMI1640 medium to terminate the reaction and wash with medium to remove residual PEG to obtain fused cells. Culture the fused cells in HAT medium, change the medium to complete medium at regular intervals, coat the ELISA plate with the target antigen, add the cell supernatant, incubate, add HRP-labeled secondary antibody, measure the OD value after color development, select the wells with OD values ​​significantly higher than the negative control, dilute the cells in the positive wells to 1 / well, plate the cells on the ELISA plate, and repeat 2-3 times until the monoclonal positive rate reaches 100%; Syngeneic mice were intraperitoneally injected with pristane, and hybridoma cells were injected 7 days later. After 10-14 days, the antibody-containing ascites was collected and purified using a Protein A affinity chromatography column to obtain seven antibodies labeled MM01, MM02, MM03, MM04, MM05, MM06, and MM07, which were used as capture antibodies or coating antibodies. Seven capture antibodies were labeled with HRP using a horseradish peroxidase-labeled antibody protein kit to obtain seven antibodies, namely MM01 / HRP, MM02 / HRP, MM03 / HRP, MM04 / HRP, MM05 / HRP, MM06 / HRP, and MM07 / HRP, which were used as enzyme-labeled antibodies or detection antibodies; Seven capture antibodies and seven enzyme-labeled antibodies were paired in pairs for sandwich validation. The capture antibody concentration was 2 μg / mL, the enzyme-labeled antibody concentration was 0.5 μg / mL, and the concentrations of the antigen standard, recombinant human kininogenase, were set to 0 ng / mL (i.e., blank), 1 ng / mL, and 10 ng / mL, respectively. The absorbance of each well was read at a wavelength of 450 nm. The absorbance of the 1 ng / mL and 10 ng / mL standard wells was subtracted from the blank well value (i.e., OD450-K) to provide the result. Combinations with a blank pairing value <0.1 and OD450-K response values ​​>4.0 for the 1 ng / mL and 10 ng / mL standard groups were selected as the capture antibody and enzyme-labeled antibody.

6. The kit according to claim 1, which optionally further comprises an ELISA plate; and / or, the kit optionally further comprises an anticoagulant tube for collecting blood and preparing test plasma; for example, the anticoagulant tube is prepared as follows: an aqueous solution containing 50 mg / mL sodium fluoride, 100 mg / mL sodium oxalate, and 10 mg / mL choline chloride is prepared with purified water, and the pH value is adjusted to 6.5 with hydrochloric acid to obtain an anticoagulant solution, which is added to an empty blood collection plastic tube, with the ratio of the labeled volume of the tube to the volume of the anticoagulant solution being 20:1, thereby obtaining the anticoagulant tube.

7. The kit according to claim 1, wherein the test method instruction document describes an operating method for determining the content of kininogenase in human plasma using the kit, comprising the following steps: 1) Coating: Dilute the capture antibody to a working concentration of 2 μg / mL in coating buffer, add 100 μL per well of the ELISA plate, and incubate at 4°C overnight. 2) Washing the plate: Wash the ELISA plate with washing solution; 3) Blocking: Add 200 μL of blocking solution to each well and incubate at room temperature for 1 hour; 4) Washing the plate: Wash the ELISA plate with washing solution; 5) Set up blank wells, wells with different dilutions of antigen recombinant human kininogenase standard, and wells with plasma test sample, add 100 μL to each well, and incubate at room temperature for 2 hours; 6) Washing the plate: Wash the ELISA plate with washing solution; 7) Add 100 μL of detection antibody diluted to 0.5 μg / mL in sample diluent to each well and incubate at room temperature for 1 hour; 8) Washing the plate: Wash the ELISA plate with washing solution; 9) Mix chromogenic solution A and chromogenic solution B at a volume ratio of 1:1, add 100 μL / well to the ELISA plate, and incubate at room temperature in the dark for 15 minutes; 10) Add 50 μL of stop solution to each well and measure the OD value of each well at 450 nm within 15 minutes; 11) Using the concentration of the antigen recombinant human kininogenase standard as the X-axis and the OD value as the Y-axis, a four-parameter logistic curve was fitted to obtain a standard curve; 12) Substitute the OD value measured in the test sample well into the standard curve and combine it with the dilution factor of the test sample to calculate the concentration of kininogenase in the test sample.

8. The kit according to claim 7, wherein in step 5), the plasma test sample is plasma appropriately diluted with a sample diluent so that the OD value falls within the range of the standard curve.

9. The kit according to claim 1, wherein the plasma is obtained by the following method: collecting venous blood into an anticoagulant tube, mixing it with an anticoagulant solution, and immediately separating the plasma by high-speed centrifugation. The plasma is then frozen and stored at -22 to -18°C until testing. For example, the anticoagulant tube is prepared by preparing an aqueous solution containing 50 mg / mL sodium fluoride, 100 mg / mL sodium oxalate, and 10 mg / mL choline chloride using purified water, adjusting the pH to 6.5 with hydrochloric acid, and adding the anticoagulant solution to an empty blood collection plastic tube. The ratio of the labeled volume of the tube to the volume of the anticoagulant solution is 20:1, thereby obtaining the anticoagulant tube.

10. A method for determining the content of kininogenase in human plasma, comprising the following steps: using the test solution, antigen standard, capture antibody, and enzyme-labeled antibody in the kit according to any one of claims 1 to 9 to determine the content of kininogenase in human plasma collected and optionally diluted. 1) Coating: Dilute the capture antibody to a working concentration of 2 μg / mL in coating buffer, add 100 μL per well of the ELISA plate, and incubate at 4°C overnight. 2) Washing the plate: Wash the ELISA plate with washing solution; 3) Blocking: Add 200 μL of blocking solution to each well and incubate at room temperature for 1 hour; 4) Washing the plate: Wash the ELISA plate with washing solution; 5) Set up blank wells, wells with different dilutions of antigen recombinant human kininogenase standard, and wells with plasma test sample, add 100 μL to each well, and incubate at room temperature for 2 hours; 6) Washing the plate: Wash the ELISA plate with washing solution; 7) Add 100 μL of detection antibody diluted to 0.5 μg / mL in sample diluent to each well and incubate at room temperature for 1 hour; 8) Washing the plate: Wash the ELISA plate with washing solution; 9) Mix chromogenic solution A and chromogenic solution B at a volume ratio of 1:1, add 100 μL / well to the ELISA plate, and incubate at room temperature in the dark for 15 minutes; 10) Add 50 μL of stop solution to each well and measure the OD value of each well at 450 nm within 15 minutes; 11) Using the concentration of the antigen recombinant human kininogenase standard as the X-axis and the OD value as the Y-axis, a four-parameter logistic curve was fitted to obtain a standard curve; 12) Substitute the OD value measured in the test sample well into the standard curve and combine it with the dilution factor of the test sample to calculate the concentration of kininogenase in the test sample.

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