A detection kit for determining the concentration of rFVIIa in an animal sample and a detection method thereof

Abstract

The present invention belongs to the field of pharmaceutical technology, and particularly relates to a detection kit for determining the concentration of rFVIIa in animal samples and a detection method thereof. The present invention constructs a detection kit by screening detection reagents such as capture proteins, detection antibodies, diluents, etc., and uses the detection kit of the present invention to detect the concentration of rFVIIa in monkey plasma. The method provided by the present invention has a good linear relationship, and there is no interference from endogenous coagulation factors, and the accuracy and selectivity meet the requirements. The detection kit and the detection method thereof of the present invention have prospects in detecting the concentration of rFVIIa in animal samples, and provide technical support for the non-clinical research of rFVIIa and the development of rFVIIa products.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical technology, and particularly relates to a detection kit for determining the concentration of rFVIIa in animal samples and a detection method thereof. Background Art

[0002] Recombinant human clotting factor VIIa (rFVIIa) is a hemostatic drug for hemophilia patients. It is a vitamin K-dependent glycoprotein composed of 406 amino acid residues and is structurally similar to activated factor VII purified from human plasma. Currently, the FDA has approved two rFVIIa products for market, namely NovoSeven approved in 1999 and SEVENFACT approved in 2020. There is no rFVIIa product on the market in China. In the non-clinical research of rFVIIa, researchers need to evaluate the efficacy and safety in animals. This means that the accurate detection of the concentration of rFVIIa in animal samples is crucial for the research and development of rFVIIa products.

[0003] At present, the drug concentration of rFVIIa in animals can only be detected by the coagulation method, lacking a method for directly detecting the protein concentration of this drug. In addition, there are some detection kits or detection antibodies for detecting FVIIa on the market. However, the detection objects of these detection products are human serum, plasma and other samples. When used for the detection samples of animals other than humans, it is difficult to distinguish the endogenous coagulation factor VIIa in animals, and the measured results do not represent the true concentration of rFVIIa drug in animals, thus affecting the accuracy of non-clinical animal pharmacokinetics and toxicokinetics results and causing obstacles to the research and development of rFVIIa products.

[0004] In addition to commercially available FVIIa detection kits or antibodies, there is natural tissue factor (TF) in animals. It forms a TF-rFⅦa complex with rFVIIa through ligand binding at the injury site. Therefore, tissue factor can be used as an alternative for detecting rFVIIa in animal samples. However, it is unknown whether tissue factor can distinguish endogenous coagulation factor VIIa in animals from rFVIIa, and the detection effect is also unpredictable compared with other commercially available FVIIa detection kits or antibodies.

[0005] In rFVIIa non-clinical studies, commonly used model animals include mice, rats, rabbits, dogs, and monkeys. Among them, monkeys are the closest to humans in terms of physiology, biochemistry, and immunology, and are ideal models for studying drug safety and pharmacodynamics. When differentiating coagulation factor VIIa from different sources, it is quite difficult to distinguish between monkey endogenous coagulation factor VIIa and human coagulation factor VIIa.

[0006] Therefore, establishing a method that can avoid species cross-reactivity and accurately detect the concentration of rFVIIa in animal samples is crucial for the non-clinical research of rFVIIa and the development of rFVIIa products. Summary of the Invention

[0007] Aiming at the defects of the prior art, the present invention provides a detection kit and a detection method for determining the concentration of rFVIIa protein in animal samples, aiming to more accurately determine the concentration of rFVIIa in monkey samples.

[0008] The present invention provides a detection kit for determining the concentration of rFVIIa in animal samples, and the detection kit for determining the concentration of rFVIIa in animal samples includes the following components in parts by weight:

[0009] Tissue factor 0.1 - 0.4 parts,

[0010] Detection antibody 0.05 - 0.2 parts,

[0011] Sample diluent 950 - 3800 parts,

[0012] Labeled antibody 0.05 - 2 parts;

[0013] The composition and parts by weight of the labeled antibody are selected from:

[0014] 1) Enzyme-labeled antibody 0.05 - 2 parts / well, chromogenic substrate 10 - 40 parts / well;

[0015] Or, 2) Fluorescently labeled antibody 0.05 - 2 parts / well.

[0016] The present invention provides a detection kit for determining the concentration of rFVIIa in animal samples, and the detection kit for determining the concentration of rFVIIa in animal samples includes the following components in parts by weight:

[0017] Tissue factor 0.1 - 0.4 parts,

[0018] Detection antibody 0.05 - 0.2 parts,

[0019] Labeled antibody 0.05 - 2 parts,

[0020] Sample diluent 950 - 3800 parts;

[0021] 10 - 40 parts of chromogenic substrate;

[0022] The labeled antibody is obtained by dissolving the labeled antibody dry powder in 0.5 ml of water. The labeled antibody dry powder is an enzyme-labeled antibody with the catalog number 170935 produced by Jackson ImmunoResearch; the chromogenic substrate is a commercially available ready-to-use ELISA chromogenic solution.

[0023] Preferably, the tissue factor is selected from one of recombinant human tissue factor proteins.

[0024] Preferably, the detection antibody is selected from one of monoclonal or polyclonal tissue factor detection antibodies.

[0025] Preferably, the sample diluent is selected from at least one of cysteine, blocking agent, and non-ionic surfactant; the blocking agent is selected from at least one of skim milk powder, bovine serum albumin, casein, and gelatin; the non-ionic surfactant is selected from at least one of Tween 20 and Triton X-100.

[0026] Preferably, the detection kit for determining the rFVIIa concentration in animal samples further includes a calibration standard; the rFVIIa concentration in the calibration standard is 250 - 16000 ng / mL; the calibration standard is prepared from animal blank samples, sample diluent, and rFVIIa.

[0027] Preferably, the enzyme-labeled antibody is selected from at least one of antibodies that are enzyme-labeled and specifically bind to the detection antibody; the fluorescently labeled antibody is selected from at least one of antibodies that are labeled with a fluorescent group and specifically bind to the detection antibody;

[0028] And / or, the enzyme-labeled antibody is selected from at least one of antibodies labeled with horseradish peroxidase, alkaline phosphatase, glucose oxidase, or biotin;

[0029] And / or, the chromogenic substrate is selected from at least one of 3,3',5,5'-tetramethylbenzidine, 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid), o-phenylenediamine, 3,3'-diaminobenzidine, QuantaBlu fluorescent substrate, QuantaRed enhanced chemifluorescent substrate, p-nitrophenyl phosphate, and glucose;

[0030] And / or, the fluorescently labeled antibody is selected from at least one of antibodies labeled with Alexa Fluor fluorescent group, Brilliant Violet fluorescent group, Cyanine fluorescent group, fluorescein isothiocyanate group, tetramethylrhodamine isothiocyanate, RhodamineRed-X, and aminomethylcoumarin acetate.

[0031] Preferably, the working solutions of the detection antibody and the labeled antibody are aqueous solutions containing 4.5 - 5.5 g / mL of a blocking agent and 0.045 - 0.055% by volume of a non-ionic surfactant;

[0032] And / or, the detection kit for determining the concentration of rFVIIa in an animal sample further includes a washing solution and a termination solution; the washing solution is an aqueous solution containing 0.045 - 0.055% by volume of a non-ionic surfactant; the termination solution is a 0.2 - 0.5 M sulfuric acid or hydrochloric acid solution;

[0033] The blocking agent is selected from at least one of skim milk powder, bovine serum albumin, casein, and gelatin; the non-ionic surfactant is selected from at least one of Tween 20 and Triton X-100.

[0034] A detection method for non-diagnostic purposes using the detection kit for determining the concentration of rFVIIa in an animal sample according to any one of the above:

[0035] Step 1, centrifuge the animal sample to be tested, take the supernatant, and then mix the supernatant to be tested with a sample diluent to prepare a sample to be tested;

[0036] Step 2, incubate the sample to be tested with tissue factor, detection antibody, and labeled antibody in sequence;

[0037] Step 3, measure the ultraviolet absorption signal or fluorescence signal of the product.

[0038] Preferably, the animal sample in Step 1 is at least one of animal serum, plasma, urine, tissue homogenate, cell culture, and cell lysate;

[0039] And / or, the animal in Step 1 is selected from at least one of mice, rats, rabbits, dogs, and monkeys;

[0040] And / or, in Step 1, the supernatant to be tested and the sample diluent are mixed at a volume ratio of 22.5 - 27.5:1;

[0041] And / or, the incubation conditions in Step 2 are 10 - 30 °C, 270 - 500 rpm, the incubation time with tissue factor is 2 h ± 20 min, and the incubation time with the detection antibody is 1.5 h ± 10 min; when the labeled antibody in Step 2 is an enzyme-labeled antibody and a chromogenic substrate, the incubation time with the enzyme-labeled antibody is 1 h ± 5 min, and the incubation time with the chromogenic substrate is 15 - 35 min; when the labeled antibody in Step 2 is a fluorescently labeled antibody, the incubation time with the fluorescently labeled antibody is 1 h ± 5 min;

[0042] And / or, the ultraviolet absorption signal described in step 3 is the ultraviolet absorption signal at 450 nm, 405 nm or 492 nm.

[0043] The present invention constructs a detection kit by screening detection reagents such as capture proteins, detection antibodies, diluents, etc., and uses the kit of the present invention to detect the concentration of rFVIIa in monkey plasma. The method provided by the present invention has a good linear relationship, and there is no interference from endogenous coagulation factors, and the accuracy and selectivity meet the requirements. The detection kit and its detection method of the present invention have prospects in detecting the concentration of rFVIIa in animal samples, and provide technical support for the non-clinical research of rFVIIa and the development of rFVIIa products.

[0044] Obviously, based on the above content of the present invention, according to the common general knowledge and conventional means in the art, without departing from the above basic technical idea of the present invention, various other forms of modifications, substitutions or changes can be made.

[0045] The following is a further detailed description of the above content of the present invention through specific embodiments in the form of examples. However, this should not be construed as limiting the scope of the above subject matter of the present invention to the following examples. All technologies implemented based on the above content of the present invention belong to the scope of the present invention. Brief Description of the Drawings

[0046] Figure 1 A typical standard curve graph for detecting the concentration of recombinant human coagulation factor a in monkey plasma by ligand binding method. Detailed Description of the Invention

[0047] In the following examples and experimental examples, the reagents and materials not specifically described are all commercially available products.

[0048] The blood samples of monkeys used in the present invention are all from the blood samples of cynomolgus monkeys.

[0049] I. Main Instrument Equipment

[0050] In the following examples and experimental examples, the main instrument equipment used is shown in Table 1 below:

[0051] Table 1 Main Instrument Equipment

[0052]

[0053] II. Software and Data Systems

[0054] In the following examples and experimental examples, the software and data systems used are shown in Table 2 below:

[0055] Table 2 Software and Data Systems Used

[0056]

[0057] III. Standard Substance Information

[0058] ① rFVIIa Information

[0059] Name or Code: Recombinant Human Coagulation Factor VIIa for Injection;

[0060] Source: Chengdu Rongsheng Pharmaceutical Co., Ltd.;

[0061] Appearance: White loose solid, colorless and clear liquid after reconstitution, may have slight opalescence;

[0062] Specification: 1 mg / vial;

[0063] Storage Conditions and Precautions: 2 - 8 °C, protected from light;

[0064] ② Standard Substance Stock Solution

[0065] Solvent: Histidine Diluent for Injection;

[0066] Preparation Method: For each vial of rFVIIa, add 1 mL of Histidine Diluent for Injection along the wall of the vial and let it flow down, avoiding direct injection onto the powder. Use after complete dissolution;

[0067] Storage Conditions after Preparation: 2 - 8 °C, protected from light.

[0068] IV. Data Processing and Analysis Methods

[0069] Use Watson LIMS v7.6.1 SP1 software to fit the standard curve equation with the OD values of the standards obtained by the multifunctional microplate reader using the 4 - parameter (weighting factor 1 / Y**2) method, calculate the concentration results of each sample, and issue the standard curve parameters, etc.

[0070] Values such as %RE, standard deviation (SD), and %CV used in the report are calculated by Office Excel 2010. The calculation formulas are as follows:

[0071] Average value: (C t is the measured concentration);

[0072] Percentage of relative error: (C t is the measured concentration, C n is the theoretical concentration);

[0073] Percentage of coefficient of variation: ( is the average value of the detected concentration);

[0074] Standard deviation: STDEV(SD)= ( is the average value of the detected concentration).

[0075] Total allowable error: %Total Error= .

[0076] Data such as concentration results, SD, standard curve parameters, etc. are retained to three significant figures, accuracy (%RE), coefficient of variation (%CV), and total allowable error (%Total Error) are retained to one decimal place, and OD values and R 2 are retained to at least three decimal places.

[0077] Example 1 Detection Kit for Measuring rFVIIa Protein Concentration in Monkey Plasma and Its Detection Method

[0078] I. Detection Kit for Measuring rFVIIa Protein Concentration in Monkey Plasma

[0079] This example provides a detection kit for measuring rFVIIa protein concentration in monkey plasma, which consists of the following components: tissue factor, detection antibody working solution, enzyme-labeled antibody working solution, calibration standard, sample diluent, washing solution, TMB chromogenic solution, termination solution, PBS, enzyme-labeled plate. The following are the relevant reagents or solutions and their preparation methods:

[0080] 1. Key reagent information

[0081] The information of the key reagents used in this example is shown in Table 3 below:

[0082] Table 3 Key reagent information

[0083]

[0084] Note: "1" indicates that the concentration of this reagent is 1.037 mg / mL; "2" this reagent is a dry powder, and 0.5 mL of pure water is added to each vial before use to dissolve it completely. After reconstitution, it is stored at -70 °C or below; the conventional concentration of TMB is 0.1 mg / mL, and the ready-to-use chromogenic solution TMB product in Table 3 is preferably used in this example.

[0085] 2. Solution preparation

[0086] (1) Blank matrix

[0087] Monkey blank plasma is stored at -60 °C or below for later use and thawed at 10 - 30 °C before use.

[0088] (2) Sample diluent

[0089] Weigh 1.0 g of L-Cysteine (Adamas beta, 62776B), and dissolve it thoroughly in 100 mL of pure water to obtain a 10 mg / mL cysteine solution.

[0090] (3)Sample buffer

[0091] Before preparing the standard curve samples, the blank plasma of cynomolgus monkeys and the sample diluent (i.e., 10 mg / mL cysteine solution) are mixed at a volume ratio of 25:1 to prepare the sample buffer.

[0092] (4)Preparation of calibration standards

[0093] Use the sample buffer to prepare calibration standards of rFVIIa with concentrations of 16000 ng / mL, 10000 ng / mL, 8000 ng / mL, 4000 ng / mL, 2000 ng / mL, 1000 ng / mL, 500 ng / mL, and 250 ng / mL respectively.

[0094] (5)Preparation of samples to be tested

[0095] Take whole animal blood, centrifuge to separate the plasma, and then mix the plasma to be tested and the sample diluent (i.e., 10 mg / mL cysteine solution) at a volume ratio of 25:1 to prepare the sample to be tested.

[0096] (6)Detergent (0.05% PBST)

[0097] Take 50 mL of 20×PBS buffer, add it to 950 mL of pure water, and mix thoroughly to prepare 1×PBS; then take 500 µL of Tween-20 and add it to 999.5 mL of 1×PBS and mix thoroughly.

[0098] (7)5% SM-1×PBST

[0099] Weigh 5.0 g of Difco TM Skim Milk (skim milk powder) and dissolve it thoroughly in 100 mL of 0.05% PBST.

[0100] (8)Working solution of detection antibody

[0101] Take Anti-Factor Ⅶ antibody [EPR22364-119]-BSA and Azide free (Detector) and dilute it 2000 times with the diluent (5% SM-1×PBST), and mix thoroughly. The preparation method is as follows:

[0102] Table 4

[0103]

[0104] Note: The concentration of the source solution is 1.037 mg / mL.

[0105] (9) Enzyme-labeled antibody working solution

[0106] For the enzyme-labeled antibody purified by affinity, the recommended concentration range is 0.5 - 5 μg / mL. In this example, the following enzyme-labeled antibody is preferred, and the preparation method is as follows: Take Peroxidase-conjugated Affini Pure TM Donkey Anti-RabbitIgG (H+L) is diluted 1000-fold with the diluent (5% SM-1×PBST) and mixed well. This product is in dry powder form. Before each use, 0.5 ml of pure water is required for each vial to fully dissolve it to make the source solution, and then it is diluted as follows.

[0107] Table 5

[0108]

[0109] The above preparation can be scaled up or down proportionally according to actual needs.

[0110] (10) Stop solution (0.5 M H2SO4)

[0111] Take 972.8 mL of pure water, slowly add 27.2 mL of concentrated H2SO4 to a certain volume of the above pure water, stir well, and after cooling, add the remaining pure water and mix well.

[0112] II. Detection method

[0113] (1) Coating the plate: Dilute the antigen Recombinant Human Tissue Factor Protein (His Tag) (hereinafter referred to as TF) to 2 μg / mL with the coating solution, i.e., 1×PBS. After mixing, add it to a 96-well enzyme-linked immunosorbent assay (ELISA) plate, 50 μL / well; after coating, place it in a refrigerator at 4°C overnight;

[0114] (2) Blocking: Discard the coating solution, wash the plate once with the washing solution and pat dry; add 150 μL of the blocking solution, i.e., 5% SM-1×PBST, to each well and incubate in an incubator at 37°C for 1 h;

[0115] (3) Adding samples: Discard the blocking solution, wash the plate 3 times with the washing solution and pat dry; take the prepared sample to be tested or calibration standard, dilute it 20-fold with the sample diluent, and then add it to the ELISA plate, 100 μL / well, cover with a film, and incubate at room temperature at 300 rpm for 2 h;

[0116] (4)Washing the plate: Wash 3 times with the washing solution, 300 μL per well, and pat dry;

[0117] (5)Adding the detection antibody: Add the prepared detection antibody working solution to the ELISA plate, 100 μL per well, cover with a film, and incubate at room temperature for 1.5 h under the condition of 300 rpm;

[0118] (5)Washing the plate: Wash 3 times with the washing solution, 300 μL per well, and pat dry;

[0119] (6)Adding the enzyme-labeled antibody: Add the prepared enzyme-labeled antibody working solution to the ELISA plate, 100 μL per well, cover with a film, and incubate at room temperature for 1 h under the condition of 300 rpm;

[0120] (7)Washing the plate: Wash 3 times with the washing solution, 300 μL per well, and pat dry;

[0121] (8)Color development: Add the TMB color development solution to the ELISA plate, 100 μL per well, cover with a film, and let it stand for color development at room temperature in the dark for 20 min;

[0122] (7)Termination: Add the termination solution, 100 μL per well, to terminate the reaction, and read the values within 20 min after termination;

[0123] (8)Detection: Use a multifunctional microplate reader to read the OD values of each well at the detection wavelength of 450 nm (reference wavelength 620 nm).

[0124] III. Data processing and analysis

[0125] Use the Watson LIMS v7.6.1 SP1 software to fit the standard curve equation for the OD values of the calibration standards obtained by the multifunctional microplate reader by the four-parameter (weighting factor 1 / Y**2) method, and calculate the rFVIIa concentration in the sample to be tested according to the external standard method.

[0126] Example 2 Detection kit for determining the rFVIIa protein concentration in monkey plasma and its detection method

[0127] Prepare the detection kit and perform the detection according to the method of Example 1, with the difference that: the sample diluent is 1% BSA - 1×PBST, and the preparation of the sample buffer, calibration standards, and samples to be tested is changed accordingly according to the preparation method of Example 1.

[0128] The preparation method of 1% BSA - 1×PBST is as follows:

[0129] Weigh 1.0 g of bovine serum albumin and dissolve it thoroughly in 100 mL of 0.05% PBST.

[0130] Example 3 Use of the detection kit of the present invention for non-diagnostic purposes

[0131] In the non-clinical study of rFVIIa, the detection kit prepared in Example 1 of the present invention was used to measure the concentration of rFVIIa in the dissected heart, liver, spleen, lung and kidney tissues of mice, so as to evaluate the distribution of rFVIIa drug in mice and measure the pharmacokinetic data.

[0132] The technical solution of the present invention will be further described below through experiments.

[0133] Experimental Example 1 Comparison with Existing Detection Reagents

[0134] In this experimental example, a screening and comparison experiment was carried out on the antibodies used for detection. Except as otherwise specified, the detection method was carried out according to the method of Example 1. The samples detected in this experimental example were calibration standards, and the concentrations of the calibration standards were set as shown in Table 6 or Table 7 respectively.

[0135] I. Detection with Commercially Available Antibody Pairs

[0136] Detection was carried out with commercially available antibody pairs (coating antibody: Anti-Factor VII antibody {EPR22364-134}-BSA and Azide free (Capture), product number: ab259697, 5 ug / mL; detection reagent: Anti-Factor VII {EPR22364-119}-BSA and Azide free (Detector), product number: ab259698, diluted 100 times with 1% BSA-1×PBST). Only monkey blank plasma was used when preparing the samples, and the samples were diluted with the self-prepared diluent (1% BSA-1×PBST) and monkey plasma with different contents before loading, so as to explore whether there was matrix interference in this detection method.

[0137] The results are shown in Table 6. When adjusting the dilution of the standard sample with monkey plasma with different contents, in the standard curve, the signal value of the high-concentration point was too low, and starting from the concentration point of 31.25 ng / mL, the signal value of the low-concentration point fluctuated too much, affecting the detection accuracy of low-concentration samples and unable to meet the high-sensitivity detection requirements. Therefore, this method could not be used to detect the concentration of rFVIIa protein in animal plasma.

[0138] Table 6

[0139]

[0140] II. Detection with Commercially Available Kits

[0141] 1. Detection of Standard Samples Prepared with Monkey Blank Plasma

[0142] Using a commercially available FVIIa detection kit (ZYMUTEST Factor VII, Aniara Diagnostica), standard samples were prepared with monkey blank plasma and diluted with the diluent provided in the kit or a self-prepared diluent (1% BSA - 1×PBST) before loading.

[0143] The results are shown in Table 7. The results indicate that the background color development of the standard curve prepared with monkey blank plasma was too high, and there was no difference between the two diluents, suggesting the presence of endogenous matrix interference. Therefore, standard samples were prepared using monkey blank plasma and a self-prepared diluent (1% BSA - 1×PBST) respectively.

[0144] Table 7

[0145]

[0146] 2. Detection of standard samples prepared with self-prepared diluent

[0147] Using a commercially available FVIIa detection kit (ZYMUTEST Factor VII, Aniara Diagnostica), standard samples were prepared with monkey blank plasma and a self-prepared diluent (1% BSA - 1×PBST) respectively, and diluted with the self-prepared diluent (1% BSA - 1×PBST) before loading.

[0148] The results are shown in Table 8. The data shows that the signal values of the standard curve measured for the samples prepared with the diluent were normal, but the background of the monkey blank plasma group was too high, indicating interference, suggesting that this kit could not distinguish endogenous coagulation factors in animal plasma.

[0149] Table 8

[0150]

[0151] The above results indicate that the commercially available rFVIIa detection antibody pair and kit still could not distinguish endogenous coagulation factors in animal plasma even after adjusting and optimizing the detection method, could not avoid species cross-reaction, and could not be used to determine the rFVIIa concentration in animal plasma.

[0152] III. Combined detection of capture protein (receptor, TF) and detection antibody

[0153] Use TF (Sino Biological, catalog number: 13133-H08B) to coat the plate as the capture protein, and the detection reagent in the commercially available antibody pair (Anti-FactorVII {EPR22364-119}-BSA and Azide free (Detector), catalog number: ab259698) as the detection antibody. Only monkey blank plasma is used when preparing the sample, and the sample is diluted with a self-prepared diluent (1% BSA - 1×PBST) before loading.

[0154] The results are shown in Table 9. The results show that the method has good linearity and can be used for the detection of rFVIIa in animal plasma.

[0155] Table 9

[0156]

[0157] The above results indicate that when the capture protein is tissue factor and the detection antibody is the commercially available detection antibody (Anti-FactorVII [EPR22364-119]), the linear relationship is good and it can be used for the detection of the concentration of rFVIIa in animal plasma.

[0158] Experimental Example 2 Optimization of Detection Conditions

[0159] I. Experimental Method

[0160] In this experimental example, the sample diluent was optimized. Except for the sample diluent and its corresponding sample buffer, other experimental methods and conditions were the same as those in Example 1.

[0161] The sample diluents in this experimental example were a self-prepared diluent (1% BSA - 1×PBST) and a 10 mg / mL cysteine solution, and the corresponding sample buffers were: (1) Monkey blank plasma and the self-prepared diluent were prepared into a sample buffer according to a volume ratio of 25:1; (2) Monkey blank plasma and the 10 mg / mL cysteine solution were prepared into a sample buffer according to a volume ratio of 25:1.

[0162] II. Experimental Results

[0163] The detection results using the self-prepared diluent are shown in Table 10, and the detection results using the diluent added with cysteine are shown in Table 11. The results show that when the capture protein and the detection antibody are the same, using the monkey blank plasma prepared with cysteine (monkey blank plasma: cysteine volume ratio of 10 mg / mL is 25:1) as the sample buffer to prepare the sample, and using 10 mg / mL cysteine as the sample diluent to dilute the prepared sample before loading. The sample detection signal value is higher, the standard curve has a good linearity, and the precision (coefficient of variation (%CV)) of different concentration samples (except 125 ng / mL) is <20% meeting the quality control standard.

[0164] Table 10

[0165]

[0166] Table 11

[0167]

[0168] Therefore, after adding cysteine to the sample diluent and the sample buffer, it can be used for the detection of rFVIIa in animal plasma, and the detection signal value is increased.

[0169] Experimental Example 4 Method Validation

[0170] In this experimental example, the linear relationship, precision, accuracy, and selectivity were investigated according to the detection method of Example 1.

[0171] I. Standard Curve

[0172] The specific standard curve results are shown in Table 12, and a typical standard curve is shown in the appendix Figure 1 . The experimental results show that when using the method of the present invention to detect the concentration of rFVIIa in monkey plasma, the linear relationship is good in the linear range of 250 - 16000 ng / mL.

[0173] Table 12 Standard Curve Results for Detecting Recombinant Human Coagulation Factor a Concentration in Monkey Plasma by Ligand Binding Method

[0174]

[0175] II. Accuracy and Precision

[0176] Prepare quality control samples at 5 concentrations of rFVIIa, namely ULOQ (Upper Limit of Quantitation, 16000 ng / mL), HQC (High Quality Control, 12800 ng / mL), MQC (Medium Quality Control, 5120 ng / mL), LQC (Low Quality Control, 1280 ng / mL), and LLOQ (Lower Limit of Quantification, 500 ng / mL), with 3 replicates for each set. According to the method of Example 1, perform the detection.

[0177] The preparation method of the quality control samples is as follows:

[0178] Use sample buffer to prepare quality control samples of rFVIIa at concentrations of 16000 ng / mL (ULOQ), 12800 ng / mL (HQC), 5120 ng / mL (MQC), 1280 ng / mL (LQC), and 500 ng / mL (LLOQ).

[0179] Table 13 Precision and accuracy results of detecting recombinant human coagulation factor for injection in monkey plasma by ligand binding method Results of precision and accuracy at concentration a

[0180]

[0181] The specific results are shown in Table 13. The experimental results show that the precision (coefficient of variation (%CV)) of detecting quality control samples at different concentrations (except LLOQ) by this method is < 20%, and for LLOQ (500 ng / mL) it is < 25%; the accuracy (recovery rate (%RE)) is between -5.3% and 13.8%, all meeting the quality control requirements. The method of the present invention can be used for detecting the concentration of rFVIIa protein in animal plasma.

[0182] III. Selectivity

[0183] 1. Selectivity under blank samples

[0184] Detect 6 monkey blank plasma samples from different sources according to the method of Example 1. The sample numbers are SE-Blank 01, SE-Blank 02, SE-Blank 03, SE-Blank 04, SE-Blank 05, and SE-Blank 06. At the same time, also detect the LLOQ (500 ng / mL) sample.

[0185] The detection results are shown in Table 14. The OD values of SE-Blank in 6 monkey blank plasma samples from different sources were all less than the LLOQ OD value, indicating that there were no obvious endogenous interfering substances affecting the determination of the analyte.

[0186] Table 14 Selective determination results of the concentration of recombinant human coagulation factor VII for injection in monkey plasma by ligand binding assay (I)

[0187]

[0188] 2. Selectivity at low and high concentration levels

[0189] According to the method of Example 1, quality control samples with concentrations of 12,800 ng / mL (SE-HQC) and 500 ng / mL (SE-LLOQ) of rFVIIa were prepared using sample buffer (where the monkey blank plasma was from three sources: SE-Blank 01, SE-Blank 02, and SE-Blank 03), and then detected according to the method of Example 1.

[0190] The detection results are shown in Table 15. The %RE of all SE-HQC samples was within ±20.0%; the %RE of all SE-LLOQ samples was within ±25.0%, meeting the selectivity accuracy requirements, indicating that the method had good selectivity.

[0191] Table 15 Selective determination results of the concentration of recombinant human coagulation factor VII for injection in monkey plasma by ligand binding assay (II)

[0192]

[0193] The above results indicate that for the detection method of Example 1 for detecting the concentration of rFVIIa in monkey plasma, the linear relationship was good within the linear range of 250 - 16,000 ng / mL; it had good precision, accuracy, and selectivity. The method of the present invention was not interfered by animal endogenous substances and could be used for the detection of rFVIIa in animal plasma.

[0194] From the above examples and experimental examples, it can be seen that the present invention screened detection reagents such as capture proteins, detection antibodies, and diluents to construct a detection kit, and used the kit of the present invention to detect the concentration of rFVIIa in monkey plasma. The method provided by the present invention had a good linear relationship, and there was no interference from endogenous coagulation factors, and the accuracy and selectivity met the requirements. The detection kit and its detection method of the present invention had prospects in detecting the concentration of rFVIIa in animal samples, providing technical support for the non-clinical research of rFVIIa and the development of rFVIIa products.

Claims

1. A detection kit for determining the concentration of rFVIIa in an animal sample, characterized in that, The detection kit for determining the concentration of rFVIIa in an animal sample comprises the following components in parts by weight: Tissue factor: 0.1 - 0.4 parts, Detection antibody: 0.05 - 0.2 parts, Sample diluent: 950 - 3800 parts, Labeled antibody: 0.05 - 2 parts; The composition and parts by weight of the labeled antibody are selected from: 1) Enzyme-labeled antibody: 0.05 - 2 parts / well, chromogenic substrate: 10 - 40 parts / well; Or, 2) Fluorescently labeled antibody: 0.05 - 2 parts / well; The detection antibody is selected from the commercially available detection antibody Anti-Factor VII antibody, clone number EPR22364-119, and the sample diluent is selected from cysteine; The rFVIIa is recombinant human coagulation factor.

2. A detection kit for determining the concentration of rFVIIa in an animal sample, characterized in that, The detection kit for determining the concentration of rFVIIa in an animal sample comprises the following components in parts by weight: Tissue factor: 0.1 - 0.4 parts, Detection antibody: 0.05 - 0.2 parts, Labeled antibody: 0.05 - 2 parts, Sample diluent: 950 - 3800 parts; Chromogenic substrate: 10 - 40 parts; The labeled antibody is obtained by dissolving the labeled antibody dry powder in 0.5 ml of water. The labeled antibody dry powder is an enzyme-labeled antibody with the product number 170935 produced by Jackson ImmunoResearch. The chromogenic substrate is a commercially available ready-to-use ELISA chromogenic solution; The detection antibody is selected from the commercially available detection antibody Anti-Factor VII antibody, clone number EPR22364-119, and the sample diluent is selected from cysteine; The rFVIIa is recombinant human coagulation factor.

3. The detection kit for determining the concentration of rFVIIa in an animal sample according to claim 1 or 2, characterized in that: The tissue factor is selected from one of recombinant human tissue factor proteins.

4. The detection kit for determining the concentration of rFVIIa in an animal sample according to claim 1 or 2, characterized in that: The detection kit for determining the concentration of rFVIIa in an animal sample further comprises a calibration standard. The concentration of rFVIIa in the calibration standard is 250 - 16000 ng / mL. The calibration standard is prepared from an animal blank sample, a sample diluent, and rFVIIa.

5. The detection kit for determining the concentration of rFVIIa in an animal sample according to claim 1, characterized in that: The enzyme-labeled antibody is selected from at least one of the antibodies that are enzyme-labeled and specifically bind to the detection antibody; the fluorescently labeled antibody is selected from at least one of the antibodies that are labeled with a fluorescent group and specifically bind to the detection antibody; And / or, the enzyme label is selected from at least one of horseradish peroxidase, alkaline phosphatase, and glucose oxidase labels; And / or, the chromogenic substrate is selected from at least one of 3,3',5,5'-tetramethylbenzidine, 2,2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid, o-phenylenediamine, 3,3'-diaminobenzidine, and p-nitrophenyl phosphate; And / or, the fluorescently labeled antibody is selected from at least one of the antibodies labeled with Alexa Fluor fluorescent group, Brilliant Violet fluorescent group, Cyanine fluorescent group, fluorescein isothiocyanate group, tetramethylrhodamine isothiocyanate, Rhodamine Red-X, and aminomethylcoumarin acetate; 6. The detection kit for determining the concentration of rFVIIa in an animal sample according to claim 1 or 2, wherein: The working solutions of the detection antibody and the labeled antibody are aqueous solutions containing 4.5 - 5.5 g / mL of a blocking agent and 0.045 - 0.055% by volume of a non-ionic surfactant; the blocking agent is selected from at least one of skim milk powder, bovine serum albumin, casein, and gelatin; and / or, the detection kit for determining the concentration of rFVIIa in an animal sample further includes a washing solution and a termination solution; the washing solution is an aqueous solution containing 0.045 - 0.055% by volume of a non-ionic surfactant; the termination solution is a 0.2 - 0.5 M sulfuric acid or hydrochloric acid solution; The non-ionic surfactant is selected from at least one of Tween 20 and Triton X-100.

7. A detection method for non-diagnostic purposes using the detection kit for determining the concentration of rFVIIa in an animal sample according to any one of claims 1 - 6, characterized in that: Step 1, centrifuge the animal sample to be tested, take the supernatant, and then mix the supernatant to be tested with a sample diluent to prepare a sample to be tested; Step 2, incubate the sample to be tested successively with tissue factor, the detection antibody, and the labeled antibody; Step 3, measure the ultraviolet absorption signal or fluorescence signal of the product.

8. A detection method for non-diagnostic purposes using the detection kit for measuring the concentration of rFVIIa in an animal sample according to claim 7, characterized in that: The animal sample in Step 1 is at least one of serum, plasma, urine, tissue homogenate, cell culture, and cell lysate of an animal; and / or, the animal in Step 1 is selected from at least one of mice, rats, rabbits, dogs, and monkeys; and / or, in Step 1, the supernatant to be tested and the sample diluent are mixed at a volume ratio of 22.5 - 27.5:1; and / or, the incubation conditions in Step 2 are 10 - 30 °C, 270 - 500 rpm, the incubation time with tissue factor is 2 h ± 20 min, and the incubation time with the detection antibody is 1.5 h ± 10 min; when the labeled antibody in Step 2 is an enzyme-labeled antibody and a chromogenic substrate, the incubation time with the enzyme-labeled antibody is 1 h ± 5 min, and the incubation time with the chromogenic substrate is 15 - 35 min; when the labeled antibody in Step 2 is a fluorescently labeled antibody, the incubation time with the fluorescently labeled antibody is 1 h ± 5 min; and / or, the ultraviolet absorption signal in Step 3 is the ultraviolet absorption signal at 450 nm, 405 nm, or 492 nm.

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