An ELISA kit for determining the concentration of rFVIIa in animal samples and a detection method thereof

Abstract

The present invention belongs to the field of pharmaceutical technology, and particularly relates to an ELISA kit for determining the concentration of rFVIIa in animal samples and a detection method thereof. The present invention constructs an ELISA kit by screening detection reagents such as combinations of capture antibodies and detection antibodies, and further optimizes the detection conditions. The ELISA kit of the present invention is used 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. The reproducibility and stability meet the requirements, and it can be used for the detection of rFVIIa in animal plasma. The ELISA 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 an ELISA 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. Structurally, it is similar to activated factor VII purified from human plasma, and its in vivo half-life is 2.5 h. rFVIIa can form a TF-rFVIIa complex with tissue factor (TF) at the injury site, and at the same time activate platelets and clotting factor X (FⅩ), ultimately leading to the activation of thrombin and the deposition of fibrin. In addition, rFVIIa binds to the surface of activated platelets with low affinity and directly activates FⅩ to generate thrombin.

[0003] At present, the FDA has approved two rFVIIa products for marketing, namely NovoSeven approved in 1999 and SEVENFACT approved in 2020. There is no marketed rFVIIa product in China. In non-clinical studies of rFVIIa, researchers need to evaluate the efficacy and safety in animals. 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 detecting samples of animals other than humans, it is difficult to distinguish the endogenous clotting factor VIIa in animals, and the measured results do not represent the true concentration of rFVIIa drug in animals. In non-clinical studies of rFVIIa, using commercially available FVIIa detection kits affects the accuracy of non-clinical animal pharmacokinetics and toxicokinetics results, and poses an obstacle to the research and development of rFVIIa products.

[0004] In non-clinical studies of rFVIIa, common 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 distinguishing clotting factor VIIa from different sources, it is difficult to distinguish endogenous clotting factor VIIa in monkeys from human clotting factor VIIa.

[0005] Therefore, establishing a method for accurately detecting the concentration of rFVIIa in monkey samples is crucial for the non-clinical research of rFVIIa and the development of rFVIIa products. Summary of the Invention

[0006] In view of the deficiencies of the prior art, the present invention provides an ELISA kit for determining the concentration of rFVIIa protein in animal samples and its detection method, aiming to more accurately determine the concentration of rFVIIa in monkey samples.

[0007] The present invention provides an ELISA kit for determining the concentration of rFVIIa in animal samples, and the ELISA kit for determining the concentration of rFVIIa in animal samples includes the following components in parts by weight:

[0008] Detection antibody: 0.025 - 0.1 part / well,

[0009] Sample diluent: 475 - 1900 parts / well,

[0010] Labeled antibody: 0.025 - 1 part / well;

[0011] The composition and parts by weight of the labeled antibody are selected from:

[0012] 1) Enzyme-labeled antibody: 0.025 - 1 part / well, chromogenic substrate: 5 - 20 parts / well;

[0013] Or, 2) Fluorescently labeled antibody: 0.025 - 1 part / well;

[0014] The ELISA kit for determining the concentration of rFVIIa in animal samples further includes a capture antibody, and the capture antibody is a human activated coagulation factor VIIa-coated plate with the product number JL17268 produced by Jianglai Biotech; the detection antibody is Anti-Factor Ⅶ antibody, and the clone number is EPR22364-119.

[0015] The present invention provides an ELISA 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:

[0016] Detection antibody: 0.025 - 0.1 part / well,

[0017] Sample diluent: 950 - 3800 parts / well,

[0018] Labeled antibody: 0.025 - 1 part / well;

[0019] Chromogenic substrate: 5 - 20 parts / well;

[0020] The ELISA kit for measuring the concentration of rFVIIa in an animal sample further includes a capture antibody, which is a human activated coagulation factor VIIa coated plate with the product number JL17268 produced by Jianglai Biotech; the detection antibody is Anti-Factor Ⅶ antibody, and the clone number is EPR22364-119;

[0021] 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.

[0022] Preferably, the sample diluent is selected from at least one of cysteine, a blocking agent, and a 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.

[0023] 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.

[0024] Preferably, the enzyme label is selected from at least one of horseradish peroxidase, alkaline phosphatase, glucose oxidase, or biotin labeling;

[0025] 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;

[0026] Or, the fluorescent group is selected from at least one of Alexa Fluor fluorescent group, Brilliant Violet fluorescent group, Cyanine fluorescent group, fluorescein isothiocyanate group, tetramethylrhodamine isothiocyanate, Rhodamine Red-X, and aminomethylcoumarin acetate.

[0027] 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 nonionic surfactant; the blocking agent is selected from at least one of skim milk powder, bovine serum albumin, casein, and gelatin; the nonionic surfactant is selected from at least one of Tween 20 and Triton X-100.

[0028] Preferably, the detection kit for determining the rFVIIa concentration 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 nonionic surfactant; the termination solution is a 0.2 - 0.5 M sulfuric acid or hydrochloric acid solution.

[0029] A detection method for non-diagnostic purposes using the ELISA kit for determining the rFVIIa concentration in an animal sample according to any one of the above.

[0030] 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.

[0031] Step 2: Incubate the sample to be tested with the capture antibody, the detection antibody, and the labeled antibody in sequence.

[0032] Step 3: Measure the ultraviolet absorption signal or fluorescence signal of the product.

[0033] Preferably, after preparing the sample to be tested in Step 1, equilibrate it at 10 - 30 °C and 270 - 500 rpm for 40 min - 60 min.

[0034] Preferably, the animal sample in Step 1 is at least one of animal serum, plasma, tissue homogenate, cell culture, and cell lysate.

[0035] 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.

[0036] And / or, the incubation conditions in Step 2 are 10 - 30 °C and 270 - 500 rpm. The incubation time with the capture antibody 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.

[0037] And / or, the ultraviolet absorption signal described in step 3 is the ultraviolet absorption signal at 450 nm, 405 nm or 492 nm.

[0038] The present invention constructs an ELISA kit by screening detection reagents such as combinations of capture antibodies and detection antibodies, and further optimizes the detection conditions. The ELISA kit of the present invention is used 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. The reproducibility and stability meet the requirements, and it can be used for the detection of rFVIIa in animal plasma. The ELISA 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.

[0039] Obviously, based on the above content of the present invention, according to the common general knowledge and customary 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.

[0040] 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 understood 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

[0041] Figure 1 It is the standard curve graph for the 4th analysis batch; among them, the standard curve fitting formula: Response = (Min - Max) / (1 + (Conc. / Ed 50 ) ** Slope) + Max, where Response is the instrument response value, Conc. is the sample concentration value, Min is the minimum value of the response value, which is -0.00547, Max is the maximum value of the response value, which is 2.68, Ed 50 is the concentration at half-maximal effect, which is 982, Slope is the slope of the curve, which is 0.761, ** represents the power operation, and the R 2 of the standard curve = 0.998. Detailed Description of the Embodiments

[0042] In the following examples and experimental examples, reagents and materials not specifically described are all commercially available products.

[0043] The blood samples of monkeys used in the present invention are all from the blood samples of cynomolgus monkeys.

[0044] I. Main Instrument and Equipment

[0045] In the following examples and experimental examples, the main instruments and equipment used are shown in Table 1 below:

[0046] Table 1 Main Instruments and Equipment

[0047]

[0048] II. Software and Data Systems

[0049] In the following examples and experimental examples, the software and data systems used are shown in Table 2 below:

[0050] Table 2 Software and Data Systems Used

[0051]

[0052] III. Information on Reference Substances

[0053] ① Information on rFVIIa

[0054] Name or Code: Recombinant Human Coagulation Factor VIIa for Injection;

[0055] Source: Chengdu Rongsheng Pharmaceutical Co., Ltd.;

[0056] Appearance: White loose mass, colorless and clear liquid after reconstitution, may have slight opalescence;

[0057] Specification: 1 mg / vial;

[0058] Storage Conditions and Precautions: 2 - 8 °C, protected from light;

[0059] ② Reference Substance Stock Solution

[0060] Solvent: Histidine Diluent for Injection;

[0061] 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;

[0062] Storage Conditions after Preparation: 2 - 8 °C, protected from light.

[0063] IV. Data Processing and Analysis Methods

[0064] The OD values of the standards obtained by the multifunctional microplate reader are fitted with a four-parameter (weighting factor 1 / Y**2) method using Watson LIMS v7.6.1 SP1 software to obtain the standard curve equation, and the concentrations of each sample are calculated, and the standard curve parameters, etc. are issued.

[0065] 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:

[0066] Average value: (C t is the measured concentration);

[0067] Percentage of relative error: (C t is the measured concentration, C n is the theoretical concentration);

[0068] Percentage of coefficient of variation: ( is the average value of the detected concentration);

[0069] Standard deviation: STDEV(SD)= ( is the average value of the detected concentration).

[0070] Total allowable error: %Total Error = ∣ ∣ + .

[0071] 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, OD values and R 2 are retained to at least three decimal places.

[0072] Example 1 ELISA kit for determining the concentration of rFVIIa protein in monkey plasma and its detection method

[0073] I. ELISA kit for determining the concentration of rFVIIa protein in monkey plasma

[0074] This example provides an ELISA kit for determining the concentration of rFVIIa protein in monkey plasma, which consists of the following components: human activated coagulation factor VIIa (Fa) coated plate, detection antibody working solution, enzyme-labeled antibody working solution, calibration standard, sample diluent, washing solution, TMB chromogenic solution, termination solution, etc. The following are the relevant reagents or solutions and their preparation methods:

[0075] 1. Key reagent information

[0076] The information of the key reagents used in this example is shown in Table 3 below:

[0077] Table 3 Key reagent information

[0078]

[0079] Note: "1" indicates that the concentration of this reagent is 1.037 mg / mL; "2" indicates that this reagent is a dry powder. Before use, add 0.5 mL of pure water to each vial to dissolve it completely. After reconstitution, store it at -70 °C or below. The conventional concentration of TMB is 0.1 mg / mL. In this example, the ready-to-use chromogenic solution TMB product in Table 3 is preferably used.

[0080] 2. Solution Preparation

[0081] (1) Blank Matrix

[0082] Monkey blank plasma is stored for standby at a temperature below -60 °C and thawed at 10 - 30 °C before use.

[0083] (2) Sample Diluent

[0084] Weigh 1.0 g of L-cysteine (Adamas beta, 62776B) and dissolve it completely in 100 mL of pure water to obtain a 10 mg / mL cysteine solution.

[0085] (3) Sample Buffer

[0086] Before preparing the standard curve samples, cynomolgus monkey blank plasma 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.

[0087] (4) Preparation of Calibration Standards

[0088] Use the sample buffer to prepare calibration standards of rFVIIa with concentrations of 8000 ng / mL, 6000 ng / mL, 4000 ng / mL, 2000 ng / mL, 1000 ng / mL, 500 ng / mL, 250 ng / mL, and 125 ng / mL (anchor points) respectively.

[0089] (5) Preparation of Test Samples

[0090] Take whole blood from animals, centrifuge to separate plasma, and then mix the test plasma with the sample diluent (i.e., 10 mg / mL cysteine solution) at a volume ratio of 25:1 to prepare the test samples.

[0091] (6) Detergent (0.05% PBST)

[0092] Take 50 mL of 20×PBS buffer and add it to 950 mL of pure water, mix well to prepare 1×PBS; then take 500 μL of Tween-20 and add it to 999.5 mL of 1×PBS and mix well.

[0093] (7) 5% SM - 1×PBST

[0094] Weigh 5.0 g of Difco TM Skim Milk, and dissolve it thoroughly in 100 mL of 0.05% PBST.

[0095] (8)Detection antibody working solution

[0096] Take Anti-Factor Ⅶ antibody[EPR22364-119]-BSA and Azide free(Detector), dilute it 2000 times with the diluent (5% SM - 1×PBST), and mix well. The preparation method is as follows:

[0097] Table 4

[0098]

[0099] Note: The concentration of the source solution is 1.037 mg / mL.

[0100] (9)Enzyme-labeled antibody working solution

[0101] 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), dilute it 1000 times with the diluent (5% SM - 1×PBST), and mix well. This product is in dry powder form. According to the instruction manual, 0.5 ml of pure water is needed for each vial to dissolve it thoroughly to make the source solution before each use, and then dilute it as follows.

[0102] Table 5

[0103]

[0104] The above preparations can be scaled up or down proportionally according to actual needs.

[0105] (10)Stop solution (0.5 M H2SO4)

[0106] 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.

[0107] II. Detection method

[0108] (1) Dilution and equilibration of the sample to be tested: Take the prepared sample to be tested or calibration standard solution, dilute it 20 times with the sample diluent, and equilibrate it at room temperature for 40 min under the condition of 300 rpm;

[0109] (2) Sample addition: Add the equilibrated sample to be tested at 100 μL / well into the human activated coagulation factor VIIa (FVIIa) - coated plate (Jianglai Biotech, product number: JL17268), cover with a film, and incubate at room temperature for 2 h under the condition of 300 rpm;

[0110] (3) Plate washing: Wash 3 times with the washing solution, 300 μL / well, and pat dry;

[0111] (4) Addition of detection antibody: Add the prepared detection antibody working solution to the enzyme - linked immunosorbent assay (ELISA) plate, 100 μL / well, cover with a film, and incubate at room temperature for 1.5 h under the condition of 300 rpm;

[0112] (5) Plate washing: Wash 3 times with the washing solution, 300 μL / well, and pat dry;

[0113] (6) Addition of enzyme - labeled antibody: Add the prepared enzyme - labeled antibody working solution to the ELISA plate, 100 μL / well, cover with a film, and incubate at room temperature for 1 h under the condition of 300 rpm;

[0114] (7) Plate washing: Wash 3 times with the washing solution, 300 μL / well, and pat dry;

[0115] (8) Color development: Add the TMB color - developing solution to the ELISA plate, 100 μL / well, cover with a film, and let it stand at room temperature in the dark for 20 min for color development;

[0116] (7) Termination: Add the termination solution, 100 μL / well, to terminate the reaction, and read the values within 20 min after termination;

[0117] (8) Detection: Use a multi - functional microplate reader to read the OD values of each well at the detection wavelength of 450 nm (reference wavelength 620 nm).

[0118] III. Data processing and analysis

[0119] Use the Watson LIMS v7.6.1 SP1 software to fit the standard curve equation with the OD values of the calibration standard obtained by the multi - functional microplate reader by the four - parameter (weighting factor 1 / Y**2) method, and calculate the rFVIIa protein concentration in the sample to be tested according to the external standard method.

[0120] Example 2 ELISA kit for determining the rFVIIa protein concentration in monkey plasma and its detection method

[0121] The ELISA kit was prepared and tested according to the method of Example 1, with the difference that the sample diluent was 1% BSA - 1×PBST, and the preparation of the sample buffer, calibration standard, and test sample was changed accordingly according to the preparation method of Example 1.

[0122] The preparation method of 1% BSA - 1×PBST is as follows:

[0123] Weigh 1.0 g of bovine serum albumin and dissolve it thoroughly in 100 mL of 0.05% PBST.

[0124] Example 3 Application of the ELISA kit of the present invention for non - diagnostic detection purposes

[0125] In the non - clinical study of rFVIIa, the ELISA kit prepared in Example 1 of the present invention was used to measure the concentration of rFVIIa in the heart, liver, spleen, lung, and kidney tissues of dissected mice, so as to evaluate the distribution of rFVIIa drug in mice and measure the pharmacokinetic data.

[0126] The technical solution of the present invention will be further described through experiments below.

[0127] Experimental Example 1 Screening of capture antibody and detection antibody

[0128] In this experimental example, a screening experiment was carried out on the capture antibody and the detection antibody. The detection method was carried out according to the method of Example 1, with the difference that the equilibration step was not carried out, the sample diluent and its sample buffer were different, and the capture antibody and the detection antibody were respectively according to the following screening conditions. The concentrations of the calibration standards in this experimental example were set as shown in Table 6 or Table 7 respectively.

[0129] I. Detection by the kit alone

[0130] In this experimental example, first, a commercially available human FVIIa detection kit (Fine Test, Wuhan Fine Biotech Co., Ltd., product number: FN - EH3030) was used to detect and verify rFVIIa in animal plasma. Standard curves were prepared using monkey blank plasma and the diluent provided with the kit respectively.

[0131] The results are shown in Table 6. The experimental results indicate that there is matrix interference, and a design with a lower blood content needs to be considered, but the sensitivity of the scheme with a lower blood content may not meet the test conditions.

[0132] Table 6

[0133]

[0134] II. Detection by self - combination of different capture antibodies and detection antibodies

[0135] Since commercially available kits cannot accurately detect rFVIIa in animal plasma, different combinations of capture and detection reagents were used to screen for antibody pairs (combinations of capture and detection antibodies) that can be used to detect rFVIIa in animal plasma, and only monkey blank plasma was used in sample preparation:

[0136] (1)The capture antibody of the commercially available Fine Test FVIIa detection kit (Fine Test, Wuhan Fine Biotech Co., Ltd., product number: FN-EH3030) was used for coating the plate. The detection antibodies of the commercially available ZYMUTEST FVIIa detection kit (ZYMUTEST Factor VII, Aniara Diagnostica) (Combination 1), the detection antibody of Jianglai FVIIa detection kit (Jianglai Biotech, product number: JL17268) (Combination 2), the detection antibody of the Fine Test kit (Combination 3), and the commercially available detection antibody (Anti-Factor VII {EPR22364-119}-BSA and Azide free (Detector), Abcam, product number: ab259698) (Combination 4) were used as detection antibodies. Before loading, the samples were diluted with 1% BSA-1×PBST as the sample diluent.

[0137] The results are shown in Table 7. The signal values of each combination were too low and fluctuated too much. The signal values were not concentration-dependent and the linearity was poor, indicating that there was matrix interference and they could not be used for the detection of rFVIIa in animal plasma.

[0138] Table 7

[0139]

[0140] (2)The capture antibody of the Jianglai FVIIa detection kit (Jianglai Biotech, product number: JL17268) was used for coating the plate. The detection antibodies of the commercially available ZYMUTEST FVIIa detection kit (ZYMUTEST Factor VII, Aniara Diagnostica) (Combination 5), the commercially available detection antibody (Anti-Factor VII {EPR22364-119}-BSA and Azide free(Detector), product number: ab259698) (Combination 6), and the detection antibody in the Jianglai kit (Combination 7) were used as detection antibodies. Before loading, the samples were diluted with 1% BSA-1×PBST as the sample diluent and then loaded.

[0141] The results are shown in Table 8. For Combination 6, the capture antibody of Jianglai kit was used for coating the plate, and a commercially available detection antibody (Anti-Factor VII {EPR22364-119}-BSA and Azide free (Detector), catalog number: ab259698) was used as the detection antibody. The linearity of detecting rFVIIa in animal plasma was better, and there was no endogenous interference.

[0142] Table 8

[0143]

[0144] The above results indicate that the combination of the capture antibody (the capture antibody in Jianglai FVIIa detection kit) and the detection antibody (the commercially available detection antibody (Anti-Factor VII {EPR22364-119}-BSA and Azide free (Detector), catalog number: ab259698)) in Combination 6 is the preferred antibody pair for detecting the concentration of rFVIIa in animals in the present invention.

[0145] Optimization of detection conditions in Experimental Example 2

[0146] Optimization of sample diluent and sample equilibration step in this experimental example

[0147] I. Experimental method

[0148] 1. Comparative experiment of sample diluents

[0149] This experiment was divided into two groups, namely Group A and Group B. Group A was carried out according to the detection method of Example 2, with the difference that: the equilibration step was not performed; Group B was carried out according to the detection method of Example 1, with the difference that: the equilibration step was not performed. The above two groups respectively detected the concentration of rFVIIa in cynomolgus monkey blank plasma (NSB), calibration standard solution or quality control sample.

[0150] 2. Comparative experiment of sample equilibration step

[0151] This experiment was divided into two groups, namely Group B and Group C. Group B was carried out according to the detection method of Example 1, with the difference that: the equilibration step was not performed; Group C was carried out according to the detection method of Example 1. The above two groups respectively detected the concentration of rFVIIa in cynomolgus monkey blank plasma (NSB), calibration standard solution or quality control sample.

[0152] 3. The preparation method of the quality control sample is as follows:

[0153] According to the grouping situation, rFVIIa was prepared into quality control samples with concentrations of 8000 ng / mL (Upper Limit of Quantitation, ULOQ), 6400 ng / mL (High Quality Control, HQC), 1280 ng / mL (Medium Quality Control, MQC), 640 ng / mL (Low Quality Control, LQC), and 250 ng / mL (Lower Limit of Quantification, LLOQ) respectively using different sample buffers.

[0154] II. Experimental Results

[0155] 1. Optimization of Sample Diluent

[0156] The experimental results of Group A are shown in the data of Combination 6 in Table 8, and the experimental results of Group B are shown in Tables 9 and 10. Using 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 samples, the sample diluent was changed from 1% BSA - 1×PBST to 10 mg / mL cysteine, and the prepared samples were diluted. This method has good linearity, and moreover, the %RE of the quality control samples is within ±25.0%, the quality control standards are good, and the accuracy and reliability are better. It shows that when cysteine is used as the sample diluent, the linear relationship and quality control standards of the detection method can be significantly improved.

[0157] Table 9

[0158]

[0159] Table 10

[0160]

[0161]

[0162] Note: "#DIV / 0!" indicates no detection signal.

[0163] 2. Optimization of Sample Equilibration Step

[0164] The experimental results of Group C are shown in Tables 11 and 12. Compared with the data of Group B (Tables 9 and 10), adding a sample room temperature equilibration step before sample addition makes the detection signal value of this method increase, and the standard curve, precision, and accuracy are further optimized.

[0165] Table 11

[0166]

[0167] Table 12

[0168]

[0169]

[0170] The above results show that using cysteine as the sample diluent and adding an equilibration step before sample addition can significantly improve the linear relationship and quality control standards of the detection method of the present invention.

[0171] Experimental Example 3 Effect of Sample Diluent on Sample Stability

[0172] I. Experimental Method

[0173] According to the detection method of Example 1, the differences are as follows: the samples to be tested are high-quality control stability samples (HSQC) and low-quality control stability samples (LSQC); before dilution and equilibration, the samples to be tested are incubated at room temperature (10 - 30 °C) or in an ice bath for 0 h, 0.5 h, 1 h, 1.5 h, and 2 h respectively.

[0174] The high-quality control stability sample (HSQC) is a sample with an rFVIIa concentration of 6400 ng / mL prepared using the sample buffer of Example 1 (added with cysteine) or monkey blank plasma (not added with cysteine).

[0175] The low-quality control stability sample (LSQC) is a sample with an rFVIIa concentration of 640 ng / mL prepared using the sample buffer of Example 1 (added with cysteine) or monkey blank plasma (not added with cysteine).

[0176] II. Experimental Results

[0177] The experimental results are shown in Tables 13 and 14. Under ice bath conditions, the samples prepared with cysteine have better stability and are stable within 2 h. It shows that adding cysteine to the animal samples to be tested can significantly improve the stability of the samples to be tested.

[0178] Table 13

[0179]

[0180] Table 14

[0181]

[0182] Experimental Example 4 Method Validation

[0183] This experimental example was used to investigate the linear relationship, precision, accuracy, selectivity, and stability according to the detection method of Example 1.

[0184] I. Standard Curve and Quantitative Range

[0185] Sixteen independent batches of calibration standards were prepared respectively according to the preparation method of Example 1, and then detected respectively according to the detection method of Example 1.

[0186] The specific standard curve results are shown in Table 15, the summary of standard curve fitting parameters is shown in Table 16, and a typical standard curve is shown in the appendix Figure 1 . The quantitative range of rFVIIa is: 250 - 8000 ng / mL, the lower limit of quantification is: 250 ng / mL, 125 ng / mL is the anchor point, the linear range of the standard curve is good, and the results all meet the requirements.

[0187] Table 15 Standard Curve Results for Detecting rFVIIa Concentration in Monkey Plasma by ELISA

[0188]

[0189] Table 16 Summary of Standard Curve Fitting Parameters

[0190]

[0191] Note: Standard curve fitting formula: Resp. = (Min - Max) / (1 + (Conc. / Ed 50 ) **Slope) + Max, where Resp. is the instrument response value, Conc. is the sample concentration value, Min and Max are the minimum and maximum response values respectively, Ed 50 is the concentration at half-maximal effect (i.e., the concentration when the response value reaches half of the maximum response value), Slope is the slope of the curve, and ** represents the power operation.

[0192] II. Accuracy and Precision

[0193] In the same analytical batch, three sets of quality control samples with five concentrations of ULOQ (8000 ng / mL), HQC (6400 ng / mL), MQC (1280 ng / mL), LQC (640 ng / mL), and LLOQ (250 ng / mL) were prepared with rFVIIa respectively. Each set was set with 2 replicates for verification. Seven batches of verification were carried out. Since the %RE of high-quality control (HQC) and upper limit of quantification (ULOQ) samples in analytical batch 1 did not meet the acceptance criteria, it was not included in the within-batch data statistics, but participated in the between-batch data statistics; the between-batch data of all accepted analytical batch quality control samples and the within-batch data of 6 analytical batch quality control samples that passed the verification items all met the average %RE within ±20.0% (the average %RE of ULOQ&LLOQ within ±25.0%); the average precision (%CV) ≤ 20.0% (the average %CV of ULOQ&LLOQ ≤ 25.0%); the total allowable error (%Total Error) ≤ 30.0% (the total allowable error of ULOQ&LLOQ ≤ 40.0%); at the same time, at least two-thirds of the quality control samples in 6 analytical batches that passed the verification items, and at least 50% of the samples at the same concentration level met the %RE of low, medium, and high concentration QC samples within ±20.0%, and ULOQ&LLOQ within ±25.0%; the above results indicate that the within-batch and between-batch precision and accuracy of this analytical method are good, and this method is applicable to the detection of rFVIIa protein concentration in monkey plasma. The specific results are shown in Table 17.

[0194] Table 17 Precision and accuracy results of detecting rFVIIa concentration in monkey plasma by ELISA method

[0195]

[0196]

[0197]

[0198] Note: " / " indicates no calculated data; "*" indicates not meeting the acceptance criteria, but participating in the between-batch data statistics.

[0199] III. Selectivity

[0200] 1% blank hemolyzed plasma was prepared by adding 1% blank monkey whole blood to blank plasma for selectivity investigation. 100% high-quality control selectivity (SE-HQC) samples were prepared with a sample buffer prepared based on 1% blank hemolyzed plasma according to the method of Example 1 to a sample with an rFVIIa concentration of 6400 ng / mL; 100% lower limit of quantification selectivity (SE-LLOQ) samples were prepared with a sample buffer prepared based on 1% blank hemolyzed plasma according to the method of Example 1 to a sample with an rFVIIa concentration of 250 ng / mL.

[0201] The OD values of 10 monkey blank plasma samples from different sources and 1 monkey blank hemolyzed plasma SE-Blank were all less than the LLOQ OD value, indicating that there were no obvious endogenous interfering substances affecting the determination of the analyte. The specific results are shown in Table 18. The %RE of the 100% high-quality control selectivity (SE-HQC) samples was within ±20.0%; the %RE of the 100% lower limit of quantification selectivity (SE-LLOQ) samples was within ±25.0%, meeting the selectivity accuracy requirements, indicating that the method had good selectivity. The specific results are shown in Table 19.

[0202] Table 18 Selectivity determination results of rFVIIa concentration in monkey plasma by ELISA method (I)

[0203]

[0204] Note: "*" indicates that the sample is a hemolyzed sample.

[0205] Table 19 Selectivity determination results of rFVIIa concentration in monkey plasma by ELISA method (II)

[0206]

[0207] Note: "*" indicates that the sample is a hemolyzed sample.

[0208] IV. Whole blood stability

[0209] High-quality control stability (HSQC) whole blood samples and low-quality control stability (LSQC) whole blood samples of rFVIIa were prepared with monkey whole blood (anticoagulant: sodium citrate). After storing at wet ice for about 1.5 hours, the plasma was separated and the mean concentration of rFVIIa was measured. Compared with the mean concentration of the plasma separated immediately, the %RE of the high-quality control stability (HSQC) concentration samples and the low-quality control stability (LSQC) concentration samples were both within ±20.0%, meeting the requirements, indicating that the samples were stable under the above conditions. The specific results are shown in Table 20.

[0210] Table 20 Whole blood stability results of rFVIIa concentration in monkey plasma by ELISA method

[0211]

[0212] The above results show that the quantitative range of the standard curve of the detection method in Example 1 is 250 - 8000 ng / mL, with 125 ng / mL as the anchor point; the lower limit of quantification (LLOQ) is 250 ng / mL, and the upper limit of quantification (ULOQ) is 8000 ng / mL; there are no endogenous interfering substances affecting the determination of rFVIIa in this method, which can specifically identify rFVIIa drugs, and has high selectivity, accuracy, precision, and whole blood stability. This method has good linearity, no interference from endogenous coagulation factors, and the reproducibility and stability meet the requirements, and can be used for the detection of rFVIIa in animal plasma.

[0213] As can be seen from the above examples and experimental examples, the present invention constructs an ELISA kit by screening detection reagents such as combinations of capture antibodies and detection antibodies, further optimizes the detection conditions, and uses the ELISA 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, no interference from endogenous coagulation factors, and the reproducibility and stability meet the requirements, and can be used for the detection of rFVIIa in animal plasma. The ELISA 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.

Claims

1. An ELISA kit for determining the concentration of rFVIIa in an animal sample, characterized in that, The ELISA kit for determining the concentration of rFVIIa in an animal sample comprises the following components in parts by weight: Detection antibody: 0.025 - 0.1 part per well, Sample diluent: 475 - 1900 parts per well, Labeled antibody; The composition and parts by weight of the labeled antibody are selected from: 1) Enzyme-labeled antibody: 0.025 - 1 part per well, chromogenic substrate: 5 - 20 parts per well; Or, 2) Fluorescently labeled antibody: 0.025 - 1 part per well; The ELISA kit for determining the concentration of rFVIIa in an animal sample further comprises a capture antibody, which is a human activated coagulation factor VIIa coated plate with the product number JL17268 produced by Jianglai Biotech; the detection antibody is Anti-Factor Ⅶ antibody with the clone number EPR22364-119; the sample diluent is selected from cysteine; The rFVIIa is recombinant human coagulation factor.

2. An ELISA 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: Detection antibody: 0.025 - 0.1 part per well, Sample diluent: 950 - 3800 parts per well, Labeled antibody: 0.025 - 1 part per well; Chromogenic substrate: 5 - 20 parts per well; The ELISA kit for determining the concentration of rFVIIa in an animal sample further comprises a capture antibody, which is a human activated coagulation factor VIIa coated plate with the product number JL17268 produced by Jianglai Biotech; the detection antibody is Anti-Factor Ⅶ antibody with the clone number EPR22364-119; 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 sample diluent is selected from cysteine; The rFVIIa is recombinant human coagulation factor.

3. The ELISA 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 used for specifically binding 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 used for specifically binding to the detection antibody.

4. The ELISA kit for determining the concentration of rFVIIa in an animal sample according to claim 3, wherein: 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; Or, the fluorescent group is selected from at least one of Alexa Fluor fluorescent group, Brilliant Violet fluorescent group, Cyanine fluorescent group, fluorescein isothiocyanate group, tetramethylrhodamine isothiocyanate, Rhodamine Red-X, and aminomethylcoumarin acetate.

5. The ELISA kit for determining the concentration of rFVIIa in an animal sample according to claim 1 or 2, characterized in that: 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; the non-ionic surfactant is selected from at least one of Tween 20 and Triton X-100.

6. The ELISA 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 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.

7. A detection method for non-diagnostic purposes using the ELISA 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 a capture antibody, a detection antibody, and a 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 ELISA kit for determining the concentration of rFVIIa in an animal sample according to claim 7, characterized in that: After preparing the sample to be tested in Step 1, equilibrate it for 40 min - 60 min at 10 - 30 °C and 270 - 500 rpm.

9. A detection method for non-diagnostic purposes using the ELISA kit for determining 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 animal serum, plasma, tissue homogenate, cell culture, and cell lysate. 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 and 270 - 500 rpm, the incubation time with the capture antibody 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 or 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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