Blood coagulation factor VIII activity determination kit and determination method

Abstract

The invention discloses a blood coagulation factor VIII activity determination kit and a determination method. The reagent comprises a reagent R1, a reagent R2, a reagent R3 and a sample diluent, the reagent R1 comprises the following components: a bovine X factor, a fibrin polymerization inhibitor, salt ions, a stabilizer, a preservative and a buffer solution; the R2 reagent comprises the following components: a bovine IXa factor, bovine thrombin, phospholipid, calcium chloride, a stabilizer, a preservative and a buffer solution; the R3 reagent comprises the following components: an FXa chromophoric substrate, a thrombin inhibitor, salt ions, a stabilizer, a preservative and a buffer solution; the sample diluent comprises the following components: a heparin antagonist, salt ions, a stabilizer, a buffer solution and a preservative; the activity of the blood coagulation factor VIII is quantitatively detected based on the principle of a chromophoric substrate method, the detection operation is simple, the sensitivity is high, the result is reliable, and the reagent stability is good.

Description

Technical Field

[0001] The present invention belongs to the technical field of in vitro detection, and in particular relates to a kit and method for determining the activity of coagulation factor VIII. Background Art

[0002] Coagulation factor VIII (FVIII) is a key coagulation factor in the intrinsic coagulation pathway. As a cofactor for the serine protease factor IXa, it participates in the activation of factor X in the coagulation cascade. Its plasma concentration is approximately 0.1 mg / L. FVIII in plasma circulates at very low concentrations as an inactive precursor, tightly and non-covalently bound to von Willebrand factor (vWF). When hydrolyzed by thrombin or factor Xa, FVIII becomes activated factor VIII (FVIIIa), dissociating from vWF and exerting its procoagulant function. In its active form, FVIIIa acts as a cofactor, forming a factor X enzyme complex with factor IXa, phospholipids, and calcium ions, enhancing the efficiency of factor IXa's catalytic activity in factor X activation.

[0003] Coagulation factor VIII is synthesized in hepatocytes. Reduced factor VIII activity in plasma can weaken the endogenous procoagulant pathway, causing the blood to be in a hypocoagulable state and leading to bleeding. Bleeding disorders caused by defects in the quantity or quality of FVIII are called hemophilia A. This disease is a recessive genetic disease on the X chromosome, and the severity of bleeding increases with the degree of FVIII deficiency. Mild hemophilia generally does not have spontaneous bleeding, but some degree of bleeding occurs after trauma and surgery. Intermediate and severe hemophilia can cause spontaneous bleeding, which is more common in joints, muscles, and other areas. With the development of hemophilia treatment, in addition to blood-derived factor products, some new therapeutic preparations have been launched on the market, such as long-acting factor preparations and emicizumab. Establishing a reliable plasma factor VIII detection method plays an important role in the diagnosis and treatment of hemophilia.

[0004] Currently, methods for detecting factor VIII in plasma fall into two main categories. One involves factor VIII antigen content, such as immunoassays. These methods can only detect the factor VIII antigen content in plasma and cannot accurately assess its functional activity, limiting their clinical use. The other involves factor VIII activity assays, such as coagulation assays (often referred to clinically as primary assays) and chromogenic substrate assays. Both assays are based on the procoagulant activity of factor VIII after activation to FVIIIa. Coagulation assays are based on the fact that factor VIII deficiency can prolong the partial thromboplastin time (PTT). Patient plasma is mixed with FVIII-deficient plasma from a commercial test kit. If the patient's plasma is deficient or has reduced factor VIII activity, the clotting time will be prolonged, and the prolongation ratio corresponds to the FVIII activity in the sample. However, this method is susceptible to interference from factors such as lupus anticoagulants and heparin in the blood, resulting in poor specificity and accuracy.

[0005] The chromogenic substrate method calculates the activity of the test substance by measuring the absorbance change of a chromogenic substrate. It has excellent sensitivity, stability, accuracy, and is not susceptible to interference. Chromogenic methods have demonstrated improved accuracy and clinical relevance in monitoring mild hemophilia and hemophilia therapeutic drugs, and are recommended by the European Pharmacopoeia and the International Society on Thrombosis and Haemostasis (ISTH). Currently, there are no commercially available chromogenic factor VIII assays in China, limiting the implementation of this program in hospitals. To address these issues, it is crucial to develop a chromogenic factor VIII activity assay, assay kit, and preparation method. Summary of the Invention

[0006] To address the above technical problems, the present invention provides a Factor VIII activity assay kit and assay method. This kit enables quantitative detection of Factor VIII activity. The assay kit offers numerous advantages, including high sensitivity, minimal inter-batch variability, excellent stability, strong anti-interference properties, and accurate and reliable test results, making it readily available for clinical use.

[0007] In order to solve the above technical problems, the present invention provides the following technical solutions: The present invention provides a coagulation factor VIII activity assay kit, which comprises a reagent R1, a reagent R2, a reagent R3 and a sample diluent; Reagent R1 includes bovine factor X, fibrin polymerization inhibitor, salt ions, stabilizer, preservative, and buffer; Reagent R2 includes bovine factor IXa, bovine thrombin, phospholipids, calcium chloride, stabilizers, preservatives, and buffer; Reagent R3 includes FXa chromogenic substrate, thrombin inhibitor, salt ions, stabilizer, preservative, and buffer; The sample diluent includes heparin antagonists, salt ions, stabilizers, buffers, and preservatives; FVIII in the test specimen is activated by thrombin in the reagent to form FVIIIa, which forms an enzyme complex with phospholipids, calcium ions, and IXa. The enzyme complex is incubated with factor X to generate factor Xa. A chromogenic substrate for factor Xa is added for detection. Factor Xa acts on the chromogenic substrate S-2765, causing it to release p-nitroaniline (pNA). The amount of pNA generated is calculated by reading the absorbance value at a wavelength of 405 nm, and this is converted into factor VIII activity through calculation.

[0008] Preferably, the bovine factor X in the reagent R1 is purified from bovine plasma, and the dosage of the bovine factor X is 0.5 IU / mL-1.5 IU / mL, which can be 0.5 IU / mL, 0.75 IU / mL, 1 IU / mL, 1.25 IU / mL, or 1.5 IU / mL; Preferably, the fibrin polymerization inhibitor of the reagent R1 is H-Gly-Pro-Arg-Pro-OH•AcOH, which can inhibit the polymerization of fibrin. The dosage of the fibrin polymerization inhibitor is 0.1 nmol / mL-1 nmol / mL, which can be 0.1 nmol / mL, 0.25 nmol / mL, 0.5 nmol / mL, 0.75 nmol / mL, and 1 nmol / mL. Preferably, the salt ions in the reagent R1 are one or more of NaCl, CaCl2, KCl, and CsCl; the inorganic salt is preferably NaCl, and the amount of NaCl is 5g / L-25g / L, which can be 5g / L, 10g / L, 15g / L, 20g / L, or 25g / L; Preferably, the stabilizer in the reagent R1 is one or more of proteins, sugars, amino acids, surfactants, suspending agents, and antioxidants; the stabilizer is preferably bovine serum albumin (BSA), and the amount of bovine serum albumin is 1g / L-10g / L, which can be 1g / L, 2.5g / L, 5g / L, 7.5g / L, and 10g / L; Preferably, the bovine factor IXa of the reagent R2 is purified from bovine plasma, and the dosage of the bovine factor IXa is 0.1 IU / mL-1 IU / mL, which can be 0.1 IU / mL, 0.25 IU / mL, 0.5 IU / mL, 0.75 IU / mL, or 1 IU / mL; Preferably, the bovine thrombin of the reagent R2 is purified from bovine plasma, and the dosage of thrombin is 0.1 NIH / mL-1 NIH / mL, which can be 0.1 NIH / mL, 0.25 NIH / mL, 0.5 NIH / mL, 0.75 NIH / mL, 1 NIH / mL; Preferably, the phospholipid of the reagent R2 is one of rabbit cephalin, bovine cephalin, and synthetic lecithin. The phospholipid is rabbit cephalin, and the dosage of rabbit cephalin is 0.05 mg / mL-0.5 mg / mL, which can be 0.05 mg / mL, 0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, or 0.5 mg / mL. Preferably, the amount of CaCl2 in the reagent R2 is 0.1g / L-1g / L, which can be 0.1g / L, 0.25g / L, 0.5g / L, 0.75g / L, 1g / L; Preferably, the stabilizer in the reagent R2 is one or more of protein, carbohydrate, amino acid, surfactant, suspending agent, and antioxidant; the stabilizer is preferably bovine serum albumin, and the amount of bovine serum albumin is 1g / L-10g / L, which can be 1g / L, 2.5g / L, 5g / L, 7.5g / L, 10g / L; Preferably, the FXa chromogenic substrate of the reagent R3 is one of CH3SO2-D-Leu-Gly-Arg-pNA•AcOH, CH3SO2-D-CHG-Gly-Arg-pNA•AcOH, CH3SO2-D-CHA-Gly-Arg-pNA•AcOH, and ZD-Arg-Gly-Arg-pNA•2HCl, and the chromogenic substrate is CH3SO2-D-Leu-Gly-Arg-pNA•AcOH, and the dosage is 0.5-2 mg / mL, which can be 0.5 mg / mL, 1 mg / mL, 1.5 mg / mL, or 2 mg / mL; Preferably, the thrombin inhibitor of the reagent R3 is one of r-Hirudin EC, Dabigatran, Civalirudin, and Argatroban, and the thrombin inhibitor is r-Hirudin EC, and the dosage is 250-1000 ng / mL, which can be 250 ng / mL, 500 ng / mL, 750 ng / mL, or 1000 ng / mL; Preferably, the salt ion in the reagent R3 is one or more of NaCl, CaCl2, KCl, and CsCl; the inorganic salt is preferably NaCl, and the amount of NaCl is 5g / L-25g / L, which can be 5g / L, 10g / L, 15g / L, 20g / L, or 25g / L; Preferably, the stabilizer in the reagent R3 is one or more of protein, carbohydrate, amino acid, surfactant, suspending agent, and antioxidant; the stabilizer is preferably bovine serum albumin, and the amount of bovine serum albumin is 1g / L-10g / L, which can be 1g / L, 2.5g / L, 5g / L, 7.5g / L, 10g / L; Preferably, the heparin antagonist of the sample diluent is one of polybrene, protamine, and heparinase, and the heparin antagonist is polybrene, and the dosage is 2 μg / mL-10 μg / mL, which can be 2.5 μg / mL, 5 μg / mL, and 10 μg / mL; Preferably, the salt ions in the sample diluent are one or more of NaCl, CaCl2, KCl, and CsCl; the inorganic salt is preferably NaCl, and the amount of NaCl is 5g / L-25g / L, which can be 5g / L, 10g / L, 15g / L, 20g / L, or 25g / L; Preferably, the stabilizer in the sample diluent is one or more of protein, sugar, amino acid, surfactant, suspending agent, and antioxidant; the stabilizer is preferably glycine, and the amount of glycine is 10g / L-50g / L, which can be 10g / L, 20g / L, 30g / L, 40g / L, or 50g / L; Preferably, the buffer used in reagents R1, R2, R3, and the sample diluent is one or more of HEPES buffer, Tris-HCl buffer, phosphate buffer, and barbiturate buffer, and the amount of the buffer is 20-50 mmol / L. The buffer used in reagents R1, R2, R3, and the sample diluent is preferably a Tris-HCl buffer with a pH of 7-9 and a concentration of 20-50 mmol / L.

[0009] Preferably, the preservative in the reagent can be one or more of sodium azide, Proclin 300, and gentamicin, wherein the preservative is Proclin 300, and the dosage is 0.1-0.5 g / L, which can be 0.1 g / L, 0.2 g / L, 0.3 g / L, 0.4 g / L, or 0.5 g / L. The preparation method of the above-mentioned kit comprises the following steps: (1) Prepare reagent R1: Prepare bovine factor X 0.75 IU / mL, fibrin polymerization inhibitor 0.5 nmol / mL, Tris-HCl buffer pH 8 50 mmol / L, BSA 10 g / L, NaCl 10 g / L, and Proclin 300 0.3 g / L. Dispense the above reagents into bottles at a volume of 3 mL / bottle for use. (2) Prepare reagent R2: Prepare bovine IXa 0.5 IU / mL, bovine thrombin 0.25 NIH / mL, rabbit cephalin 0.1 mg / mL, CaCl2 0.5 g / L, Tris-HCl buffer 50 mmol / L (pH 8), BSA 10 g / L, and Proclin 300 0.3 g / L. Dispense the above reagents into bottles at a volume of 3 mL / bottle for use. (3) Prepare reagent R3: Prepare FXa chromogenic substrate 1 mg / mL, thrombin inhibitor 250 ng / mL, Tris-HCl buffer pH 7 20 mmol / L, BSA 5 g / L, NaCl 10 g / L, and Proclin 300 0.3 g / L. Dispense the above reagents into bottles at 3 mL / bottle for use. (4) Prepare sample diluent: heparin antagonist 5 μg / mL, Tris-HCl buffer pH 7 20 mmol / L, NaCl 20 g / L, glycine 30 g / L, Proclin 300 0.3 g / L. After the above reagents are prepared, dispense them into bottles at 3 mL / bottle for use.

[0010] A method for determining the activity of coagulation factor VIII, comprising the following steps: (1) First, establish a standard curve for the activity of coagulation factor VIII. Dilute the factor VIII calibrator five times with the sample diluent to obtain six calibrators with activities of 160%, 80%, 40%, 20%, 10%, and 5%, respectively. (2) Reagent R1, reagent R2, and reagent R3 were reconstituted with 3 mL of purified water at room temperature for 30 min, and the sample dilution solution was taken out for later use; (3) Take out 30uL of the calibrator of different activities and add it to the reaction cup, add 135uL of sample diluent, take out 30uL, add 40uL of reagent R1, incubate for 100 seconds, add 40uL of reagent R2, incubate for another 100 seconds, add 120uL of reagent R3, and measure the absorbance change value OD between 15 seconds and 300 seconds; (4) Each calibrator to be tested is measured three times, and a standard curve is drawn with the horizontal axis representing the activity of the calibrator and the vertical axis representing the mean OD values of the calibrators to be tested with different activities; (5) The plasma sample to be tested is tested using the same method. The measured OD is substituted into the above standard curve to obtain the coagulation factor VIII activity value of the plasma sample to be tested.

[0011] Beneficial effects: The present invention adds a fibrin polymerization inhibitor to the R1 reagent to inhibit fibrin polymerization and avoid interference from fibrin polymerization. The present invention also adds a thrombin inhibitor to the R3 reagent to avoid competition between thrombin and substrate Xa, thereby improving detection accuracy. The present invention adds a heparin antagonist to the sample diluent to antagonize the effects of ordinary heparin and low molecular weight heparin on the test results, thereby improving the anti-interference ability of the reagent. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 Schematic diagram of the standard curve of the assay reagent of the present invention; Figure 2 Schematic diagram of the linear range of the assay reagent of the present invention; Figure 3 Schematic diagram of the stability evaluation results of the assay reagent of the present invention; Figure 4 Schematic diagram of correlation analysis between the assay reagent of the present invention and a first-phase coagulation factor VIII activity detection reagent; Figure 5 Schematic diagram of consistency analysis between the assay reagent of the present invention and a first-phase method coagulation factor VIII activity detection reagent. DETAILED DESCRIPTION

[0013] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for further improvements by those skilled in the art and are not intended to limit the present invention in any way.

[0014] Unless otherwise specified, the experimental methods in the following examples are conventional methods and were performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials and reagents used in the following examples, unless otherwise specified, were all commercially available.

[0015] The assay method is compatible with various commercially available brands and models of fully automated coagulation analyzers, such as the Shandong Alkeda Biotechnology GW-6000 Coagulation Analyzer, the Japanese Sysmex CS-5100 Fully Automated Coagulometer, and the Werfen ACL TOP-700 Fully Automated Coagulometer. Specific reaction parameters should be adjusted appropriately for each coagulation analyzer. The fibrin polymerization inhibitor was purchased from DSM Pentapharm, Switzerland, under the product name Pefabloc FG, catalog number 099-01, CAS number 157009-81-9.

[0016] Example 1 Reference Figure 1-5 This specific embodiment adopts the following technical solution: a coagulation factor VIII activity assay kit, the kit comprising reagent R1, reagent R2, reagent R3 and a sample diluent; reagent R1 comprises bovine factor X, a fibrin polymerization inhibitor, salt ions, a stabilizer, a preservative, and a buffer; reagent R2 comprises bovine factor IXa, bovine thrombin, phospholipids, calcium chloride, a stabilizer, a preservative, and a buffer; reagent R3 comprises a FXa chromogenic substrate, a thrombin inhibitor, salt ions, a stabilizer, a preservative, and a buffer; the sample diluent comprises a heparin antagonist, a buffer, salt ions, a stabilizer, and a preservative; It is worth noting that the bovine factor X in reagent R1 is purified from bovine plasma. The dosage of bovine factor X is 0.5 IU / mL-1.5 IU / mL, which can be 0.5 IU / mL, 0.75 IU / mL, 1 IU / mL, 1.25 IU / mL, and 1.5 IU / mL; It is worth noting that the fibrin polymerization inhibitor of the reagent R1 is H-Gly-Pro-Arg-Pro-OH•AcOH, which can inhibit the polymerization of fibrin. The dosage of the fibrin polymerization inhibitor is 0.1 nmol / ml-1 nmol / ml, which can be 0.1 nmol / ml, 0.25 nmol / ml, 0.5 nmol / ml, 0.75 nmol / ml, and 1 nmol / ml; The salt ion in the reagent R1 is NaCl, and the amount of NaCl is 5g / L-25g / L, which can be 5g / L, 10g / L, 15g / L, 20g / L, 25g / L; The stabilizer in the reagent R1 is bovine serum albumin, and the amount of bovine serum albumin is 1g / L-10g / L, which can be 1g / L, 2.5g / L, 5g / L, 7.5g / L, 10g / L; It is noteworthy that the bovine factor IXa of the reagent R2 is purified from bovine plasma, and the dosage of the bovine factor IXa is 0.1 IU / mL-1 IU / mL, which can be 0.1 IU / mL, 0.25 IU / mL, 0.5 IU / mL, 0.75 IU / mL, and 1 IU / mL; It is worth noting that the bovine thrombin of the reagent R2 is purified from bovine plasma, and the dosage of thrombin is 0.1 NIH / mL-1 NIH / mL, which can be 0.1 NIH / mL, 0.25 NIH / mL, 0.5 NIH / mL, 0.75 NIH / mL, 1 NIH / mL; It is noteworthy that the phospholipid of the reagent R2 is one of rabbit cephalin, bovine cephalin, and synthetic lecithin. The phospholipid is rabbit cephalin, and the dosage of rabbit cephalin is 0.05 mg / mL-0.5 mg / mL, which can be 0.05 mg / mL, 0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, 0.5 mg / mL; The dosage of CaCl2 in the reagent R2 is 0.1g / L-1g / L, which can be 0.1g / L, 0.25g / L, 0.5g / L, 0.75g / L, 1g / L; The stabilizer in the reagent R2 is bovine serum albumin, and the amount of bovine serum albumin is 1g / L-10g / L, which can be 1g / L, 2.5g / L, 5g / L, 7.5g / L, 10g / L; It is noteworthy that the FXa chromogenic substrate of the reagent R3 is one of CH3SO2-D-Leu-Gly-Arg-pNA•AcOH, CH3SO2-D-CHG-Gly-Arg-pNA•AcOH, CH3SO2-D-CHA-Gly-Arg-pNA•AcOH, and ZD-Arg-Gly-Arg-pNA•2HCl, and the chromogenic substrate is CH3SO2-D-Leu-Gly-Arg-pNA•AcOH, and the dosage is 0.5-2 mg / mL, which can be 0.5 mg / mL, 1 mg / mL, 1.5 mg / mL, or 2 mg / mL; It is noteworthy that the thrombin inhibitor of the reagent R3 is one of r-Hirudin EC, Dabigatran, Civalirudin, and Argatroban, and the thrombin inhibitor is r-Hirudin EC, and the dosage is 250-1000 ng / mL, which can be 250 ng / mL, 500 ng / mL, 750 ng / mL, and 1000 ng / mL; The salt ion in the reagent R3 is NaCl, and the amount of NaCl is 5g / L-25g / L, which can be 5g / L, 10g / L, 15g / L, 20g / L, 25g / L; The stabilizer in the reagent R3 is bovine serum albumin, and the amount of bovine serum albumin is 1g / L-10g / L, which can be 1g / L, 2.5g / L, 5g / L, 7.5g / L, 10g / L; The heparin antagonist of the sample diluent is one of polybrene, protamine, and heparinase. The heparin antagonist is polybrene, and the dosage is 2 μg / mL-10 μg / mL, which can be 2.5 μg / mL, 5 μg / mL, and 10 μg / mL; The salt ion in the reagent sample diluent is NaCl, and the amount of NaCl is 5g / L-25g / L, which can be 5g / L, 10g / L, 15g / L, 20g / L, 25g / L; The stabilizer in the sample diluent is glycine, and the amount of glycine used is 10g / L-50g / L, which can be 10g / L, 20g / L, 30g / L, 40g / L, or 50g / L; The buffer used in reagents R1, R2, R3, and the sample diluent is one or more of HEPES buffer, Tris-HCl buffer, phosphate buffer, and barbiturate buffer, and the amount of the buffer is 20-50 mmol / L. The buffer used in reagents R1, R2, R3, and the sample diluent is preferably Tris-HCl buffer with a pH of 7-9 and a concentration of 20-50 mmol / L.

[0017] The preservative in the reagent can be one or more of sodium azide, Proclin 300, and gentamicin. The preservative is Proclin 300, and the dosage is 0.1-0.5 g / L, which can be 0.1 g / L, 0.2 g / L, 0.3 g / L, 0.4 g / L, or 0.5 g / L. The preparation method of the activity assay kit includes a coagulation factor VIII activity assay reagent comprising reagent R1, reagent R2, reagent R3, and a sample diluent, wherein reagents R1, R2, and R3 are lyophilized reagents and the sample diluent is a liquid reagent. The preparation steps are as follows: (1) Prepare reagent R1: Prepare bovine factor X 0.75 IU / mL, fibrin polymerization inhibitor 0.5 nmol / mL, Tris-HCl buffer 50 mmol / L, pH 8, BSA 10 g / L, NaCl 10 g / L, and Proclin 300 0.3 g / L. Dispense the above reagents into bottles at a volume of 3 mL / bottle for use. (2) Prepare reagent R2: Prepare bovine IXa 0.5 IU / mL, bovine thrombin 0.25 NIH / mL, rabbit cephalin 0.1 mg / mL, CaCl2 0.5 g / L, Tris-HCl buffer 50 mmol / L (pH 8), BSA 10 g / L, and Proclin 300 0.3 g / L. Dispense the above reagents into bottles at a volume of 3 mL / bottle for use. (3) Prepare reagent R3: Prepare FXa chromogenic substrate 1 mg / mL, thrombin inhibitor 250 ng / mL, Tris-HCl buffer pH 7 20 mmol / L, BSA 5 g / L, NaCl 10 g / L, and Proclin 300 0.3 g / L. Dispense the above reagents into bottles at a volume of 3 mL / bottle for use. (4) Prepare sample diluent: heparin antagonist 5 μg / mL, Tris-HCl buffer pH 7 20 mmol / L, NaCl 20 g / L, glycine 30 g / L, Proclin 300 0.3 g / L. After the above reagents are prepared, dispense them into bottles at 3 mL / bottle for use.

[0018] A method for determining the activity of coagulation factor VIII, comprising the following steps: (1) First, establish a standard curve for coagulation factor VIII activity (such as Figure 1 As shown), the Factor VIII calibrator was serially diluted five times with the sample diluent to obtain six calibrators with activities of 160%, 80%, 40%, 20%, 10%, and 5%, respectively; (2) Reagent R1, reagent R2, and reagent R3 were reconstituted with 3 mL of purified water at room temperature for 30 min, and the sample dilution solution was taken out for later use; (3) Take out 30uL of the calibrator of different activities and add it to the reaction cup, add 135uL of sample diluent, take out 30uL, add 40uL of reagent R1, incubate for 100 seconds, add 40uL of reagent R2, incubate for another 100 seconds, add 120uL of reagent R3, and measure the absorbance change value OD between 15 seconds and 300 seconds; (4) Each calibrator to be tested is measured three times, and a standard curve is drawn with the horizontal axis representing the activity of the calibrator and the vertical axis representing the mean OD values of the calibrators to be tested with different activities; (5) Use the same method to detect the plasma sample to be tested, and substitute the measured OD into the above standard curve to obtain the factor VIII activity value of the plasma sample to be tested.

[0019] Example 2 This specific embodiment performs imprecision evaluation, linear range evaluation, stability evaluation, correlation and consistency analysis with existing commercially available first-phase reagents, and anti-interference ability test on the assay reagents. Specifically: (1) Imprecision assessment: The quality control plasma with normal and pathological values used for imprecision assessment was purchased from Siemens, Germany, batch numbers 507936 and 556744. The normal human pooled plasma was prepared by mixing the plasma of 40 apparently healthy subjects with normal physical examinations and normal coagulation-related tests.

[0020] ① Intra-batch imprecision assessment: Two levels of quality control plasma and normal mixed plasma were used to repeatedly measure plasma FVIII activity 20 times using the assay reagent of the present invention. The arithmetic mean (X), standard deviation (SD), and intra-assay coefficient of variation (CV) were calculated (CV = (SD / X) * 100%). The results are shown in Table 1: Table 1 Results of intra-batch imprecision evaluation of assay reagents

[0021] As shown in Table 1, the intra-batch imprecision evaluation results of the assay reagents of the present invention have a small variation, with a CV of 1-3%, and a CV of ≤3%.

[0022] ②Inter-day imprecision assessment: Take two levels of quality control plasma and normal mixed plasma, a total of three levels, each divided into 20 portions, and stored in a -80°C refrigerator. Take one portion of each of the three levels of plasma every day, and use this assay reagent to measure two batches per day. Each batch is measured twice, and the interval between the two batches should be greater than 4 hours. The measurement is carried out for a total of 20 days, and the SD and CV are calculated. The results are shown in Table 2: Table 2 Inter-day imprecision evaluation results of assay reagents

[0023] As shown in Table 2, the inter-day imprecision evaluation results of the assay reagent of the present invention have a small variation, with a CV of 2-8%, and a CV of ≤8%.

[0024] (2) Linear range evaluation The high-value and low-value plasmas used for linear range assessment were both obtained from the clinic, and their activity was measured by the German Siemens FVIII factor reagent. A high-value plasma H (FVIII%: 148%) close to the expected upper limit and a low-value plasma L (FVIII%: 20.0%) close to the expected lower limit were selected, and diluted into 7 points according to the proportions of H, 5H+L, 4H+2L, 3H+3L, 2H+4L, H+5L, and L. Each dilution was repeated twice, and the mean was calculated. After checking the data, outliers were eliminated, the repeatability was judged, and finally the average value of the measurement results was compared with the theoretical value for linear regression analysis. Theoretical value = (CL*VL+CH*VH) / (VL+VH). The results of the linear range assessment are shown in Figure 2 It can be seen that there are no obvious outliers in the above test results. The regression equation obtained is: Y=1.016X-0.2427, R2=0.9992≥0.99, indicating that the linear range is about 20%-150%.

[0025] (3) Stability assessment Open bottle stability assessment The three levels of plasma used for the stability assessment of the bottle opening were all taken from the clinic, and the FVIII activity was measured using this assay reagent. The measured value obtained was the initial measured value. On the first day, the assay reagent was re-dissolved and placed in the reagent position of the coagulometer. It was tested once at 8:00, 12:00, and 16:00 each day. The average value was taken for each test three times. The test was continued for 7 days. After the test, the reagent was covered with the bottle cap and returned to the refrigerator at 2-8°C for refrigeration until the end of the experiment. The relative deviation between each measured value and the initial measured value was calculated, and the relative deviation within ±10% was considered stable. The stability assessment results are as follows Figure 3 As shown, after opening the vial of the assay reagent of the present invention, plasma FVIII activity at all three levels showed a gradual downward trend, with the relative deviations reaching their highest level on day 7 after opening. On day 7, the relative deviations of FVIII activity in high / intermediate and low plasma compared to the initial measured value were 4.88%, 8.57%, and 8.9%, respectively, all less than 10%. This assay reagent exhibits excellent open-vial stability, remaining stable for at least 7 days after opening.

[0026] (4) Correlation and consistency analysis with currently available reagents This assay was used to test 30 plasma samples, including 20 apparently healthy subjects and 10 patients with bleeding symptoms. Siemens FVIII factor reagent was also used for testing. A scatter plot was drawn and the correlation coefficient between the two test systems was estimated. The scatter plot characteristics were analyzed to select the best regression model and perform linear regression. The test results are shown in the figure. Figure 4The correlation coefficient of the two kits was r=0.98, and the linear regression equation was Y=0.9412X+1.5413. The Bland-Altman analysis was used to evaluate the consistency of the test results of the two detection systems. The test results are shown in Figure 5 .

[0027] (5) Evaluation of anti-heparin interference ability Plasma used for anti-interference ability assessment was obtained from the clinic. 15 mL of mixed plasma samples were collected from the clinic and divided into 12 portions, each 1 mL. Unfractionated heparin 0.2 U, 0.4 U, 0.6 U, 0.8 U, 1.0 U, 1.2 U and low molecular weight heparin 0.3 U, 0.6 U, 0.9 U, 1.2 U, 1.5 U were added, respectively. The remaining 1 tube of plasma was blank plasma. 12 specimens were prepared and tested using Siemens FVIII coagulation reagent and the reagent of the present invention. The test results are shown in Table 3: Table 3 Results of anti-heparin interference ability test

[0028] As shown in Table 3, the assay reagent of the present invention is not interfered by heparin.

[0029] This specific embodiment is a reagent developed based on the principle of chromogenic substrate method to detect the activity of human plasma coagulation factor VIII. Its technical advantages are: (1) The assay reagent uses a chromogenic substrate method, which has high specificity and is not easily interfered with by other substances such as heparin in plasma; (2) The assay reagents have passed performance evaluation, the test results are reliable, the test performance is excellent, and the coefficient of variation (CV) of inter-batch and intra-batch imprecision meets the industry standard requirements; (3) The assay reagent has a wide linear range and good sensitivity for both high-value and low-value samples; (4) The assay reagent is easy to operate and can be used with various brands and models of coagulation analyzers, thus having broad market application prospects.

[0030] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. A kit for determining the activity of coagulation factor VIII, characterized in that: The kit includes reagent R1, reagent R2, reagent R3 and sample diluent; Reagent R1 includes bovine factor X, fibrin polymerization inhibitor, salt ions, stabilizer, preservative, and buffer; Reagent R2 includes bovine factor IXa, bovine thrombin, phospholipids, calcium chloride, stabilizers, preservatives, and buffer; Reagent R3 includes FXa chromogenic substrate, thrombin inhibitor, salt ions, stabilizer, preservative, and buffer; The sample diluent includes a heparin antagonist, salt ions, stabilizers, preservatives, and buffer.

2. A coagulation factor VIII activity assay kit according to claim 1, characterized in that: The bovine factor X in the reagent R1 is purified from bovine plasma, and the dosage is 0.5 IU / mL-1.5 IU / mL; the fibrin polymerization inhibitor in the reagent R1 is H-Gly-Pro-Arg-Pro-OH•AcOH, and the dosage is 0.1 nmol / ml-1 nmol / ml.

3. A coagulation factor VIII activity assay kit according to claim 1, characterized in that: The bovine factor IXa in the reagent R2 is purified from bovine plasma, and the dosage is 0.1 IU / mL-1 IU / mL; the bovine thrombin in the reagent R2 is purified from bovine plasma, and the dosage is 0.1 NIH / mL-1 NIH / mL; the phospholipid in the reagent R2 is one of rabbit cephalin, bovine cephalin, and synthetic lecithin, and the dosage is 0.05 mg / mL-0.5 mg / mL; the calcium chloride in the reagent R2 is 0.1 g / L-1 g / L.

4. A coagulation factor VIII activity assay kit according to claim 1, characterized in that: The FXa chromogenic substrate of the reagent R3 is one of CH3SO2-D-Leu-Gly-Arg-pNA•AcOH, CH3SO2-D-CHG-Gly-Arg-pNA•AcOH, CH3SO2-D-CHA-Gly-Arg-pNA•AcOH, and ZD-Arg-Gly-Arg-pNA•2HCl, and the dosage is 0.5-2 mg / mL; The thrombin inhibitor of the reagent R3 is one of r-Hirudin EC, Dabigatran, Civalirudin, and Argatroban, and the dosage is 250-1000 ng / mL.

5. A coagulation factor VIII activity assay kit according to claim 1, characterized in that: The heparin antagonist in the sample diluent is one of polybrene, protamine and heparinase, and the dosage is 2 μg / mL-10 μg / mL.

6. A kit for determining the activity of coagulation factor VIII according to claim 1, characterized in that: The buffer in the reagents R1, R2, R3 and the sample diluent is one or more of HEPES buffer, Tris-HCl buffer, phosphate buffer and barbiturate buffer, with a dosage of 20-50 mmol / L; The stabilizers in the reagents R1, R2, R3 and the sample diluent are one or more of proteins, sugars, amino acids, surfactants, suspending agents, and antioxidants; the stabilizer is one or more of bovine serum albumin, mannitol, trehalose, and glycine, with an amount of 1-50 g / L. The salt ions in the reagent R1, reagent R3 and sample diluent are one or more of NaCl, CaCl2, KCl, and CsCl, with an amount of 5-25 g / L; The preservative in the reagent is one or more of sodium azide, Proclin 300, and gentamicin, with a dosage of 0.1-0.5 g / L.

7. A kit for determining the activity of coagulation factor VIII according to claim 6, characterized in that: The buffer solution of the reagent R1, reagent R2, reagent R3 and sample diluent is a Tris-HCl buffer solution with a pH value of 7-9 and a concentration of 20-50 mmol / L.

8. Use of the kit according to claims 1 to 7 in the preparation of a product for detecting the activity of coagulation factor VIII.

9. A method for determining the activity of coagulation factor VIII, characterized in that: The following steps are included: (1) First, establish a standard curve for the activity of coagulation factor VIII. Dilute the coagulation factor VIII calibrator five times with the sample diluent to obtain six calibrators with activities of 160%, 80%, 40%, 20%, 10%, and 5%, respectively. (2) Reagent R1, reagent R2, and reagent R3 were reconstituted with 3 mL of purified water at room temperature for 30 min, and the sample dilution solution was taken out for later use; (3) Take out 30uL of the calibrator with different activities and add it to the reaction cup. Add 135uL of sample diluent. After taking out 30uL, add 40uL of reagent R1. After incubation for 100 seconds, add 40uL of reagent R2. After incubation for another 100 seconds, add 120uL of reagent R3. Measure the absorbance change value OD between 15 seconds and 300 seconds. (4) Each calibrator to be tested is measured three times, and a standard curve is drawn with the horizontal axis representing the activity of the calibrator to be tested and the vertical axis representing the mean OD values of the calibrators to be tested with different activities; (5) The plasma sample to be tested is tested using the same method. The measured OD value is substituted into the above standard curve to obtain the coagulation factor VIII activity value of the plasma sample to be tested.

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