CN115327128A - VIII factor activity determination kit and preparation method thereof - Google Patents
VIII factor activity determination kit and preparation method thereof Download PDFInfo
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/745—Assays involving non-enzymic blood coagulation factors
- G01N2333/755—Factors VIII, e.g. factor VIII C [AHF], factor VIII Ag [VWF]
Abstract
The invention discloses a VIII factor activity determination kit and a preparation method thereof, wherein the kit comprises VIII factor poor plasma, standard plasma and sample diluent; factor VIII-poor plasma includes: amino acid, preservative, freeze-drying protective agent, and the balance of VIII factor-poor plasma raw material which is added with anticoagulant and prepared by monoclonal antibody; the standard plasma includes: amino acid, preservative, freeze-drying protective agent, and the balance of standard blood plasma raw material added with anticoagulant; the sample diluent comprises: stabilizer, preservative, buffer solution and the balance of water; the preparation method comprises the following steps: preparing affinity immune gel, preparing VIII-deficient factor plasma, preparing standard plasma and preparing a sample diluent. The factor VIII-poor plasma kit has high biological safety, wide linear range, good test correlation in the linear range, high precision and simple and easy preparation method.
Description
Technical Field
The invention relates to the technical field of medical in-vitro diagnosis, in particular to a VIII factor activity determination kit and a preparation method thereof.
Background
Analysis of the coagulation activity of FVIII is of great importance in the clinical diagnosis of haemophilia a and in the production of concentrated formulations of factor VIII for therapeutic use.
Measurement of FVIII: there are two major methods of C activity, the first-phase method and the second-phase method. The second-stage method is complicated to operate and not easy to popularize. The first-stage method is relatively simple to operate and is quite popular. However, this approach requires a factor VIII deficient plasma as a substrate. This agent needs to be obtained from severe haemophilia patients, but the source is very limited and there is a risk of infection with hepatitis and aids, so that artificially prepared factor VIII deficient plasma needs to be provided. The artificially prepared factor VIII deficient plasma is safe to use and can be produced in large quantities so as to meet the clinical and research requirements.
Disclosure of Invention
The invention aims to solve the technical problems that in the prior art, artificially prepared factor VIII-deficient plasma is very limited in source and high in biological risk, and provides a factor VIII activity determination kit and a preparation method thereof.
The technical scheme adopted by the invention for solving the technical problem is as follows: a factor VIII activity assay kit comprises factor VIII-poor plasma, standard plasma and sample diluent; the factor VIII-poor plasma comprises the following components in terms of the total volume of 1L: 20-50 g of amino acid, 0.5-1.5 g of preservative, 15-40 g of freeze-drying protective agent and the balance of VIII factor plasma raw material which is added with anticoagulant and prepared by monoclonal antibody; the standard plasma comprises the following components in terms of total volume of 1L: 20-50 g of amino acid, 0.5-1.5 g of preservative, 15-40 g of freeze-drying protective agent and the balance of standard blood plasma raw material added with anticoagulant; the anticoagulant comprises 30-40 g of sodium citrate and water based on the total volume of 1L, and the volume ratio of the anticoagulant to the normal human plasma is 1 (8-10); the sample diluent comprises the following components in percentage by total volume of 1L: 11 to 25g of stabilizer, 0.5 to 1.5g of preservative, buffer solution with the content of 1mol and the pH value of 6.0 to 8.0, and the balance of water.
Preferably, the preservative is DN, proclin-300, thimerosal, gentamicin or cason.
Preferably, the lyoprotectant includes 10-30 g mannitol and 5-10 g trehalose.
Preferably, the amino acid is glycine, alanine or arginine.
Preferably, the buffer is Tris buffer, HEPES buffer or PBS buffer.
Preferably, the stabilizer comprises 3 to 5g of barbiturate sodium, 3 to 5g of an inorganic salt and 5 to 15g of albumin.
Preferably, the inorganic salt is sodium chloride, calcium chloride or ammonium sulfate.
Preferably, the albumin is bovine serum albumin, horse serum albumin or human serum albumin.
The preparation method of the factor VIII activity assay kit comprises the following steps:
s1, preparing an affinity immune gel: respectively coupling the antibody A, the antibody B and the antibody C with cyanogen bromide activated agarose gel to obtain affinity immune gel, loading the affinity immune gel into an affinity chromatographic column, connecting the affinity chromatographic column in series, and washing with a balance liquid flow; the antibody A is an anti-vWF monoclonal antibody, the antibody B is an anti-FVIII heavy chain monoclonal antibody, and the antibody C is an anti-FVIII light chain monoclonal antibody;
s2, preparation of plasma with factor VIII deficiency: mixing the anticoagulant with normal human plasma according to the volume ratio of 1 (8-10), centrifuging, taking supernatant plasma, filling the supernatant plasma into the affinity chromatography column obtained in the step S1, and collecting effluent liquid; adding 20-50 g of amino acid, 0.5-1.5 g of preservative and 15-40 g of freeze-drying protective agent into the effluent liquid according to the total volume of the effluent liquid as 1L, subpackaging and freeze-drying to obtain VIII-poor factor plasma freeze-dried powder;
s3, preparing standard plasma: mixing the anticoagulant with normal human plasma according to the volume ratio of 1 (8-10), centrifuging, and taking supernatant plasma; adding 20-50 g of amino acid, 0.5-1.5 g of preservative and 15-40 g of freeze-drying protective agent into supernatant plasma by taking the total volume of the standard plasma as 1L, subpackaging and freeze-drying to obtain standard plasma freeze-dried powder;
s4, preparation of a sample diluent: based on the total volume of the diluent of 1L, 11 to 25g of stabilizer, 0.5 to 1.5g of preservative, 1mol of buffer solution with the pH value of 6.0 to 8.0 and water are mixed to obtain the sample diluent.
Preferably, the volume ratio of the antibody A to the cyanogen bromide-activated sepharose in step S1 is (0.9-1.1): 1, the volume ratio of the antibody B to the cyanogen bromide-activated sepharose is (0.8-1.2): 1, and the volume ratio of the antibody C to the cyanogen bromide-activated sepharose is (0.8-1.2): 1.
Preferably, the equilibrium solution is composed of 8-10 g of sodium chloride and 1mol of Tris buffer solution with the pH value of 6.0-8.0 by taking the total volume of the equilibrium solution as 1L; the volume ratio of the balancing liquid to the affinity immune gel is (5-10): 1.
Preferably, in steps S2 and S3, the temperature of centrifugation is 2-6 ℃, the rotation speed is 2000-4000 rpm, and the time is 10-20 min.
Preferably, the flow rate of the normal human plasma cold supernatant from the affinity chromatography column in the step S2 is 20-40 mL/h.
Preferably, the volume ratio of the immunoaffinity gum to the normal human plasma cold supernatant is 1 (4-5).
The invention has the beneficial effects that:
according to the invention, the VIII factor-poor plasma is prepared by purifying normal human plasma through a monoclonal antibody coupling affinity chromatography technology, the biological risk is low, and the industrial production can be realized; the invention prepares the freeze-dried blood plasma by a freeze-drying process. The VIII factor activity determination kit of the invention has the advantages of ensured yield, wider linear range, good test correlation in the linear range and higher precision, and is used for diagnosing hemophilia A.
The invention provides a preparation method of a VIII factor activity assay kit, which has the advantages of simple operation process, capability of being used for industrial production, quality guarantee, strong practicability and easiness in popularization.
Drawings
FIG. 1 is a graph of the linearity of theoretical and test values of a test calibrator in accordance with example 1 of the present invention;
FIG. 2 is a graph showing the linearity of the theoretical and test values of the test calibrators in example 2 of the present invention.
Detailed Description
In order to clearly understand the technical features, objects and effects of the present invention, the present invention will be further described in detail with reference to the following embodiments, which are only used for explaining the present invention and are not to be construed as limiting the scope of the present invention.
A factor VIII activity assay kit comprises factor VIII-poor plasma, standard plasma and sample diluent; the factor VIII-poor plasma comprises the following components in percentage by total volume of 1L: 20-50 g of amino acid, 0.5-1.5 g of preservative, 15-40 g of freeze-drying protective agent and the balance of VIII factor-poor plasma raw material which is added with anticoagulant and prepared by monoclonal antibody; the standard plasma comprises the following components in terms of the total volume of 1L: 20-50 g of amino acid, 0.5-1.5 g of preservative, 15-40 g of freeze-drying protective agent and the balance of standard blood plasma raw material added with anticoagulant; the anticoagulant comprises 30-40 g of sodium citrate and water based on the total volume of 1L, and the volume ratio of the anticoagulant to the normal human plasma is 1 (8-10); the sample diluent comprises the following components in percentage by total volume of 1L: 11 to 25g of stabilizer, 0.5 to 1.5g of preservative, buffer solution with the content of 1mol and the pH value of 6.0 to 8.0, and the balance of water.
Wherein the anticoagulant is sodium citrate solution for delaying blood coagulation time.
The antiseptic is DN, proclin-300, thimerosal, gentamicin or cason.
The freeze-drying protective agent comprises 10-30 g of mannitol and 5-10 g of trehalose.
The amino acid is glycine, alanine or arginine, and can improve the stability of blood plasma.
The buffer solution is Tris buffer solution, HEPES buffer solution or PBS buffer solution.
The stabilizer comprises 3-5 g of barbital sodium, 3-5 g of inorganic salt and 5-15 g of albumin. Wherein the barbiturate sodium can be replaced by imidazole, the inorganic salt is sodium chloride, calcium chloride or ammonium sulfate, and the albumin is bovine serum albumin, horse serum albumin or human serum albumin. The stabilizer can stabilize blood plasma and reduce matrix effect.
The VIII factor activity determination kit needs to be matched with an APTT (activated partial thromboplastin time) determination kit for use.
According to the invention, the VIII factor-poor plasma is prepared by purifying normal human plasma through a monoclonal antibody coupling affinity chromatography method, the biological risk is low, and the industrial production can be realized; the invention prepares the freeze-dried blood plasma by a freeze-drying process. The VIII factor activity determination kit of the invention has the advantages of ensured yield, wider linear range, good test correlation in the linear range and higher precision, and is used for diagnosing hemophilia A.
The preparation method of the factor VIII activity assay kit comprises the following steps:
s1, preparing an affinity immune gel: coupling the antibody with cyanogen bromide activated agarose gel to obtain affinity immune gel, and filling the affinity immune gel into an affinity chromatographic column;
step S1 includes the following substeps:
s1.1, coupling the anti-vWF monoclonal antibody and cyanogen bromide activated agarose gel according to the volume ratio of (0.9-1.1) to 1, and filling the coupled agarose gel into an affinity chromatography column;
s1.2, coupling the anti-FVIII heavy chain monoclonal antibody and cyanogen bromide activated sepharose gel according to the volume ratio of (0.8-1.2) to 1, and loading the coupled sepharose gel into an affinity chromatography column;
s1.3, coupling the anti-FVIII light chain monoclonal antibody and cyanogen bromide activated agarose gel according to the volume ratio of (0.8-1.2) to 1, and filling the coupled product into an affinity chromatography column;
s1.4, connecting affinity chromatographic columns in series, and washing with an equilibrium liquid flow.
Wherein the equilibrium solution is composed of 8-10 g of sodium chloride and Tris buffer solution with the content of 1mol and the pH value of 6.0-8.0 by taking the total volume of the equilibrium solution as 1L; the volume ratio of the balancing liquid to the affinity immune gel is (5-10): 1.
Studies have shown that in fresh plasma, FVIII: c is mostly bound to VWF, and a small fraction is cleaved into unequal-sized fragments by the action of FIIa, FXa and APC (activated protein C). These fragments vary in molecular weight from 80KD to 310KD and all exhibit FVIII: c, so that there is not only FVIII in bound form: c, but also fragmented FVIII: c, and FVIII in bound form: the epitope portion of C is masked or modified such that FVIII: c monoclonal, unrecognizable, part of FVIII: c was removed using vWF mab. Since a monoclonal antibody recognizes only one epitope of an antigen, the affinity chromatography preparation of matrix plasma monoclonal antibodies including at least 3 antibodies recognizing heavy chain, light chain and vWF is effective in removing FVIII from plasma: C. therefore, the present invention adopts anti-vWF monoclonal antibody, anti-FVIII heavy chain monoclonal antibody and anti-FVIII light chain monoclonal antibody to prepare affinity immune gel, which is used for preparing factor VIII-poor plasma in the next step.
S2, preparation of factor VIII-poor plasma: mixing an anticoagulant with normal human plasma according to a volume ratio of 1 (8-10), centrifuging at the temperature of 2-6 ℃ at the rotating speed of 2000-4000 rpm for 10-20 min, taking supernatant blood plasma, filling the supernatant blood plasma into an affinity chromatography column obtained in the step S1, wherein the flow rate is 20-40 mL/h, the volume ratio of immunoaffinity glue to normal human plasma cold supernatant is 1 (4-5), collecting effluent, and removing the front and back 3mL; adding 20-50 g of amino acid, 0.5-1.5 g of preservative and 15-40 g of freeze-drying protective agent into the effluent liquid by taking the total volume of the effluent liquid as 1L, packaging into 1 mL/piece, and freeze-drying by using a freeze dryer to obtain VIII-poor factor plasma freeze-dried powder; the freeze-dried blood plasma can be preserved for 1 year at the temperature of 2-8 ℃.
S3, preparing standard plasma: mixing the anticoagulant with normal human plasma according to the volume ratio of 1 (8-10), centrifuging at the rotating speed of 2000-4000 rpm for 10-20 min at the temperature of 2-6 ℃, and taking supernatant plasma; adding 20-50 g of amino acid, 0.5-1.5 g of preservative and 15-40 g of freeze-drying protective agent into the supernatant plasma by taking the total volume of the standard plasma as 1L, subpackaging the mixture into 1 mL/branch, and freeze-drying by using a freeze dryer to obtain the standard plasma freeze-dried powder. And (4) carrying out blood coagulation factor activity assignment on the standard plasma according to a tracing process recommended by WHO.
In steps S1 and S2, an anticoagulant is first prepared: adding 30-40 g of sodium citrate into 1L of water based on the total volume of the anticoagulant being 1L; then blood sampling is carried out: mixing anticoagulant with normal human plasma, centrifuging, and collecting cold supernatant as raw material of VIII-poor factor plasma or standard plasma.
S4, preparation of a sample diluent: based on the total volume of the diluent of 1L, 11 to 25g of stabilizer, 0.5 to 1.5g of preservative, 1mol of buffer solution with the pH value of 6.0 to 8.0 and water are mixed to obtain the sample diluent.
The using method of the VIII factor activity determination kit comprises the following steps:
taking 1 frozen standard plasma and factor VIII-poor plasma, dissolving and shaking the frozen standard plasma and the factor VIII-poor plasma respectively with 1mL of purified water, dissolving the factor VIII-poor plasma with a sample diluent, diluting the standard plasma by multiple times with the factor VIII-poor plasma according to a calibration point arranged in a coagulation analyzer, testing with an APTT kit, and making a standard curve according to the measured time and a theoretical activity value. And putting the plasma to be tested and the factor VIII plasma into corresponding sample positions and reagent positions, testing according to parameters set in the instrument, and correspondingly obtaining the activity value of the factor VIII in the plasma to be tested on a calibration curve according to the APTT test value. The content of coagulation factors was read from the reference curve and expressed as% normal.
The invention provides a preparation method of a VIII factor activity assay kit, which has the advantages of simple operation process, capability of being used for industrial production, quality guarantee, strong practicability and easiness in popularization.
The following is illustrated by specific examples:
example 1
A factor VIII activity assay kit comprises factor VIII-poor plasma, standard plasma and sample diluent; the factor VIII-poor plasma comprises the following components in percentage by total volume of 1L: 20g of glycine, 0.5g of DN, 10g of mannitol and 5g of trehalose, and the balance of VIII factor-poor plasma raw material which is added with anticoagulant and is prepared by monoclonal antibody; the standard plasma comprises the following components in terms of total volume of 1L: 20g of glycine, 0.5g of DN, 10g of mannitol and 5g of trehalose, and the balance of standard plasma raw materials added with anticoagulant; the anticoagulant comprises 30g of sodium citrate and water based on the total volume of 1L, and the volume ratio of the anticoagulant to normal human plasma is 1:8; the sample diluent comprises the following components in percentage by total volume of 1L: 3g of barbital sodium, 3g of sodium chloride, 5g of bovine serum albumin, 0.5g of DN, 1mol of Tris buffer solution with the pH value of 6.0, and the balance of water.
The preparation method of the factor VIII activity assay kit comprises the following steps:
s1, preparing an affinity immune gel: coupling the antibody with cyanogen bromide activated sepharose gel 4B to obtain an affinity immune gel, and filling the affinity immune gel into an affinity chromatography column;
step S1 includes the following substeps:
s1.1, coupling 4.5mg of anti-vWF monoclonal antibody and 5mL of cyanogen bromide activated sepharose gel 4B, and filling the coupled sepharose gel into an affinity chromatography column;
s1.2, coupling 2mg of anti-FVIII heavy chain monoclonal antibody with 2.5mL of cyanogen bromide activated sepharose gel 4B, and filling the sepharose gel into an affinity chromatography column;
s1.3, coupling 2mg of anti-FVIII light chain monoclonal antibody with 2.5mL of cyanogen bromide activated sepharose gel 4B, and loading the coupled sepharose gel into an affinity chromatography column;
s1.4, connecting affinity chromatographic columns in series, and washing with an equilibrium liquid flow.
The equilibrium solution is calculated by the total volume of 1L and consists of 8g of sodium chloride and Tris buffer solution with the content of 1mol and the pH value of 6.0; the volume ratio of the equilibrium solution to the affinity immune gel is 5:1.
The equilibrium solution was prepared as follows: preparing 1mol/L Tris buffer solution with the pH value of 6.0, and adding 8g of sodium chloride into 1L of buffer solution for uniformly mixing. The following examples were prepared in the same manner as described above.
S2, preparation of factor VIII-poor plasma: mixing the anticoagulant with normal human plasma according to a volume ratio of 1:8, centrifuging at 2 ℃ for 20min at 2000rpm, loading supernatant plasma into an affinity chromatography column obtained in the step S1 at a flow rate of 20mL/h, adsorbing 40mL of normal human plasma cold supernatant by 10mL of immunoaffinity glue, collecting about 34mL of effluent, and removing front and back 3mL; adding 20g of glycine, 0.5g of DN, 10g of mannitol and 5g of trehalose into the effluent liquid by taking the volume of the effluent liquid as 1L, packaging into 1 mL/piece, and freeze-drying by using a freeze dryer to obtain the VIII-poor factor plasma freeze-dried powder;
s3, preparing standard plasma: mixing the anticoagulant with normal human plasma according to a volume ratio of 1:8, centrifuging at 2 ℃ and 2000rpm for 20min, and taking supernatant plasma; adding 20g of glycine, 0.5g of DN, 10g of mannitol and 5g of trehalose into supernatant plasma by taking the total volume of the standard plasma as 1L, packaging into 1 mL/branch, and freeze-drying by using a freeze dryer to obtain standard plasma freeze-dried powder; performing factor activity assignment on standard plasma according to a tracing process recommended by WHO;
s4, preparation of a sample diluent: and mixing 3g of barbital sodium, 3g of sodium chloride, 5g of bovine serum albumin, 0.5g of DN, 1mol of Tris buffer solution with the pH value of 6.0 and water according to the total volume of the diluent of 1L to obtain the sample diluent.
Example 2
A factor VIII activity assay kit comprises factor VIII-poor plasma, standard plasma and sample diluent; the factor VIII-poor plasma comprises the following components in percentage by total volume of 1L: 50g of alanine, 1.5g of Proclin-300, 30g of mannitol and 10g of trehalose, and the balance of VIII factor-poor plasma raw material which is added with an anticoagulant and is prepared by using a monoclonal antibody; the standard plasma comprises the following components in terms of total volume of 1L: 50g of alanine, 1.5g of Proclin-300, 30g of mannitol and 10g of trehalose, and the balance of standard plasma raw materials added with anticoagulant; the anticoagulant comprises 40g of sodium citrate and water, wherein the total volume of the anticoagulant is 1L, and the volume ratio of the anticoagulant to normal human plasma is 1; the sample diluent comprises the following components in percentage by total volume of 1L: 5g of imidazole, 5g of calcium chloride, 15g of horse serum albumin, 1.5g of Proclin-300, 1mol of HEPES buffer solution with the pH value of 8.0, and the balance of water.
The preparation method of the factor VIII activity assay kit comprises the following steps:
s1, preparing an affinity immune gel: coupling the antibody with cyanogen bromide activated sepharose gel 4B to obtain an affinity immune gel, and filling the affinity immune gel into an affinity chromatography column;
step S1 includes the following substeps:
s1.1, coupling 5.5mg of anti-vWF monoclonal antibody and 5mL of cyanogen bromide activated sepharose gel 4B, and filling the coupled sepharose gel into an affinity chromatography column;
s1.2, coupling 3mg of anti-FVIII heavy chain monoclonal antibody with 2.5mL of cyanogen bromide activated sepharose gel 4B, and filling the sepharose gel into an affinity chromatography column;
s1.3, coupling 3mg of anti-FVIII light chain monoclonal antibody with 2.5mL of cyanogen bromide activated sepharose gel 4B, and loading the coupled sepharose gel into an affinity chromatography column;
s1.4, connecting affinity chromatographic columns in series, and washing with an equilibrium liquid flow.
Wherein the equilibrium solution is composed of 10g of sodium chloride and HEPES buffer solution with the content of 1mol and the pH value of 8.0 by taking the total volume of the equilibrium solution as 1L; the volume ratio of the equilibrium solution to the affinity immune gel is 10.
S2, preparation of plasma with factor VIII deficiency: mixing the anticoagulant with normal human plasma according to a volume ratio of 1 to 10, centrifuging at 4000rpm for 10min at 6 ℃, taking supernatant plasma, filling the supernatant plasma into an affinity chromatography column obtained in the step S1, enabling the flow rate to be 40mL/h, adsorbing 50mL of normal human plasma cold supernatant by 10mL of immunoaffinity glue, collecting about 44mL of effluent, and removing front and back 3mL; adding 50g of alanine, 1.5g of Proclin-300, 30g of mannitol and 10g of trehalose into the effluent, packaging into 1mL per branch, and freeze-drying by using a freeze-dryer to obtain factor VIII-deficient plasma freeze-dried powder, wherein the total volume of the effluent is 1L;
s3, preparing standard plasma: mixing the anticoagulant with normal human plasma according to a volume ratio of 1; adding 50g of alanine, 1.5g of Proclin-300, 30g of mannitol and 10g of trehalose into supernatant plasma by taking the total volume of the standard plasma as 1L, packaging into 1 mL/branch, and freeze-drying by using a freeze dryer to obtain standard plasma freeze-dried powder; performing factor activity assignment on standard plasma according to a tracing process recommended by WHO;
s4, preparation of a sample diluent: based on the total volume of the diluent being 1L, 5g of imidazole, 5g of calcium chloride, 15g of horse serum albumin, 1.5g of Proclin-300, 1mol of HEPES buffer solution with the pH value of 8.0 and water are mixed to obtain a sample diluent.
Example 3
A factor VIII activity assay kit comprises factor VIII-poor plasma, standard plasma and sample diluent; the factor VIII-poor plasma comprises the following components in terms of the total volume of 1L: 35g of arginine, 1g of thimerosal, 20g of mannitol and 7.5g of trehalose, and the balance of VIII factor-poor plasma raw materials which are added with an anticoagulant and prepared by using a monoclonal antibody; the standard plasma comprises the following components in terms of total volume of 1L: 35g of arginine, 1g of merthiolate, 20g of mannitol and 7.5g of trehalose, and the balance of standard plasma raw materials added with an anticoagulant; the anticoagulant comprises 35g of sodium citrate and water based on the total volume of 1L, and the volume ratio of the anticoagulant to normal human plasma is 1:9; the sample diluent is counted by the total volume of 1L and comprises the following components: 4g of imidazole, 4g of ammonium sulfate, 10g of human serum albumin, 1g of thimerosal, 1mol of PBS buffer solution with the pH value of 7.0, and the balance of water.
A preparation method of a VIII factor activity assay kit comprises the following steps:
s1, preparing an affinity immune gel: coupling the antibody with cyanogen bromide activated sepharose gel 4B to obtain an affinity immune gel, and filling the affinity immune gel into an affinity chromatography column;
step S1 includes the following substeps:
s1.1, coupling 5mg of anti-vWF monoclonal antibody and 5mL of cyanogen bromide activated sepharose gel 4B, and filling the coupled sepharose gel into an affinity chromatography column;
s1.2, coupling 2.5mg of anti-FVIII heavy chain monoclonal antibody with 2.5mL of cyanogen bromide activated sepharose gel 4B, and loading the coupled sepharose gel into an affinity chromatography column;
s1.3, coupling 2.5mg of anti-FVIII light chain monoclonal antibody with 2.5mL of cyanogen bromide activated sepharose 4B, and loading into an affinity chromatography column;
s1.4, connecting affinity chromatographic columns in series, and washing with an equilibrium liquid flow.
Wherein the equilibrium solution is composed of 9g of sodium chloride and 1mol of PBS buffer solution with the pH value of 7.0 by taking the total volume of 1L; the volume ratio of the equilibrium solution to the affinity immune gel is 7:1.
S2, preparation of plasma with factor VIII deficiency: mixing the anticoagulant with normal human plasma according to a volume ratio of 1:9, centrifuging at 4 ℃ for 15min at 3000rpm, loading supernatant plasma into an affinity chromatography column obtained in the step S1 at a flow rate of 30mL/h, adsorbing 45mL of normal human plasma cold supernatant by 10mL of immunoaffinity glue, collecting about 39mL of effluent liquid, and removing front and back 3mL; adding 35g of arginine, 1g of thimerosal, 20g of mannitol and 7.5g of trehalose into the effluent, wherein the total volume of the effluent is 1L, packaging into 1 mL/piece, and freeze-drying by using a freeze dryer to obtain the VIII-deficient factor plasma freeze-dried powder;
s3, preparing standard plasma: mixing the anticoagulant with normal human plasma according to a volume ratio of 1:9, centrifuging at 4 ℃ and 3000rpm for 15min, and taking supernatant plasma; adding 35g of arginine, 1g of thimerosal, 20g of mannitol and 7.5g of trehalose into supernatant plasma by taking the total volume of the standard plasma as 1L, packaging into 1 mL/branch, and freeze-drying by using a freeze dryer to obtain standard plasma freeze-dried powder; performing factor activity assignment on standard plasma according to a tracing process recommended by WHO;
s4, preparation of a sample diluent: and mixing 4g of imidazole, 4g of ammonium sulfate, 10g of human serum albumin, 1g of thimerosal, 1mol of PBS buffer solution with the pH value of 7.0 and water according to the total volume of the diluent of 1L to obtain the sample diluent.
It will be appreciated that in the above examples and alternatives thereof, the preservative may also be replaced with gentamicin or cason.
Performance test:
1. coagulation factor activity assay
The test instrument: coagulation analyzer CA520 produced by Shenzhen Di Mey Biotechnology Limited
The test steps are as follows: the factor VIII-poor plasma effluents of examples 1 and 2 were tested for factor clotting activity using factor clotting test reagents and the results are shown in tables 1 and 2.
Table 1 factor VIII-poor plasma effluent and raw plasma coagulation factor levels of example 1 (n = 3%)
| Blood coagulation factor Activity | Raw plasma (%) | Factor VIII-poor plasma (%) |
| FII：C | 98.35 | 92.00 |
| FV：C | 85.76 | 81.24 |
| FVII：C | 102.21 | 98.33 |
| FVIII：C | 86.23 | ＜1 |
| FIX：C | 105.12 | 87.16 |
| FX：C | 111.98 | 89.67 |
Table 2 factor VIII-poor plasma effluent and raw plasma coagulation factor levels of example 2 (n = 3%)
| Blood coagulation factor Activity | Raw plasma (%) | Factor VIII-poor plasma (%) |
| FII：C | 96.5 | 93.1 |
| FV：C | 82.7 | 78.4 |
| FVII：C | 100.1 | 93.3 |
| FVIII：C | 81.5 | ＜1 |
| FIX：C | 95.2 | 78.5 |
| FX：C | 102.98 | 85.7 |
The test result shows that the activity of FVIII in the effluent of the factor VIII-poor plasma obtained by adsorption of the affinity immune gel is less than 1 percent, and the activities of other coagulation factors are all maintained at normal values, so that the effluent is the factor VIII-poor plasma and can be used for preparing the factor VIII-poor plasma kit.
2. Measurement of precision
The test instrument: coagulation analyzer CA520 produced by Shenzhen Di Mey Biotechnology Limited
The test steps are as follows: the same quality control was tested 20 times using the factor VIII activity assay kits of example 1 and example 2, and the precision was calculated, and the test results are shown in tables 3 and 4.
Table 3 results of precision measurements of example 1
Table 4 results of precision measurements of example 2
The test results show that the precision of 20 times of testing the same quality control product in the example 1 and the example 2 is less than or equal to 5 percent. Therefore, the factor VIII activity assay kit has high precision.
3. Linear range test
The test instrument: coagulation analyzer CA520 produced by Shenzhen Di Mey Biotechnology Limited
The test steps are as follows: the factor VIII activity assay kits of example 1 and example 2 were used to test different levels of calibrators, and linear regression analysis was performed using the test values as x-axis and the theoretical values as y-axis, and the test results are shown in table 5 and table 6 and fig. 1 and fig. 2. The test result is required to be: linear correlation coefficient R 2 ≥0.990。
Table 5 example 1 linear range test results
Table 6 example 2 linear range test results
| Theoretical value (%) | Test value (%) |
| 200.0 | 198.30 |
| 180.1 | 186.50 |
| 160.2 | 160.30 |
| 140.3 | 141.80 |
| 120.4 | 125.70 |
| 100.5 | 89.50 |
| 80.5 | 78.90 |
| 60.6 | 50.70 |
| 40.7 | 46.90 |
| 0.9 | 0.98 |
| Coefficient of correlation R 2 | 0.9916 |
The test result shows that when the activity of the factor VIII is in a linear range of 0-200%, the linear correlation coefficients are all larger than 0.990, and the test result shows that the linear correlation is better. Therefore, the factor VIII assay kit of the present invention has a wide linear range.
The test results show that the factor VIII activity determination kit has high precision, wide linear range and good test correlation in the linear range.
The above description is only an embodiment of the present invention, and not intended to limit the scope of the present invention, and all modifications of equivalent structures and equivalent processes, which are made by the present specification, or directly or indirectly applied to other related technical fields, are included in the scope of the present invention.
Claims (14)
1. A VIII factor activity assay kit is characterized by comprising VIII factor-poor plasma, standard plasma and sample diluent;
the factor VIII-poor plasma comprises the following components in percentage by volume of 1L: 20-50 g of amino acid, 0.5-1.5 g of preservative, 15-40 g of freeze-drying protective agent and the balance of VIII factor plasma raw material which is added with anticoagulant and prepared by monoclonal antibody;
the standard plasma comprises the following components in a total volume of 1L: 20-50 g of amino acid, 0.5-1.5 g of preservative, 15-40 g of freeze-drying protective agent and the balance of standard blood plasma raw material added with anticoagulant;
the anticoagulant comprises 30-40 g of sodium citrate and water based on the total volume of 1L, and the volume ratio of the anticoagulant to the normal human plasma is 1 (8-10);
the sample diluent comprises the following components in a total volume of 1L: 11 to 25g of stabilizer, 0.5 to 1.5g of preservative, buffer solution with the content of 1mol and the pH value of 6.0 to 8.0, and the balance of water.
2. The factor VIII activity assay kit according to claim 1, wherein the preservative is DN, proclin-300, thimerosal, gentamicin or cason.
3. The factor VIII activity assay kit according to claim 1, wherein said lyoprotectant comprises 10-30 g of mannitol and 5-10 g of trehalose.
4. The factor VIII activity assay kit of claim 1, wherein the amino acid is glycine, alanine, or arginine.
5. The factor VIII activity assay kit of claim 1, wherein the buffer is Tris buffer, HEPES buffer, or PBS buffer.
6. The factor VIII activity assay kit of claim 1, wherein said stabilizer comprises 3-5 g of barbiturate sodium, 3-5 g of inorganic salts, and 5-15 g of albumin.
7. The factor VIII activity assay kit of claim 6, wherein said inorganic salt is sodium chloride, calcium chloride or ammonium sulfate.
8. The kit for determining the activity of factor VIII according to claim 6, wherein the albumin is bovine serum albumin, horse serum albumin or human serum albumin.
9. A preparation method of a VIII factor activity assay kit is characterized by comprising the following steps:
s1, preparing an affinity immune gel: respectively coupling the antibody A, the antibody B and the antibody C with cyanogen bromide activated agarose gel to obtain affinity immune gel, loading the affinity immune gel into an affinity chromatographic column, connecting the affinity chromatographic column in series, and washing with a balance liquid flow; the antibody A is an anti-vWF monoclonal antibody, the antibody B is an anti-FVIII heavy chain monoclonal antibody, and the antibody C is an anti-FVIII light chain monoclonal antibody;
s2, preparation of plasma with factor VIII deficiency: mixing the anticoagulant with normal human plasma according to the volume ratio of 1 (8-10), centrifuging, taking supernatant plasma, filling the supernatant plasma into the affinity chromatography column obtained in the step S1, and collecting effluent liquid; adding 20-50 g of amino acid, 0.5-1.5 g of preservative and 15-40 g of freeze-drying protective agent into the effluent liquid according to the total volume of the effluent liquid as 1L, subpackaging and freeze-drying to obtain VIII-poor factor plasma freeze-dried powder;
s3, preparing standard plasma: mixing the anticoagulant with normal human plasma according to the volume ratio of 1 (8-10), centrifuging, and taking supernatant plasma; adding 20-50 g of amino acid, 0.5-1.5 g of preservative and 15-40 g of freeze-drying protective agent into supernatant plasma by taking the total volume of the standard plasma as 1L, subpackaging and freeze-drying to obtain standard plasma freeze-dried powder;
s4, preparation of a sample diluent: based on the total volume of the diluent of 1L, 11 to 25g of stabilizer, 0.5 to 1.5g of preservative, 1mol of buffer solution with the pH value of 6.0 to 8.0 and water are mixed to obtain the sample diluent.
10. The method for preparing a factor VIII activity assay kit according to claim 9, wherein in step S1, the volume ratio of the antibody A to the cyanogen bromide-activated sepharose is (0.9-1.1): 1, the volume ratio of the antibody B to the cyanogen bromide-activated sepharose is (0.8-1.2): 1, and the volume ratio of the antibody C to the cyanogen bromide-activated sepharose is (0.8-1.2): 1.
11. The method for preparing a factor VIII activity assay kit according to claim 9, wherein the balance solution comprises 8 to 10g of sodium chloride and 1mol of Tris buffer with pH value of 6.0 to 8.0, based on 1L of the total volume of the balance solution; the volume ratio of the balancing liquid to the affinity immune gel is (5-10): 1.
12. The method for preparing a factor VIII activity assay kit according to claim 9, wherein in steps S2 and S3, the centrifugation temperature is 2-6 ℃, the rotation speed is 2000-4000 rpm, and the time is 10-20 min.
13. The method for preparing a factor VIII activity assay kit according to claim 9, wherein the flow rate of the normal human plasma cold supernatant from the affinity chromatography column in step S2 is 20-40 mL/h.
14. The method for preparing the factor VIII activity assay kit according to claim 9, wherein the volume ratio of the immunoaffinity gum to the normal human plasma cold supernatant is 1 (4-5).
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