CN104181313A - Preparation method of blood coagulation factor IX quality control product - Google Patents
Preparation method of blood coagulation factor IX quality control product Download PDFInfo
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- CN104181313A CN104181313A CN201410449813.9A CN201410449813A CN104181313A CN 104181313 A CN104181313 A CN 104181313A CN 201410449813 A CN201410449813 A CN 201410449813A CN 104181313 A CN104181313 A CN 104181313A
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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
- G01N33/96—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood or serum control standard
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N2001/2893—Preparing calibration standards
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2496/00—Reference solutions for assays of biological material
- G01N2496/05—Reference solutions for assays of biological material containing blood cells or plasma
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Abstract
The invention relates to a preparation method of a clinical blood coagulation inspection preparation and particularly relates to a preparation method of a blood coagulation factor IX quality control product. The preparation method comprises the following steps: carrying out affinity chromatography on the mixed blood plasma of multiple persons by using an anti-human blood coagulation factor IX monoclonal antibody immunoaffinity chromatography column, removing a blood coagulation factor IX in the mixed blood plasma of multiple persons to obtain a blood plasma in shortage of the blood coagulation factor IX; mixing the mixed blood plasma of multiple persons with the blood plasma in shortage of the blood coagulation factor IX according to a certain proportion to prepare a blood coagulation factor IX quality control product with the content of the blood coagulation factor IX at different concentration levels, adding a freeze-drying protective additive, carrying out sub-packaging, and carrying out freeze drying so as to obtain the blood coagulation factor IX quality control product. According to the blood coagulation factor IX quality control product prepared by the preparation method, the uniformity, the stability and the stability of freeze-dried aquatic product subjected to re-melting are good, and the quality control product can replace an imported product to be used for quality control on detection of blood coagulation factor IX, so that the reduction of the detection cost is facilitated and the capability of detecting the blood coagulation factor IX in China can be promoted.
Description
Technical field
The present invention relates to the preparation method of clinical detection of blood coagulation preparation, concrete plasma thromboplastin component quality-control product preparation method.
Background technology
Plasma thromboplastin component is measured has important clinical meaning.Along with the active Interventions Requested of plasma thromboplastin component are constantly extended to the every field in medical diagnosis on disease and treatment, use plasma thromboplastin component quality-control product to carry out Internal Quality Control and chamber interstitial and just comment and seem and be even more important active detection of plasma thromboplastin component.The reagent, the instrument that detect for plasma thromboplastin component are at present more, and different reagent and detecting instrument, detection principle are different, cause its result difference very large.No matter adopt which kind of method, carrying out Internal Quality Control and participating in that chamber interstitial comments is to ensure that testing result has the necessary means of good reproducibility and comparability, and carrying out of these quality assurance measures all be unable to do without plasma thromboplastin component quality-control product.
The blood coagulation Quality Control thing that clinical labororatory is used is at present generally multiparameter blood coagulation quality-control product, its exceptional value blood coagulation quality-control product other parameter except plasma thromboplastin component horizontal abnormality is also abnormal, be difficult to ensure the accuracy of pathologic sample plasma thromboplastin component measurement result, be also difficult to the validity that ensures that plasma thromboplastin component Internal Quality Control and chamber interstitial are commented simultaneously.
Domestic blood coagulation quality-control product is almost monopolized by external reagent manufacturer, valuable product and level are single, cannot meet the demand of the many activity levels of clinical examination, therefore, in research and development producing country, the plasma thromboplastin component quality-control product of multiple concentration levels of independent brand is extremely urgent.
Summary of the invention
For above-mentioned technical matters, the invention provides a kind of method and acquisition plasma thromboplastin component quality-control product of preparing plasma thromboplastin component quality-control product.Plasma thromboplastin component quality-control product prepared by the inventive method is the multiple concentration levels of plasma thromboplastin component, and other clotting factor content is in normal range; This product homogeneity and stability are better, meet the clinical quality control requirement to the inspection of plasma thromboplastin component quality-control product.
In order to realize foregoing invention object, the invention provides following technical scheme: plasma thromboplastin component quality-control product preparation method, comprises the following steps:
(1) blood of people's vein collection was added and mixed as 9: 1 taking volume ratio with anti-coagulants, under 2 DEG C～8 DEG C conditions centrifugal 15 minutes, relative centrifugal force(RCF) was 2500g, separated and drew upper plasma, many person-portions blood plasma is mixed, many person-portions pooled plasma; Anti-coagulants is citric acid three sodium solutions, and its concentration is 0.109mol/L～0.129mol/L.
(2) immune affinity chromatographic column is used damping fluid balance under 10 DEG C～18 DEG C conditions in advance, and damping fluid comprises 0.02mol/L Tris, 0.15mol/L NaCl, 8mmol/L MgCl
2, pH7.25.
(3) raw blood plasma adds MgCl
2after, MgCl in raw blood plasma
2concentration is 4.5～5.5mmol/L.Upper immune affinity chromatographic column, collects the blood plasma that flow out immune affinity chromatographic column below, is and lacks plasma thromboplastin component blood plasma.
(4) will lack plasma thromboplastin component blood plasma and many person-portions pooled plasma as requested ratio mix, can be prepared into the plasma thromboplastin component quality-control product of variable concentrations level.
(5) the plasma thromboplastin component quality-control product of preparation is added to freeze drying protectant, packing, freeze drying.The freeze drying protectant adding, comprises that mass ratio accounts for 0.5～1.5%Hepes of plasma thromboplastin component quality-control product, l～5% glycocoll, 0.5～5% sweet mellow wine.
Plasma thromboplastin component quality-control product preparation method provided by the invention, many person-portions pooled plasma is carried out to affinity chromatography with anti-human coagulation factor IX monoclonal antibody immunity affinity column, remove plasma thromboplastin component in many person-portions pooled plasma and be and lack plasma thromboplastin component blood plasma; Many person-portions pooled plasma is mixed according to a certain percentage with shortage plasma thromboplastin component blood plasma, being prepared into plasma thromboplastin component content is the plasma thromboplastin component quality-control product of variable concentrations level, adds freeze drying protectant again, packing, and freeze drying, obtains plasma thromboplastin component quality-control product.
In preparation method provided by the invention, the preparation method of anti-human coagulation factor IX monoclonal antibody immunity affinity column is coupled to a kind of monoclonal antibody of connection plasma thromboplastin component that can high degree of specificity on the Sepharose4B Ago-Gel of CNBr activation, composition immune affinity chromatographic column.At Mg
2+under the condition existing, in the time that blood plasma passes through this immune affinity chromatographic column, plasma thromboplastin component is adsorbed only, and the activity of other clotting factor does not become substantially.
Plasma thromboplastin component quality-control product prepared by the present invention, it is good that its product homogeneity, stability and dried frozen aquatic products melt rear stability again, can replace imported product to be used for the quality control that plasma thromboplastin component detects, be conducive to reduce testing cost, promote the ability that China is detected plasma thromboplastin component.
Embodiment
In conjunction with the embodiments, further set forth the present invention:
Embodiment is to prepare the plasma thromboplastin component quality-control product of activity as 30% as example.
(1) blood of people's vein collection was added and mixed as 9: 1 taking volume ratio with anti-coagulants, under 2 DEG C～8 DEG C conditions centrifugal 15 minutes, relative centrifugal force(RCF) was 2500g, separated and drew upper plasma, many person-portions blood plasma is mixed, many person-portions pooled plasma; Anti-coagulants is citric acid three sodium solutions, and its concentration is 0.109mol/L～0.129mol/L.
(2) immune affinity chromatographic column is used damping fluid balance under 10 DEG C～18 DEG C conditions in advance, and damping fluid comprises 0.02mol/L Tris, 0.15mol/L NaCl, 8mmol/L MgCl
2, pH7.25.
(3) raw blood plasma adds MgCl
2after, MgCl in raw blood plasma
2concentration is 4.5～5.5mmol/L.Upper immune affinity chromatographic column, collects the blood plasma that flow out immune affinity chromatographic column below, is and lacks plasma thromboplastin component blood plasma.
(4) will lack plasma thromboplastin component blood plasma and many person-portions pooled plasma as requested ratio mix, preparing plasma thromboplastin component activity is the plasma thromboplastin component quality-control product of 30% left and right concentration level.
(5) the plasma thromboplastin component quality-control product of preparation is added to freeze drying protectant.Protective agent comprises 1%Hepes, 2% glycocoll, 2% sweet mellow wine.Carry out packing, freeze drying.
Test of product performance test:
(1) Product Activity detects
Detecting reagent with the full-automatic coagulo meter of Sysmex CA-1500 and supporting plasma thromboplastin component detects embodiment plasma thromboplastin component quality-control product activity.
The coagulation factor activity of table 1 plasma thromboplastin component quality-control product is measured table
| Test item | II | V | VII | X | VIII | IX | XI | XII |
| Factor active (%) | 99.8 | 92.6 | 98.3 | 96.5 | 93.4 | 1.2 | 100.5 | 93.2 |
As seen from the above table, this preparation method products obtained therefrom specificity is good, can specific removal plasma thromboplastin component and on other clotting factor content substantially without impact.
(2) product homogeneity is measured
In the plasma thromboplastin component quality-control product of preparing at embodiment, randomly draw respectively 10, redissolve accurately reconstruction.
Detecting reagent with the full-automatic coagulo meter of Sysmex CA-1500 and supporting plasma thromboplastin component detects embodiment plasma thromboplastin component quality-control product activity, result is as follows successively: percentage activity (%) 29.9,30.2,30.0,29.8,29.0,29.5,30.3,30.1,30.2,29.7, mean value 29.9.
Get 3 bottles of plasma thromboplastin component quality-control products prepared by embodiment, redissolve accurately reconstruction, active 10 times of continuous detecting FIX after mixing, result is as follows: percentage activity (%) 29.4,30.5,29.9,29.8,30.0,30.5,30.6,30.1,30.3,29.9; Mean value 30.1.
As calculated, between embodiment plasma thromboplastin component quality-control product bottle, between repeated CV value CV bottle, be 0.39%, illustrate that this horizontal plasma thromboplastin component quality-control product reaches the General Requirement (between batch interior bottle CV≤5.0%) of clinical testing for quality-control product homogeneity.
(3) product dried frozen aquatic products Detection of Stability
Plasma thromboplastin component quality-control product prepared by embodiment and SIEMENS P quality-control product are placed in 37 DEG C of preservations, respectively at sampling in 1 day, 3 days, 5 days, 7 days, redissolve accurately reconstruction simultaneously.
Detect reagent with the full-automatic coagulo meter of Sysmex CA-1500 and supporting plasma thromboplastin component embodiment plasma thromboplastin component quality-control product activity is detected, result is as follows:
37 DEG C of stability of table 2 dried frozen aquatic products
37 DEG C of placements of embodiment plasma thromboplastin component quality-control product 7 days, Activity Fluctuation range L EssT.LTssT.LT 5% before and after placing, suitable with the abnormal Quality Control blood plasma of commercially available import SIEMENS, can think that 37 DEG C of placements of blood coagulation factor VIII quality-control product prepared by the present invention are stable in 7 days.
(4) product redissolution Detection of Stability
Plasma thromboplastin component quality-control product prepared by embodiment and SIEMENS P quality-control product redissolve reconstruction accurately, be divided into two parts and be placed in respectively 2 DEG C-8 DEG C and room temperature (20 DEG C-25 DEG C) preservation, the active detection of plasma thromboplastin component is carried out in sampling at regular intervals.
Detect reagent with the full-automatic coagulo meter of Sysmex CA-1500 and supporting plasma thromboplastin component embodiment plasma thromboplastin component quality-control product activity is detected, result is as follows:
Table 3 redissolves stability
Embodiment plasma thromboplastin component quality-control product redissolves and is placed under 2 DEG C～8 DEG C and room temperature condition, 24 hours with 0 hour FIX: C without significant change, and the equal < 5% of Activity Fluctuation scope, suitable with the abnormal Quality Control blood plasma of commercially available import SIEMENS.Can think that this product redissolves rear 2 DEG C～8 DEG C and room temperature and deposits and in 24 hours, can keep stable.As can be seen here, the plasma thromboplastin component that prepared by the present invention has good uncork stability.
The above is only taking a concentration level as example; it is the preferred embodiment of the present invention; what the present invention protected is whole technical process and product; should be understood that; for those skilled in the art; under the premise without departing from the principles of the invention, can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.
Claims (4)
1. plasma thromboplastin component quality-control product preparation method, is characterized in that: comprise the following steps:
(1) blood of people's vein collection was added and is mixed as 9: 1 taking volume ratio with anti-coagulants, centrifugal under 2 DEG C～8 DEG C conditions, separate and draw upper plasma, the upper plasma after many person-portions are separated mixes, many person-portions pooled plasma;
(2) immune affinity chromatographic column is used damping fluid balance under 10 DEG C～18 DEG C conditions in advance;
(3) raw blood plasma adds MgCl
2after, MgCl in raw blood plasma
2concentration is 4.5～5.5mmol/L, and upper immune affinity chromatographic column is collected the blood plasma that flow out immune affinity chromatographic column below, is and lacks plasma thromboplastin component blood plasma;
(4) will lack plasma thromboplastin component blood plasma and many person-portions pooled plasma as requested ratio mix, be prepared into the plasma thromboplastin component quality-control product of variable concentrations level;
(5) the plasma thromboplastin component quality-control product of preparation is added to freeze drying protectant, packing, freeze drying.
2. plasma thromboplastin component quality-control product preparation method according to claim 1, is characterized in that: described anti-coagulants is citric acid three sodium solutions, and concentration is 0.109mol/L～0.129mol/L.
3. plasma thromboplastin component quality-control product preparation method according to claim 1, is characterized in that: described damping fluid comprises 0.02mol/L Tris, 0.15mol/L NaCl, 8mmol/L MgCl
2, pH7.25.
4. plasma thromboplastin component quality-control product preparation method according to claim 1, is characterized in that: described freeze drying protectant comprises that mass ratio accounts for 0.5～1.5%Hepes of plasma thromboplastin component quality-control product, 1～5% glycocoll, 0.5～5% sweet mellow wine.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410449813.9A CN104181313B (en) | 2014-09-04 | 2014-09-04 | Factor IX quality-control product preparation method |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410449813.9A CN104181313B (en) | 2014-09-04 | 2014-09-04 | Factor IX quality-control product preparation method |
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| CN104181313B CN104181313B (en) | 2015-11-18 |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105175486A (en) * | 2015-10-20 | 2015-12-23 | 上海洲跃生物科技有限公司 | Preparation method of high-purity human coagulation factor IX |
| CN105330736A (en) * | 2015-11-06 | 2016-02-17 | 上海洲跃生物科技有限公司 | Method for preparing human blood coagulation factors IX and VII subcutaneously from cold-glue-removed blood plasma |
| CN107467013A (en) * | 2017-09-20 | 2017-12-15 | 上海贞元诊断用品科技有限公司 | Preparation method of quality control product universal plasma matrix capable of being stored for long time |
| CN110257358A (en) * | 2019-06-10 | 2019-09-20 | 广东双林生物制药有限公司 | A kind of production method of high-purity Complex |
| CN110346582A (en) * | 2019-07-15 | 2019-10-18 | 三诺生物传感股份有限公司 | A kind of compound quality-control product of blood coagulation and preparation method thereof |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105175486A (en) * | 2015-10-20 | 2015-12-23 | 上海洲跃生物科技有限公司 | Preparation method of high-purity human coagulation factor IX |
| CN105330736A (en) * | 2015-11-06 | 2016-02-17 | 上海洲跃生物科技有限公司 | Method for preparing human blood coagulation factors IX and VII subcutaneously from cold-glue-removed blood plasma |
| CN107467013A (en) * | 2017-09-20 | 2017-12-15 | 上海贞元诊断用品科技有限公司 | Preparation method of quality control product universal plasma matrix capable of being stored for long time |
| CN110257358A (en) * | 2019-06-10 | 2019-09-20 | 广东双林生物制药有限公司 | A kind of production method of high-purity Complex |
| CN110257358B (en) * | 2019-06-10 | 2023-05-19 | 广东双林生物制药有限公司 | Production method of high-purity human coagulation factor IX preparation |
| CN110346582A (en) * | 2019-07-15 | 2019-10-18 | 三诺生物传感股份有限公司 | A kind of compound quality-control product of blood coagulation and preparation method thereof |
| CN110346582B (en) * | 2019-07-15 | 2022-10-25 | 三诺生物传感股份有限公司 | Blood coagulation composite quality control product and preparation method thereof |
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