CN101445793A - Method for rapidly separating supernatant from human whole blood or suspended red blood cells by utilizing lyophilized preparation - Google Patents
Method for rapidly separating supernatant from human whole blood or suspended red blood cells by utilizing lyophilized preparation Download PDFInfo
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- CN101445793A CN101445793A CNA2008102445038A CN200810244503A CN101445793A CN 101445793 A CN101445793 A CN 101445793A CN A2008102445038 A CNA2008102445038 A CN A2008102445038A CN 200810244503 A CN200810244503 A CN 200810244503A CN 101445793 A CN101445793 A CN 101445793A
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Abstract
The invention relates to a method for rapidly separating supernatant from human whole blood or suspended red blood cells by utilizing lyophilized preparation. The method comprises the following steps: the phytoagglutinin or the anti erythrocyte antibody is pre-coated on a magnetic polymer microsphere to be prepared into isolating reagent for the liquid whole blood or suspended red blood cells; the isolating reagent for the liquid whole blood or suspended red blood cells is absorbed by insoluble polymer, and after lyophylization, the isolating reagent is cut into thin slices to be prepared into isolating reagent for lyophilized whole blood or suspended red blood cells; and the isolating reagent for the lyophilized whole blood or suspended red blood cells is added to the human whole blood or suspended red blood cells to be uniformly mixed and then placed above a magnetic body, and the supernatant is rapidly separated. The lyophilized preparation preserved for long term at the normal temperature can be prepared by the method, and the supernatant can be rapidly separated form the human whole blood or suspended red blood cells under the conditions without ensuing the electric power and the centrifugal machine.
Description
Technical field
The present invention relates to the method with a kind of freeze-dried preparation sharp separation people's whole blood or Red Blood Cells Suspension supernatant, particularly a kind of relating to, use freeze-drying to wrap by the method for supernatant in the polymer microballoon separation of human whole blood that contains magnetic material of phytohemagglutinin or anti erythrocyte antibody or the Red Blood Cells Suspension in advance.The present invention can be widely used in fields such as Clinical Laboratory and medical scientific.
Background technology
The supernatant of separating whole blood or Red Blood Cells Suspension is clinical and one of maximum a kind of technology is used in scientific research, and separation method commonly used comprises natural subsidence and centrifugal, and the former need expend the long time, and the latter needs the guarantee of whizzer and electric power.
Summary of the invention
The purpose of this invention is to provide a kind of method that does not need supernatant in whole blood of separation of human rapidly and efficiently that electric power and whizzer ensure or the Red Blood Cells Suspension, particularly provide a kind of freeze dried use to wrap in advance by the polymer microballoon that contains magnetic material of phytohemagglutinin or anti erythrocyte antibody, utilize outside magnetic force from people's whole blood or Red Blood Cells Suspension, to separate the method for supernatant as carrier.
Separation method of the present invention may further comprise the steps: phytohemagglutinin or anti erythrocyte antibody are wrapped in advance by on magnetic polymer microsphere, make the separation agent of liquid whole blood or Red Blood Cells Suspension; Adsorb the separation agent of this liquid state whole blood or Red Blood Cells Suspension with the non-solubility polymer, after freeze-drying, cut into the separation agent that thin slice is made freeze-drying whole blood or Red Blood Cells Suspension, separately packing.The separation agent of this freeze-drying whole blood or Red Blood Cells Suspension is added in people's whole blood or the Red Blood Cells Suspension mixes; Mixing is placed on the magnet top, separates supernatant rapidly.Utilization of the present invention is wrapped by the magnetic polymer microsphere of phytohemagglutinin or anti erythrocyte antibody in advance by the magnetic force absorption that permanent magnet or electromagnet provide, the red corpuscle that will be adsorbed by microballoon is the out-phase separate stage from the homogeneous phase state-transition of dissolving each other, thereby reaches the purpose of supernatant in separating whole blood or the Red Blood Cells Suspension.
The present invention's said " homogeneous phase dissolve each other state " is meant that removing exo-erythrocytic other compositions in Red Blood Cells Suspension and the whole blood is in evenly mixed state.
The present invention's said " out-phase separate stage " is meant that red corpuscle spatially is enriched in together and separates with other most compositions of whole blood or Red Blood Cells Suspension, is in the state of relative separation.
The present invention's said " people's whole blood " is meant from human body and gathers, without natural separation or artificial isolating whole blood sample.
The present invention's said " Red Blood Cells Suspension " is meant the red cell suspension that has added alserver's solution after whole blood goes out separating plasma.
The present invention's said " anti erythrocyte antibody " is to use the HRBC immune mouse, the anti-erythrocyte monoclonal antibody of conventional preparation.
The present invention's said " magnetic polymer microsphere " is meant that the Z 250 surface coats one layer of polymeric, and this polymkeric substance can combine with physical adsorption or chemical b ` with phytohemagglutinin.Wherein, preferred polystyrene of described polymkeric substance or polyethylene.
The said magnet of the present invention is permanent magnet or electromagnet, can provide magnetic force by permanent magnet or electro-magnet.
The present invention's said " phytohemagglutinin " derives from material such as Semen Phaseoli (semen Phaseoli), the sword bean (Semen Canavaliae) etc. that contain phytohemagglutinin, also is commercially available.The phytohemagglutinin that the present invention adopts can be cleaned Semen Phaseoli and soak that mechanical disintegration becomes Mag after about 24 hours with pure water, centrifugal then, and (6000g 30min) isolates and adopts conventional saturated sulfuric acid ammonia process further to purify behind the supernatant to obtain.
Non-solubility polymer of the present invention is meant analogues such as glass fibre or non-woven fabrics.In addition, percentage composition of the present invention is weight percentage.
Pre-bag of the present invention can be dissolved in magnetic polymer microsphere in the PBS damping fluid, stir fast and add the stirring of lectin or anti erythrocyte antibody and room temperature down, reacted 25-35 minute, add the final concentration 0.15-0.25% of bovine serum albumin then to bovine serum albumin, stir under the room temperature once more, reacted 10-20 minute, abandon supernatant after the centrifugal 15-25 of 10000g minute at last, use the resuspended precipitation of PBS damping fluid again, promptly obtain liquid whole blood or Red Blood Cells Suspension separation agent.
According to a preferred embodiment of the invention, can use lectin or anti erythrocyte antibody to wrap by magnetic polymer microsphere in advance as follows: in advance magnetic polymer microsphere to be dissolved in the PBS damping fluid (phosphate buffered saline buffer (50mM that contains the pH7.4 of 0.05% tween 20, pH7.4)) in, microballoon concentration in damping fluid is 10% (m/v), stir fast and add lectin or anti erythrocyte antibody down, 100 rev/mins rotating speed room temperature is stirred reaction 30 minutes, add bovine serum albumin (BSA) then, make bovine serum albumin final concentration 0.2%, once more to stir reaction 15 minutes under 100 rev/mins the rotating speed room temperature, last 10000g abandons supernatant after centrifugal 20 minutes, use PBS damping fluid (50mM again, pH7.4) resuspended precipitation, obtain the good magnetic polymer microsphere solution of mark, promptly liquid whole blood or Red Blood Cells Suspension separation agent.
Now at several key steps of the present invention, details are as follows respectively:
1. the preparation of Ye Tai whole blood or Red Blood Cells Suspension supernatant separation agent
Its major technique invention is that phytohemagglutinin or anti erythrocyte antibody are wrapped by on magnetic polymer microsphere according to a conventional method in advance.What the present invention adopted is the pre-method for coating of optimizing: at first optimized pre-bag by the pH value, compared pre-bag when pH5.0, pH6.0, pH7.0, pH8.0, pH9.0, pH10.0 by efficient, best when found that pH7-8, continue to optimize relatively, wrap in advance by most effective when finding pH7.4, during use PBS damping fluid salt concn is brought up to 50mM from the 10mM of routine, help pre-bag by after stability, especially join the stability behind the whole blood sample, reduce coming off of phytohemagglutinin or anti erythrocyte antibody, improve adsorption efficiency.Adopt polymkeric substance (polystyrene or the polyethylene) microballoon of high-magnetism ferroferric oxide preparation in addition, can improve speed greatly by magneticseparation, also compared simultaneously the pre-bag quilt and the separating effect of different microsphere diameter (40nm, 60nm, 100nm, 150nm, 200nm, 300nm), the pre-bag quilt and the separation efficiency of the microballoon of 100nm are best as a result.
2. the freeze-drying of whole blood or Red Blood Cells Suspension supernatant separation agent
At first sheet non-solubility polymer is soaked in the magnetic polymer microsphere solution, soaks half an hour, make polymer fully adsorb whole blood or Red Blood Cells Suspension separation agent.The sheet non-solubility polymer that fully adsorbs whole blood or Red Blood Cells Suspension separation separation agent is lain against on the pallet, place Freeze Drying Equipment, close opening for feed, start Freeze Drying Equipment, open refrigerating function, reduce the temperature to-60 ℃, continue freezing 4 hours.Stop refrigeration, open vacuum pump, continue to vacuumize 24 hours.Close vacuum pump, open the Freeze Drying Equipment opening for feed.Be lower than in relative humidity under 20% the drying conditions, freeze dried separation of whole blood reagent is taken out from Freeze Drying Equipment, cut into 1cm
2Big small thin slices.Each thin slice is packed separately with aluminium foil bag.Finished product can be preserved 2 years down by normal temperature.Open aluminium foil bag during use, place 0.5ml anticoagulated whole blood or Red Blood Cells Suspension, shake, and place the magnet top, can separate supernatant rapidly.
3. utilize the magneticseparation supernatant
The magnetic force source that the present invention adopts can be permanent magnet or electromagnet, and permanent magnet comprises the common magnet and the alloy magnet of high magnetic force.Electro-magnet can be controlled the magnetic force break-make, can realize under the situation of mobile containers not that co-located separates supernatant.Magnet can place a plurality of positions of container, comprises bottom, sidewall, top even internal tank etc., can be an end, also can be surrounded on container.
Beneficial effect of the present invention compared with the prior art:
Present method will be wrapped by the magnetic polymer microsphere of phytohemagglutinin or anti erythrocyte antibody in advance with the freeze-dried preparation of making normal temperature preservation for a long time after the non-solubility polymer absorption.This freeze-dried preparation is mixed with whole blood or Red Blood Cells Suspension, under the situation that does not need electric power and whizzer to ensure, can the shortlyest use the magnetic force phase-splitting to reach the purpose of sharp separation whole blood or Red Blood Cells Suspension supernatant in 2～3 minutes, its separating effect and centrifuging be suitable.
In addition, the present invention has adopted the permanent magnet of ferromagnetic polymer microballoon and strong magnetic force, makes the speed of magneticseparation accelerate greatly.In addition, the present invention also can adopt various magnets, is convenient to design the device of different costs and performance, satisfies different application and demand, is beneficial to and applies.
Description of drawings
Fig. 1 is the equipment for magnetic separation separating whole blood of demonstration bottom fixed magnets or the synoptic diagram of Red Blood Cells Suspension supernatant.
Among the figure, in whole blood, add whole blood or Red Blood Cells Suspension separation agent.
Embodiment
Below in conjunction with accompanying drawing, further understand the present invention by embodiments of the invention given below, but the present invention is not subjected to the qualification of embodiment.
Embodiment 1: adopt phytohemagglutinin to wrap by the method for magnetic-particle separating whole blood or Red Blood Cells Suspension supernatant in advance
Get the 100g Semen Phaseoli, clean with pure water, be soaked in the 200ml pure water 24 hours, with juice extractor Semen Phaseoli is broken into the soya-bean milk slurry, smash and add the 50ml pure water in the process to improve mill efficiency, serum centrifugal 30 minutes in 6000g keeps supernatant and also further purifies with conventional saturated sulfuric acid ammonia process, and it is standby to obtain the pure product of phytohemagglutinin.
Magnetic polymer microsphere (mean diameter 100nm) solution according to every milliliter 10% concentration wraps in advance by the ratio of 20 μ g phytohemagglutinins, in advance magnetic polystyrene microsphere is dissolved in PBS (50mM, pH7.4) in the damping fluid, microballoon is concentration 10% (m/v) in solution, adding phytohemagglutinin under the condition of stirring fast, the rotating speed with 100 rev/mins under the room temperature stirs reaction 30 minutes.Add BSA to BSA final concentration 0.2% then, to stir reaction 15 minutes under 100 rev/mins the rotating speed room temperature, last 10000g abandons supernatant after centrifugal 20 minutes, with the resuspended precipitation of PBS (50mM pH7.4) damping fluid, obtain the magnetic polystyrene microsphere solution of the good phytohemagglutinin of mark, microballoon final concentration 10% (m/v), promptly liquid whole blood or Red Blood Cells Suspension separation agent.
The foliated glass fiber is soaked in liquid whole blood or the Red Blood Cells Suspension separation agent, soaks half an hour, make polymer fully adsorb liquid whole blood or Red Blood Cells Suspension separation agent.The foliated glass fiber that fully adsorbs liquid whole blood or Red Blood Cells Suspension separation agent is lain against on the pallet, place Freeze Drying Equipment, close opening for feed, start Freeze Drying Equipment, open refrigerating function, reduce the temperature to-60 ℃, continue freezing 4 hours.Stop refrigeration, open vacuum pump, continue to vacuumize 24 hours.Close vacuum pump, open the Freeze Drying Equipment opening for feed.Be lower than in relative humidity under 20% the drying conditions, freeze-drying whole blood or Red Blood Cells Suspension separation agent are taken out from Freeze Drying Equipment, cut into 1cm
2Big small thin slices.
Gather fresh normal people's whole blood sample 5ml, use the Citric Acid anti-freezing, standby.Prepare the test-tube stand that a bottom presets magnet simultaneously, every 0.5ml whole blood adds 1 freeze-drying whole blood or Red Blood Cells Suspension separation agent, mixing is placed on the magnetic force frame, and leave standstill visible significantly demixing phenomenon after 2～3 minutes: the upper strata is limpid blood plasma, and lower floor is red red corpuscle.
Supernatant after red blood cell count(RBC) detect to separate calculates this method and removes erythrocytic effect and reach 99.6%.
Embodiment 2: adopt anti erythrocyte antibody to wrap by the method for magnetic-particle separate out suspended red corpuscle supernatant in advance
Get available from newly the create anti erythrocyte antibody 1mg of thing Engineering Co., Ltd of Beijing.Magnetic polymer microsphere according to every milliliter 10% concentration, microballoon mean diameter 100nm, solution wraps in advance by the ratio of 20 μ g anti erythrocyte antibodies, in advance magnetic polymer microsphere is dissolved in the PBS damping fluid (50mM pH7.4), the concentration 10% (m/v) of microballoon in damping fluid, adding anti erythrocyte antibody under the condition of stirring fast, rotating speed with 100 rev/mins under the room temperature stirs reaction 30 minutes, add the final concentration 0.2% of BSA to BSA in solution, rotating speed with 100 rev/mins under the room temperature stirs reaction 15 minutes, last 10000g abandons supernatant after centrifugal 20 minutes, with the resuspended precipitation of PBS (50mM pH7.4) damping fluid, obtain the magnetic polymer microsphere solution (microballoon final concentration 10% (m/v)) of the good anti erythrocyte antibody of mark, promptly liquid whole blood or Red Blood Cells Suspension separation agent.Gather fresh normal people's whole blood sample 5ml, do not add any lectin or other reagent, standby.
Non-woven fabrics is soaked in liquid whole blood or the Red Blood Cells Suspension separation agent, soaks half an hour, make non-woven fabrics fully adsorb liquid whole blood or Red Blood Cells Suspension separation agent.The non-woven fabrics that fully adsorbs liquid whole blood or Red Blood Cells Suspension separation agent is lain against on the pallet, place Freeze Drying Equipment, close opening for feed, start Freeze Drying Equipment, open refrigerating function, reduce the temperature to-60 ℃, continue freezing 4 hours.Stop refrigeration, open vacuum pump, continue to vacuumize 24 hours.Close vacuum pump, open the Freeze Drying Equipment opening for feed.Be lower than in relative humidity under 20% the drying conditions, freeze-drying whole blood or Red Blood Cells Suspension separation agent are taken out from Freeze Drying Equipment, cut into 1cm
2Big small thin slices.
Prepare the test-tube stand that a bottom presets magnet, add 2 freeze-drying whole bloods or Red Blood Cells Suspension separation agent according to every milliliter of whole blood, leave standstill 10 minutes after, visible significantly demixing phenomenon: the upper strata is limpid blood plasma, lower floor is red red corpuscle.
Supernatant after red blood cell count(RBC) detect to separate calculates this method and removes erythrocytic effect and reach 99.8%.
Claims (8)
1, a kind of method of utilizing freeze-dried preparation sharp separation people's whole blood or Red Blood Cells Suspension supernatant is characterized in that this method may further comprise the steps:
(1) phytohemagglutinin or anti erythrocyte antibody are wrapped in advance by on magnetic polymer microsphere, make the separation agent of liquid whole blood or Red Blood Cells Suspension;
(2) adsorb the separation agent of this liquid state whole blood or Red Blood Cells Suspension with the non-solubility polymer, after freeze-drying, cut into the separation agent that thin slice is made freeze-drying whole blood or Red Blood Cells Suspension;
(3) separation agent of above-mentioned freeze-drying whole blood or Red Blood Cells Suspension is added in people's whole blood or the Red Blood Cells Suspension mixes;
(4) mixing is placed on magnet top, standing separation supernatant.
2, the method for utilizing freeze-dried preparation sharp separation people's whole blood or Red Blood Cells Suspension supernatant according to claim 1, it is characterized in that described phytohemagglutinin is Semen Phaseoli to be cleaned and soaked after 24 hours to be ground into Mag, the supernatant that obtains of centrifugation adopts saturated sulfuric acid ammonia process to purify then.
3, the method of utilizing freeze-dried preparation sharp separation people's whole blood or Red Blood Cells Suspension supernatant according to claim 1, it is characterized in that described pre-bag is that magnetic polymer microsphere is dissolved in the PBS damping fluid, stir fast and add the stirring of lectin or anti erythrocyte antibody and room temperature down, reacted 25-35 minute, add the final concentration 0.15-0.25% of bovine serum albumin then to bovine serum albumin, stir under the room temperature once more, reacted 10-20 minute, at last abandon supernatant after the centrifugal 15-25 of 10000g minute, use the resuspended precipitation of PBS damping fluid again, promptly obtain liquid whole blood or Red Blood Cells Suspension separation agent.
4, the method for utilizing freeze-dried preparation sharp separation people's whole blood or Red Blood Cells Suspension supernatant according to claim 1, it is characterized in that described magnetic polymer microsphere is that the Z 250 surface coats one layer of polymeric, this polymkeric substance can combine with physical adsorption or chemical b ` with phytohemagglutinin.
5, the method for utilizing freeze-dried preparation sharp separation people's whole blood or Red Blood Cells Suspension supernatant according to claim 4 is characterized in that described polymkeric substance is polystyrene or polyethylene.
6, the method for utilizing freeze-dried preparation sharp separation people's whole blood or Red Blood Cells Suspension supernatant according to claim 1 is characterized in that described non-solubility polymer is glass fibre or non-woven fabrics.
7, the method for utilizing freeze-dried preparation sharp separation people's whole blood or Red Blood Cells Suspension supernatant according to claim 3 is characterized in that described PBS damping fluid salt concn is 50mM, pH7-8.
8, the method for utilizing freeze-dried preparation sharp separation people's whole blood or Red Blood Cells Suspension supernatant according to claim 1 is characterized in that described magnet is permanent magnet or electromagnet.
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| CN200810244503A CN100584942C (en) | 2008-12-09 | 2008-12-09 | Method for quickly separating human total blood or suspended red cell supernatant |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103344755A (en) * | 2013-07-19 | 2013-10-09 | 中国科学院苏州生物医学工程技术研究所 | Method for preparing magnetized and hydroformyled sheep red blood cell |
| CN104111333A (en) * | 2013-11-13 | 2014-10-22 | 叶森 | Fast erythrocyte removal method applicable to whole blood detection |
| CN107436318A (en) * | 2017-08-17 | 2017-12-05 | 武汉璟泓万方堂医药科技股份有限公司 | A kind of electrochemistry blood testing and device for reducing red blood cell interference |
| CN109541215A (en) * | 2018-12-20 | 2019-03-29 | 中国人民解放军陆军军医大学 | A kind of lesion detection object and the preparation method and application thereof |
| CN111308070A (en) * | 2020-02-28 | 2020-06-19 | 厦门宝太生物科技有限公司 | Kit for separating red blood cells from whole blood and method for separating red blood cells from whole blood |
| CN111662869A (en) * | 2020-06-02 | 2020-09-15 | 北京康思润业生物技术有限公司 | Immunomagnetic bead for separating red blood cells and preparation method and application thereof |
| WO2022166230A1 (en) * | 2021-02-03 | 2022-08-11 | 杭州欣捷生物技术有限公司 | Composition and method for rapid separation of whole blood |
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| EP3694322A1 (en) | 2017-10-09 | 2020-08-19 | Terumo BCT Biotechnologies, LLC | Lyophilization container and method of using same |
| EP3938742A1 (en) | 2019-03-14 | 2022-01-19 | Terumo BCT Biotechnologies, LLC | Multi-part lyophilization container and method of use |
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- 2008-12-09 CN CN200810244503A patent/CN100584942C/en not_active Expired - Fee Related
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103344755A (en) * | 2013-07-19 | 2013-10-09 | 中国科学院苏州生物医学工程技术研究所 | Method for preparing magnetized and hydroformyled sheep red blood cell |
| CN103344755B (en) * | 2013-07-19 | 2015-06-24 | 中国科学院苏州生物医学工程技术研究所 | Method for preparing magnetized and hydroformyled sheep red blood cell |
| CN104111333A (en) * | 2013-11-13 | 2014-10-22 | 叶森 | Fast erythrocyte removal method applicable to whole blood detection |
| CN107436318A (en) * | 2017-08-17 | 2017-12-05 | 武汉璟泓万方堂医药科技股份有限公司 | A kind of electrochemistry blood testing and device for reducing red blood cell interference |
| CN109541215A (en) * | 2018-12-20 | 2019-03-29 | 中国人民解放军陆军军医大学 | A kind of lesion detection object and the preparation method and application thereof |
| CN111308070A (en) * | 2020-02-28 | 2020-06-19 | 厦门宝太生物科技有限公司 | Kit for separating red blood cells from whole blood and method for separating red blood cells from whole blood |
| CN111662869A (en) * | 2020-06-02 | 2020-09-15 | 北京康思润业生物技术有限公司 | Immunomagnetic bead for separating red blood cells and preparation method and application thereof |
| WO2022166230A1 (en) * | 2021-02-03 | 2022-08-11 | 杭州欣捷生物技术有限公司 | Composition and method for rapid separation of whole blood |
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