CN101957366A - Human erythrocyte membrane antigen coated microsphere and application thereof - Google Patents
Human erythrocyte membrane antigen coated microsphere and application thereof Download PDFInfo
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- CN101957366A CN101957366A CN2009101581684A CN200910158168A CN101957366A CN 101957366 A CN101957366 A CN 101957366A CN 2009101581684 A CN2009101581684 A CN 2009101581684A CN 200910158168 A CN200910158168 A CN 200910158168A CN 101957366 A CN101957366 A CN 101957366A
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- DAJSVUQLFFJUSX-UHFFFAOYSA-M sodium;dodecane-1-sulfonate Chemical compound [Na+].CCCCCCCCCCCCS([O-])(=O)=O DAJSVUQLFFJUSX-UHFFFAOYSA-M 0.000 description 4
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- PYWVYCXTNDRMGF-UHFFFAOYSA-N rhodamine B Chemical compound [Cl-].C=12C=CC(=[N+](CC)CC)C=C2OC2=CC(N(CC)CC)=CC=C2C=1C1=CC=CC=C1C(O)=O PYWVYCXTNDRMGF-UHFFFAOYSA-N 0.000 description 1
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Abstract
The invention provides a method for preserving the activity of a human cell membrane blood group antigen, which comprises the steps of: preparing an erythrocyte membrane blood group antigen extract, and then coating the prepared erythrocyte membrane blood group antigen on a solid microsphere so as to replace a fresh erythrocyte to be used for detecting a blood group antibody in a sample.
Description
Invention field
The present invention relates to blood group test material and the permanent method that keeps the red cell antigens activity, particularly relate to antigen coated microballoon of human erythrocyte membrane and the application in the antibody test of human blood type thereof.
Background of invention
It to the research of human body cell membranous antigen and specific antibody thereof and detection one of substance of bio-science, immunological experiment research and clinical detection.People ABO and Rh blood group antigens/antibody test are routine clinical test items.Blood group is identified and is based on the cell agglutination reaction that antigen-antibody reaction causes.The blood group antibody that uses erythrocyte surface antigen to detect in the serum is referred to as reverse type.In clinical blood transfusion, must guarantee positive and negative typing method qualification result unanimity, just can guarantee the security of transfusing blood.
According to the blood group antigens that are carried on erythrocyte surface and be present in immunochemistry agglutinating reaction between the homologous antibody in the tested serum, further identify blood group (reverse type detection), be a laboratory examination must finishing before the blood transfusion.At present, most reverse type test all is as standard antigen with great-hearted erythrocyte surface antigen.Yet fresh red blood cell is difficult for preserving, and the antigenicity of erythrocyte surface antigen prolonged and reduces gradually with the holding time.This class reagent not only needs low temperature (about 2-8 ℃) to preserve, and long storage life has only 3 to 6 months.
For a long time, how keeping the antigenicity of red cell antigens is that life science, immune science are still failed the difficult problem of fine solution.In order to solve this difficult problem, people once taked cryogenic freezing technology, but the cryogenic freezing method is difficult to preserve a large amount of red blood cells.And erythrocytic antigenicity can be cut down with frozen time lengthening.In addition, the problem that red blood cell is preserved difficulty has also limited its application in other respects, as quality-control product as positive definite form reagent, or field such as anti erythrocyte antibody titration.At present, fresh red blood cell reagent existence is difficult to obtain in batches, problems such as the term of validity is short, preservation condition is harsh, quality instability.Therefore, be necessary the store method of traditional blood group antibody authentication method and red cell antigens is done the improvement of essence.
Summary of the invention
According to the blood group antigens that are carried on erythrocyte surface and be present in immunochemistry agglutinating reaction between the homologous antibody in the tested serum, further identify blood group (reverse type detection), be a laboratory examination must finishing before the blood transfusion.At present, most reverse type test all is as standard antigen with great-hearted blood group antigen.Yet fresh red blood cell is difficult for preserving, and the antigenicity of blood group antigen prolonged and reduces gradually with the holding time.This class reagent not only needs low temperature (about 2-8 ℃) to preserve, and long storage life has only 3 to 6 months.
In order to address this problem, the invention provides a kind of method of depositary's cell membrane blood group antigens activity, this method comprises preparation erythrocyte membrane blood group antigens extract, the erythrocyte membrane blood group antigens with preparation are coated on the microspheres with solid then.
According to a preferred embodiment of the invention, wherein said microspheres with solid is the colored nonmagnetic microballoon of synthetic, for example comprises but is not only limited to organic microballoons such as polystyrene-acrylic acid microballoon, polystyrene-acrylamide, PMMA microballoon, PVC microballoon; Inorganic microspheres such as silicon dioxide microsphere, calcium carbonate microspheres, titanium dioxide microballoon sphere, transient metal sulfide microballoon; And the inorganic/organic composite microballoon etc.
Microballoon by the inventive method preparation can make human erythrocyte membrane surface blood group antigens preserve at least more than 1 year, and does not lose or reduce erythrocytic antigen active.
According to the microballoon of the erythrocyte membrane blood group antigens bag quilt of method of the present invention preparation, can keep and keep erythrocytic antigen active effectively, can be widely used in the human blood type detection of antibodies as reagent.For example, in clinical blood group analysis practice, the reagent that can be used as the experiment of blood group reverse type uses.Briefly, at first erythrocyte membrane fixedly is coated on the microballoon, and replaces red blood cell to carry out the detection of blood group antibody with this microballoon that is coated with erythrocyte membrane.In addition, so the microballoon that is coated with erythrocyte membrane of preparation also can be used in other laboratories and industrial practice of using red cell antigenses, as quality-control product as positive definite form reagent, or field such as anti erythrocyte antibody titration.
Said erythrocyte membrane blood group antigens, can be with low osmotic pressure or surfactant handle obtain, be attached to the lip-deep blood group antigens of complete erythrocyte membrane, or the blood group antigens extract of the removal impurity of handling with organic solvent.
Said erythrocyte membrane blood group antigens bag quilt is meant with modes such as physisorption or chemical couplings, and erythrocyte membrane or blood group antigens extract are fixed on the microspheres with solid.
In order to prepare erythrocyte membrane antigen, can at first under room temperature, in the red blood cell of hematocrit, add the bloated molten red blood cell of surfactant, perhaps utilize Hyposmolality method swelling red blood cell, centrifugal collecting precipitate repeatedly then, promptly needed erythrocyte membrane antigen extract.
In order to prepare the microballoon that can wrap by the erythrocyte membrane blood group antigens, for example can in the potpourri of ethanol/water, add ammonium persulfate, styrene and acrylic acid successively, feed high-purity nitrogen behind the mixing.Place water-bath (60 ℃) to stir and heating (about 48 hours) then, promptly obtain required polystyrene-acrylic acid microballoon.
Get the as above polystyrene-acrylic acid microballoon of preparation, the centrifugal supernatant that goes.Get an amount of sodium dodecylsulphonate (SDS) thin up.Again in another container, in absolute ethyl alcohol, add the dyestuff methyl red, dissolve and shake up after join in the liquid of preparing above.Place water-bath (60 ℃) heating back (about 24 hours), promptly obtain red the polystyrene that dyes-acrylic acid microballoon.Certainly, also can use other dye substance, the polystyrene-acrylic acid microballoon of so preparation is dyed other required colors.
In order to prepare the microballoon of erythrocyte membrane bag quilt, desirable a certain amount of erythrocyte membrane adds 10mM PBS solution, after vibration mixes, adds an amount of solution of potassium carbonate and regulates the pH value to about 9.In the PBS of this erythrocyte membrane solution, add an amount of as above microballoon suspension of preparation then, vibration and mixing, centrifugal back collecting precipitation promptly obtains the microballoon of needed erythrocyte membrane bag quilt.
So utilize the microballoon of erythrocyte membrane bag quilt of the lipophilicity preparation of cell membrane, can make firm the combining of cell membrane and microballoon.Like this, both preserve the blood group antigens on erythrocyte membrane surface, and made the erythrocyte membrane blood group antigens break away from living cells again and exist, prolonged the pot-life of erythrocyte membrane blood group antigens greatly.
According to a preferred embodiment of the invention, the microsphere particle granularity of red cell antigens material bag quilt is generally 10nm-50 μ m.
Our preliminary test observations shows, microballoon according to the erythrocyte membrane blood group antigens bag quilt of method of the present invention preparation, can make the active reservation of erythrocyte surface antigen (comprising blood group antigens) more than 1 year, and the forfeiture or the decay of surface antigen activity do not take place.Can under low temperature or normal temperature condition, preserve microballoon according to the erythrocyte membrane blood group antigens bag quilt of method preparation of the present invention.
Can use the microballoon that is coated with erythrocyte membrane antigen, replace remaining with the fresh red blood cell of surface antigen activity, be used for clinical blood group reverse type and detect, or blood group antibody detect.So the microballoon that is coated with erythrocyte membrane of preparation also can be used in other laboratories and industrial practice of using red cell antigens.
According to a preferred embodiment of the invention, being used to wrap by the microballoon of red cell antigens can be polystyrene-acrylic acid, silicon dioxide, or the microballoon made of other organic or inorganic polymeric materials.
According to a preferred embodiment of the invention, the blood group authentication method that is adopted can be a slide method, or other conventional blood group authentication methods.
Erythrocyte membrane antigen store method of the present invention, and use the erythrocyte membrane antigen of so preserving to detect the reverse type detection method of human blood type, be particularly suitable for the blood sampling of blood station blood group antibody examination and street corner and use.The erythrocyte membrane antigen term of validity that this method is preserved is long, and antigenicity is stable.In addition, this method is simple, is fit to large-scale industrial production.Erythrocyte membrane antigen with this method preparation is used for the detection of human blood type reverse type, can reflect the blood group antibody kind accurately, has reached the purpose that replaces fresh red blood cell.
Moreover, according to the microballoon of the erythrocyte membrane antigen bag quilt of the inventive method preparation, other researchs and the demand of production, for example quality-control product that detects as the blood group positive definite form that also can satisfy the demand and use the fresh red blood cell membranous antigen.
Description of drawings
Fig. 1 shows the microballoon replacement fresh red blood cell for preparing A type and Type B erythrocyte membrane antigen bag quilt with the present invention, and people's abo blood group reverse type of finishing detects.From left to right, each file is followed successively by A type, Type B, AB type, O type serum.As shown in the figure, in the A type serum only the Type B microballoon aggegation appears; In the Type B serum only A type microballoon aggegation appears; Aggegation does not appear in AB type serum two Kong Jun; Aggegation all appears in O type serum two holes.
Fig. 2 shows that the microballoon with the positive erythrocyte membrane antigen bag of the RhD quilt of the present invention's preparation replaces fresh red blood cell, the detection of Antibodies Against Rhesus D Antigen in the human serum of finishing.The left side is for adding the centrifuge tube that contains anti--D serum, and the right is for adding the centrifuge tube that does not contain anti--D serum.As shown in the figure, in the serum that contains anti--D, RhD is positive, and macroscopic aggegation has appearred in the erythrocyte membrane microballoon; Do not contain on the right in the serum of anti--D, RhD is positive, and the erythrocyte membrane microballoon is uniformly dispersed, and does not see aggegation.
Embodiment
Following examples are intended to further describe for example the present invention, and these embodiment also do not limit the present invention in any way.Under the prerequisite that does not deviate from the technology of the present invention solution, any change or change that those of ordinary skills that the present invention did are realized easily all will fall within the claim scope of the present invention.
The preparation of the positive blood group erythrocyte membrane antigen of embodiment 1:A type, Type B and RhD extract
Get fresh A/B type venous blood 1ml, 3000rpm removed supernatant in centrifugal 5 minutes, and the long-pending red blood cell of pressure is for further processing.In packed red cells, add 10ml 5% sodium dodecylsulphonate (SDS), place after 3 minutes the centrifugal supernatant that goes of 3000rpm under the room temperature.Add 10ml pure water and the mixing that vibrates, the centrifugal supernatant that goes of 3000rpm once more to sediment.5 times so repeatedly, resulting sediment is A/B type erythrocyte membranous antigen extract.
Get the positive venous blood 1ml of fresh RhD, centrifugal 5 minutes collecting cell precipitations of 3000rpm.In packed red cells, add 10ml sodium dodecylsulphonate (SDS).Place under the room temperature after 3 minutes, the centrifugal supernatant that goes of 3000rpm adds absolute ethyl alcohol to gained cell precipitation thing.Behind the vibration mixing, 3000rpm is centrifugal and remove supernatant once more, adds the pure water and the mixing that vibrates to sediment.Then, centrifugal with 3000rpm, go supernatant to stay precipitation, obtain the positive erythrocyte membrane antigen extract of RhD.
Embodiment 2: red the preparation of the polystyrene that dyes-acrylic acid microballoon
It is stand-by to get 70ml absolute ethyl alcohol adding 30ml pure water mixing.In the gained hydrous ethanol, add 1.0g ammonium persulfate, 10ml styrene and 5ml acrylic acid successively.Feed high pure nitrogen (10 minutes) behind the mixing.Place 60 ℃ of stirred in water bath then, heat continuously and finish reaction after 48 hours, obtain polystyrene-acrylic acid microballoon.
Get the as above polystyrene-acrylic acid microballoon of preparation, the centrifugal supernatant that goes.In precipitation, add the 100ml pure water, add 1g sodium dodecylsulphonate (SDS) again.Get another bottle and add 10ml absolute ethyl alcohol, 0.01g methyl red, 1ml styrene, 0.03g azo isobutyronitrile.Dissolving and shake up after join as above in the liquid mixture of preparation.Place the heating of 70 ℃ of water-baths to finish reaction after 24 hours, promptly obtain red the polystyrene that dyes-acrylic acid microballoon.
The preparation of the silicon dioxide microsphere of the positive type erythrocyte membrane antigen of embodiment 3:RhD bag quilt
Get the 100ml absolute ethyl alcohol and add 10ml ethyl orthosilicate and 10ml ammoniacal liquor, 24 hours afterreactions of stirring at room finish, and obtain silicon dioxide microsphere.Get the silicon dioxide microsphere 20ml of preparation like this, the centrifugal supernatant that goes stays precipitation.
In the silicon dioxide microsphere sediment, add the positive O type of the RhD erythrocyte membrane antigen of embodiment 1 preparation, regulate pH value to 10, mixing and appropriateness vibration.The centrifugal supernatant that goes promptly obtains the silicon dioxide microsphere sediment of the positive erythrocyte membrane antigen bag of RhD quilt.
The rhodamine B solution of 10ml 10mg/ml is joined in the silicon dioxide microsphere precipitation of the positive erythrocyte membrane antigen bag of above-mentioned RhD quilt.37 ℃ were dyeed 2 hours, promptly obtained the silicon dioxide microsphere of the positive type erythrocyte membrane antigen of the RhD bag quilt of shiny red.
The preparation of the polystyrene-acrylic acid microballoon of embodiment 4:A/B type erythrocyte membrane antigen bag quilt
Get the redness of preparation among the embodiment 2 and the polystyrene-acrylic acid microballoon 1.0g that dyes, the centrifugal supernatant that goes.In the microballoon precipitation, add the A/B type erythrocyte membrane antigen of embodiment 1 preparation, regulate pH value to 10, mixing and appropriateness vibration.Centrifugal remove supernatant after, promptly obtain the polystyrene-acrylic acid microballoon of A/B type erythrocyte membrane antigen bag quilt.
Embodiment 5: the reverse type that uses the polystyrene-acrylic acid microballoon of erythrocyte membrane antigen bag quilt of the present invention to finish people's abo blood group detects
Get the recessed formula blood group in 8 holes and identify paper, 4 holes of first row respectively add the red blood cells of type A antigen microballoon of preparation among the 25 μ l embodiment 4, and 4 holes of second row respectively add the Type B red cell antigens microballoon of preparation among the 25 μ l embodiment 4.Then, get known A type, Type B, AB type, O type 4 routine serum, longitudinally respectively add 50 μ l respectively in two holes up and down that contain red blood cells of type A antigen microballoon and Type B red cell antigens microballoon.After stirring evenly, result of determination in 5 minutes.The result as seen, in the A type serum only the Type B microballoon macroscopic aggegation appears; In the Type B serum only A type microballoon macroscopic aggegation appears; Aggegation does not appear in AB type serum two Kong Jun; Aggegation (referring to Fig. 1) all appears in O type serum two holes.
Embodiment 6: use the polystyrene-acrylic acid microballoon of the positive erythrocyte membrane antigen bag of RhD of the present invention quilt to finish the detection of people's Antibodies Against Rhesus D Antigen
Get known each 200 μ l of two parts of fresh human serums that contain anti--D and do not contain anti--D in the 1.5ml centrifuge tube, and add the polystyrene-acrylic acid microballoon suspension of the positive erythrocyte membrane antigen bag of the RhD quilt of 50 μ l embodiment, 3 preparations at every pipe.Fully after the mixing concussion, 37 ℃ are incubated 60 minutes.
Take out the reaction centrifuge tube, leave the heart with 4000 and separate and supernatant discarded.With physiological saline washing precipitate 3 times.Every pipe adds 100 μ l wide spectrum antiglobulin reagents, after fully mixing, and with 2000rpm centrifugal 1 minute.Observations then.The result as seen, in the serum that contains anti--D, RhD is positive, and macroscopic aggegation has appearred in the erythrocyte membrane microballoon; In the serum that does not contain anti--D, the positive erythrocyte membrane microballoon of RhD disperses very even, and not seeing has agglutination phenomenon (referring to Fig. 2).
Claims (5)
1. the method for depositary's cell membrane blood group antigens activity, this method comprise preparation erythrocyte membrane blood group antigens extract, then prepared erythrocyte membrane blood group antigens are coated on the microspheres with solid.
2. according to the process of claim 1 wherein that said microballoon is the nonmagnetic microballoon of synthetic.
3. according to the method for claim 1, said erythrocyte membrane blood group antigens, can be with hypotonic solution or surfactant handle obtain be attached to the lip-deep blood group antigens of complete erythrocyte membrane, or the blood group antigens extract of the removal impurity of handling with organic solvent.
4. according to the process of claim 1 wherein said erythrocyte membrane blood group antigens bag quilt, be erythrocyte membrane or blood group antigens extract to be fixed on the microspheres with solid with physisorption or chemical coupling method.
5. the application of erythrocyte membrane blood group antigens in the human blood type detection of antibodies of preserving according to the method for claim 1.
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| CN103487590A (en) * | 2013-09-03 | 2014-01-01 | 华南农业大学 | Indirect Dot-ELISA (enzyme-linked immunosorbent assay) method and application of blood group antibody |
| CN103926415A (en) * | 2014-03-24 | 2014-07-16 | 上海市血液中心 | System for combined and rapid detection of blood type antibody in human blood |
| CN105445449A (en) * | 2015-04-04 | 2016-03-30 | 吉林双正医疗科技有限公司 | Apparatus for rapidly and semi-quantitatively detecting uric acid in saliva, and making method thereof |
| CN105561346A (en) * | 2015-12-31 | 2016-05-11 | 常州市长宇实用气体有限公司 | Preparation method of ultrasonic contrast agent |
| CN105699667A (en) * | 2016-01-26 | 2016-06-22 | 北京中科圆融生物科技发展有限公司 | Bacterial magnetic particle-red cell membrane composite particles and clinical application thereof |
| CN107014992A (en) * | 2016-11-10 | 2017-08-04 | 王毅 | A kind of foundation for quantitatively detecting human blood type antibody concentration method |
| CN107643409A (en) * | 2017-09-19 | 2018-01-30 | 汪德清 | A kind of blood group antigens chip and its application in red blood cell accident antibody test |
| CN114397464A (en) * | 2022-03-24 | 2022-04-26 | 天津德祥生物技术有限公司 | Coupling compound of erythrocyte membrane fragments and carrier, coupling method and application thereof |
| CN114660305A (en) * | 2022-03-31 | 2022-06-24 | 中国人民解放军军事科学院军事医学研究院 | Reagent, preparation method, test strip and application for rapid detection of blood group antibody titer |
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| CN107014992A (en) * | 2016-11-10 | 2017-08-04 | 王毅 | A kind of foundation for quantitatively detecting human blood type antibody concentration method |
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| CN114660305A (en) * | 2022-03-31 | 2022-06-24 | 中国人民解放军军事科学院军事医学研究院 | Reagent, preparation method, test strip and application for rapid detection of blood group antibody titer |
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