CN104714034A - Blood type detection method based on membrane structure - Google Patents
Blood type detection method based on membrane structure Download PDFInfo
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- CN104714034A CN104714034A CN201310689909.8A CN201310689909A CN104714034A CN 104714034 A CN104714034 A CN 104714034A CN 201310689909 A CN201310689909 A CN 201310689909A CN 104714034 A CN104714034 A CN 104714034A
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- G—PHYSICS
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- 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
- G01N33/80—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood groups or blood types or red blood cells
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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
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54306—Solid-phase reaction mechanisms
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Abstract
The present invention discloses a blood type detection method based on a membrane structure. According to the method, a filtration membrane allowing a single red blood cell to pass through is selected as a core material, blood type antibodies are bound to the filtration membrane, a water absorption material is surrounding the periphery of the filtration membrane, red blood cells with a certain concentration are added on the filtration membrane surface in a dropwise manner during detecting, rinsing is performed with a rinsing liquid, the red blood cells with no antigen-antibody binding exude along with the rinsing liquid, the red blood cells with the antigen-antibody binding are remained on the membrane surface so as to form the visible red clumps, and an operator can judge the result according to whether the red blood cells form the visible red clumps on the filtration membrane so as to determine the blood type of red blood cells. The method of the present invention has beneficial effects of short detection time and objective result.
Description
Technical field
The present invention relates to a kind of blood group and judge detection technique, particularly a kind of blood type testing methods based on membrane structure.
Background technology
Blood group examination has been widely used in medical institutions at different levels, and wherein the detection of ABO blood group system and anti-Rh (D) is as conventional sense.The anti-A of usual IgM type, anti-B and anti-D reagent respectively with red blood cell reaction, come to red cell grouping according to whether forming aggregation.There is Staphylococal Protein A on red blood cells of type A surface, meeting and anti-A antibody generation aggregation.Type B erythrocyte surface has B antigen, meeting and anti-B antibody generation aggregation.Result according to these two reactions can give erythrocytic ABO blood group system somatotype.Determine that Rh (D) is positive and negative with anti-D reagent.The detection of other blood group system also has application in medical institutions.As the DCcEe somatotype of Rh system, MN system, the detection etc. of duffy and lewis system.
In current clinical detection with flat band method and post agglutination technology maximum.Flat band method lacks remaining unchanged of objective judgement, sometimes easily causes misjudgment.Post agglutination result is objective, is easy to standardization, is substituting the former gradually at present.Post agglutination needs to use hydro-extractor, length consuming time, limits application in an emergency situation.
Summary of the invention
The object of the invention is to solve the problem, devising a kind of blood type testing methods based on membrane structure.
Realizing above-mentioned purpose technical scheme of the present invention is, a kind of blood type testing methods based on membrane structure, the method selects the filter membrane that single red blood cell can be allowed to pass through as core material, blood group antibody is attached on filter membrane, the periphery of filter membrane is surrounded by absorbent material, during detection, certain density red blood cell is dripped at filter membrane surface, rinse with washing fluid again, the red blood cell that antigen-antibody combination does not occur will ooze out with washing fluid liquid, the red blood cell that antigen-antibody combination occurs then can stay the macroscopic red agglomerate of film surface formation, whether operating personnel can form macroscopic red agglomerate at filter membrane according to red blood cell is carried out judged result, determine erythrocytic blood group.
Described the method selects the filter membrane that single red blood cell can be allowed to pass through as core material, and filter membrane material can be the solid that glass fibre membrane or nitrocellulose membrane or silicate granules etc. can form specific continuous flow passage, and the internal diameter of filter membrane is between 2-20 micron.
The periphery of described filter membrane is surrounded by absorbent material, and the shape of filter membrane is square or circular, and the mode that absorbent material surrounds filter membrane is all surround or part encirclement.
Described blood group antibody is attached on filter membrane, and the blood group antibody method be attached on filter membrane is physisorphtion or chemical crosslink technique.
During described detection drip certain density red blood cell at filter membrane surface, wherein certain density red blood cell is the red cell suspension of whole blood or variable concentrations.And the liquid preparing red cell suspension can be physiological saline or other isotonic solution, it also can be alserver's solution.
The method also comprises detection kit, the filter membrane that single red blood cell can be allowed to pass through is provided with in detection kit, blood group antibody is attached on filter membrane, adsorptive pads is lined with below filter membrane, the area of adsorptive pads is greater than filter membrane, and filter membrane and adsorptive pads are all fixed by the plastic solidification structure in detection kit and supported;
During detection, certain density red blood cell is added drop-wise in detection kit the filter membrane surface combining blood group antibody, is added drop-wise to by washing fluid on same filter membrane surface subsequently, the red blood cell that antigen-antibody combination does not occur rinses out from reaction zone by washing fluid.The red blood cell that antigen-antibody combination occurs then is trapped in filter membrane surface and forms macroscopic red agglomerate.
Described washing fluid is physiological saline.
Utilize the blood type testing methods based on membrane structure that technical scheme of the present invention makes, the advantages such as have without the need to specific installation, detection time is short, and result is objective, have good clinical generalization value.
Accompanying drawing explanation
Fig. 1 is the structural representation of the embodiment 1 of the blood type testing methods based on membrane structure of the present invention;
In figure, 1, kit base plate; 2, filter membrane; 3, adsorptive pads; 4, with the filter membrane region of antigen or antibody.
Fig. 2 is the structural representation of the embodiment 2 of the blood type testing methods based on membrane structure of the present invention;
In figure, 1, filter membrane; 2, adsorptive pads.
Fig. 3 is the structural representation of the embodiment 3 of the blood type testing methods based on membrane structure of the present invention;
In figure, 1, kit base plate; 2, with the filter membrane of ABD antibody; 3, adsorptive pads.
Fig. 4 is two kinds of structural representations of the embodiment 4 of the blood type testing methods based on membrane structure of the present invention;
In figure, 1, kit base plate; 2, with the filter membrane of antihuman globulin; 3, adsorptive pads.
Fig. 5 is the structural representation of the embodiment 5 of the blood type testing methods based on membrane structure of the present invention;
In figure, 1, kit base plate; 2, with the filter membrane of different MN antibody; 3, adsorptive pads.
Fig. 6 is the structural representation of the embodiment 6 of the blood type testing methods based on membrane structure of the present invention;
In figure, 1, kit base plate; 2, with the filter membrane hanging Staphylococal Protein A and extension B antigen; 3, adsorptive pads; A is for hanging Staphylococal Protein A; B is for hanging B antigen.
Embodiment
Be specifically described of the present invention below in conjunction with accompanying drawing, a kind of blood type testing methods based on membrane structure, the method selects the filter membrane that single red blood cell can be allowed to pass through as core material, blood group antibody is attached on filter membrane, the periphery of filter membrane is surrounded by absorbent material, during detection, certain density red blood cell is dripped at filter membrane surface, rinse with washing fluid again, the red blood cell that antigen-antibody combination does not occur will ooze out with washing fluid liquid, the red blood cell that antigen-antibody combination occurs then can stay the macroscopic red agglomerate of film surface formation, whether operating personnel can form macroscopic red agglomerate at filter membrane according to red blood cell is carried out judged result, determine erythrocytic blood group.Wherein, described the method selects the filter membrane that single red blood cell can be allowed to pass through as core material, and filter membrane material can be the solid that glass fibre membrane or nitrocellulose membrane or silicate granules etc. can form specific continuous flow passage, and the internal diameter of filter membrane is between 2-20 micron; The periphery of described filter membrane is surrounded by absorbent material, and the shape of filter membrane is square or circular, and the mode that absorbent material surrounds filter membrane is all surround or part encirclement; Described blood group antibody is attached on filter membrane, and the blood group antibody method be attached on filter membrane is physisorphtion or chemical crosslink technique; During described detection drip certain density red blood cell at filter membrane surface, wherein certain density red blood cell is the red cell suspension of whole blood or variable concentrations.And the liquid preparing red cell suspension can be physiological saline or other isotonic solution, it also can be alserver's solution; The method also comprises detection kit, the filter membrane that single red blood cell can be allowed to pass through is provided with in detection kit, blood group antibody is attached on filter membrane, adsorptive pads is lined with below filter membrane, the area of adsorptive pads is greater than filter membrane, and filter membrane and adsorptive pads are all fixed by the plastic solidification structure in detection kit and supported; During detection, certain density red blood cell is added drop-wise in detection kit the filter membrane surface combining blood group antibody, is added drop-wise to by washing fluid on same filter membrane surface subsequently, the red blood cell that antigen-antibody combination does not occur rinses out from reaction zone by washing fluid.The red blood cell that antigen-antibody combination occurs then is trapped in filter membrane surface and forms macroscopic red agglomerate; Described washing fluid is physiological saline.
Embodiment 1
A kind of blood type testing methods based on membrane structure, the method selects the filter membrane that single red blood cell can be allowed to pass through as core material, blood group antibody is attached on filter membrane, the periphery of filter membrane is surrounded by absorbent material, during detection, certain density red blood cell is dripped at filter membrane surface, rinse with washing fluid again, the red blood cell that antigen-antibody combination does not occur will ooze out with washing fluid liquid, the red blood cell that antigen-antibody combination occurs then can stay the macroscopic red agglomerate of film surface formation, whether operating personnel can form macroscopic red agglomerate at filter membrane according to red blood cell is carried out judged result, determine erythrocytic blood group.Wherein, described the method selects the filter membrane that single red blood cell can be allowed to pass through as core material, and filter membrane material can be the solid that glass fibre membrane or nitrocellulose membrane or silicate granules etc. can form specific continuous flow passage, and the internal diameter of filter membrane is between 2-20 micron; The periphery of described filter membrane is surrounded by absorbent material, and the shape of filter membrane can be square or circular, and the mode that absorbent material surrounds filter membrane is all surround or part encirclement; Described blood group antibody is attached on filter membrane, and the blood group antibody method be attached on filter membrane is physisorphtion or chemical crosslink technique; During described detection drip certain density red blood cell at filter membrane surface, wherein certain density red blood cell is the red cell suspension of whole blood or variable concentrations.And the liquid preparing red cell suspension can be physiological saline or other isotonic solution, it also can be alserver's solution; The method also comprises detection kit, the filter membrane that single red blood cell can be allowed to pass through is provided with in detection kit, blood group antibody is attached on filter membrane, adsorptive pads is lined with below filter membrane, the area of adsorptive pads is greater than filter membrane, and the width of adsorptive pads is identical with filter membrane, but length is greater than filter membrane, as shown in Figure 1, and filter membrane and adsorptive pads all fix by the plastic solidification structure in detection kit and support; During detection, certain density red blood cell is added drop-wise in detection kit the filter membrane surface combining blood group antibody, is added drop-wise to by washing fluid on same filter membrane surface subsequently, the red blood cell that antigen-antibody combination does not occur rinses out from reaction zone by washing fluid.The red blood cell that antigen-antibody combination occurs then is trapped in filter membrane surface and forms macroscopic red agglomerate; Described washing fluid is physiological saline.
Embodiment 2
The method also comprises detection kit, the filter membrane that single red blood cell can be allowed to pass through is provided with in detection kit, blood group antibody is attached on filter membrane, adsorptive pads is lined with below filter membrane, the area of adsorptive pads is greater than filter membrane, and length and the width of adsorptive pads are all greater than filter membrane, and namely filter membrane is positioned at the central area of adsorptive pads, as shown in Figure 2, other are identical with embodiment 1.
Embodiment 3
ABO and Rh (D) blood type detection kit, choosing glass fibre membrane is filter membrane;
1 fixes blood group antibody (anti-A, anti-B, anti-D) on glass fibre membrane
Blood group antibody is fixed: the Blood group McAb (anti-A, anti-B, anti-D) getting high-titer, to add antibody protective agent.Antibody protective agent can be containing 5% bovine serum albumin(BSA), 5% sucrose, 5%PVP, 5% the 5mM phosphate buffer (pH7.4) of calf serum.Antibody protection liquid can be fixed on glass fibre membrane by auxiliary antibody, and strengthens the term of validity of antibody.With antibody protection liquid as dilution, dilution antibody, making it tire eventually is 128.Antibody after the dilution of 150 μ L is added drop-wise to the center of 1.5cm × 1.5cm glass fibre membrane, allows antibody is rounded to be sprawled on glass fibre membrane, then 37 DEG C of drying and processings seal preservation.
The assembling of 2 quick detection reagent bars:
Stick the 1.5cm × 1.5cm glass fibre membrane with 4 antibody one with the square plastic plate back of 4 each and every one holes (aperture is 0.5cm), make the hole on plastic duplicate plate and the antibody center mapping on glass fibre membrane.Then shown in the periphery (as Fig. 3 A) thieving paper wide for 2.5cm being attached to glass fibre membrane.Glass fibre membrane and thieving paper are all use marine glue to paste in polyester version, prevent application of sample and flushing process glass fibre membrane and thieving paper comes off or displacement is put, finally buckle bonnet.
After assembling, ABO and Rh (D) blood type detection kit as shown in Figure 1, and wherein well I is anti-A antibody, and well II is anti-B antibody, and well III is anti-D, and well IV is Positive control wells, as shown in Figure 3.
3 sample detection
Whole blood (or red cell suspension of 3% of the 50 μ l) sample of 150 μ L is dripped at the well place of detection kit.After dripping detected sample, wait for 30s to 1min, then slowly add the washing fluid of 500 μ L to each sample well.After liquid to be rinsed is absorbed by thieving paper, observe the color of well.
Well there is significant red material remain as positive reaction, application of sample pad does not have red material remain as negative reaction.In conjunction with the relation that ABO and Rh (D) blood group antibody and red blood cell react, the blood group learning detected sample can be easy to.Or, also can utilize red residue matter residual on light signal or electrical signal collection system acquisition application of sample pad, then can to result automatization judgement, thus realize detecting ABO and Rh (D) the blood group positive definite of batch blood sample.
Embodiment 4
Antihuman globulin detection kit, choosing glass fibre membrane is filter membrane;
1 fixes antihuman globulin antibody on glass fibre membrane
Blood group antibody is fixed: the antihuman globulin antibody getting high-titer, adds antibody protective agent dilution.Antibody protective agent can be containing 5% bovine serum albumin(BSA), 5% sucrose, 5%PVP, 5% the 5mM phosphate buffer (pH7.4) of calf serum.Antibody protection liquid can be fixed on glass fibre membrane by auxiliary antibody, and strengthens the term of validity of antibody.With antibody protection liquid as dilution, dilution antibody.Antibody after the dilution of 150 μ L is added drop-wise to the center of 1.5cm × 1.5cm glass fibre membrane, allows rounded the sprawling on glass fibre membrane of antibody, then 37 DEG C of drying and processings seal preservation.
The assembling of 2 quick detection reagent bars:
Stick the 1.5cm × 1.5cm glass fibre membrane with antihuman globulin antibody one with the rectangular plastic plate back of a hole (aperture is 0.5cm), make the hole on plastic duplicate plate and the antibody center mapping on glass fibre membrane.Then thieving paper wide for 2.5cm is attached to the both sides of glass fibre membrane, as shown in Figure 4.Glass fibre membrane and thieving paper are all use marine glue to paste in polyester version, prevent application of sample and flushing process glass fibre membrane and thieving paper comes off or displacement is put, finally buckle bonnet.After assembling, antihuman globulin detection kit as shown in Figure 4.
3 sample detection
Drip at the well place of detection kit 15 μ L whole blood (or red blood cell of 50 3%) sample or through external and red blood cell that is sera incubation.After dripping detected sample, wait for 30s to 1min, then slowly add the washing fluid of 500 μ L to each sample well.After liquid to be rinsed is absorbed by thieving paper, observe the color of well.
There is at well place significant red material to remain as positive reaction, application of sample pad does not have red material remain as negative reaction.In conjunction with antihuman globulin experimental principle, can be easy to learn that whether the haemocyte of detected sample is by sensitization.Or, also can utilize red residue matter residual on light signal or electrical signal collection system acquisition application of sample pad, then can to result automatization judgement, thus realize detecting the direct antihuman globulin of batch blood sample.
Embodiment 5
MN system blood type detection kit, choosing glass fibre membrane is filter membrane;
1 blood group antibody fixing MN system on glass fibre membrane
Blood group antibody is fixed: the Blood group McAb (anti-M, anti-N, anti-S, anti-s etc.) getting high-titer, to add antibody protective agent.Antibody protective agent can be containing 5% bovine serum albumin(BSA), 5% sucrose, 5%PVP, 5% the 5mM phosphate buffer (pH7.4) of calf serum.Antibody protection liquid can be fixed on glass fibre membrane by auxiliary antibody, and strengthens the term of validity of antibody.With antibody protection liquid as dilution, dilution antibody.Antibody after the dilution of 150 μ L is added drop-wise to the center of 1.5cm × 1.5cm glass fibre membrane, allows antibody is rounded to be sprawled on glass fibre membrane, then 37 DEG C of drying and processings seal preservation.
The assembling of 2 quick detection reagent bars:
Stick the 1.5cm × 1.5cm glass fibre membrane with 4 antibody one with the rectangular plastic plate back of each and every one hole of porous (aperture is 0.5cm), make the hole on plastic duplicate plate and the antibody center mapping on glass fibre membrane.Then thieving paper wide for 2.5cm is attached to the periphery of glass fibre membrane, as shown in Figure 5.Glass fibre membrane and thieving paper are all use marine glue to paste in polyester version, prevent application of sample and flushing process glass fibre membrane and thieving paper comes off or displacement is put, finally buckle bonnet.After assembling, antihuman globulin detection kit as shown in Figure 5.
3 sample detection
Whole blood (or red cell suspension of 3% of the 50 μ l) sample of 15 μ L is dripped at the well place of detection kit.After dripping detected sample, wait for 30s to 1min, then slowly add the washing fluid of 500 μ L to each sample well.After liquid to be rinsed is absorbed by thieving paper, observe the color of well.
Well there is significant red material remain as positive reaction, application of sample pad does not have red material remain as negative reaction.In conjunction with the relation that MN blood group antibody and red blood cell react, the MN blood group learning detected sample can be easy to.Or, also can utilize red residue matter residual on light signal or electrical signal collection system acquisition application of sample pad, then to result automatization judgement, thus the MN Blood grouping to batch blood sample can be realized.
Embodiment 6
The anti-of abo blood group determines detection kit, and choosing glass fibre membrane is filter membrane;
1 on glass fibre membrane immobilized antigen (Staphylococal Protein A, B antigen)
Blood group antigens are fixed: use broken A type (or Type B) red blood cell of hypotonic solution, centrifugally remove supernatant, re-use identical hypotonic solution washing precipitation, until supernatant water white transparency, collecting precipitation is erythrocyte membrane, as blood group A (or B) Antigen extraction thing.Use adds antibody protective agent dilution Antigen extraction thing.Antigen extraction thing after dilution is added drop-wise to the center of 1.5cm × 1.5cm glass fibre membrane, then 37 DEG C of drying and processings seal preservation.
The assembling of 2 quick detection reagent bars:
Stick the 1.5cm × 1.5cm glass fibre membrane with antigen one with the rectangular plastic plate back of two holes (aperture is 0.5cm), make the hole in polyester version and the antigen center mapping on glass fibre membrane.Then thieving paper wide for 2.5cm is attached to the both sides of glass fibre membrane.Glass fibre membrane and thieving paper are all use marine glue to paste in polyester version, prevent application of sample and flushing process glass fibre membrane and thieving paper comes off or displacement is put, finally buckle bonnet.Antihuman globulin detection kit after assembling, as shown in Figure 6.
3 sample detection
Drip at two anti-regular inspections survey well places and add 50 μ L serum or plasma samples respectively, then drip type A cell suspension and the type B cell suspension of 50 μ L3% respectively at two well places, after waiting for 30s to 1min, slowly add the reaction rinse liquid of 500 μ L.After liquid to be rinsed is absorbed by thieving paper, observe the color of well.
Well there is significant red material remain as positive reaction, application of sample pad does not have red material remain as negative reaction.The AB antibody of abo blood group antigen in serum or blood plasma is combined, and then reacts with the cell suspension passed through, thus can determine the Antibody types that contains in serum, the blood group of indirect determination sample.Or, also can utilize red residue matter residual on light signal or electrical signal collection system acquisition application of sample pad, then can to result automatization judgement, thus realize surveying the anti-regular inspection of the abo blood group of batch blood sample.
Technique scheme only embodies the optimal technical scheme of technical solution of the present invention, and those skilled in the art all embody principle of the present invention to some variations that wherein some part may be made, and belong within protection scope of the present invention.
Claims (6)
1. the blood type testing methods based on membrane structure, the method selects the filter membrane that single red blood cell can be allowed to pass through as core material, blood group antibody is attached on filter membrane, the periphery of filter membrane is surrounded by absorbent material, during detection, certain density red blood cell is dripped at filter membrane surface, rinse with washing fluid again, the red blood cell that antigen-antibody combination does not occur will ooze out with washing fluid liquid, the red blood cell that antigen-antibody combination occurs then can stay the macroscopic red agglomerate of film surface formation, whether operating personnel can form macroscopic red agglomerate at filter membrane according to red blood cell is carried out judged result, determine erythrocytic blood group.
2. the blood type testing methods based on membrane structure according to claim 1, it is characterized in that, described the method selects the filter membrane that single red blood cell can be allowed to pass through as core material, and filter membrane material can be the solid that glass fibre membrane or nitrocellulose membrane or silicate granules etc. can form specific continuous flow passage, and the internal diameter of filter membrane is between 2-20 microns.
3. the blood type testing methods based on membrane structure according to claim 1, is characterized in that, the periphery of described filter membrane is surrounded by absorbent material, and the shape of filter membrane is square or circular, and the mode that absorbent material surrounds filter membrane is all surround or part encirclement.
4. the blood type testing methods based on membrane structure according to claim 1, is characterized in that, described blood group antibody is attached on filter membrane, and the blood group antibody method be attached on filter membrane is physisorphtion or chemical crosslink technique.
5. the blood type testing methods based on membrane structure according to claim 1, is characterized in that, during described detection drip certain density red blood cell at filter membrane surface, wherein certain density red blood cell is the red cell suspension of whole blood or variable concentrations.
6. the blood type testing methods based on membrane structure according to claim 1, it is characterized in that, the method also comprises detection kit, the filter membrane that single red blood cell can be allowed to pass through is provided with in detection kit, blood group antibody is attached on filter membrane, be lined with adsorptive pads below filter membrane, the area of adsorptive pads is greater than filter membrane, and filter membrane and adsorptive pads are all fixed by the plastic solidification structure in detection kit and supported;
During detection, certain density red blood cell is added drop-wise in detection kit the filter membrane surface combining blood group antibody, is added drop-wise to by washing fluid on same filter membrane surface subsequently, the red blood cell that antigen-antibody combination does not occur rinses out from reaction zone by washing fluid.The red blood cell that antigen-antibody combination occurs then is trapped in filter membrane surface and forms macroscopic red agglomerate.
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| CN106290921A (en) * | 2016-08-03 | 2017-01-04 | 中山生物工程有限公司 | A kind of blood type test card based on microporous membrane, Blood grouping system |
| CN107202899A (en) * | 2017-07-10 | 2017-09-26 | 四川省医学科学院·四川省人民医院实验动物研究所 | A kind of authentication method of good chief of a tribe monkey blood group |
| CN109459575A (en) * | 2018-09-19 | 2019-03-12 | 厦门为正生物科技股份有限公司 | A kind of ABO&RhD blood group detection device |
| CN110702902A (en) * | 2019-11-04 | 2020-01-17 | 珠海丽珠试剂股份有限公司 | Reagent strip and using method thereof |
| CN115684611A (en) * | 2022-12-30 | 2023-02-03 | 天津德祥生物技术股份有限公司 | Erythrocyte antigen detection test strip and application |
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