CN104950114A - Screening method for serum (plasma) antibody based on membrane structure and preparation method of screening and detecting kit - Google Patents
Screening method for serum (plasma) antibody based on membrane structure and preparation method of screening and detecting kit Download PDFInfo
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Abstract
The invention discloses a screening method for a serum (plasma) antibody based on a membrane structure and a preparation method of a screening and detecting kit. The screening method is characterized by comprising the steps of with a filter membrane capable of allowing single erythrocyte to pass through as a core material, combining the erythrocyte, a cell debris or a purified antigen onto the filter membrane; enclosing water absorbent materials at the periphery of the filter membrane; during detecting, dropwise adding serum or plasma to be detected onto the surface of the filter membrane; after incubating for a period of time, washing with a trace amount of washing liquid; then, dropwise adding a specific indicator cell onto the surface of the filter membrane; and finally, washing with a washing liquid. The screening method has the beneficial effects that the detecting time is short, and the result is objective.
Description
Technical field
The present invention relates to a kind of blood group and judge detection technique, the particularly preparation of a kind of serum based on membrane structure (blood plasma) Antibody screening method and examination detection kit.
Background technology
Serum or blood plasma have suffered except the abo antibody of routine, also containing a large amount of irregular antibodies and platelet antibody.The irregular antibody with clinical meaning can cause hemolytic blood transfusion reaction, neonatal hemolytic disease etc.Therefore, in clinical detection, irregular antibody examination is carried out to the serum of blood donor or blood plasma, blood syringe containing irregular antibody can be prevented to patient, cause the hemolytic blood transfusion reaction of patient; By examination to the blood of irregular antibody be prepared into antibody serum, be conducive to the detection of rare blood type; To needing the patient of blood transfusion through the examination of row irregular antibody, contributing to blood and selecting, the input of the antigenemia cell relative with irregular antibody can be prevented, ensureing transfusion safety; Irregular antibody examination is carried out to pregnant woman, can prevention and therapy neonatal hemolytic disease, improve the fitness of fetus an d neonate.And platelet antibody can cause that platelet transfusion is invalid, post-transfusion purpura etc., clinically, for the patient of platelet transfusion repeatedly, the platelet antibody in results of regular determination serum can predict platelet transfusion effect; Examination is carried out for the platelet antibody in the serum of the patient of Inefficacy of Platelets Transfusion or blood plasma, the immunity factor of patient's Inefficacy of Platelets Transfusion can be caused by Timeliness coverage; Carry out platelet antibody examination to pregnant woman is antenatal, the risk that can be early abortion generation provides Diagnosis and Treat foundation.Therefore, in order to ensure transfusion safety, before blood transfusion, the Antibody screening in serum (blood plasma) is more and more drawn attention.
Because irregular antibody, platelet antibody are mainly by transfusing blood or the generation of the immunostimulation such as blood platelet or gestation, these antibody directly can not produce cell agglutination reaction in physiological saline medium, therefore, the specific process such as antihuman globulin method, cohesion amine method must be passed through detect.
In current clinical detection serum or blood plasma antibody screening method with flat band method and post agglutination technology in the majority.Flat band method and post agglutination complicated operation; The condition such as centrifugal has impact to experimental result; Easily cause misjudgment time more for sample; Need utility appliance, length consuming time, limit application in an emergency situation.
Summary of the invention
The object of the invention is to solve the problem, devising the preparation of a kind of serum based on membrane structure (blood plasma) Antibody screening method and examination detection kit.
Realizing above-mentioned purpose technical scheme of the present invention is, the preparation of a kind of serum based on membrane structure (blood plasma) Antibody screening method and examination detection kit, the method selects the filter membrane that single red blood cell can be allowed to pass through as core material, by cell, cell fragment or purifying antigen are attached on filter membrane, the periphery of filter membrane is surrounded by absorbent material, during detection, serum to be checked or blood plasma are added drop-wise to filter membrane surface, after hatching certain hour, after trace washing fluid rinses, again specific indicator cell is added drop-wise to filter membrane surface, washing fluid is finally used to rinse.After the antigentic specificity on film is in conjunction with antibody in serum (blood plasma), can with specific indicator cells generation red cell agglutination, and then form macroscopic red agglomerate.When the antigen on film is not combined with antibody in serum (blood plasma), would not there is aggegation in indicator cell, and red blood cell can be rinsed liquid dispersion, does not have naked eyes red color visible aggregation block.Whether operating personnel can form macroscopic red agglomerate at filter membrane according to red blood cell is carried out judged result, determines the antibody in serum or blood plasma.The combination of indicator red blood cell and membrane structure, for reaction provides the mode of result display.
Described serum (blood plasma) antibody comprises blood group antibody, hla antibody, platelet antibody and other antibody.
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 absorbent material can be thieving paper, cotton etc., and the mode that absorbent material surrounds filter membrane is all surround or part encirclement.
Describedly be attached on filter membrane by cell, cell fragment or purifying antigen, method cell, cell fragment or purifying antigen are attached on filter membrane is physisorphtion or chemical crosslink technique.
Described serum to be checked or blood plasma are added drop-wise to filter membrane surface, and serum to be checked wherein or blood plasma are fresh human serum or the blood plasma that volume is about 10-200 μ L.
Described is added drop-wise to filter membrane surface by specific indicator cell, and wherein indicator cell is the erythrocyte of the antihuman globulin bag quilt be kept in alserver's solution or other isotonic solution.The erythrocytic preparation of indicator can make according to the method for red blood cell sensitization.Experimentally requirement, indicator cell concentration is between 0.5%-20%.
This detection kit is made up of test card and indicator cell.Wherein be provided with the filter membrane that single red blood cell can be allowed to pass through in test card, cell, cell fragment or purifying antigen are attached on filter membrane, filter membrane periphery is lined with adsorptive pads, and the area of adsorptive pads is greater than filter membrane, and filter membrane and adsorptive pads are all fixed by plastic solidification structure and support.Indicator cell is then the erythrocyte of antihuman globulin bag quilt.
During detection, serum to be checked or blood plasma are added drop-wise to the filter membrane surface of test card, after hatching certain hour, after micro-washing fluid rinses, more specific indicator cell are added drop-wise to filter membrane surface, finally use washing fluid to rinse.If contain the antibody with antigen generation specific reaction on filter membrane in serum or blood plasma, in serum or blood plasma, antibody will be incorporated on filter membrane, and then be combined with indicator cell generation antigen-antibody, instruction red blood cell then can be stayed film surface and form macroscopic red agglomerate; Otherwise, if do not contain the antibody with antigen generation specific reaction on filter membrane in serum or blood plasma, can not be there is antigen-antibody and combine in corresponding reverse type indicator cell, indicator red blood cell will ooze out with washing fluid liquid, then filter membrane can not be formed macroscopic red agglomerate.
Described washing fluid is physiological saline or other isotonic damping fluids.
(blood plasma) the Antibody screening method of the serum based on membrane structure utilizing technical scheme of the present invention to make and the preparation of examination detection kit, have without the need to specific installation, the advantages such as 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 Antibody screening method in the serum based on membrane structure of the present invention or blood plasma;
In figure, 1, kit base plate; 2, with the filter membrane region of blood group antigens, platelet antigen or other antigens; 3, adsorptive pads.
Fig. 2 is the schematic diagram that in serum or blood plasma, irregular antibody detects reagent card, and wherein A is the overall diagram that irregular antibody detects reagent card; B is the lid figure that irregular antibody detects reagent card; C is the water absorbing box that irregular antibody detects reagent card; D be irregular antibody detect reagent card add model, and 1 is plastic bottom board, and 2 is filter membrane, with blood group antigens; 3 is thieving paper.
Fig. 3 is the schematic diagram that in serum or blood plasma, platelet antibody detects reagent card, and wherein A is the overall diagram that platelet antibody detects reagent card; B is the lid figure that platelet antibody detects reagent card; C is the water absorbing box that platelet antibody detects reagent card; D be platelet antibody detect reagent card add model, and 1 is plastic bottom board; 2 is filter membrane, with platelet antigen; 3 is thieving paper.
Embodiment
Be specifically described of the present invention below in conjunction with accompanying drawing, the invention provides method and the preparation of its detection kit of Antibody screening in a kind of serum based on filter membrane structure or blood plasma.This kit comprises the test card of the filter membrane after with modification and corresponding indicator cell.To illustrate further description the present invention below in conjunction with case study on implementation.
Case study on implementation 1
Antibody screening detection method in a kind of serum based on filter membrane structure or blood plasma
The method selects the filter membrane that single red blood cell can be allowed to pass through as core material, cell, cell fragment or purifying antigen are attached on filter membrane, the periphery of filter membrane is surrounded by absorbent material, during detection, serum to be checked or blood plasma are added drop-wise to filter membrane surface, after hatching certain hour, after micro-washing fluid rinses, again specific indicator cell is added drop-wise to filter membrane surface, finally uses washing fluid to rinse.After the antigentic specificity on film is in conjunction with antibody in serum (blood plasma), can reacts with specific indicator cell generation cell agglutination, and then form macroscopic red agglomerate.When the antigen on film is not combined with antibody in serum (blood plasma), would not there is aggegation in indicator cell, and red blood cell can be rinsed liquid dispersion, does not have naked eyes red color visible aggregation block.Whether operating personnel can form macroscopic red agglomerate at filter membrane according to red blood cell is carried out judged result, determines the antibody in serum or blood plasma.
The kit of the detection method described in realization comprises two parts, and a part is by the test card (as shown in Figure 1) formed with the filter membrane of cell, cell fragment or purifying antigen, thieving paper, plastic solidification support; A part is the anti-human ball indicator cell for detecting.Wherein, the filter membrane that selecting described in said method can allow single red blood cell to pass through is as core material, filter membrane material can be the solid that glass fibre membrane, nitrocellulose membrane, silicate granules etc. can form specific continuous flow passage, and the aperture of filter membrane is between 2-20 micron; The periphery of described filter membrane is surrounded by absorbent material, and absorbent material can be thieving paper, cotton etc., and the mode that absorbent material surrounds filter membrane is all surround or part encirclement; Described cell, cell fragment or purifying antigen are attached on filter membrane, and cell, cell fragment or purifying antigen are physisorphtion or chemical crosslink technique to the method on filter membrane; Described serum to be checked or blood plasma are added drop-wise to filter membrane surface, and serum to be checked wherein or blood plasma are that volume is about the fresh human serum of 10-200 μ l or blood plasma; Described is added drop-wise to filter membrane surface by specific indicator cell, and wherein indicator cell is the erythrocyte of the antihuman globulin bag quilt be kept in alserver's solution or other isotonic solution.Experimentally requirement, indicator cell concentration is between 0.5%-20%; Described washing fluid is physiological saline or other isotonic damping fluids.
Case study on implementation 2
The preparation of irregular antibody examination detection kit and detecting step in serum or blood plasma
1 fixes blood group antigens on filter membrane
The preparation of blood group antigens: select the O type red blood cell of three groups of known antigens collection of illustrative plates and one group by the O type red blood cell of IgG sensitization respectively as detectable antigens group and positive controls, use Hyposmolality solution smudge cells, centrifugally remove supernatant, re-use identical Hyposmolality solution washing precipitation, until supernatant water white transparency, collection erythrocyte membrane precipitates, as blood group antigens extract.Add antigen protective agent dilution Antigen extraction thing.Antigen protective agent can be containing 5% bovine serum albumin(BSA), 5% sucrose, 5%PVP, 5% the 5mM phosphate buffer (pH7.4) of calf serum.Namely antigen protection liquid can protect antigen originality, the term of validity of enhancement antigen; Also can be fixed on worry film by helper antigen.Antigen extraction thing after dilution is added drop-wise to the center of 1.5cm × 1.5cm filter membrane that activation process is crossed, then 37 DEG C of drying and processings seal preservation.
The preparation of 2 indicator cells:
Certain density O type red blood cell mixes with anti-D (IgG) antibody equal-volume, 37 DEG C of couveuses, hatch 30min, obtain anti-D (IgG) antibody sensitized cell, use 5 times of mL normal saline washings for several times, remove unnecessary IgG, resuspended to desired concn with alserver's solution, finally add the mixing of certain volume antihuman globulin reagent, obtain anti-igg indicator cell.
The assembling of 3 detection reagent cards:
Detect reagent card and mainly contain the filter membrane processed, absorbent material and plastic stent composition.Wherein plastic stent is made up of three parts, and one is lid, and two is add model, and three is water absorbing boxes.Adding model is one with the square plastic flitch of 4 holes (aperture is 0.5cm), can remove from reagent card.Filter membrane with not synantigen test set and cellular control unit film is attached to the back adding model, considers the antigen center on film and the sample well phase mapping on plastic duplicate plate.The square absorbent material of 4cm × 4cm is filled with inside lid and water absorbing box.Filter membrane and absorbent material are all use marine glue to paste on plastic duplicate plate, prevent application of sample and flushing process filter membrane and thieving paper comes off or displacement is put.
Irregular antibody after assembling detects reagent card with as shown in Figure 2, and wherein well I, II, III is the filter membrane with three groups of known antigens, and well IV is Positive control wells, namely on filter membrane with the quick cell membrane of system.As shown in Figure 2.
The sample detection of irregular antibody kit in 4 serum or blood plasma
First test card (Fig. 2 A) is checked card, extract out and add scale (Fig. 2 D).Then 10-200 μ l serum or plasma sample are dripped on I, II, III film adding scale.After dripping detected sample, be placed in 37 DEG C of incubators, hatch 1-40min, take out and add scale, scale will be added and be placed on lid, and then slowly add the washing fluid of 50-500ul to each sample film, rinse removing unreacted or unnecessary antibody.Then will add scale and turn back to water absorbing box, then in well, drip 10-200 μ l indicator cell, and wait for 30s to 1min, slowly add the washing fluid of 200-1000ul 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, illustrate that serum contains the antibody corresponding with antigen on film; Application of sample pad does not have red material remain as negative reaction, then illustrate in serum not containing antibody corresponding with it.Conjugated antigen and antibody specific reaction, antihuman globulin experimental principle, can be easy to the antibody learnt in detected 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 detecting the irregular antibody of batch serum or plasma sample.
Case study on implementation 3
The preparation of platelet antibody examination detection kit and detection method
1 fixes platelet antigen on filter membrane
After making chemically to process activation filter membrane, the platelet antigen (or blood platelet) after protective agent dilution is then used to be added drop-wise to the center of 1.5cm × 1.5cm filter membrane, then 37 DEG C of drying and processings seal preservation.
The assembling of 3 detection reagent cards:
Detect reagent card and comprise filter membrane, absorbent material and the plastic stent composition processed.Plastic stent is made up of three parts, and one is lid, and two is add model, and three is water absorbing boxes.Adding model is one with the square plastic flitch of 4 holes (aperture is 0.5cm), can remove from reagent card.Filter membrane with platelet antigen is attached to the back adding model, considers the antigen center on film and the sample well phase mapping on plastic duplicate plate.The square thieving paper of 4cm × 4cm is filled with inside lid and water absorbing box.Filter membrane and thieving paper are all use marine glue to paste on plastic duplicate plate, prevent application of sample and flushing process filter membrane and thieving paper comes off or displacement is put.
Platelet antibody after assembling detects reagent card with as shown in Figure 3, and wherein well I, II is detect aperture, and well III is Positive control wells, and well IV is negative control hole.
The preparation of 3 indicator cells:
Certain density O type red blood cell mixes with anti-D (IgG) antibody equal-volume, 37 DEG C of couveuses, hatch 30min, obtain anti-D (IgG) antibody sensitized cell, use 5 times of mL normal saline washings for several times, remove unnecessary IgG, resuspended to desired concn with alserver's solution, finally add the mixing of certain volume antihuman globulin reagent, obtain anti-igg indicator cell.
4 sample detection:
Take out the sample panel of detection kit, mark detect aperture, Positive control wells and negative control hole, test serum or the blood plasma of 10-200 μ L is dripped to detect aperture, heliotropism control wells drips 10-200 μ L positive control (containing anti human platelet antibody), negative control hole drips 10-200 μ L negative control (not containing anti human platelet antibody), couveuse sample panel being placed in 37 DEG C hatches 1-40min, again drip the physiological saline of 50-500 μ L, rinse and remove unnecessary serum or blood plasma.Drip 10-200 μ l indicator cell in the most backward reacting hole, wait for 30s to 1min, slowly add the washing fluid of 200-1000ul to each reacting hole, after liquid to be rinsed is absorbed by thieving paper, observe the color of well.
Add positive control reacting hole on should have significant red material remain, its principle be in positive control containing anti human platelet antigen can platelet antibody combine, and then with indicator Cell binding, by cell retention at filter membrane surface; Add the reacting hole of negative control, have anti human platelet antibody because do not conform in negative control, therefore can not retain indicator cell, do not have red material to remain.According to conjugated antigen and antibody specific reaction, antihuman globulin experimental principle, and with reference to positive control wells and cloudy control wells result, whether can be easy to learn in detected sample containing anti human platelet antibody.
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 (8)
1. the preparation of (blood plasma) the Antibody screening method of the serum based on membrane structure and examination detection kit, it is characterized in that, the method selects the filter membrane that single red blood cell can be allowed to pass through as core material, cell, cell fragment or purifying antigen are attached on filter membrane, the periphery of filter membrane is surrounded by absorbent material, during detection, serum to be checked or blood plasma are added drop-wise to filter membrane surface, after hatching certain hour, after trace washing fluid rinses, again specific indicator cell is added drop-wise to filter membrane surface, finally uses washing fluid to rinse.After the antigentic specificity on film is in conjunction with antibody in serum (blood plasma), can with specific indicator cells generation red cell agglutination, and then form macroscopic red agglomerate.When the antigen on film is not combined with antibody in serum (blood plasma), would not there is aggegation in indicator cell, and red blood cell can be rinsed liquid dispersion, does not have naked eyes red color visible aggregation block.Whether operating personnel can form macroscopic red agglomerate at filter membrane according to red blood cell is carried out judged result, determines the antibody in serum or blood plasma.The combination of indicator red blood cell and membrane structure, for reaction provides the mode of result display.
2. the serum based on membrane structure according to claim 1 (blood plasma) Antibody screening method, is characterized in that, described serum (blood plasma) antibody comprises blood group antibody, hla antibody, platelet antibody.
3. the serum based on membrane structure according to claim 1 (blood plasma) Antibody screening method, 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 micron.
4. the serum based on membrane structure according to claim 1 (blood plasma) Antibody screening method, it is characterized in that, the periphery of described filter membrane is surrounded by absorbent material, absorbent material can be thieving paper, cotton etc., and the mode that absorbent material surrounds filter membrane is all surround or part encirclement.
5. the serum based on membrane structure according to claim 1 (blood plasma) Antibody screening method, it is characterized in that, describedly be attached on filter membrane by cell, cell fragment or purifying antigen, method cell, cell fragment or purifying antigen are attached on filter membrane is physisorphtion or chemical crosslink technique.
6. the serum based on membrane structure according to claim 1 (blood plasma) Antibody screening method, it is characterized in that, described serum to be checked or blood plasma are added drop-wise to filter membrane surface, and serum to be checked wherein or blood plasma are fresh human serum or the blood plasma that volume is about 10-200 μ L.
7. the serum based on membrane structure according to claim 1 (blood plasma) Antibody screening method, it is characterized in that, described is added drop-wise to filter membrane surface by specific indicator cell, and wherein indicator cell is the erythrocyte of the antihuman globulin bag quilt be kept in alserver's solution or other isotonic solution.The erythrocytic preparation of indicator can make according to the method for red blood cell sensitization.Experimentally requirement, indicator cell concentration is between 0.5%-20%.
8. for completing the detection kit of the serum based on membrane structure according to claim 1 (blood plasma) Antibody screening method, it is characterized in that, this detection kit is made up of test card and indicator cell.Wherein be provided with the filter membrane that single red blood cell can be allowed to pass through in test card, cell, cell fragment or purifying antigen are attached on filter membrane, filter membrane periphery is lined with adsorptive pads, and the area of adsorptive pads is greater than filter membrane, and filter membrane and adsorptive pads are all fixed by plastic solidification structure and support.Indicator cell is then the erythrocyte of antihuman globulin bag quilt.
During detection, serum to be checked or blood plasma are added drop-wise to the filter membrane surface of test card, after hatching certain hour, after micro-washing fluid rinses, more specific indicator cell are added drop-wise to filter membrane surface, finally use washing fluid to rinse.If contain the antibody with antigen generation specific reaction on filter membrane in serum or blood plasma, in serum or blood plasma, antibody will be incorporated on filter membrane, and then be combined with indicator cell generation antigen-antibody, instruction red blood cell then can be stayed film surface and form macroscopic red agglomerate; Otherwise, if do not contain the antibody with antigen generation specific reaction on filter membrane in serum or blood plasma, can not be there is antigen-antibody and combine in corresponding reverse type indicator cell, indicator red blood cell will ooze out with washing fluid liquid, then filter membrane can not be formed macroscopic red agglomerate.
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| CN116539872A (en) * | 2023-04-25 | 2023-08-04 | 上海润普生物技术有限公司 | Fluorescent immunochromatography detection kit for quantitative platelet antibody and preparation method thereof |
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| CN116539872B (en) * | 2023-04-25 | 2024-02-09 | 上海润普生物技术有限公司 | Fluorescent immunochromatography detection kit for quantitative platelet antibody and preparation method thereof |
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