WO2021007738A1 - 一种功能化生物多层孔隙膜免疫载体、制备方法、应用 - Google Patents

一种功能化生物多层孔隙膜免疫载体、制备方法、应用 Download PDF

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WO2021007738A1
WO2021007738A1 PCT/CN2019/095958 CN2019095958W WO2021007738A1 WO 2021007738 A1 WO2021007738 A1 WO 2021007738A1 CN 2019095958 W CN2019095958 W CN 2019095958W WO 2021007738 A1 WO2021007738 A1 WO 2021007738A1
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antibody
porous membrane
multilayer porous
biological multilayer
red blood
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PCT/CN2019/095958
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English (en)
French (fr)
Inventor
李勇
唐玉国
王红梅
段生宝
丁少华
陈晔洲
魏双施
李剑平
Original Assignee
中国科学院苏州生物医学工程技术研究所
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Priority to CN201980001171.7A priority Critical patent/CN110520736B/zh
Priority to EP19937668.2A priority patent/EP3971574A4/en
Priority to PCT/CN2019/095958 priority patent/WO2021007738A1/zh
Publication of WO2021007738A1 publication Critical patent/WO2021007738A1/zh

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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
    • 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
    • G01N33/5302—Apparatus specially adapted for immunological test procedures
    • 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
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • 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
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G01N33/54386—Analytical elements
    • G01N33/54387—Immunochromatographic test strips
    • G01N33/54388—Immunochromatographic test strips based on lateral flow
    • 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
    • G01N33/544—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being organic
    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6854—Immunoglobulins
    • 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
    • G01N33/80—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

Definitions

  • the present invention relates to the field of immune detection, in particular to a functionalized biological multilayer porous membrane immune carrier, a preparation method and an application.
  • Incompatible blood types between blood donors and recipients can cause severe hemolytic transfusion reactions.
  • Blood type compatibility testing is one of the most important ways to ensure safe blood transfusion. Compatible means harmonious coexistence, referring to patient serum There is no specific antibody corresponding to the imported red blood cell antigen, and no immune response is called compatible blood transfusion. On the contrary, it is called incompatible blood transfusion, that is, there are specific antibodies corresponding to the imported red blood cell antigen in the patient's serum, and an immune reaction occurs, and the red blood cells imported into the patient are damaged and even hemolyzed. The specific antigens on the surface of the Red Blood Cell and the antibodies in the serum determine the blood type.
  • the pre-transfusion blood group compatibility test not only includes ABO positive and negative typing, RhD blood type typing, and the detection of irregular antibodies in the blood recipient serum, which is clinically meaningful Detection and identification of antibodies and cross-matching tests, etc.
  • MGIA microcolumn gel immunoassay
  • the microplate solid phase detection method improves the sensitivity of detection, but also requires complicated incubation and washing steps.
  • the fiber filter paper has the fiber characteristics that can filter out red blood cells and retain the agglutinated red blood cells, the method of filter paper for detecting blood types has been widely reported.
  • the filter paper detection technology mainly fixes the antibody to the filter paper through physical adsorption, and the adsorption capacity is limited, so the detection sensitivity is low, and it has not been widely used in clinical practice.
  • IgG monoclonal antibodies cannot be used as fixed antibodies on this type of fiber paper, because even if there is a positive reaction, but there is no agglutination, the positive reaction of red blood cells (red blood cells) will still be washed away, resulting in the occurrence of false negative reactions. At the same time, it is restricted by antibody storage conditions and is not suitable for emergency use in the field.
  • an object of the present invention is to provide a functionalized biological multilayer porous membrane immune carrier, including a biological multilayer porous membrane, attached to the biological multilayer porous membrane through specific binding of antigen and antibody
  • the specific anti-biological multilayer porous membrane antibody on the surface, and the cross-linked antibody coupled with the specific anti-biological multilayer porous membrane antibody can pass Coupling or antibody antigen specific binding and then binding to red blood cell blood group antibody or sensitized red blood cell blood group antigen, thereby further used for blood group detection; the functionalized biological multilayer porous membrane immune carrier provided by the present invention can also be selected according to needs. Capturing antibodies, performing immune response or continuing immune selection and binding, so as to detect and analyze pathogenic microbial cell antigen antibodies, bacterial cell antigen antibodies, tumor cell antigen antibodies and various cell antigen antibodies.
  • the present invention provides a functionalized biological multilayer porous membrane immune carrier, including:
  • a specific biological multilayer porous membrane antibody wherein the specific anti-biological multilayer porous membrane antibody specifically binds to the biological multilayer porous membrane with an antigen antibody to attach to the surface of the biological multilayer porous membrane;
  • a cross-linked antibody wherein the cross-linked antibody is coupled to the specific anti-biological multilayer porous membrane antibody;
  • the specific anti-biological multilayer porous membrane antibody is an antibody that can specifically react with the antigen-antibody of the biological multilayer porous membrane
  • One Fab end of the cross-linked antibody can be coupled to the specific anti-biological multilayer porous membrane antibody, and the other Fab end can be combined with the specific antibody.
  • the specific biological multilayer porous membrane antibody includes murine monoclonal antibody, goat-derived polyclonal antibody, rabbit-derived monoclonal antibody/polyclonal antibody, chicken-derived polyclonal antibody;
  • the cross-linked antibody includes anti-mouse polyclonal antibody, anti-goat polyclonal antibody Polyclonal antibodies, anti-rabbit polyclonal antibodies, and anti-chicken polyclonal antibodies;
  • the specific antibodies capable of binding to the Fab end of the cross-linked antibody include murine monoclonal antibodies, goat polyclonal antibodies, rabbit monoclonal antibodies/polyclonal antibodies, and chicken polyclonal antibodies .
  • the second object of the present invention is to provide a method for preparing a functionalized biological multilayer porous membrane immune carrier, which includes the following steps:
  • the specific anti-biological multilayer porous membrane antibody is an antibody that can specifically react with the antigen-antibody of the biological multilayer porous membrane
  • One Fab end of the cross-linked antibody can be coupled to the specific anti-biological multilayer porous membrane antibody, and the other Fab end can be combined with the specific antibody.
  • the specific anti-biological multilayer porous membrane antibody is diluted with a diluent to a final concentration of 1-100 ⁇ g/ml; the cross-linked antibody is diluted with a diluent to a final concentration of 1-100 ⁇ g/ml; wherein, the dilution The solution is 0.01mol/L, PH 7.0-7.2 phosphate buffer;
  • the treatment solution is a 0.01 mol/L phosphate buffer containing 1% bovine serum albumin and a pH of 7.0-7.2;
  • the lotion is 0.01 mol/L and a phosphate buffer with a pH of 7.0-7.2;
  • the blocking solution is 5% skimmed milk powder prepared with a phosphate buffer with a pH of 7.0-7.2 and 0.01 mol/L; wherein the unit% represents the mass of the solute in 100 ml of the solution.
  • the third objective of the present invention is to provide a biological multilayer porous membrane immune carrier for blood group compatibility detection, including:
  • the red blood cell blood group antibody or the sensitized red blood cell blood group antigen is attached to the functionalized biological multilayer porous membrane immune carrier by specific binding with the cross-linked antibody on the functionalized biological multilayer porous membrane immune carrier;
  • the sensitized red blood cell blood group antigen is a red blood cell blood group antigen that specifically binds a red blood cell membrane antibody of a non-blood group antibody.
  • the red blood cell blood group antibody includes IgM, IgG, and IgM-IgG mixed red blood cell A, B, Rh, MNS, Kell, Kidd blood group antibodies, and the red blood cell blood group antibody has a titer greater than 1:32.
  • the sensitized red blood cell blood group antigen includes sensitized type A red blood cell antigen, sensitized type B red blood cell antigen, sensitized irregular antibody screening red blood cell antigen, and sensitized red blood cell spectrum cell antigen.
  • the fourth object of the present invention is to provide a method for preparing a biological multilayer porous membrane immune carrier for blood group compatibility detection, which includes the following steps:
  • red blood cell blood group antibody or sensitized red blood cell blood group antigen is specifically combined with the cross-linked antibody on the functionalized biological multilayer porous membrane immune carrier to prepare the blood group compatibility test as described above Biological multilayer porous membrane immune carrier;
  • the specific anti-biological multilayer porous membrane antibody is an antibody that can specifically react with the antigen-antibody of the biological multilayer porous membrane
  • One Fab end of the cross-linked antibody can be coupled to the specific anti-biological multilayer porous membrane antibody, and the other Fab end can bind to a specific antibody, and the specific antibody is capable of binding to the Fab end of the cross-linked antibody Red blood cell blood group antibody or sensitized red blood cell blood group antigen;
  • the sensitized red blood cell blood group antigen is a red blood cell blood group antigen that specifically binds to a red blood cell membrane antibody of a non-blood group antibody.
  • sensitized red blood cell blood group antigens Preferably, it also includes the step of preparing sensitized red blood cell blood group antigens.
  • the specific steps of preparing sensitized red blood cell blood group antigens are as follows: use red blood cell membrane antibodies other than blood group antibodies to react with fresh red blood cells, and use a hypotonic solution when 1+ agglutination strength appears The red blood cells were lysed and centrifuged to collect the sensitized red blood cell antigen.
  • the hypotonic solution is a 0.1%-0.7% NaCl solution
  • the rotation speed of the centrifugation is 12000 rpm, and the centrifugation time is 5 min.
  • the fifth object of the present invention is to provide a blood type test card, including:
  • a solid-phase plastic carrier the solid-phase plastic carrier includes a card slot outer shell and a card slot inner shell, the card slot outer shell and the card slot inner shell are detachably connected;
  • the biological multilayer porous membrane immune carrier used for blood group compatibility detection as described above, wherein the biological multilayer porous membrane immune carrier used for blood group compatibility detection is located at the bottom of the solid-phase plastic carrier card slot shell;
  • Absorbent cotton located at the bottom of the inner shell of the solid-phase plastic carrier card slot;
  • the isolation film is placed above the absorbent cotton.
  • the preparation material of the solid-phase plastic carrier includes polyethylene, polyethylene, polystyrene, styrene-butadiene-acrylonitrile; the solid-phase plastic carrier further includes a detection hole, and the detection hole includes
  • the isolation membrane includes filter paper, cellulose fiber, glass fiber pad, and fiber pad or test paper made of porous polymer material; the absorbent cotton includes cotton pulp pad and filter paper.
  • the temperature required for storage or detection of the blood type test card is 4-50°C.
  • the sixth object of the present invention is to provide a blood type detection method, including the following steps:
  • the blood type sample to be detected has an antigen-antibody specific reaction with the biological multilayer porous membrane immune carrier used for blood type compatibility detection in the blood type test card, which is positive; otherwise, it is negative.
  • the washing liquid is a buffer solution containing 0.05%-0.5% Tween-20, the pH of the washing liquid is 7.0-7.2, and the dosage is 200-1000 ⁇ l.
  • the seventh object of the present invention is to provide an application of a functionalized biological multilayer porous membrane immune carrier in the preparation of blood group detection products.
  • the eighth object of the present invention is to provide a functionalized biological multilayer porous membrane immune carrier in the preparation of products for detecting and analyzing diseases including but not limited to protomicrobial cell antigen antibodies, bacterial cell antigen antibodies, tumor cell antigen antibodies Applications.
  • the preparation principle of the functionalized biological multilayer porous membrane immune carrier is that the Fab segment of the specific anti-biological multilayer porous membrane antibody binds to the biological multilayer porous membrane, and the Fc segment is free to outward; the cross-linked antibody One of the Fab segments can bind to the free Fc segment of the specific anti-biological multilayer pore membrane antibody, and the other Fab segment of the cross-linked antibody can bind to the specific antibody of the same species as the biological multilayer pore membrane specific antibody to form a functionalized Biological multilayer porous membrane immune carrier.
  • the preparation principle of the biological multilayer porous membrane immune carrier used for blood group compatibility detection is to first prepare the functionalized biological multilayer pore by the preparation method of the functionalized biological multilayer porous membrane immune carrier provided by the present invention
  • Membrane immune carrier is prepared by coupling cross-linked antibody and erythrocyte blood group antibody or sensitized erythrocyte blood group antigen or antigen-antibody specific binding reaction on a functionalized biological multilayer porous membrane immune carrier, and can be used for blood group detection.
  • the biological multilayer porous membrane immune carrier for blood group compatibility detection provided by the present invention can realize rapid and accurate detection of red blood cell ABO, Rh, MNS, Kell, Kidd and other blood type systems, and can simultaneously perform positive type, Anti-typing, antibody screening, antibody identification and other red blood cell blood group compatibility tests can also be selected according to the needs of real-time testing to suit various blood types and other testing requirements such as hospitals, families, and field emergencies.
  • the present invention utilizes the immunogenicity and multi-layer porosity of biological multilayer porous membranes, and uses biological techniques such as antigen-antibody specific affinity binding interaction to combine thick cotton cloth, wool fabric, silkworm cocoons (silk fabric), or plant fiber fabric
  • biological techniques such as antigen-antibody specific affinity binding interaction to combine thick cotton cloth, wool fabric, silkworm cocoons (silk fabric), or plant fiber fabric
  • the hydrophilic biological multilayer pore membrane is coupled with a biological multilayer pore membrane specific antibody and then combined with a cross-linked antibody to prepare a functional biological multilayer Pore membrane immune carrier, functionalized biological multilayer porous membrane immune carrier is coupled to red blood cell blood group antibody or sensitized red blood cell blood group antigen to prepare a biological multilayer porous membrane immune carrier for blood group compatibility testing, used for blood group compatibility testing
  • the biological multilayer porous membrane immune carrier and isolation membrane, absorbent cotton and plastic shell are assembled in order to form a blood type test card for blood type compatibility testing.
  • red blood cells react with the antibody on the carrier, whether they are agglutinated or not, they will be captured by the antibody on the carrier. When the membrane becomes red after washing, it is a positive result. On the contrary, if there is no specific antigen-antibody reaction between the red blood cells and the antibody on the carrier, the red blood cells will pass through the porous biomembrane and be sucked away by the absorbent cotton when washing with the washing liquid, and the membrane will show its natural color, which is a negative result, thus achieving rapid Perform positive blood type testing.
  • the blood group antibody in the blood sample reacts with the sensitized red blood cell blood group antigen coupled on the biological multilayer pore membrane, and the reverse-typed cell or Screen cells or spectrum cells.
  • the red blood cells that have undergone antigen-antibody reaction are captured and fixed on the porous biofilm and cannot be washed away.
  • the biological multilayer pore membrane is red, and the red blood cells without antigen-antibody reaction are washed by washing liquid.
  • the biological multi-layer porous membrane membrane shows the true color, so as to realize the anti-type detection, antibody screening and antibody identification.
  • the present invention has the following beneficial effects:
  • the biological multilayer porous membrane used in the present invention is derived from natural materials such as thick cotton cloth, wool fabric, silkworm cocoons (silk fabric), plant fiber woven, etc., using the immunogenicity of these natural materials, and its corresponding biological multilayer porous membrane
  • the antibody is conjugated and then combined with the cross-linked antibody to prepare a functionalized biological multilayer porous membrane immune carrier.
  • the specificity is The specific binding of antigen and antibody on the surface of the biological multilayer porous membrane carrier is stronger, the site is clearer, and the specificity is better.
  • the biological multilayer porous membrane used in the present invention is a multilayer membrane. Therefore, the surface area that binds to red blood cell blood group antigen or sensitized red blood cell blood group antibody is large, which is much larger than the surface area of the 96-well plate in the traditional ELISA experiment. Increased sensitivity.
  • the biological multilayer porous membrane immune carrier for blood group compatibility detection provided by the present invention can specifically bind to the blood group antigen or blood group antibody in the blood group sample to be tested, and has high adsorption capacity and firmness, which shortens the time for blood group detection and improves The sensitivity of blood group detection.
  • the blood type detection card provided by the present invention combines solid phase method and agglutination method to achieve blood type compatibility detection.
  • the porous feature of the multilayer porous membrane can directly wash away unreacted red blood cells. At the same time, as many single red blood cells that have a positive reaction are captured as much as possible, and the agglutination reaction is intercepted, making the detection more sensitive, specific and accurate.
  • the natural protein has a protective effect on biomolecules, the most important biologically active components in the test card of the present invention are protected, which is coupled to the biological multilayer porous membrane carrier.
  • Antigen and antibody, storage temperature is 4-50 °C, can maintain stable antigen activity and antibody titer, strong stability of the test card, storage temperature limit is small, can be stored at room temperature, limited by outdoor conditions, more suitable for harsh environments use.
  • the isolation membrane, the absorbent pad, the plastic shell and the inner shell included in the blood type detection card structure are all common consumable substrates, which are easy to store, low in cost and high in availability.
  • the isolation membrane used includes but is not limited to filter paper, cellulose fiber, glass fiber mat, or fiber mat or test paper made of porous polymer material, and its polymer material properties and porosity It can effectively separate red blood cells to prevent reverse osmosis, make the detection background clear, and accurately and quickly interpret blood group results.
  • the absorbent pad used can be cotton pulp pad, filter paper or other dense absorbent materials, which can effectively absorb excess liquid, facilitate immediate observation of the reaction results, and shorten the blood type detection time, 1 minute Perform blood type judgment and realize POCT (Immediate Detection) blood type detection.
  • the blood type detection card provided by the present invention adopts the vertical percolation method, which can greatly reduce the time required for blood chromatography and cleaning, simplify the operation steps, and shorten the detection time.
  • the blood type test card provided by the present invention is used to detect a positive type of blood type sample to be tested, and the required amount of blood type sample to be tested is 5-50 ⁇ l of fresh human whole blood, which requires less blood for testing and saves samples the amount.
  • the detection holes on the solid-phase plastic carrier of the blood type detection card include, but are not limited to, double holes, triple holes, and multiple holes. Red blood cell ABO, Rh, Kell, and Kell can be detected as needed. Kidd and others have discovered the blood group system and anti-typing, antibody screening and antibody identification recognized by the International Blood Transfusion Association.
  • the result judgment can be manually interpreted by the naked eye, and it can also be combined with conventional spectral detection for automatic judgment.
  • manual interpretation the difference between a positive result and a negative result is whether there is red or not, and the color difference is obvious, which can be quickly and accurately judged.
  • batch detection of blood types can be realized to meet the needs of blood group detection of a large number of samples.
  • the functionalized biological multilayer porous membrane immune carrier provided by the present invention can be used for blood group detection, and can also select corresponding specific capture antibodies for immune response, or continue to perform immune connection and binding according to needs. It includes but is not limited to pathogenic microorganisms, bacteria, tumors and various cell antigen antibody immunoassay detection and analysis.
  • Figure 1 is a schematic diagram of the ABO positive setting and RhD detection of the present invention
  • Figure 2 is a schematic diagram of the ABO reverse stereotype detection of the present invention.
  • Figure 3 is a process flow diagram of the preparation of the functionalized biological multilayer porous membrane immune carrier of the present invention.
  • FIG. 4 is a process flow diagram of the preparation process of the multilayer porous membrane immune carrier for blood group compatibility detection of the present invention
  • FIG. 5 is an exploded view of the structure of the blood type detection card of the present invention.
  • Fig. 6 is a diagram of the ABO positive setting detection result in an embodiment of the present invention.
  • Spensitized red blood cells refers to incomplete antibodies or autoantibodies that bind to red blood cell surface antigens or attach to red blood cell membranes to sensitize them.
  • Red blood cell profile cells refer to a group of red blood cells that carry most of the clinically meaningful blood group antigens and are used for red blood group identification.
  • Erythrocyte membrane antibodies other than blood group antibodies refers to red blood cell membrane antibodies but not antibodies against the red blood cell blood group system.
  • the preparation of a functionalized biological multilayer porous membrane immune carrier includes the following steps:
  • step 1 To the biological multilayer pore membrane washed in step 1) was added the final concentration of 20 ⁇ g/ml anti-silk protein antibody (antibody dosage about 200 ⁇ l/piece) for 2 hours at room temperature and overnight at 4°C. Wash the lotion for 3 times, through the commercially available HRP-anti-mouse Ig (G+A+M) detection and identification, after reacting at 37°C for 1 hour, wash the lotion for 3 times, add 200 ⁇ l/piece of precipitation substrate After 15 minutes of color development, the diaphragm is completely dark purple.
  • the preparation of a biological multilayer porous membrane immune carrier for the detection of human ABO and Rh(D) blood type positive setting includes the following steps:
  • the preparation of a biological multi-layer porous membrane immune carrier for the detection of human ABO blood group reverse typing includes the following steps:
  • red blood cells Take fresh type A, type B and type O red blood cells, respectively prepare 50% red blood cell suspension with normal saline, respectively add equal volumes of monoclonal anti-A, anti-B and anti-H (titer 8), and react at room temperature for 5 minutes At this time, the red blood cells will agglomerate like fine sand (agglutination intensity 1+), add 5 times the volume of hypotonic solution (0.1%-0.7% Nacl solution), stand at room temperature for 1 minute, and centrifuge at 12000 rpm for 5 minutes. The supernatant was discarded, and the sensitized red blood cell blood group antigen was collected and suspended to the original volume with normal saline.
  • step 2 Add the sensitized type A, type B and type O red blood cell antigens prepared in step 2) to the functionalized biological multilayer porous membrane immune carrier obtained in step 1), and react overnight at 4°C. Take it out, dry it at 20-25°C, and store it at 4°C for later use to prepare a biological multilayer porous membrane immune carrier for the detection of human ABO blood group reverse typing.
  • the preparation of a biological multilayer porous membrane immune carrier for red blood cell blood group antibody screening includes the following steps:
  • red blood cells I, II, and III Take the irregular antibody screening red blood cells I, II, and III from 3 individuals, respectively prepare 50% red blood cell suspension with normal saline, add an equal volume of monoclonal anti-H (titer 8), and react at room temperature for 5 minutes. At this time, the red blood cells will agglomerate like fine sand (agglutination intensity 1+), add 5 times the volume of hypotonic solution (0.1%-0.7% Nacl solution), stand at room temperature for 1 minute and centrifuge at 12000 rpm for 5 minutes. The supernatant was discarded, and the sensitized irregular antibody was collected for screening red blood cell antigens I, II, and III. Suspend to the original volume with normal saline.
  • step 2 Add the irregular antibody prepared in step 2) to screen red blood cell antigens I, II, and III to the functionalized biological multilayer porous membrane immune carrier prepared in step 1), and react overnight at 4°C. Take it out, dry at 20-25°C, and store at 4°C for later use to prepare a biological multilayer porous membrane immune carrier for red blood cell blood group antibody screening.
  • the preparation of a biological multilayer porous membrane immune carrier used for red blood cell blood group antibody identification includes the following steps:
  • red blood cell spectrum cells Take 12 groups of red blood cell spectrum cells, respectively prepare 50% red blood cell suspension with normal saline, add an equal volume of monoclonal anti-H (titer 8), and react at room temperature for 5 minutes. At this time, the red blood cells will appear fine sand-like agglutination (agglutination). Intensity 1+), add 5 times the volume of hypotonic solution (0.1%-0.7% NaCl solution), leave it at room temperature for 1 minute, and centrifuge at 12000 rpm for 5 minutes. The supernatant was discarded, and 12 groups of sensitized red blood cell profile cell antigens were collected and suspended to the original volume with normal saline.
  • the 12 sets of red blood cell profiling cell antigens prepared in step 2) were added to 12 sets of functionalized biological multilayer porous membrane immune carrier couplings prepared in step 1), and reacted overnight at 4°C. Take it out, dry it at 20-25°C, and store it at 4°C for later use to prepare a biological multilayer porous membrane immune carrier for red blood cell blood group antibody identification.
  • the specific biological multilayer porous membrane antibody is diluted with a diluent to a final concentration of 1-100 ⁇ g/ml;
  • the cross-linked antibody is diluted with a diluent to a final concentration of 1-100 ⁇ g/ml. ml;
  • the diluent is 0.01mol/L, PH 7.0-7.2 phosphate buffer;
  • the treatment solution is a 0.01 mol/L phosphate buffer solution with a pH of 7.0-7.2 containing 1% bovine serum albumin;
  • the washing solution is a phosphate buffer solution with a pH of 7.0-7.2 and 0.01 mol/L;
  • the blocking solution is a 5% skimmed milk powder prepared with a phosphate buffer with a pH of 7.0-7.2 and 0.01 mol/L; wherein the unit% represents the mass of the solute in 100 ml of the solution.
  • a blood type detection card includes a solid-phase plastic carrier made of polyethylene.
  • the solid-phase plastic carrier includes a card slot outer shell 1 and a card slot inner shell 2, a card slot outer shell 1 and a card slot inner shell 2 Removably connected;
  • the blood type detection card slot shell 1 is also provided with a first detection hole 3, a second detection hole 4, and a third detection hole 5;
  • the first detection hole 3, the second detection hole 4, and the Three detection holes 5 are placed at the bottom of the biological multilayer porous membrane immune carrier prepared in any one of Example 2 to Example 5 for blood group compatibility testing;
  • the card slot inner shell 2 is provided with the first
  • the three grooves 6 corresponding to the detection hole 3, the second detection hole 4, and the third detection hole 5 are placed on the bottom of the groove 6 with absorbent cotton, which is a cotton pad;
  • the isolation membrane is a glass fiber mat.
  • the number of the detection holes is not limited to three, and can be specifically set according to the detection object and detection conditions of the detection card, and may be double holes, three holes, and multiple holes.
  • the number of test holes is 3.
  • the bottoms of the first test hole 3, the second test hole 4, and the third test hole 5 are respectively equipped with the anti-conjugate prepared in Example 2 A.
  • Fig. 6 is the display result of the test card in this embodiment.
  • the RBC that has undergone antigen-antibody reaction is captured and fixed and blocked on the porous biofilm and cannot be washed away.
  • the red blood cells in the detection hole are enriched and appear red, which is a positive result.
  • the RBC that has not undergone antigen-antibody reaction is washed through the washing fluid. If the pore membrane is sucked away by absorbent cotton, and the detection hole has no red blood cell enrichment, it is a negative result.
  • the natural color of the detection hole is blue; when anti-B is bound, the natural color of the detection hole is yellow; In combination with anti-D, the color of the detection hole is colorless.
  • Figure 6 shows the color development of the color hole. Since Figure 6 is not a color image, the color observation is not obvious.
  • the color display results of this embodiment can also be referred to Table 1.
  • the number of test holes is 3.
  • the bottoms of the first test hole 3, the second test hole 4, and the third test hole 5 are respectively equipped with the specific binding prepared in Example 3 Biological multilayer porous membrane immune carrier for sensitized red blood cell A blood group antigen, sensitized red blood cell B blood group antigen, and sensitized red blood cell H antigen;
  • red blood cells that have undergone antigen-antibody reaction are captured and fixed on the porous biofilm and cannot be washed away. If red blood cells are enriched in the detection hole and appear red, it is a positive result; on the contrary, the red blood cells that have not undergone antigen-antibody reaction are washed through the porous by washing fluid If the membrane is sucked away by absorbent cotton, and the detection hole has no red blood cell enrichment and appears as the natural color of the membrane, it is a negative result. Finally, the ABO anti-sizing result is determined according to the identification of the test hole with the positive result.
  • Example 6 Using the blood type test card provided in Example 6, the number of test holes is 3, and the bottoms of the first test hole 3, the second test hole 4, and the third test hole 5 are respectively equipped with the irregular antibodies of Example 4 for 3 individuals
  • the biological multilayer porous membrane immune carrier prepared by screening red blood cells I, II, and III for the identification of red blood cell blood group antibodies;
  • the RBC that has undergone antigen-antibody reaction is captured and fixed on the porous biofilm and cannot be washed away. If the red blood cell is enriched in the detection hole and appears red, it is a positive result; on the contrary, the RBC that has not undergone the antigen-antibody reaction is washed through the porous by the washing fluid If the membrane is sucked away by absorbent cotton, and the detection hole has no red blood cell enrichment, it is a negative result. Finally, the antibody screening result is determined according to the identification of the test hole with a positive result.
  • the number of test holes is 12, and the bottoms of the first test holes 3-12 are respectively equipped with antibodies for red blood cell blood group prepared in Example 5 using 12 sets of sensitized red blood cell profile cell antigens.
  • red blood cells that have undergone antigen-antibody reaction are captured and fixed on the porous biofilm and cannot be washed away. If red blood cells are enriched in the detection hole and appear red, it is a positive result; on the contrary, the red blood cells that have not undergone antigen-antibody reaction are washed through the porous by washing fluid If the membrane is sucked away by absorbent cotton, and the detection hole has no red blood cell enrichment, it is a negative result. Finally, the antibody identification result is judged according to the identification of the detection hole with the positive result.
  • the detection method disclosed in the present invention is beneficial to promote clinical red blood cell blood group compatibility detection, including red blood cell positive typing, reverse typing, antibody screening, antibody identification, etc., and can realize rapid and accurate detection of red blood cell ABO, Rh, MNS, Kell, Kidd, etc.
  • the blood types of multiple blood type systems can also be selected according to the needs of POCT detection (real-time detection) to suit various blood type detection situations such as hospitals, families, and field emergencies.
  • the temperature required for storage or detection of the detection card provided by the present invention is 4°C-50°C, which overcomes the strict requirements for storage or detection temperature of existing blood type detection products.

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Abstract

本发明提供了一种功能化生物多层孔隙膜免疫载体，包括生物多层孔隙膜、与生物多层孔隙膜结合的特异性抗生物多层孔隙膜抗体、与膜抗体结合的交联抗体。还提供了功能化生物多层孔隙膜免疫载体的制备方法，利用生物多层孔隙膜的免疫原性及多层多孔性，结合抗体-抗原和抗体-抗体特异性结合进行制备。本发明提供的功能化生物多层孔隙膜免疫载体可以用于制备血型相容性检测的免疫载体，进而可以被安装在血型检测卡中进行血型检测。本发明还提供了血型检测卡进行血型检测的方法。本发明提供的用于血型相容性检测的免疫载体可以用于红细胞正定型、反定型、抗体筛检、抗体鉴定等，可实现快速、准确检测红细胞ABO、Rh、MNS、Kell、Kidd等多种血型系统血型。

Description

一种功能化生物多层孔隙膜免疫载体、制备方法、应用 技术领域
本发明涉及免疫检测领域，特别涉及一种功能化生物多层孔隙膜免疫载体、制备方法以及应用。
背景技术
血液供者和受者之间不相容的血型会导致严重的溶血性输血反应，血型相容性检测是确保安全输血的最重要途径之一，相容(Compatible)即和谐共存，指病人血清中不存在与输入的红细胞抗原相对应的特异性抗体，不发生免疫反应称为相容性输血。反之称为不相容输血，即病人血清中存在与输入的红细胞抗原相对应的特异性的抗体，发生免疫反应，输入病人体内的红细胞受损以至溶血。红细胞(Red Blood Cell)表面的特定抗原和血清中的抗体决定了血型，输血前血型相容性检测不仅包括ABO正反定型、RhD血型定型、受血者血清中不规则抗体检测、临床有意义抗体的检测和鉴定以及交叉配血试验等。
血型检测最传统的金标准检测方法是试管法，试管法需要繁琐的离心和洗涤步骤，以及细胞悬浮液的制备。其它常规的血型检测方法包括玻片法、微柱凝胶免疫检测法(Micrcolumn gel immunoassay，MGIA)以及微孔板固相检测法等。玻片检测技术是一种廉价、快速的血型方法。但是这种方法对检测弱抗原亚群不够敏感，而且只能进行正定型检测。MGIA可进行正定型和反定型检测，而且能够避免清洗步骤，但是待检样品中纤维蛋白原或明显的白细胞增多，则会导致假阳性结果，同时需要孵化和离心步骤，因此不适用 发展落后的地区，特别是在大多数发展中国家，不方便用作床边检测血型。此外，原材料凝胶颗粒的高价格垄断和有限的检测通量也是限制MGIA在临床和血站进一步广泛使用的不利因素。微孔板固相检测法则提高了检测的灵敏度，但也同样需要复杂的孵育和洗涤步骤。除上述方法外，由于纤维滤纸具有可以过滤出红细胞并保留凝集红细胞的纤维特性，因此滤纸用于检测血型的方法得到了广泛的报道。滤纸检测技术主要是通过物理吸附将抗体固定到滤纸上，吸附能力有限，所以检测的敏感性较低，在临床上没有得到广泛的应用。此外，IgG类的单克隆抗体不能作为固定抗体在这类纤维纸上使用，因为即使有阳性反应，但没有凝集，阳性反应的红细胞(红细胞)仍将被冲走，导致假阴性反应的发生，同时受抗体保存条件限制，不适用于野外紧急条件使用。
发明内容
为了克服现有技术的不足，本发明的一个目的是提供一种功能化生物多层孔隙膜免疫载体，包括生物多层孔隙膜、通过抗原抗体特异性结合而依附在所述生物多层孔隙膜的表面的特异性抗生物多层孔隙膜抗体、与所述特异性抗生物多层孔隙膜抗体偶联结合在一起的交联抗体，本发明提供的功能化生物多层孔隙膜免疫载体可以通过偶联或者抗体抗原特异性结合再与红细胞血型抗体或致敏红细胞血型抗原结合，从而进一步用于血型检测；本发明提供的功能化生物多层孔隙膜免疫载体还可以根据需要，选择相应的特异性捕获抗体，进行免疫反应或继续进行免疫选择和结合，从而用于检测和分析病原微生物细胞抗原抗体、细菌细胞抗原抗体、肿瘤细胞抗原抗体以及各种细胞抗原抗体。
本发明提供一种功能化生物多层孔隙膜免疫载体，包括：
生物多层孔隙膜；
特异性生物多层孔隙膜抗体，所述特异性抗生物多层孔隙膜抗体与所述 生物多层孔隙膜发生抗原抗体特异性结合从而依附在所述生物多层孔隙膜的表面；
交联抗体，所述交联抗体与所述特异性抗生物多层孔隙膜抗体偶联结合在一起；
其中，所述特异性抗生物多层孔隙膜抗体为能够与所述生物多层孔隙膜发生抗原抗体特异性反应的抗体；
所述交联抗体一Fab端能与所述特异性抗生物多层孔隙膜抗体偶联，另一Fab端能够与特异性抗体结合。
优选地，所述特异性生物多层孔隙膜抗体包括鼠源单抗、羊源多抗、兔源单抗/多抗、鸡源多抗；所述交联抗体包括抗鼠多抗、抗羊多抗、抗兔多抗、抗鸡多抗；所述能够与交联抗体Fab端结合的特异性抗体包括鼠源单抗、羊源多抗、兔源单抗/多抗、鸡源多抗。
本发明的第二个目的是提供一种功能化生物多层孔隙膜免疫载体的制备方法，包括以下步骤：
S1、将清洗干净的厚棉布或羊毛织物或真丝织物或植物纤维织物剪切成圆形或方形膜片，然后将膜片浸入处理液中，在40-70℃水浴中浸泡2-4小时，制得生物多层孔隙膜；
S2、将所述生物多层孔隙膜与特异性抗生物多层孔隙膜抗体进行抗原抗体特异性结合，洗液清洗，再与交联抗体进行偶联后，洗液清洗，用封闭液封闭后再用洗液清洗，制得如上所述的功能化生物多层孔隙膜免疫载体；
其中，所述特异性抗生物多层孔隙膜抗体为能够与所述生物多层孔隙膜发生抗原抗体特异性反应的抗体；
所述交联抗体一Fab端能与所述特异性抗生物多层孔隙膜抗体偶联，另一Fab端能够与特异性抗体结合。
优选地，所述特异性抗生物多层孔隙膜抗体用稀释液稀释为终浓度1-100μg/ml；所述交联抗体用稀释液稀释为终浓度1-100μg/ml；其中，所 述稀释液为0.01mol/L，PH 7.0-7.2的磷酸盐缓冲液；
优选地，所述处理液为含有1％牛血清白蛋白的0.01mol/L，PH为7.0-7.2的磷酸盐缓冲液；
优选地，所述洗液为0.01mol/L，PH为7.0-7.2的磷酸盐缓冲液；
优选地，所述封闭液为用0.01mol/L，PH为7.0-7.2的磷酸盐缓冲液配制的5％脱脂奶粉；其中，单位％表示100ml溶液中的溶质的质量。
本发明的第三个目的是提供一种用于血型相容性检测的生物多层孔隙膜免疫载体，包括：
本发明如上所述的功能化生物多层孔隙膜免疫载体；
红细胞血型抗体或致敏红细胞血型抗原，通过与所述功能化生物多层孔隙膜免疫载体上的所述交联抗体进行特异性结合而依附在所述功能化生物多层孔隙膜免疫载体上；
其中，所述致敏红细胞血型抗原为特异性结合了非血型抗体的红细胞膜抗体的红细胞血型抗原。
优选地，所述红细胞血型抗体包括IgM型、IgG型、IgM-IgG型混合型的红细胞A、B、Rh、MNS、Kell、Kidd血型的抗体，所述红细胞血型抗体效价大于1:32。
优选地，所述致敏红细胞血型抗原包括致敏A型红细胞抗原、致敏B型红细胞抗原、致敏不规则抗体筛检红细胞抗原、致敏红细胞谱细胞抗原。
本发明的第四个目的是提供一种用于血型相容性检测的生物多层孔隙膜免疫载体的制备方法，包括以下步骤：
S1、将清洗干净的厚棉布或羊毛织物或真丝织物或植物纤维织物剪切成圆形或方形膜片，然后将膜片浸入处理液中，在40-70℃水浴中浸泡2-4小时，制得生物多层孔隙膜；
S2、将所述生物多层孔隙膜与特异性抗生物多层孔隙膜抗体进行抗原抗体特异性结合，洗液清洗，再与交联抗体进行偶联后，洗液清洗，用封闭液 封闭后再用洗液清洗，制得如上所述功能化生物多层孔隙膜免疫载体；
S3、将红细胞血型抗体或致敏红细胞血型抗原与所述功能化生物多层孔隙膜免疫载体上的所述交联抗体进行特异性结合，制得如上所述的用于血型相容性检测的生物多层孔隙膜免疫载体；
其中，所述特异性抗生物多层孔隙膜抗体为能够与所述生物多层孔隙膜发生抗原抗体特异性反应的抗体；
所述交联抗体一Fab端能与所述特异性抗生物多层孔隙膜抗体偶联，另一Fab端能够与特异性抗体结合，所述特异性抗体为能够与交联抗体Fab端结合的红细胞血型抗体或致敏红细胞血型抗原；
所述致敏红细胞血型抗原为特异性结合了非血型抗体的红细胞膜抗体的红细胞血型抗原。
优选地，还包括步骤致敏红细胞血型抗原的制备，具体制备致敏红细胞血型抗原的步骤如下：用非血型抗体的红细胞膜抗体与新鲜红细胞反应，待出现1+凝集强度时，用低渗溶液将红细胞裂解，离心收集获得致敏红细胞抗原。
优选地，所述低渗溶液为0.1％-0.7％Nacl溶液；
优选地，所述离心的转速为12000rpm，离心时间为5min。
本发明的第五个目的是提供一种血型检测卡，包括：
固相塑料载体，所述固相塑料载体包括卡槽外壳和卡槽内壳，所述卡槽外壳与所述卡槽内壳可拆卸连接；
如上所述的用于血型相容性检测的生物多层孔隙膜免疫载体，所述用于血型相容性检测的生物多层孔隙膜免疫载体位于所述固相塑料载体卡槽外壳的底部；
吸水棉，位于固相塑料载体卡槽内壳的底部；
隔离膜，放置在所述吸水棉的上方。
优选地，所述固相塑料载体的制备材料包括聚乙烯、聚乙烯、聚苯乙烯、 苯乙烯-丁二烯-丙烯腈；所述固相塑料载体，还包括检测孔，所述检测孔包括双孔、三孔、多孔；所述隔离膜包括滤纸、纤维素纤维、玻璃纤维垫、多孔高分子材料制成的纤维垫或试纸；所述吸水棉包括棉浆垫、滤纸。
优选地，所述血型检测卡的保存或检测所需温度为4-50℃。
本发明的第六个目的是提供一种血型的检测方法，包括以下步骤：
将待检测血型样品加入到如上所述的血型检测卡的检测孔中，放置至少1分钟；洗液冲洗，观察检测孔的显色情况，显色为红色则为阳性，显色为本色则为阴性；
其中，待检测血型样品与血型检测卡中的所述用于血型相容性检测的生物多层孔隙膜免疫载体发生了抗原抗体特异性反应，为阳性；反之，为阴性。
优选地，所述洗液为含有0.05％-0.5％Tween-20的缓冲液，洗液PH值为7.0-7.2，用量为200-1000μl。
本发明的第七个目的是提供一种功能化生物多层孔隙膜免疫载体在制备血型检测产品中的应用。
本发明的第八个目的是提供一种功能化生物多层孔隙膜免疫载体在制备用于检测和分析病包括但不限于原微生物细胞抗原抗体、细菌细胞抗原抗体、肿瘤细胞抗原抗体的产品中的应用。
本发明中，功能化生物多层孔隙膜免疫载体的制备原理是所述特异性抗生物多层孔隙膜抗体的Fab段结合所述生物多层孔隙膜，Fc段游离向外；交联抗体的其中一个Fab段能结合特异性抗生物多层孔隙膜抗体游离Fc段，交联抗体的另一个Fab段能结合与生物多层孔隙膜特异性抗体同种属的特异性抗体，形成功能化的生物多层孔隙膜免疫载体。
本发明中，用于血型相容性检测的生物多层孔隙膜免疫载体的制备原理是先通过本发明所提供的功能化生物多层孔隙膜免疫载体的制备方法制备得到功能化生物多层孔隙膜免疫载体，在通过功能化生物多层孔隙膜免疫载体上结合的交联抗体与红细胞血型抗体或致敏红细胞血型抗原发生偶联或抗原 抗体特异性结合反应制得，可用于血型检测。
本发明提供的用于血型相容性检测的生物多层孔隙膜免疫载体，能够实现快速、准确检测红细胞ABO、Rh、MNS、Kell、Kidd等多种血型系统的血型，可同时进行正定型、反定型、抗体筛检、抗体鉴定等红细胞血型相容性检测，还可根据即时检测需要进行项目选择，以适合医院、家庭以及野外紧急等多种血型以及其它检测要求。
本发明利用生物多层孔隙膜的免疫原性以及多层多孔性，利用抗原抗体特异性的亲和结合相互作用等生物学技术将厚棉布、羊毛织物，蚕茧(真丝织物)，或者植物纤维织物制备成具有生物学活性的亲水性生物多层孔隙膜；亲水性生物多层孔隙膜通过与生物多层孔隙膜特异性抗体偶联后再与交联抗体结合制备成功能化生物多层孔隙膜免疫载体，功能化生物多层孔隙膜免疫载体偶联红细胞血型抗体或者致敏红细胞血型抗原后制备成血型相容性检测的生物多层孔隙膜免疫载体，用于血型相容性检测的生物多层孔隙膜免疫载体与隔离膜、吸水棉以及塑料外壳，按顺序组装形成血型检测卡，用于血型相容性检测。红细胞与载体上的抗体若是发生反应，无论是否凝集，都会被载体上的抗体捕获，当膜在洗涤后呈红色时，为阳性结果。相反，如果红细胞和载体上的抗体之间没有特异性抗原抗体反应时，用洗液洗涤时，红细胞穿过多孔隙生物膜并被吸水棉吸走，膜显示本色，为阴性结果，从而实现快速进行血型正定型检测。对于其它血型相容性检测，则是将待检测血浆或者血清加入到反应孔中后，血样中的血型抗体与生物多层孔隙膜上偶联的致敏红细胞血型抗原反应，加入反定型细胞或者筛检细胞或者谱细胞，洗液冲洗后，发生抗原抗体反应的红细胞被捕获固定在多孔隙生物膜上冲洗不掉，生物多层孔隙膜显红色，没发生抗原抗体反应的红细胞经冲洗液冲洗通过多孔隙膜被吸水棉吸走，生物多层孔隙膜膜显示本色，从而实现反定型检测、抗体筛检以及抗体鉴定。
相比现有技术，本发明的有益效果在于：
(1)本发明所采用生物多层孔隙膜来自于厚棉布、羊毛织物、蚕茧(真丝织物)、植物纤维织等天然材料，利用这些天然材料的免疫原性，与其相应的生物多层孔隙膜抗体偶联，再与交联抗体结合制备成了功能化生物多层孔隙膜免疫载体，相对于传统ELISA(酶联免疫吸附)实验中96孔板塑料表面的无序、非特异性结合，特异性生物多层孔隙膜载体表面抗原抗体特异性结合更牢固，位点更明确，特异性更好。
(2)本发明所采用的生物多层孔隙膜，是多层膜，因此与红细胞血型抗原或致敏红细胞血型抗体结合的表面积大，远大于传统ELISA实验中96孔板微孔表面积，使反应敏感性增强。
(3)本发明所提供的血型相容性检测的生物多层孔隙膜免疫载体能够特异性结合待检测血型样品中的血型抗原或血型抗体，吸附能力高且牢固，缩短血型检测的时间，提高血型检测的灵敏度。
(4)本发明所提供的的血型检测卡，是将固相法和凝集法结合，实现对血型相容性检测，多层孔隙膜的多孔性特点可实现将未反应的红细胞直接冲走，同时尽可能多的捕获发生阳性反应的单个红细胞，截留凝集反应的凝集块，使检测更为敏感、特异、准确。
(5)本发明所提供的血型检测卡，由于天然蛋白对生物分子具有保护作用，所以本发明的检测卡中最主要生物活性组分都被保护，偶联在生物多层孔隙膜载体上的抗原抗体，保存温度在4-50℃，可保持稳定的抗原活性以及抗体效价，检测卡的稳定性强，保存温度限制小，可常温保存，受户外条件限制小，更适合在恶劣环境中使用。
(6)本发明的一些优选方案中，血型检测卡结构中包括的所述隔离膜、吸水垫、塑料外壳和内壳，其基质均为常见耗材，易保存，成本低，可获得性高。
(7)本发明的一些优选方案中，所采用的隔离膜包括但不限于滤纸、纤 维素纤维、玻璃纤维垫或者多孔高分子材料制成的纤维垫或试纸，其高分子材料性能以及多孔性可有效分离红细胞防止反渗，使检测背景清晰，准确快速进行血型结果判读。
(8)本发明的一些优选方案中，所采用的吸水垫可为棉浆垫、滤纸或其他致密的吸水性材料，可有效吸取多余液体，便于立即观察反应结果，缩短血型检测时间，1分钟进行血型判断，实现POCT(即时检测)血型检测。
(9)本发明所提供的血型检测卡，所采用的垂直渗滤法，可极大减少血液层析以及清洗所需时间，简化操作步骤，缩短检测时间。
(10)本发明所提供的血型检测卡，用于检测正定型待检测血型样品时，所需待检血型样品的量为5-50μl的人新鲜全血，检测所需血量少，节约样本量。
(11)本发明的一些优选方案中，所提供的血型检测卡采取的固相塑料载体上的检测孔包括但不限于双孔、三孔、多孔，可根据需要检测红细胞ABO、Rh、Kell、Kidd等已经发现并被国际输血协会承认的血型系统以及反定型、抗体筛检和抗体鉴定。
(12)本发明所提供的检测方法，结果判定可以采用肉眼进行人工判读，同时也可以结合常规光谱检测进行自动判断。人工判读时，阳性结果与阴性结果区别在于有无红色，颜色差异明显，可快速准确判断。结合常规光谱检测时，可实现血型的批量化检测，以满足大量样本血型检测需求。
(13)本发明所提供的功能化生物多层孔隙膜免疫载体，除了用于血型检测，还可根据需要，选择相应的特异性捕获抗体，进行免疫反应，或者继续进行免疫连接和结合，用于包括但不限于病原微生物、细菌、肿瘤以及各种细胞抗原抗体免疫实验检测和分析。
上述说明仅是本发明技术方案的概述，为了能够更清楚了解本发明的技术手段，并可依照说明书的内容予以实施，以下以一些实施例来详细说明。本发明的具体实施方式由以下实施例详细给出。
附图说明
此处所说明的附图用来提供对本发明的进一步理解，构成本申请的一部分，本发明的示意性实施例及其说明用于解释本发明，并不构成对本发明的不当限定。在附图中：
图1为本发明ABO正定型及RhD检测原理图；
图2为本发明的ABO反定型检测原理图；
图3为本发明的功能化生物多层孔隙膜免疫载体的制备工艺流程图；
图4为本发明的用于血型相容性检测的多层孔隙膜免疫载体的制备工艺流程图；
图5为本发明的血型检测卡结构分解图；
图6为本发明一实施例中的ABO正定型检测结果图。
其中，图中：1、卡槽外壳；2、卡槽内壳；3、第一检测孔；4、第二检测孔；5、第三检测孔；6、凹槽。
具体实施方式
为了可以更充分地理解本文描述的发明，阐述以下实施例。应当理解这些实施例仅用于说明性目的并且不应被理解为以任何方式限制本发明。
先对本文中所使用的的一些术语进行解释。
“致敏红细胞”是指不完全抗体或自身抗体与红细胞表面抗原结合或附着在红细胞膜上使其致敏。
“红细胞谱细胞”指一组携有绝大多数临床有意义血型抗原的，用于红细胞血型鉴定的多人份红细胞。
“非血型抗体的红细胞膜抗体”是指红细胞膜抗体但不是针对红细胞血型系统的抗体。
为了可以更充分地理解本文描述的发明，阐述以下实施例。
实施例1
如图3所示，功能化生物多层孔隙膜免疫载体的制备，包括如下步骤：
1)制备生物多层孔隙膜
首先将蚕茧(真丝织物)从长轴方向剪开，去除蚕蛹、杂丝及其他杂质。用纯水清洗两遍，放入100℃水中浸泡15min，取出后室温晾干。然后用打孔器打成5-9mm圆形生物多层孔隙膜。加入处理液浸泡蚕片，50℃水浴中孵育2小时。用真空干燥机真空除气泡一次，所有圆形生物多层孔隙膜都沉入处理液底部，50℃水浴中继续孵育2小时，洗液洗涤2次，制备得到所需生物多层孔隙膜。
2)制备功能化生物多层孔隙膜免疫载体
向步骤1)中洗好的生物多层孔隙膜中加入终浓度20μg/ml抗蚕丝蛋白抗体(抗体用量约200μl/片)室温振荡反应2小时，4℃过夜。洗液洗涤3次，通过商业途径可采购的HRP-anti-mouse Ig(G+A+M)检测鉴定，37℃反应1小时后，洗液洗涤3次，加入200μl/片的沉淀性底物显色15min，膜片完全显深紫色。向偶联好蚕丝蛋白抗体的生物多层孔隙膜中加入羊抗鼠IgG交联抗体，终浓度20μg/ml，室温振荡反应2小时，4℃过夜。洗液洗涤3次，用HRP-anti-GST检测鉴定，37℃反应1小时后，37℃用封闭液封闭2小时，洗液洗涤3次，膜片完全显深紫色，制备得到功能化生物多层孔隙膜免疫载体；其中，在偶联和封闭过程中需将膜完全浸泡。
实施例2
如图4所示，用于人ABO、Rh(D)血型正定型检测的生物多层孔隙膜免疫载体的制备，包括如下步骤：
1)按照实施例1所述方法制备功能化生物多层孔隙膜免疫载体；
2)功能化生物多层孔隙膜免疫载体偶联红细胞血型抗体
向步骤1)所得的功能化生物多层孔隙膜免疫载体中分别加入效价为128的通过商业途径可采购的单克隆抗-A，抗-B，4℃过夜。20-25℃室温晾干，4℃保存备用，制得用于人ABO血型检测的生物多层孔隙膜免疫载体。
向另一部分生物多层孔隙膜免疫载体中加入单克隆抗人IgG终浓度20μg/ml，4℃过夜后，洗液洗涤3次，加入效价为512抗-D，4℃过夜。室温20-25℃晾干，4℃保存备用，制得用于人Rh(D)血型检测的生物多层孔隙膜免疫载体。
实施例3
用于人ABO血型反定型检测的生物多层孔隙膜免疫载体的制备，包括如下步骤：
1)按照实施例1所述方法制备功能化生物多层孔隙膜免疫载体。
2)致敏红细胞血型抗原的制备
取新鲜A型、B型和O型红细胞，分别用生理盐水配制成50％红细胞悬液，分别对应加入等体积的单克隆抗A、抗B和抗H(效价8)，室温反应5分钟，此时红细胞将出现细沙状凝集(凝集强度1+)，加入5倍体积的低渗溶液(0.1％-0.7％Nacl溶液)，室温静置1分钟后12000rpm离心5分钟。弃去上清，收集获得致敏红细胞血型抗原，并用生理盐水悬浮至原体积。
3)功能化生物生物多层孔隙膜免疫载体与致敏红细胞血型抗原发生抗体-抗体特异性结合制备用于人ABO血型反定型检测的生物多层孔隙膜免疫载体
向步骤1)所得的功能化生物多层孔隙膜免疫载体中加入步骤2)制备的致敏A型、B型和O型红细胞抗原，4℃反应过夜。取出，20-25℃晾干，4℃保存备用，制得用于人ABO血型反定型检测的生物多层孔隙膜免疫载体。
实施例4
用于红细胞血型抗体筛检的的生物多层孔隙膜免疫载体的制备，包括如下步骤：
1)按照实施例1所述方法制备功能化生物多层孔隙膜免疫载体。
2)不规则抗体筛检红细胞抗原的制备
取分别来自3个人的不规则抗体筛检红细胞I、II、III，分别用生理盐水配制成50％红细胞悬液，分别加入等体积的单克隆抗H(效价8)，室温反应5分钟，此时红细胞将出现细沙状凝集(凝集强度1+)，加入5倍体积的低渗溶液(0.1％-0.7％Nacl溶液)，室温静置1分钟后12000rpm离心5分钟。弃去上清，收集获得致敏不规则抗体筛检红细胞抗原I、II、III。并用生理盐水悬浮至原体积。
3)功能化生物多层孔隙膜免疫载体和不规则抗体筛检红细胞抗原发生抗体-抗体特异性结合
向步骤1)制得的功能化生物多层孔隙膜免疫载体中加入步骤2)制备的不规则抗体筛检红细胞抗原I、II、III，4℃反应过夜。取出，20-25℃晾干，4℃保存备用，制得用于红细胞血型抗体筛检的的生物多层孔隙膜免疫载体。
实施例5
用于红细胞血型抗体鉴定的的生物多层孔隙膜免疫载体的制备，包括如下步骤：
1)按照实施例1所述方法制备功能化生物多层孔隙膜免疫载体。
2)致敏红细胞谱细胞抗原的制备
取12组红细胞谱细胞，分别用生理盐水配制成50％红细胞悬液，分别加入等体积的单克隆抗H(效价8)，室温反应5分钟，此时红细胞将出现细沙状凝集(凝集强度1+)，加入5倍体积的低渗溶液(0.1％-0.7％Nacl溶液)，室温静置1分钟后12000rpm离心5分钟。弃去上清，收集获得12组致敏红细胞谱细胞抗原，并用生理盐水悬浮至原体积。
3)功能化生物多层孔隙膜免疫载体和致敏红细胞谱细胞抗原发生抗原抗体特异性结合
向12份步骤1)制得的功能化生物多层孔隙膜免疫载体偶联中分别加入 步骤2)制备的12组红细胞谱细胞抗原，4℃反应过夜。取出，20-25℃晾干，4℃保存备用，制得用于红细胞血型抗体鉴定的生物多层孔隙膜免疫载体。
应当解释的，实施例1-5中，所述特异性生物多层孔隙膜抗体用稀释液稀释为终浓度1-100μg/ml；所述交联抗体用稀释液稀释为终浓度1-100μg/ml；其中，所述稀释液为0.01mol/L，PH 7.0-7.2的磷酸盐缓冲液；
所述处理液为含有1％牛血清白蛋白的0.01mol/L，PH为7.0-7.2的磷酸盐缓冲液；所述洗液为0.01mol/L，PH为7.0-7.2的磷酸盐缓冲液；所述封闭液为用0.01mol/L，PH为7.0-7.2的磷酸盐缓冲液配制的5％脱脂奶粉；其中，单位％表示100ml溶液中的溶质的质量。
实施例6
如图5所示，一种血型检测卡，包括聚乙烯材质的固相塑料载体，所述固相塑料载体包括卡槽外壳1和卡槽内壳2，卡槽外壳1和卡槽内壳2可拆卸地连接；所述血型检测卡卡槽外壳1还设有第一检测孔3、第二检测孔4、第三检测孔5；所述第一检测孔3、第二检测孔4、第三检测孔5的底部放置实施例2-实施例5任一实施例所制备得到的用于血型相容性检测的生物多层孔隙膜免疫载体；所述卡槽内壳2设有与第一检测孔3、第二检测孔4、第三检测孔5相对应的3个凹槽6，所述凹槽6的底部放置吸水棉，所述吸水棉为棉浆垫；所述吸水棉上放置隔离膜，所述隔离膜为玻璃纤维垫。
应当理解的，所述检测孔的数量不仅仅局限于为3个，根据检测卡检测对象和检测条件具体设置，可以为双孔、三孔、多孔。
实施例7
ABO正定型及RhD血型检测及检测结果
采用实施例6所提供的血型检测卡，检测孔的数量为3，第一检测孔3、第二检测孔4、第三检测孔5底部分别装有实施例2所制备的偶联了抗-A、抗-B、抗-D的生物多层孔隙膜免疫载体；
取新鲜全血，向第一检测孔3、2、3中分别滴加10μl全血，室温放置1分钟。每孔分别滴加3滴冲洗液(PH值为7.0的0.05％Tween-20的缓冲液)，然后观察红细胞分布情况并判定检测结果，检测原理如图1所示。
如图6所示，图6为本实施例检测卡的显示结果。发生抗原抗体反应的RBC被捕获固定阻滞在多孔隙生物膜上冲洗不掉，检测孔中有红细胞富集呈现红色，为阳性结果；反之，没发生抗原抗体反应的RBC经冲洗液冲洗通过多孔隙膜被吸水棉吸走，检测孔无红细胞富集呈现为本色，则为阴性结果。其中，若检测孔底部的用于血型相容性检测的生物多层孔隙膜免疫载体中结合有抗-A，则检测孔本色为蓝色；结合有抗-B，则检测孔本色为黄色；结合有抗-D，则检测孔本色为无色。最后按照出现阳性结果的检测孔标识判定结果。
图6为显色孔显色情况，由于图6不是彩图，色彩观察不明显，本实施例的颜色显示结果还可以参表1。
表1 检测结果显色情况表
Figure PCTCN2019095958-appb-000001
实施例8
ABO反定型检测
采用实施例6所提供的血型检测卡，检测孔的数量为3，第一检测孔3、第二检测孔4、第三检测孔5底部分别装有实施例3所制得的特异性结合了致敏红细胞A血型抗原、致敏红细胞B血型抗原、致敏红细胞H抗原的生物多层孔隙膜免疫载体；
取新鲜待检样本(血浆或血清)，向第一检测孔3、2、3中分别滴加一滴 样本，室温放置1分钟。向对应孔中分别加入1滴ABO反定型红细胞，室温放置1分钟。然后再向相应孔中分别滴加每孔分别滴加3滴冲洗液(PH值为7.0的0.05％Tween-20的缓冲液)，观察红细胞分布情况并判定检测结果，检测原理如图2所示。
发生抗原抗体反应的红细胞被捕获固定在多孔隙生物膜上冲洗不掉，检测孔中有红细胞富集呈现红色，则为阳性结果；反之，没发生抗原抗体反应的红细胞经冲洗液冲洗通过多孔隙膜被吸水棉吸走，检测孔无红细胞富集呈现为膜本色，则为阴性结果。最后按照出现阳性结果的检测孔标识判定ABO反定型结果。
实施例9
不规则抗体筛检
采用实施例6所提供的血型检测卡，检测孔的数量为3，第一检测孔3、第二检测孔4、第三检测孔5底部分别装有实施例4分别用3个人的不规则抗体筛检红细胞I、II、III所制得的用于红细胞血型抗体鉴定的生物多层孔隙膜免疫载体；
取新鲜待检样本(血浆或血清)，向检测孔中分别滴加一滴样本，室温放置1分钟。向对应孔中分别加入1滴不规则抗体筛检红细胞I、II、III，室温放置1分钟。然后再向相应孔中分别滴加每孔分别滴加3滴冲洗液(PH值为7.0的0.05％Tween-20的缓冲液)，观察红细胞分布情况并判定检测结果，检测原理如图2所示。
发生抗原抗体反应的RBC被捕获固定在多孔隙生物膜上冲洗不掉，检测孔中有红细胞富集呈现红色，则为阳性结果；反之，没发生抗原抗体反应的RBC经冲洗液冲洗通过多孔隙膜被吸水棉吸走，检测孔无红细胞富集呈现为本色，则为阴性结果。最后按照出现阳性结果的检测孔标识判定抗体筛检结果。
实施例10
红细胞抗体鉴定
采用实施例6所提供的血型检测卡，检测孔的数量为12，第一检测孔3-12底部分别装有实施例5分别使用12组致敏红细胞谱细胞抗原制得的用于红细胞血型抗体鉴定的生物多层孔隙膜免疫载体；
取新鲜待检样本(血浆或血清)，向12个检测孔中分别滴加一滴样本，室温放置1分钟。向对应孔中分别加入1滴红细胞谱细胞，室温放置1分钟。然后再向相应孔中分别每孔分别滴加3滴冲洗液，观察红细胞分布情况并判定检测结果，检测原理如图2所示。
发生抗原抗体反应的红细胞被捕获固定在多孔隙生物膜上冲洗不掉，检测孔中有红细胞富集呈现红色，则为阳性结果；反之，没发生抗原抗体反应的红细胞经冲洗液冲洗通过多孔隙膜被吸水棉吸走，检测孔无红细胞富集呈现为本色，则为阴性结果。最后按照出现阳性结果的检测孔标识判定抗体鉴定结果。
本发明公开的检测方法有利于促进临床红细胞血型相容性检测，包括红细胞正定型、反定型、抗体筛检、抗体鉴定等，可实现快速、准确检测红细胞ABO、Rh、MNS、Kell、Kidd等多种血型系统血型，还可根据POCT检测(即时检测)需要进行不同检测项目选择，以适合医院、家庭以及野外紧急等多种血型检测情况。
本发明所提供的检测卡的保存或检测所需温度为4℃-50℃，克服现有的血型检测产品对保存或检测温度的严格要求。
尽管本发明的实施方案已公开如上，但其并不仅仅限于说明书和实施方式中所列运用，它完全可以被适用于各种适合本发明的领域，对于熟悉本领域的人员而言，可容易地实现另外的修改，因此在不背离权利要求及等同范围所限定的一般概念下，本发明并不限于特定的细节和这里示出的实施例。

Claims (14)

  1. 一种功能化生物多层孔隙膜免疫载体，其特征在于，包括：
    生物多层孔隙膜；
    特异性抗生物多层孔隙膜抗体，所述特异性抗生物多层孔隙膜抗体与所述生物多层孔隙膜发生抗原抗体特异性结合从而依附在所述生物多层孔隙膜的表面；
    交联抗体，所述交联抗体与所述特异性抗生物多层孔隙膜抗体偶联结合在一起；
    其中，所述交联抗体一Fab端能与所述特异性抗生物多层孔隙膜抗体偶联，另一Fab端能够与特异性抗体结合。
  2. 根据权利要求1所述的一种功能化生物多层孔隙膜免疫载体，其特征在于，所述特异性生物多层孔隙膜抗体包括鼠源单抗、羊源多抗、兔源单抗/多抗、鸡源多抗；所述交联抗体包括抗鼠多抗、抗羊多抗、抗兔多抗、抗鸡多抗；所述能够与交联抗体Fab端结合的特异性抗体为鼠源单抗、羊源多抗、兔源单抗/多抗、鸡源多抗。
  3. 一种功能化生物多层孔隙膜免疫载体的制备方法，其特征在于，包括以下步骤：
    S1、将清洗干净的厚棉布或羊毛织物或真丝织物或植物纤维织物剪切成圆形或方形膜片，然后将膜片浸入处理液中，在40-70℃水浴中浸泡2-4小时，制得生物多层孔隙膜；
    S2、将所述生物多层孔隙膜与特异性抗生物多层孔隙膜抗体进行抗原抗体特异性结合，洗液清洗，再与交联抗体进行偶联后，洗液清洗，用封闭液封闭后再用洗液清洗，制得如权利要求1所述的功能化生物多层孔隙膜免疫载体；
    其中，所述特异性抗生物多层孔隙膜抗体为能够与所述生物多层孔隙膜 发生抗原抗体特异性反应的抗体；
    所述交联抗体Fab一端能与所述特异性抗生物多层孔隙膜抗体偶联，另一Fab端能够与特异性抗体结合。
  4. 一种用于血型相容性检测的生物多层孔隙膜免疫载体，其特征在于，包括：
    权利要求1或2所述的功能化生物多层孔隙膜免疫载体；
    红细胞血型抗体或致敏红细胞血型抗原，通过与所述功能化生物多层孔隙膜免疫载体上的所述交联抗体进行特异性结合而依附在所述功能化生物多层孔隙膜免疫载体上；
    其中，所述致敏红细胞血型抗原为特异性结合了非血型抗体的红细胞膜抗体的红细胞血型抗原。
  5. 根据权利要求4所述的用于血型相容性检测的生物多层孔隙膜免疫载体，其特征在于，所述红细胞血型抗体包括IgM型、IgG型、IgM-IgG型混合型的红细胞ABO、Rh、MNS、Kell、Kidd血型系统的血型抗体，所述红细胞血型抗体效价大于1:32。
  6. 根据权利要求4所述的用于血型相容性检测的生物多层孔隙膜免疫载体，其特征在于，所述致敏红细胞血型抗原包括致敏A型红细胞抗原、致敏B型红细胞抗原、致敏不规则抗体筛检红细胞抗原、致敏红细胞谱细胞抗原。
  7. 一种用于血型相容性检测的生物多层孔隙膜免疫载体的制备方法，其特征在于，包括以下步骤：
    S1、将清洗干净的厚棉布或羊毛织物或真丝织物或植物纤维织物剪切成圆形或方形膜片，然后将膜片浸入处理液中，在40-70℃水浴中浸泡2-4小时，制得生物多层孔隙膜；
    S2、将所述生物多层孔隙膜与特异性抗生物多层孔隙膜抗体进行抗原抗体特异性结合，洗液清洗，再与交联抗体进行偶联后，洗液清洗，用封闭液封闭后再用洗液清洗，制得如权利要求1所述的功能化生物多层孔隙膜免疫 载体；
    S3、将红细胞血型抗体或致敏红细胞血型抗原与所述功能化生物多层孔隙膜免疫载体上的所述交联抗体进行特异性结合，制得如权利要求4所述的用于血型相容性检测的生物多层孔隙膜免疫载体；
    其中，所述特异性抗生物多层孔隙膜抗体为能够与所述生物多层孔隙膜发生抗原抗体特异性反应的抗体；
    所述交联抗体一Fab端能与所述特异性抗生物多层孔隙膜抗体偶联，另一Fab端能够与特异性抗体结合，所述特异性抗体为能够与交联抗体Fab端结合的红细胞血型抗体或致敏红细胞血型抗原；
    所述致敏红细胞血型抗原为特异性结合了非血型抗体的红细胞膜抗体的红细胞血型抗原。
  8. 根据权利要求7所述的用于血型相容性检测的生物多层孔隙膜免疫载体的制备方法，其特征在于，还包括步骤致敏红细胞血型抗原的制备，如下：用非血型抗体的红细胞膜抗体与新鲜红细胞反应，待出现1+凝集强度时，用低渗溶液将红细胞裂解，离心收集获得致敏红细胞抗原。
  9. 一种血型检测卡，其特征在于，包括：
    固相塑料载体，所述固相塑料载体包括卡槽外壳和卡槽内壳，所述卡槽外壳与所述卡槽内壳可拆卸连接；
    权利要求4-6任意一项所述用于血型相容性检测的生物多层孔隙膜免疫载体，所述用于血型相容性检测的生物多层孔隙膜免疫载体位于所述固相塑料载体卡槽外壳的底部；
    吸水棉，位于固相塑料载体卡槽内壳的底部；
    隔离膜，放置在所述吸水棉的上方。
  10. 根据权利要求9所述的血型检测卡，其特征在于，所述固相塑料载体的制备材料包括聚乙烯、聚乙烯、聚苯乙烯、苯乙烯-丁二烯-丙烯腈；所述固相塑料载体，还包括检测孔，所述检测孔包括双孔、三孔、多孔；所述隔 离膜包括滤纸、纤维素纤维、玻璃纤维垫、多孔高分子材料制成的纤维垫或试纸；所述吸水棉包括棉浆垫、滤纸。
  11. 根据权利要求9所述的血型检测卡，其特征在于，所述血型检测卡的保存或检测温度为4-50℃。
  12. 一种血型的检测方法，其特征在于，包括以下步骤：
    将待检测血型样品加入到如权利要求9-11任意一项所述的血型检测卡的检测孔中，放置至少1分钟；洗液冲洗，观察检测孔的显色情况，显色为红色则为阳性，显色为本色则为阴性；
    其中，待检测血型样品与血型检测卡中的所述用于血型相容性检测的生物多层孔隙膜免疫载体发生了抗原抗体特异性反应，为阳性；反之，为阴性。
  13. 权利要求1或2所述的功能化生物多层孔隙膜免疫载体在制备血型相容性检测产品中的应用。
  14. 权利要求1或2所述的功能化生物多层孔隙膜免疫载体在制备用于检测和分析病原微生物细胞抗原抗体、细菌细胞抗原抗体、肿瘤细胞抗原抗体的产品中的应用。
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