WO2019057014A1 - 一种血型抗原芯片及其在红细胞意外抗体检测中的应用 - Google Patents

一种血型抗原芯片及其在红细胞意外抗体检测中的应用 Download PDF

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WO2019057014A1
WO2019057014A1 PCT/CN2018/106040 CN2018106040W WO2019057014A1 WO 2019057014 A1 WO2019057014 A1 WO 2019057014A1 CN 2018106040 W CN2018106040 W CN 2018106040W WO 2019057014 A1 WO2019057014 A1 WO 2019057014A1
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antibody
blood group
antigen
microspheres
detection
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PCT/CN2018/106040
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English (en)
French (fr)
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汪德清
杨璐
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汪德清
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Priority to CN201880003173.5A priority Critical patent/CN110121649A/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
    • 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
    • G01N33/545—Synthetic resin
    • G01N33/546—Synthetic resin as water suspendable particles
    • 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
    • G01N33/551—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being inorganic
    • 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 invention relates to the field of biomedical technology, in particular to a serological detection technology of blood group antibodies, in particular to a blood type antigen chip, a preparation method thereof and application thereof in detecting an unexpected antibody of red blood cells.
  • the detection rate of erythrocyte allogeneic antibody in Chinese Han patients is 0.38%-2.38%, which is caused by non-infectious transfusion adverse reactions, neonatal hemolytic disease, blood type identification and difficult matching, unexplained anemia, red blood cell transfusion The main reason for invalidity.
  • Precise testing of erythrocyte allotypes of irregular blood group antibodies prior to transfusion is essential for transfusion safety and transfusion efficacy.
  • the detection of erythrocyte allogeneic antibodies has become a routine test item before transfusion. If there is a red blood cell accidental antibody in the patient's body, once the corresponding antigen is present on the donor's red blood cells, an adverse reaction of hemolytic transfusion will occur, and even severe death may occur.
  • the method for evaluating the quality of rare blood type antibody reagents is to characterize the specificity of the antibody by using the reagent red blood cells of known antigens based on the principle of agglutination reaction, but the concentration of the red blood cell reagent is low, the antigenicity is weak, and the cells are different before different batches of different manufacturers; At the same time, the agglutination reaction has low sensitivity and low specificity, and multiple detections cannot be achieved. Therefore, there are certain disadvantages in evaluating the quality of rare blood type antibody reagents.
  • Patent CN00105438.4 detects platelet blood group antigen antibodies by microcolumn gel method and is a platelet type.
  • Patent CN200710049305.1 provides a kit for detecting blood type by using microfluidic technology, a preparation method and a detection method.
  • the detection of unexpected antibodies to red blood cells in clinical blood generally has technical problems such as poor sensitivity, narrow linear range of dynamics, low ability of screening and screening, serious false negative problems, and inconvenient storage of detection reagents.
  • Patent CN200880019301.1 achieves the identification of individual anti-erythrocyte antibodies in vitro by immobilizing red blood cell or erythrocyte membrane fragments onto beads, however the conditional parameters described in this patent are not repeatable, and the method is applied to the detection of erythrocyte accidental antibodies, antigenic The coating method, the concentration of the secondary antibody, and the dilution of the sample will affect the accuracy of the test, so the method needs to be redesigned and evaluated.
  • the inventors of the present invention provide a blood type antigen chip with high sensitivity and accuracy by optimizing the detection method and detection conditions of the red blood cell accidental antibody, and the use thereof in detecting the accidental antibody or blood transfusion type of red blood cells.
  • the present invention provides a blood group antigen chip, the chip comprising a purified blood group antigen and a carrier, the purified blood group antigen coated onto the carrier, the purified blood group antigen capable of binding to an erythrocyte accidental antibody.
  • the purified blood group antigen of the present invention is selected from the group consisting of D, C, E, c, e, JK a , JK b , M, N, S, s, Kp a , Kp b , Lu a , Lu b , Fy a , One or a combination of two or more of Fy b , K, k, Le a , Le b , P 1 , Dob, CROM, Cha, Rga, Doa, Lwa, CR1, Xga, Yta, SC1.
  • the carrier of the present invention is a microsphere, and the microsphere is selected from one or a combination of two or more of silica microspheres, polystyrene microspheres, magnetic microspheres and biomacropolymer microspheres.
  • the carrier of the present invention is a polystyrene microsphere, and particularly preferably, the carrier of the present invention is a polyphenylene microsphere to which a fluorescent dye is incorporated.
  • the purified blood group antigen is coated to the carrier in an amount of from 1 to 200 ⁇ g / 1.25 ⁇ 10 7 microspheres. More preferably, the purified blood group antigen is coated to the carrier in an amount of 50-200 ⁇ g / 1.25 ⁇ 10 7 microspheres, and more preferably, the purified blood group antigen is coated to the carrier in an amount of 50-150 ⁇ g / 1.25 ⁇ 10 7 microspheres, particularly preferably, the purified blood group antigen is coated to the carrier in an amount of 50-100 ⁇ g / 1.25 ⁇ 10 7 microspheres.
  • the blood group antigen chip is a liquid chip, and the microspheres coated with the purified blood group antigen in the blood group antigen chip are suspended in the preservation solution.
  • the invention provides a blood type antigen chip for use in the following applications: (1) screening and identification of erythrocyte accidental antibodies; (2) quantitative detection of erythrocyte accidental antibodies; (3) blood transfusion matching.
  • the blood group antibody of the present invention is a red blood cell accidental antibody
  • the red blood cell accidental antibody is selected from the group consisting of Rh blood type system, MNS blood type system, P blood type system, Kell blood type system, Kidd blood type system, Lewis blood type system, Duffy blood type system. , Lutheran blood type system, etc.
  • the invention provides a preparation method of a blood group antigen chip, the method comprising:
  • the microspheres are resuspended ultrasonically;
  • the microspheres are sequentially added to N-hydroxythiosuccinimide (Sulfo-NHS), carbodiimide (EDC) for activation;
  • Sulfo-NHS N-hydroxythiosuccinimide
  • EDC carbodiimide
  • Coupling is carried out by adding a purified blood group antigen to the microsphere solution, and the purified blood group antigen is capable of binding to an unexpected antibody against red blood cells.
  • the purified blood group antigen is coated to the carrier in an amount of from 1 to 200 ⁇ g / 1.25 ⁇ 10 7 microspheres. More preferably, the purified blood group antigen is coated to the carrier in an amount of 50-200 ⁇ g / 1.25 ⁇ 10 7 microspheres, and more preferably, the purified blood group antigen is coated to the carrier in an amount of 50-150 ⁇ g / 1.25 ⁇ 10 7 microspheres, particularly preferably, the purified blood group antigen is coated to the carrier in an amount of 50-100 ⁇ g / 1.25 ⁇ 10 7 microspheres.
  • the preparation method of the blood group antigen chip comprises: after the microsphere is resuspended by ultrasonic, the supernatant is removed; the microsphere is resuspended by shaking with deionized water, and the supernatant is removed; Resuspend the microspheres in the disodium hydrogen phosphate buffer, add the Sulfo-NHS solution, mix well by shaking, add to the EDC solution, mix by shaking, incubate for 5-30 minutes at room temperature, preferably for 20 minutes; remove the supernatant, use MES Solution, resuspend the microspheres, remove the supernatant, repeat the two times, then add the MES solution to resuspend the microspheres; add the purified blood group antigen to the suspended microsphere solution for coupling reaction, incubate for 0.5-4 hours, preferably 2 After the supernatant, the conjugated microspheres were resuspended in PBS-TBN (1% BSA, 0.05% twe
  • the invention provides a method for identifying or detecting a blood group antibody, comprising:
  • the purified blood group antigen of the present invention is selected from the group consisting of D, C, E, c, e, JK a , JK b , M, N, S, s, Kp a , Kp b , Lu a , Lu b , Fy a , One or a combination of two or more of Fy b , K, k, Le a , Le b , P 1 , Dob, CROM, Cha, Rga, Doa, Lwa, CR1, Xga, Yta, SC1.
  • the carrier of the present invention is a microsphere, and the microsphere is selected from one or a combination of two or more of silica microspheres, polystyrene microspheres, magnetic microspheres and biomacropolymer microspheres.
  • the carrier of the present invention is a polystyrene microsphere, and particularly preferably, the carrier of the present invention is a polyphenylene microsphere to which a fluorescent dye is incorporated.
  • the purified blood group antigen is coated to the carrier in an amount of from 1 to 200 ⁇ g / 1.25 ⁇ 10 7 microspheres. More preferably, the purified blood group antigen is coated to the carrier in an amount of 50-200 ⁇ g / 1.25 ⁇ 10 7 microspheres, and more preferably, the purified blood group antigen is coated to the carrier in an amount of 50-150 ⁇ g / 1.25 ⁇ 10 7 microspheres, particularly preferably, the purified blood group antigen is coated to the carrier in an amount of 50-100 ⁇ g / 1.25 ⁇ 10 7 microspheres.
  • the test sample according to the present invention may be whole blood, serum, plasma or an antibody.
  • the test sample is plasma.
  • the test sample of the present invention is diluted and then contacted with an antigen coated to a carrier, and the dilution ratio of the test sample is 25-200 times. More preferably, the dilution ratio of the test sample is 50-100. In many times, it is particularly preferred that the test sample has a dilution factor of 100 times.
  • the time during which the test sample is incubated with the antigen coated to the carrier in the step (2) is 15-60 min. More preferably, the incubation time is 30-45 min. Most preferably, the incubation time is 45 min.
  • the labeled secondary antibody of the present invention is selected from the group consisting of a protein or antibody labeled with fluorescein or an enzyme and a conjugate thereof; the protein or antibody and its conjugate are selected from avidin, streptavidin , a digoxigenin antibody, a histidine antibody, a Ni-containing affinity molecule, a fluorescent molecule antibody; the fluorescein is selected from the group consisting of anthocyanin Cy series fluorescein, Alexa Fluor series fluorescein or fluorescein isothiocyanate;
  • the enzyme is alkaline phosphatase or peroxidase.
  • the secondary antibody labeled with the marker is a Cy3-labeled anti-human IgG antibody.
  • the secondary antibody of the present invention is added at a concentration of 1-8 ug/ml, and more preferably, the secondary antibody of the present invention is added at a concentration of 2-6 ug/ml, particularly preferably, the present invention The secondary antibody was added at a concentration of 4 ug/ml.
  • the signal detection according to the present invention includes fluorescence detection, color detection, electrochemical detection, mechanical detection, and the like.
  • the signal detection according to the present invention is a fluorescence detection, and more preferably, the signal detection according to the present invention performs fluorescence detection at wavelengths of 532 nm and 635 nm.
  • the method for detecting a blood group antibody comprises: taking a 96-well microplate, each well is added with a PBS-TBN solution or a 1% BSA solution at 37 ° C for 0.5-3 h, and blocking; Sample and purified blood group antigen-coated vector, incubate, remove supernatant; wash 96-well plate with PBS-TBN solution; add fluorescent-labeled secondary antibody and PBS-TBN; wash 96-well plate with PBS-TBN solution; The microspheres were resuspended in PBS-TBN and detected by flow cytometry.
  • the sensitivity of the microsphere, chip and blood group antibody detecting method of the invention is much higher than that of the conventional atypical lectin screening method (AAS) and microcolumn gel method, and the detection limit is far lower than the microcolumn gel method.
  • the sensitivity is increased by more than 1000 times; in addition, the linear range of the invention is wide, and the dynamic range is increased by 2-3 orders of magnitude, which is especially suitable for detecting low-concentration rare blood type antibodies and erythrocyte accidental antibodies.
  • the blood group antigen chip prepared by the invention has good specificity, and different antigen chips can detect corresponding antibodies, and no cross reaction occurs between different antigen chips, and the blood type antibody detection method of the invention has a very high Good repeatability.
  • the blood group antibody detecting method of the invention does not need to use red blood cells, and the microspheres coupled with the antigen can be stably stored for a long period of time, and solves the technical problem of insufficient source of red blood cells carrying rare antigens, difficulty in storage, and easy hemolysis.
  • the microspheres, chips, and blood group antibody detecting methods of the present invention can be appropriately integrated according to needs and costs. It can integrate a variety of rare blood group antigens to achieve a comprehensive analysis of all blood group antigens in one test. It can also integrate the same rare blood group antigen classification markers to achieve large-scale screening of certain rare blood group antigens.
  • Figure 1 Flow chart of blood group antigen chip detection of blood group antibodies.
  • the antigen-coated microspheres were added to a 96-well plate, serum was added to form a microsphere-antigen-antibody complex, and then a fluorescently labeled secondary antibody was added to read the signal, and the fluorescence detection value at 532 nm was detected by flow cytometry. The 635 nm microspheres detect the common read signal results.
  • Fig. 2 Kinetic curves of four blood group antigen chips of Fy a , Fy b , Lu a and Lu b .
  • Figure 3 Fy a , Fy b , Lu a , Lu b , K, k six blood group antigen chip specific detection map.
  • Fig. 4 Intra-assay repeatability of blood group antigen chip for blood group antibody detection, the curve from top to bottom is Fy b -1, Fy b -2, Fy a -1, Fy a -2, Lu a -1, Lu a - 2. Lu b -1 and Lu b -2.
  • Fig. 5 Inter-assay repeatability of blood group antigen chip for blood group antibody detection, the curve from top to bottom is Fy b -D1, Fy b -D2, Fy a -D1, Fy a -D2, Lu a -D1, Lu a - D2, Lu b -D1, Lu b -D2.
  • Fig. 6 Results of detection of erythrocyte accidental antibodies in 20 clinical serum samples, the horizontal axis is the detection result of clinical microcolumn agglutination method of serum and corresponding blood group antigen, the vertical axis is the name of different antigen coated on the microsphere, 0 to 3000 represents serum Antibody binding signals range from low to high.
  • Figure 7 Results of the detection of erythrocyte accidental antibodies in 114 clinical samples.
  • the horizontal axis is the sample number
  • the vertical axis is the different antigen name coated on the microspheres
  • the 0.8721 to 0.8721058 represents the serum antibody binding signal from low to high.
  • Fig. 8 Results of accidental antibody detection of 100 samples of serum in clinical samples.
  • the horizontal axis is the sample number, the vertical axis is the different antigen name coated on the microspheres, and the 0.6166731 to 0.61671 represents the serum antibody binding signal from low to high.
  • the unconjugated microspheres were ultrasonically resuspended, and 200 uL (concentration: 1.25 ⁇ 10 7 /mL) microspheres were taken into the EP tube, and the EP tube was placed on a magnetic plate for 30 s, the microspheres were separated from the solution, and the supernatant was removed. Liquid, remove the magnetic separator and resuspend the microspheres with 100 ⁇ l of deionized water, place the EP tube on a magnetic plate for 30 s, separate the microspheres from the solution, and remove the supernatant.
  • the EP tube was placed on a magnetic plate for 30 s, the microspheres were separated from the solution, and the supernatant was removed twice.
  • the magnetic separator was removed and the activated and washed microspheres were resuspended by shaking with 100 ⁇ l of 50 mM MES, pH 5.0 solution.
  • Add 12.5 ⁇ g (concentration 0.5mg/ml 25 ⁇ l) pure antigen Fy a to the suspension microsphere solution increase the total volume to 500 ⁇ l with 50mM MES, pH5.0 solution, shake and mix for coupling reaction, at room temperature Incubate for 2 hours while inverting the mixture.
  • the EP tube was placed on a magnetic plate for 30 s, the microspheres were separated from the solution, and the supernatant was removed.
  • the magnetic separator was removed and the coupled microspheres were resuspended by shaking with 500 ⁇ l of PBS-TBN solution, and incubated at room temperature for 30 min.
  • the EP tube was placed on a magnetic plate for 30 s, the microspheres were separated from the solution, and the supernatant was removed.
  • the microspheres were washed twice with 1 mL PBST solution.
  • the magnetic separator was removed and the coupled and washed microspheres were resuspended in 1 ml of PBST solution and stored in a dark environment at 4 °C.
  • a 96-well plate was taken and each well was incubated with 200 ⁇ l of 1% BSA at 37 ° C for 1 h. 50 ⁇ l of the diluted test sample Fy a antibody and 2500 blood group antigen chips prepared in Example 1 were added, and 100 ⁇ l was made up with PBS-TBN, and incubated at room temperature for a certain period of time. The 96-well plate was placed on a magnetic plate for 5 min, the microspheres were separated from the solution, and the supernatant was removed.
  • the 96-well plate was washed 3 times with 150 ⁇ l of PBS-TBN solution (150 ⁇ l PBS-TBN solution was added to the plate, shaken for 5 min in the shaker, and the 96-well plate was placed on a magnetic plate for 5 min after removal, and the microspheres were separated from the solution. Go to the supernatant).
  • Add 50 ⁇ l of fluorescently labeled secondary anti-human IgG add 50 ⁇ l of PBS-TBN, incubate for 0.5 h at room temperature, wash the 96-well plate twice with 150 ⁇ l PBS-TBN solution, resuspend the magnetic beads with 200 ⁇ l PBS-TBN, and perform flow cytometry. Detection.
  • Test sample dilution factor 25 times 50 times 100 times 200 times Incubation time 15min 30min 45min 60min Secondary antibody concentration 2ug/ml 4ug/ml 6ug/ml 8ug/ml
  • Test sample dilution factor 25 times, 50 times, 100 times, 200 times are different, as the dilution factor increases, the signal decreases, so the dilution factor is selected 100 times according to the actual situation.
  • the concentration of secondary antibody 2ug/ml and 4ug/ml, 6ug/ml, 8ug/ml are different P ⁇ 0.05; 4ug/ml and 6ug/ml, 8ug/ml no difference P>0.05; 6ug/ml and 4ug /ml, 8ug/ml no difference P>0.05; 8ug/ml and 4ug/ml, 6ug/ml no difference P>0.05; therefore 4ug/ml was finally selected.
  • the optimal detection conditions are 100 times dilution of the antibody, incubation of the antibody with the blood group antigen chip for 45 minutes, and concentration of the secondary antibody of 4 ug/ml.
  • PBS-TBN was supplemented with 100 uL of the system volume for 45 min at room temperature; then the 96-well plate was placed on a magnetic plate for 5 minutes, and the supernatant was removed; the 96-well plate was washed 3 times with 150 ⁇ l of PBS-TBN solution; 50 ⁇ M was added.
  • Increasing Cy3-labeled anti-human IgG adding 50 ⁇ l of PBS-TBN, incubating for 0.5 hour at room temperature; washing 96-well plates twice with 100 ⁇ l of PBS-TBN solution; resuspending the microspheres with 200 ⁇ l of PBS-TBN Flow cytometry was performed for detection.
  • the kinetic curves of four blood group antigen chips of Fy a , Fy b , Lu a and Lu b are shown in Fig. 2. It can be seen from the experimental results that the blood type antigen chip prepared by the invention still remains when the detection antibody is diluted 163840 times. The antibody can be detected, indicating that the blood type antigen chip has high sensitivity, and when the dilution factor of the detection antibody is 25-200 times, the detection effect of the antibody is optimal.
  • Example 2 Six kinds of blood type antigen chips of Fy a , Fy b , Lu a , Lu b , K and k were prepared according to the preparation method described in Example 1, and the six coupled microspheres were mixed together according to Example 2. The best detection method described was carried out for antibody detection, and five different antibodies of Fy a , Fy b , Lu b , K and k were added respectively to verify whether there was a cross reaction between different antigen chips and antibodies, and the results are shown in FIG. 3 . According to the experimental results, it can be seen that the blood group antigen chip prepared by the invention has good specificity, and different antigen chips can detect the corresponding antibodies, and no cross reaction occurs between different antigen chips.
  • Example 1 four blood type antigen chips of Fy a , Fy b , Lu a and Lu b were prepared, and the corresponding four antibodies were respectively detected according to the optimal detection method described in Example 2, and the antibodies were diluted 40,960 respectively.
  • the repeatability of the two microplate data on the same day represents the repeatability in the experiment.
  • the two microplate data for two consecutive days represent the repeatability between experiments.
  • the experimental results are shown in Fig. 4 and Fig. 5. According to the experimental results, it can be seen that the blood group antigen chip prepared by the invention has good reproducibility both in the experiment and in the experiment when detecting the blood group antibody.
  • Example 6 Clinical small-scale application of blood type antigen chip for detecting unexpected antibodies of red blood cells
  • Example 2 According to the preparation method described in Example 1, six blood type antigen chips of Fy a , Fy b , Lu a , Lu b , K and k were prepared, and 20 clinical serum samples were detected respectively, and the detection method was as described in Example 2. The best test method. Among them, 20 clinical specimens were detected by conventional microcolumn gel method: 3 anti-Fy b , 1 anti-Fy a , 1 anti-K, 1 anti-D, 1 anti-M antibody, 3 anti-E combined with other Antibody, 3 unknown antibodies, and 7 negative samples. The results obtained by blood group antigen chip detection are shown in Fig. 6.
  • Fy b , Dob, K, Lu a , Lu b , k, CROM, Fy a , Cha, Rga, Doa, Lwa, CR1, Xga, Yta, SC1 were prepared.
  • the blood type antigen chip is used for the detection of 100 clinical serum samples, and the detection method is the best detection method as described in Embodiment 2, and the detection result is shown in FIG. 8.
  • the blood group antigen chip prepared by the invention still detects some antibodies, indicating that the blood type antigen chip has high sensitivity, and can detect antibodies not detected by clinical routine methods, thereby avoiding The hemolytic transfusion adverse reaction caused by this antibody.

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Abstract

一种血型抗原芯片及其在红细胞意外抗体检测中的应用，所述的检测包括(1)将纯化血型抗原包被至载体；(2)使检测样品与包被至载体的抗原接触；(3)加入标记有标记物的二抗；(4)针对标记物进行信号检测。

Description

一种血型抗原芯片及其在红细胞意外抗体检测中的应用 技术领域
本发明涉及生物医学技术领域，具体涉及血型抗体的血清学检测技术，特别是涉及一种血型抗原芯片、其制备方法及在红细胞意外抗体检测中的应用。
背景技术
输血安全已成为医疗卫生工作中的一个重要问题，并引起全社会的高度关注。世界卫生组织(WHO)一贯重视输血安全工作，近年来进一步加强了血液安全工作的力度，血液安全已被WHO列为了全球卫生工作七项重点工作之一。输血安全问题包括传染性输血不良反应的问题和非传染性输血不良反应的问题。英国、美国、法国均启动了输血严重危害报告***，用来收集和报告各所医院自愿报告的与血液成分输注相关的主要不良事件。从欧美国家统计的数据可以看出，非传染性输血不良反应的发生率很高，并且一旦发生，后果极其严重。
红细胞同种意外抗体在中国汉族患者中检出率为0.38％-2.38％，是引起非传染性输血不良反应、新生儿溶血病、血型鉴定困难以及疑难配型、不明原因的贫血、红细胞输注无效等的主要原因。输血前进行红细胞同种不规则血型抗体精准检定，对于输血安全和输血疗效至关重要。为确保输血安全和输血疗效，红细胞同种意外抗体检测已成为输血前常规检测项目。若患者体内存在红细胞意外抗体，一旦输入的献血员红细胞上存在相应抗原，即会发生溶血性输血不良反应，严重者甚至死亡。
由于用来免疫生成单克隆抗体的抗原的精确结构是未知的，来源于多种异体材料包括卵巢囊液及唾液糖蛋白的可溶性抗原和人类细胞，因此为了减少由于抗原变异导致漏检的风险，这些用于常规血清学检测的克隆株反应较广泛，这同样增加了结构相似的抗原之间的交叉反应风险。有文章报道，ABO单克隆抗体与非ABO抗原的交叉反应尽管在血清学反应中很少出现，但经常出现于非血清学反应如酶免疫分析法和抑制试验中，因此抗体应用于此类试验时必须考虑交叉反应的情况。常规血清学试验检测灵敏度较低，容易导致献血员弱抗原的漏检，输注至患者体内引起溶血性输血不良反应，威胁患者生命，应用灵敏度较高的非血清学试验检测献血员血型抗原已成为发展趋势，若使用特异性不 强的稀有血型抗体试剂进行献血员血型抗原分型可能导致错误的分型结果，进而无法确保安全有效的输血。因此用于献血员血型抗原检测的稀有血型抗体试剂的质量评价尤为重要。目前用于稀有血型抗体试剂质量评价的方法是基于凝集反应原理利用已知抗原的试剂红细胞表征抗体的特异性，但由于红细胞试剂浓度低、抗原性弱，且不同厂家不同批号之前细胞有差异；同时凝集反应灵敏度低，特异性低，无法实现多重检测，因此评价稀有血型抗体试剂的质量存在一定弊端。
红细胞意外抗体检定一直是输血安全关注的焦点，为此研发了很多种检测红细胞意外抗体的方法，如：经典抗人球蛋白法、柱凝集法、Capture捕获法等。专利CN00105438.4中通过微柱凝胶法对血小板血型抗原抗体进行检测，为血小板配型。专利CN200710049305.1提供一种利用微流技术检测血型的试剂盒、制备方法及检测方法。然而上述专利中临床血液中红细胞意外抗体的检测普遍存在灵敏度差、动力学线性范围窄、通量化筛选能力低、假阴性问题严重、检测试剂不便于储存等技术问题。
专利CN200880019301.1通过将红细胞或红细胞膜片段固定到珠子上实现体外鉴定个体的抗红细胞抗体，然而该专利中所描述的条件参数不可重复，并且该方法应用于红细胞意外抗体的检测时，抗原的包被方式、二抗的加入浓度以及样品的稀释浓度的变化都会影响检测的准确性，因此需要对该方法进行重新设计和评估。
发明内容
为解决上述问题，本发明发明人通过对红细胞意外抗体的检测方法和检测条件的优化，提供一种灵敏度、准确性好的血型抗原芯片及其在检测红细胞意外抗体或输血配型中的用途。
本发明提供一种血型抗原芯片，所述的芯片包括纯化血型抗原和载体，所述的纯化血型抗原包被至所述的载体上，所述的纯化血型抗原能够结合红细胞意外抗体。
本发明所述的纯化血型抗原选自：D、C、E、c、e、JK a、JK b、M、N、S、s、Kp a、Kp b、Lu a、Lu b、Fy a、Fy b、K、k、Le a，Le b、P 1、Dob、CROM、Cha、Rga、Doa、Lwa、CR1、Xga、Yta、SC1中的一种或两种以上的组合。
本发明所述的载体为微球，所述的微球选自二氧化硅微球、聚苯乙烯微球、磁性微球和生物大分子聚合物微球中的一种或两种以上的组合，优选的，本发明所述的载体为聚苯乙烯微球，特别优选的，本发明所述的载体为结合有荧光染料的聚苯烯微球。
优选的，所述的纯化血型抗原包被至载体的量为1-200μg/1.25×10 7个微球。更优选的，所述纯化血型抗原包被至载体的量为50-200μg/1.25×10 7个微球，更优选的，所述纯化血型抗原包被至载体的量为50-150μg/1.25×10 7个微球，特别优选的，所述纯化血型抗原包被至载体的量为50-100μg/1.25×10 7个微球。
在本发明的一个具体实施方式中，所述的血型抗原芯片为液体芯片，所述的血型抗原芯片中包被有纯化血型抗原的微球悬浮于保存液中。
本发明提供了一种血型抗原芯片在如下用途中的应用：(1)红细胞意外抗体的筛查及鉴定；(2)红细胞意外抗体的定量检测；(3)输血配型。
优选的，本发明所述的血型抗体为红细胞意外抗体，所述的红细胞意外抗体选自Rh血型***、MNS血型***、P血型***、Kell血型***、Kidd血型***、Lewis血型***、Duffy血型***、Lutheran血型***等。
本发明提供了一种血型抗原芯片的制备方法，所述的方法包括：
(1)微球的预处理：将微球超声重悬；
(2)微球的活化：将微球依次加入N-羟基硫代琥珀酰亚胺(Sulfo-NHS)、碳二亚胺(EDC)活化；
(3)向微球溶液中加入纯化血型抗原进行偶联，所述的纯化血型抗原能够结合红细胞意外抗体。
优选的，所述的纯化血型抗原包被至载体的量为1-200μg/1.25×10 7个微球。更优选的，所述纯化血型抗原包被至载体的量为50-200μg/1.25×10 7个微球，更优选的，所述纯化血型抗原包被至载体的量为50-150μg/1.25×10 7个微球，特别优选的，所述纯化血型抗原包被至载体的量为50-100μg/1.25×10 7个微球。
在本发明的另一个具体实施方式中，所述的血型抗原芯片的制备方法包括：微球超声重悬后，去上清液；用去离子水震荡重悬微球，去上清液；用磷酸氢二钠缓冲液震荡重悬微球，加入Sulfo-NHS溶液，震荡混匀，加入EDC溶液中，震荡混匀，室温孵育5-30分钟，优选孵育20分钟；去上清液，用MES 溶液，重悬微球，去上清液，重复两次后再加入MES溶液重悬微球；加入纯化血型抗原到悬浮的微球溶液中进行偶联反应，孵育0.5-4小时，优选为2小时；去上清液，用PBS-TBN(1％BSA，0.05％tween-20)溶液重悬偶联后的微球，室温混合孵育30min后去上清；用PBST清洗微球两次后，再用PBST重悬，并在2-8℃的黑暗环境中保存。
本发明提供一种血型抗体的鉴定或检测方法，包括：
(1)将纯化血型抗原包被至载体，所述的纯化血型抗原能够结合红细胞意外抗体；
(2)使检测样品与包被至载体的抗原孵育；
(3)加入标记有标记物的二抗；
(4)针对标记物进行信号检测。
本发明所述的纯化血型抗原选自：D、C、E、c、e、JK a、JK b、M、N、S、s、Kp a、Kp b、Lu a、Lu b、Fy a、Fy b、K、k、Le a，Le b、P 1、Dob、CROM、Cha、Rga、Doa、Lwa、CR1、Xga、Yta、SC1中的一种或两种以上的组合。
本发明所述的载体为微球，所述的微球选自二氧化硅微球、聚苯乙烯微球、磁性微球和生物大分子聚合物微球中的一种或两种以上的组合，优选的，本发明所述的载体为聚苯乙烯微球，特别优选的，本发明所述的载体为结合有荧光染料的聚苯烯微球。
优选的，所述的纯化血型抗原包被至载体的量为1-200μg/1.25×10 7个微球。更优选的，所述纯化血型抗原包被至载体的量为50-200μg/1.25×10 7个微球，更优选的，所述纯化血型抗原包被至载体的量为50-150μg/1.25×10 7个微球，特别优选的，所述纯化血型抗原包被至载体的量为50-100μg/1.25×10 7个微球。
本发明所述的检测样品可以是全血、血清、血浆或抗体，优选的，所述的检测样品为血浆。优选的，本发明所述检测样品稀释后再与包被至载体的抗原接触，所述检测样品的稀释倍数为25-200倍，更优选的，所述的检测样品的稀释倍数为50-100倍，特别优选的，所述的检测样品的稀释倍数为100倍。
优选的，所述步骤(2)中检测样品与包被至载体的抗原孵育的时间为15-60min。更优选的，所述孵育时间为30-45min。最优选的，所述孵育时间为45min。
本发明所述的标记有标记物的二抗选自标记有荧光素或酶的蛋白或抗体及 其偶联物；所述蛋白或抗体及其偶联物选自亲和素、链霉亲和素、地高辛抗体、组氨酸抗体、含Ni亲和分子、荧光分子抗体；所述的荧光素选自花青素Cy系列荧光素、Alexa Fluor系列荧光素或异硫氰酸荧光素；所述的酶为碱性磷酸酶或过氧化物酶。优选的，所述的标记有标记物的二抗为Cy3标记的抗人IgG抗体。优选的，本发明所述的二抗的加入浓度为1-8ug/ml，更优选的，本发明所述的二抗的加入浓度为2-6ug/ml，特别优选的，本发明所述的二抗的加入浓度为4ug/ml。
本发明所述的信号检测包括荧光法检测、显色法检测、电化学检测、力学检测等。优选的，本发明所述的信号检测为荧光法检测，更优选的，本发明所述的信号检测的在波长为532nm和635nm进行荧光检测。
在本发明的一个具体实施方式中，所述的血型抗体的检测方法包括：取96孔微孔板，每孔加入PBS-TBN溶液或1％BSA溶液37℃孵育0.5-3h，封闭；加入检测样品和纯化血型抗原包被的载体，孵育，去上清液；用PBS-TBN溶液洗涤96孔板；加入荧光标记的二抗和PBS-TBN孵育；用PBS-TBN溶液洗涤96孔板；用PBS-TBN重悬微球，用流式细胞仪进行检测。
与现有技术相比，本发明的技术方案具有以下优点：
(1)灵敏度高、线性范围广。本发明所述微球、芯片、血型抗体检测方法的灵敏度远高于现有技术常用的非典型凝集素筛选法(AAS)、微柱凝胶法，检测限远远低于微柱凝胶法，灵敏度提高1000倍以上；另外本发明线性范围广，动力学范围提高2-3个数量级，尤其适用于低浓度稀有血型抗体、红细胞意外抗体的检测。
(2)特异性强，重复性好。本发明所制备的血型抗原芯片具有很好的特异性，不同的抗原芯片均能检测出相应的抗体，不同的抗原芯片之间没有发生交叉反应，且本发明所述的血型抗体检测方法具有很好的重复性。
(3)不依赖红细胞、储存方便。本发明所述血型抗体检测方法无需使用红细胞，偶联有抗原的微球可长期稳定储存，解决了携带稀有抗原红细胞来源不足、难储存、易溶血的技术问题。
(4)集成度高、可实现高通量全面筛查。本发明所述微球、芯片、血型抗体检测方法可根据需要和成本进行适当集成。既可以将多种稀有血型抗原集成， 实现一次检测全面分析所有血型抗原的目的；也可以将同种稀有血型抗原分类标记集成，实现对某种稀有血型抗原的大规模筛查。
(5)可实现抗体的精准鉴定。由于抗原为纯化抗原，与抗体进行特异性结合，因此可直接判断抗体的具体种类，无需根据细胞谱进行核对排除，可确保结果的单一性，避免由于人员资质问题导致的结果误判，实现抗体的精准鉴定。
附图说明
通过阅读下文优选实施方式的详细描述，各种其他的优点和益处对于本领域普通技术人员将变得清楚明了。附图仅用于示出优选实施方式的目的，而并不认为是对本发明的限制。而且在整个附图中，用相同的参考符号表示相同的部件。
图1 血型抗原芯片检测血型抗体的流程图。在96孔板中加入抗原包被的微球，加入血清形成微球-抗原-抗体复合物，然后再加入荧光标记的二抗读取信号，通过流式细胞仪检测，532nm的荧光检测值与635nm的微球检测共同读取信号结果。
图2 Fy a、Fy b、Lu a、Lu b四种血型抗原芯片的动力学曲线。
图3 Fy a、Fy b、Lu a、Lu b、K、k六种血型抗原芯片特异性检测图。
图4 血型抗原芯片对血型抗体检测的实验内重复性，曲线从上至下依次为Fy b-1、Fy b-2、Fy a-1、Fy a-2、Lu a-1、Lu a-2、Lu b-1、Lu b-2。
图5 血型抗原芯片对血型抗体检测的实验间重复性，曲线从上至下依次为Fy b-D1、Fy b-D2、Fy a-D1、Fy a-D2、Lu a-D1、Lu a-D2、Lu b-D1、Lu b-D2。
图6 临床20例血清样本红细胞意外抗体检测结果，横轴为血清和相应血型抗原的临床微柱凝集法的检测结果，纵轴为包被到微球上的不同抗原名称，0到3000代表血清抗体结合信号由低到高。
图7 临床114例血清样本红细胞意外抗体检测结果，横轴为样本标号，纵轴为包被到微球上的不同抗原名称，0.8721到0.8721058代表血清抗体结合信号由低到高。
图8 临床100例血清样本红细胞意外抗体检测结果，横轴为样本标号，纵轴为包被到微球上的不同抗原名称，0.6166731到0.61671代表血清抗体结合信号由低到高。
具体实施方式
下面将参照附图更详细地描述本公开的示例性实施方式。虽然附图中显示了本公开的示例性实施方式，然而应当理解，可以以各种形式实现本公开而不应被这里阐述的实施方式所限制。相反，提供这些实施方式是为了能够更透彻地理解本公开，并且能够将本公开的范围完整的传达给本领域的技术人员。
实施例1 血型抗原芯片的制备
将未偶联的微球超声重悬，取200uL(浓度为1.25×10 7个/mL)微球到EP管内，将EP管放置在磁力板上30s，将微球与溶液分开，去上清液，移走磁力分离器并用100μl去离子水震荡重悬微球，将EP管放置在磁力板上30s，将微球与溶液分开，去上清液。移走磁力分离器并用80uL 100mM，PH6.2磷酸氢二钠缓冲液，震荡重悬微球，加入10uL的50mg/ml的Sulfo-NHS溶液(溶于水中)，轻柔震荡混匀，加入10μl的50mg/ml的EDC溶液中(溶于水中)，轻柔震荡混匀，在室温孵育20分钟，每隔10分钟轻柔震荡混匀一次，将EP管放置在磁力板上30s，将微球与溶液分开，去上清液。移走磁力分离器并用250uL的50mM MES，PH5.0溶液，震荡重悬微球，将EP管放置在磁力板上30s，将微球与溶液分开，去上清液，重复两次。移走磁力分离器并用100μl的50mM MES，PH5.0溶液，震荡重悬活化并清洗后的微球。加入12.5μg(浓度0.5mg/ml 25μl)纯抗原Fy a到悬浮的微球溶液中，用50mM MES，PH5.0的溶液将总体积增至500μl，震荡混匀进行偶联反应，在室温中边翻转混合边孵育2个小时。将EP管放置在磁力板上30s，将微球与溶液分开，去上清液。移走磁力分离器并用500μl的PBS-TBN溶液震荡重悬偶联后的微球，在室温中翻转混合孵育30min。将EP管放置在磁力板上30s，将微球与溶液分开，去上清液。用1mL PBST溶液清洗微球2次。移走磁力分离器并用1ml的PBST溶液重悬偶联并清洗后的微球，将其保存在4℃的黑暗环境中。
实施例2 抗体的稀释倍数、孵育时间及二抗浓度对检测的影响
取96孔板，每孔加入200μl 1％BSA 37℃孵育1h封闭。加入50μl稀释后的检测样品Fy a抗体和2500个实施例1制备的血型抗原芯片，用PBS-TBN补足100μl，室温孵育一定时间。将96孔板放置在磁力板上5min，将微球与溶液分开，去上清液。用150μlPBS-TBN溶液洗涤96孔板3次(洗板时加入150 μlPBS-TBN溶液，置于振荡器中震荡5min，取出后将96孔板放置在磁力板上5min，将微球与溶液分开，去上清液)。加入50μl的荧光标记的二抗抗人IgG，加入50μlPBS-TBN，室温震荡孵育0.5h，用150μlPBS-TBN溶液洗涤96孔板2次，用200μlPBS-TBN重悬磁珠，用流式细胞仪进行检测。
此实验在样品检测步骤设置三个影响因素，分别是：检测样品稀释倍数、孵育时间和二抗浓度，每种影响因素有4个水平，因此设计三因素、4水平正交试验，利用spss软件进行正交试验设计。
检测样品稀释倍数 25倍 50倍 100倍 200倍
孵育时间 15min 30min 45min 60min
二抗浓度 2ug/ml 4ug/ml 6ug/ml 8ug/ml
实验结果如下：
1)检测样品稀释倍数：25倍、50倍、100倍、200倍均有差异，随稀释倍数增加，信号降低，因此根据实际情况选取稀释倍数100倍。
2)孵育时间：15min与30min、45min、60min均有差异P＜0.05；30min与15min、45min、60min均有差异P＜0.05；45min与15min、30min均有差异，与60min无差异P＞0.05；60min与15min、30min均有差异，与45min无差异P＞0.05；因此最终选取45min。
3)二抗浓度：2ug/ml与4ug/ml、6ug/ml、8ug/ml均有差异P＜0.05；4ug/ml与6ug/ml、8ug/ml无差异P＞0.05；6ug/ml与4ug/ml、8ug/ml无差异P＞0.05；8ug/ml与4ug/ml、6ug/ml无差异P＞0.05；因此最终选取4ug/ml。
综上所述，在血型抗体检测操作中，最佳的检测条件为抗体稀释100倍、抗体与血型抗原芯片孵育45min、二抗浓度为4ug/ml。
实施例3 血型抗原芯片对血型抗体检测的灵敏度
根据实施例1所述的制备方法制备出Fy a、Fy b、Lu a、Lu b四种血型抗原芯片。按照图1所示检测流程，取96孔板，每孔加入200微升1％BSA溶液37℃孵育1h封闭。加入50uL稀释后的抗体，抗体分别稀释163840倍、40960倍、10240倍、2560倍、640倍、160倍、40倍、10倍、5倍和2500个上述四种偶联后的微球，用PBS-TBN将体系体积补足100uL进行室温孵育45min，；然后将96孔板放置在磁力板上5分钟，去上清液；用150微升PBS-TBN溶液 洗涤96孔板3次；加入50微升Cy3标记的抗人IgG，加入50微升PBS-TBN，室温震荡孵育0.5小时；用100微升PBS-TBN溶液洗涤96孔板2次；用200微升PBS-TBN重悬微球，用流式细胞仪进行检测。Fy a、Fy b、Lu a、Lu b四种血型抗原芯片的动力学曲线如图2所示，由实验结果可以看出，当检测抗体稀释163840倍时，本发明所制备的血型抗原芯片依然能检测出抗体，说明血型抗原芯片具有较高的灵敏度，当所述检测抗体的稀释倍数为25-200倍时，抗体的检测效果最佳。
实施例4 血型抗原芯片对血型抗体检测的特异性
根据实施例1所述的制备方法制备出Fy a、Fy b、Lu a、Lu b、K、k六种血型抗原芯片，将六种偶联后的微球混合在一起，按照实施例2所述的最佳检测方法进行抗体检测，分别加入Fy a、Fy b、Lu b、K、k五种不同抗体，验证不同抗原芯片和抗体之间是否存在交叉反应，结果如图3所示。根据实验结果可以看出，本发明所制备的血型抗原芯片具有很好的特异性，不同的抗原芯片均能检测出相应的抗体，不同的抗原芯片之间没有发生交叉反应。
实施例5 血型抗原芯片对血型抗体检测的重复性
根据实施例1所述的制备方法制备出Fy a、Fy b、Lu a、Lu b四种血型抗原芯片，按照实施例2所述的最佳检测方法分别检测相应四种抗体，抗体分别稀40960倍、10240倍、2560倍、640倍、160倍、40倍、10倍，连续两天做相同的实验，每次做两块微孔板，检测本发明所制备的血型抗原芯片对血型抗体检测时的重复性，其中同一天的两块微孔板数据代表实验内重复性，连续两天的两块微孔板数据代表实验间重复性，实验结果如图4和图5所示。根据实验结果可以看出，本发明所制备的血型抗原芯片在对血型抗体进行检测时，实验间及实验内均存在很好的重复性。
实施例6 血型抗原芯片对红细胞意外抗体检测的临床小规模应用
根据实施例1所述的制备方法制备出Fy a、Fy b、Lu a、Lu b、K、k六种血型抗原芯片，分别对临床20例血清标本进行检测，检测方法如实施例2所述的最佳检测方法。其中，临床20例标本经常规微柱凝胶法检测结果为3例抗Fy b、1例抗Fy a、1例抗K、1例抗D、1例抗M抗体、3例抗E联合其他抗体、3例未知抗体、7例阴性标本。而应用血型抗原芯片进行检测所得结果见图6，其 中3例Fy b，1例Fy a、1例K，6例阴性，与常规微柱凝胶法结果一致，有1例经微柱凝胶法检测阴性的标本经血型抗原芯片检测为阳性，3例未知抗体及3例抗E联合其他抗体的标本中经血型抗原芯片检测出现除相应抗原外的其它抗原的阳性反应。结果表明，由于血型抗原芯片灵敏度高，因此可发现常规检测方法中无法检出的弱阳性反应，同时由于包被的为纯化抗原，因此可明确鉴定出所含抗体类型无需再根据细胞谱进行推测，准确性极高。
实施例7 血型抗原芯片对红细胞意外抗体检测的临床大规模应用
根据实施例1所述的制备方法制备出Fy b、Dob、K、Lu a、Lu b、k、CROM、Fy a、Cha、Rga、Doa、Lwa、CR1、Xga、Yta、SC1十六种血型抗原芯片，用上述芯片分别检测114例临床血清标本，检测方法如实施例2所述的最佳检测方法，检测结果如图7所示。需要说明，上述114例临床标本在常规检测方法检测时结果均为阴性，由图7可以看出，利用本申请所制备的血型抗原芯片及检测检测方法能检测到常规方法检测不到的抗体。为验证本发明所述的检测方法的可靠性，进行下一批临床样本的试验。
同样，根据实施例1所述的制备方法制备出Fy b、Dob、K、Lu a、Lu b、k、CROM、Fy a、Cha、Rga、Doa、Lwa、CR1、Xga、Yta、SC1十六种血型抗原芯片用于100例临床血清标本的检测，检测方法如实施例2所述的最佳检测方法，检测结果如图8所示。在常规检测方法检测结果均为阴性的情况下，本发明所制备的血型抗原芯片仍检测到一些抗体，说明所述血型抗原芯片灵敏度高，可检测到临床常规方法检测不到的抗体，从而避免该种抗体导致的溶血性输血不良反应。
以上所述，仅为本发明较佳的具体实施方式，但本发明的保护范围并不局限于此，任何熟悉本技术领域的技术人员在本发明揭露的技术范围内，可轻易想到的变化或替换，都应涵盖在本发明的保护范围之内。因此，本发明的保护范围应以所述权利要求的保护范围为准。

Claims (10)

  1. 一种血型抗原芯片，包括纯化血型抗原和载体，所述的纯化血型抗原包被至所述的载体上；所述的载体为微球，所述的微球选自二氧化硅微球、聚苯乙烯微球、磁性微球和生物大分子聚合物微球中的一种或两种以上的组合。
  2. 根据权利要求1所述的血型抗原芯片，其特征在于，所述纯化血型抗原选自：D、C、E、c、e、JK a、JK b、M、N、S、s、Kp a、Kp b、Lu a、Lu b、Fy a、Fy b、K、k、Le a，Le b、P 1、Dob、CROM、Cha、Rga、Doa、Lwa、CR1、Xga、Yta、SC1中的一种或两种以上的组合。
  3. 根据权利要求1所述的血型抗原芯片，其特征在于，所述的纯化血型抗原包被至载体的量为1-200μg/1.25×10 7个微球。
  4. 一种权利要求1-3任一所述的血型抗原芯片的制备方法，包括如下步骤：
    (1)微球的预处理：将微球超声重悬；
    (2)微球的活化：将微球依次加入N-羟基硫代琥珀酰亚胺(Sulfo-NHS)、碳二亚胺(EDC)活化；
    (3)向微球溶液中加入纯化血型抗原进行偶联，所述的纯化血型抗原能够结合红细胞意外抗体。
  5. 一种权利要求1-3任一所述的血型抗原芯片在如下用途中的应用：(1)红细胞意外抗体的筛查及鉴定；(2)红细胞意外抗体的定量检测；(3)输血配型。
  6. 一种用权利要求1-3任一所述的血型抗原芯片进行血型抗体的鉴定或检测方法，包括：
    (1)将纯化血型抗原包被至载体，所述的纯化血型抗原能够结合红细胞意外抗体；
    (2)使检测样品与包被至载体的抗原孵育；
    (3)加入标记有标记物的二抗；
    (4)针对标记物进行信号检测。
  7. 根据权利要求6所述的方法，其特征在于，所述的检测样品为全血、血清、血浆或抗体，所述检测样品稀释后再与包被至载体的抗原接触，稀释倍数为25-200倍。
  8. 根据权利要求6所述的方法，其特征在于，所述步骤(2)中检测样品 与包被至载体的抗原孵育的时间为15-60min。
  9. 根据权利要求6所述的方法，其特征在于，所述的标记有标记物的二抗选自标记有荧光素或酶的蛋白或抗体及其偶联物；所述蛋白或抗体及其偶联物选自亲和素、链霉亲和素、地高辛抗体、组氨酸抗体、含Ni亲和分子、荧光分子抗体；所述的荧光素选自花青素Cy系列荧光素、Alexa Fluor系列荧光素或异硫氰酸荧光素；所述的酶为碱性磷酸酶或过氧化物酶；所述二抗的加入浓度为1-8ug/ml。
  10. 根据权利要求6所述的方法，其特征在于，所述的信号检测包括荧光法检测、显色法检测、电化学检测、力学检测。
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Families Citing this family (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107643409B (zh) * 2017-09-19 2021-05-04 中国人民解放军总医院 一种血型抗原芯片及其在红细胞意外抗体检测中的应用
JP2021514060A (ja) * 2018-02-16 2021-06-03 ディアガストＤｉａｇａｓｔ ビーズを含むＩｎ Ｖｉｔｒｏ診断デバイス及びその使用
CN109725145A (zh) * 2019-01-03 2019-05-07 北京蛋白质组研究中心 一种Inb血型抗原芯片及其在红细胞意外抗体检测中的应用
CN109725144A (zh) * 2019-01-03 2019-05-07 北京蛋白质组研究中心 一种jmh血型抗原芯片及其在红细胞意外抗体检测中的应用
CN113092793A (zh) * 2021-04-16 2021-07-09 江苏力博医药生物技术股份有限公司 一种检测Rh血型抗原的方法、***及其应用
CN113640528A (zh) * 2021-06-28 2021-11-12 青岛益柏生物科技有限公司 全自动rh***血型检测仪检测方法
CN114137231B (zh) * 2022-01-29 2022-04-29 北京大有天弘科技有限公司 一种血型不规则抗体的检测试剂盒及其应用
CN114113639B (zh) * 2022-01-29 2022-04-19 北京大有天弘科技有限公司 一种血型抗体检测方法及其应用
CN114397464B (zh) * 2022-03-24 2022-06-10 天津德祥生物技术有限公司 一种红细胞膜碎片与载体的偶联复合物、其偶联方法和应用
CN114660305A (zh) * 2022-03-31 2022-06-24 中国人民解放军军事科学院军事医学研究院 一种快速检测血型抗体滴度的试剂、制备方法、试纸条及应用

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101387648A (zh) * 2008-10-24 2009-03-18 李勇 红细胞膜抗原磁珠试剂盒及在血型抗体检测方面的应用
CN101711363A (zh) * 2007-06-08 2010-05-19 博瑞巴斯德公司 血液样品的多重分析
CN101957366A (zh) * 2009-07-15 2011-01-26 英科新创(厦门)科技有限公司 人红细胞膜抗原包被的微球及其应用
CN104360087A (zh) * 2014-12-10 2015-02-18 中国科学院苏州生物医学工程技术研究所 一种检测Miltenberger血型抗体的方法
CN105137096A (zh) * 2015-08-27 2015-12-09 中国人民解放军总医院 一种用于血型抗体检测的试剂盒及其应用
CN105699667A (zh) * 2016-01-26 2016-06-22 北京中科圆融生物科技发展有限公司 细菌磁颗粒红细胞膜复合颗粒及其临床应用
CN107643409A (zh) * 2017-09-19 2018-01-30 汪德清 一种血型抗原芯片及其在红细胞意外抗体检测中的应用

Family Cites Families (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH0954091A (ja) * 1995-08-09 1997-02-25 Nippon Zoki Pharmaceut Co Ltd 抗赤血球抗体の測定法
NL1003570C2 (nl) * 1996-07-11 1998-01-15 Stichting Centraal Lab Methode voor antigeen- en antistofbepaling in de bloedgroepserologie.
US5776711A (en) * 1996-11-12 1998-07-07 The Regents Of The University Of California Simultaneous human ABO and RH(D) blood typing or antibody screening by flow cytometry
CN1282871A (zh) * 2000-03-29 2001-02-07 长春生物制品研究所 微柱凝胶法血小板血型抗原抗体检测技术和试剂盒
GB0706820D0 (en) * 2007-04-10 2007-05-16 Common Services Agency Blood grouop antibody screening
CN101118239A (zh) * 2007-06-14 2008-02-06 四川省迈克科技有限责任公司 一种利用微流体技术检测血型的试剂盒及其制备方法
JP5210551B2 (ja) * 2007-06-19 2013-06-12 ベックマン コールター バイオメディカル リミテッド 赤血球標識用磁性粒子
CN101109756A (zh) * 2007-08-20 2008-01-23 陕西省血液中心 一种用于检测血清中不规则抗体的试剂盒及其制备方法
CN103344772B (zh) * 2013-07-19 2015-06-24 中国科学院苏州生物医学工程技术研究所 Miltenberger血型抗体检测新方法
CN106754692B (zh) * 2016-12-15 2020-03-13 汪德清 一种红细胞膜及其制备方法和应用

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101711363A (zh) * 2007-06-08 2010-05-19 博瑞巴斯德公司 血液样品的多重分析
CN101387648A (zh) * 2008-10-24 2009-03-18 李勇 红细胞膜抗原磁珠试剂盒及在血型抗体检测方面的应用
CN101957366A (zh) * 2009-07-15 2011-01-26 英科新创(厦门)科技有限公司 人红细胞膜抗原包被的微球及其应用
CN104360087A (zh) * 2014-12-10 2015-02-18 中国科学院苏州生物医学工程技术研究所 一种检测Miltenberger血型抗体的方法
CN105137096A (zh) * 2015-08-27 2015-12-09 中国人民解放军总医院 一种用于血型抗体检测的试剂盒及其应用
CN105699667A (zh) * 2016-01-26 2016-06-22 北京中科圆融生物科技发展有限公司 细菌磁颗粒红细胞膜复合颗粒及其临床应用
CN107643409A (zh) * 2017-09-19 2018-01-30 汪德清 一种血型抗原芯片及其在红细胞意外抗体检测中的应用

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