WO2009149615A1 - 快速检测人abo/rh/mn血型的方法及试剂盒 - Google Patents

快速检测人abo/rh/mn血型的方法及试剂盒 Download PDF

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Publication number
WO2009149615A1
WO2009149615A1 PCT/CN2009/000322 CN2009000322W WO2009149615A1 WO 2009149615 A1 WO2009149615 A1 WO 2009149615A1 CN 2009000322 W CN2009000322 W CN 2009000322W WO 2009149615 A1 WO2009149615 A1 WO 2009149615A1
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blood
sample
pad
antibody
tested
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PCT/CN2009/000322
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English (en)
French (fr)
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WO2009149615A8 (zh
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汪大明
杨文�
肖江群
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英科新创(厦门)科技有限公司
王保丹
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Priority to EP09761240.2A priority Critical patent/EP2315024B1/en
Priority to MX2010013460A priority patent/MX2010013460A/es
Priority to JP2011510804A priority patent/JP5181058B2/ja
Priority to KR1020107028483A priority patent/KR101333654B1/ko
Priority to US12/995,288 priority patent/US20110076698A1/en
Priority to RU2010150683/15A priority patent/RU2475758C2/ru
Priority to BRPI0914801A priority patent/BRPI0914801A2/pt
Priority to ES09761240.2T priority patent/ES2444430T3/es
Publication of WO2009149615A1 publication Critical patent/WO2009149615A1/zh
Publication of WO2009149615A8 publication Critical patent/WO2009149615A8/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
    • G01N33/80—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 a method for rapidly detecting a human ABO/Rh/MN blood type and a corresponding kit. Background of the invention
  • the blood typing method is a conventional method of dividing human blood red blood cells into four different types of B, AB, and 0, and the R blood type system and the MN blood type system.
  • a general method for identifying a blood type is to mix the test red blood cells with a reagent containing a blood type antibody, and after reacting the two, determine the blood type of the red blood cells to be examined by observing whether or not the red blood cells are agglutinated.
  • One such method is to first isolate red blood cells from the patient's serum. Mix appropriate amount of red blood cells with the test solution (containing anti-A serum, anti-B serum or anti-D serum or anti-M serum, anti-N serum) introduced into the test tube or slide, and add other reagents for the immune reaction and keep warm. , centrifugation and agitation. Then visually observe to determine if the cells have accumulated.
  • the sample is aggregated in the presence of an anti-A agent, and no aggregation occurs in the presence of anti-B, it is identified as type A blood.
  • the sample was aggregated in the presence of an anti-B agent, but was identified as type B blood without aggregation in the presence of anti-A.
  • the sample was aggregated with anti-A and anti-B reagents, and the sample was type AB blood. Samples were not classified as anti-A and anti-B reagents and were classified as type 0 blood.
  • the Rh-positive blood type is aggregated between the sample and the anti-D reagent, and the blood sample in which the sample and the anti-D reagent do not agglutinate is Rh-negative blood type.
  • the MN blood group system can be divided into three blood types: 1 ⁇ 1, N and MN depending on whether or not the corresponding agglutination reaction occurs.
  • the techniques for detecting human ABO/Rh/MN blood type can be divided into two types, one is to detect blood group antigens bound on the cell membrane, including red blood cell saline agglutination method, neutralization experiment, absorption experiment, dissociation experiment and mixed agglutination reaction.
  • the other type is the direct detection of free blood group antigens, including synergistic agglutination, radioimmunoassay, and the like.
  • the commonly used erythrocyte saline agglutination method is to bind a blood group antigen on the surface of fresh red blood cells by a known IgM or IgG type antibody, thereby causing agglutination of red blood cells, thereby judging the blood type of the blood to be tested.
  • This method is widely used for blood group testing (matching) before clinical transfusion.
  • this method requires a corresponding supporting reagent, and the reagent needs to be cryopreserved, and the result is difficult to judge.
  • the neutralization test method takes a long time and the sensitivity is relatively low.
  • the dissociation experiment and the mixed agglutination reaction method have high operation requirements, and each experiment requires repeated exploration conditions, and the sensitivity thereof is not high;
  • the synergistic coagulation reaction method is to sensitize Staphylococcus aureus A protein (SPA) with an IgG type antibody.
  • SPA Staphylococcus aureus A protein
  • the blood type of the test sample is judged by observing whether the staphylococcus is agglutinated, but the method has high requirements for the preservation of the reagent; the radioimmunoassay and the enzyme-linked immunosorbent method have high sensitivity, but it takes a long time and is not suitable for the test. Detection.
  • An object of the method is to provide a method for detecting a human ABO Rh/MN blood type, the method comprising: adding a blood sample to be tested to a pre-coated anti-A or anti-B or anti-D or anti-M or anti-N blood group antibody On the test strip, after the rinse solution is added to one end of the test strip, the red blood cells which have not undergone agglutination reaction with the antibody are moved away from the sample point by the rinsing liquid, and the red blood cells which are immunoagulated with the antibody are left in the addition. At the sample point, the blood type of the sample to be tested can then be determined based on whether there is agglutinated red blood cell residue at the reaction site.
  • the volume of the blood sample to be tested dropped on the test strip can be as small as 1-10 ⁇ l.
  • the rinsing solution is 10 mM phosphate buffered saline (PBS), physiological saline or other isotonic solution.
  • PBS phosphate buffered saline
  • the blood sample to be tested is dropped on the loading pad of the test reagent strip.
  • the method of immobilizing the blood group antibody may be a method such as physical adsorption or chemical crosslinking.
  • the blood sample to be tested may be red blood cells or whole blood or a red blood cell or whole blood suspension diluted with a physiological solution of saline, phosphate buffer or the like.
  • the blood sample may also include a blood sample with partial hemolysis or high blood lipid content.
  • the judgment of the detection result may be visually observed or judged by using an optical signal or an electric signal collecting instrument.
  • Another object of the present invention is to provide a kit for performing the above detection method, which comprises a reagent strip, The rinsing liquid and the blood collecting tool are composed, wherein the detecting reagent strip is composed of a rinsing pad, a loading pad, a cushion pad, an absorbent pad, a waterproof tape, and a polyester plate used for the backing.
  • FIG. 1 is a schematic view showing a strip of ABO blood group rapid detecting reagent in the blood type detecting kit of the present invention.
  • Fig. 2 is a view showing the further schematic structural decomposition of the ABO blood group rapid test reagent strip in the blood group detecting kit of the present invention.
  • 1 is a rinse pad
  • 2 is a waterproof tape
  • 3 is a sample pad containing blood type antibodies
  • 4 is a cushion
  • 5 is an absorbent pad
  • 6 is a polyester backing.
  • Fig. 3 is a schematic view showing a Rh blood type rapid detection reagent strip in the blood group detecting kit of the present invention. among them
  • 1 is a rinse pad
  • 2 is a waterproof tape
  • 3 is a sample pad containing an anti-D antibody
  • 4 is a cushion
  • 5 is an absorbent pad.
  • Fig. 4 is a schematic view showing the MN blood type rapid detection reagent strip in the blood type detecting kit of the present invention.
  • Fig. 5 is a view showing the reaction after a positive blood sample was dropped on a glass fiber mat to which a blood group antibody was immobilized.
  • 1 is red blood cells
  • 2 is blood type antibody
  • 3 is a glass fiber loading pad.
  • the invention relates to a method for rapidly detecting ABO/R/MN blood type of a blood sample to be tested in humans and a corresponding kit.
  • the present invention first provides a method of detecting the ABO/Rh/MN blood type of a human blood sample to be tested.
  • the method comprises: adding a blood sample to be tested on a test strip pre-coated with anti-A or anti-B or anti-D or anti-M or anti-N blood type antibody, and adding a rinse liquid to one end of the test strip to make
  • the red blood cells that have not undergone agglutination reaction with the antibody are far away from the sample point under the rinsing of the rinsing liquid, and the red blood cells which are immunoagglutinated with the antibody are left at the loading point, and then the erythrocyte residue can be agglutinated according to the reaction site.
  • the blood sample to be tested may be red blood cells or whole blood or a red blood cell suspension or a whole blood diluent diluted with a physiological solution of saline, phosphate buffered saline (PBS) or the like.
  • the blood sample may also include a hemolyzed sample or a blood sample having a high blood lipid content.
  • the volume of the blood sample to be tested to be dropped is not more than 10 ⁇ l.
  • the rinsing solution is 10 mM phosphate buffer, physiological saline or other solution.
  • the method of immobilizing the blood group antibody may be a method such as physical adsorption or chemical crosslinking.
  • the invention further provides a kit for completing the above detection method, the kit is composed of a detection reagent strip, a rinsing liquid and a blood stasis tool, wherein the detection reagent strip is composed of a rinsing pad, a loading pad, a cushion pad, an absorbent pad, and a waterproof tape. And the composition of the polyester board used to make the backing (see Figure 1).
  • the rinsing pad, the loading pad, the cushion, and the absorbent pad are made of a porous polymer material having a certain pore diameter such as glass fiber, and the pore size thereof allows at least a single red blood cell. by.
  • the rinse pad, sample pad and cushion are sometimes collectively referred to as fiberglass mats.
  • said blood sample to be tested is dropped on the loading pad, and said rinsing liquid is dropped on the rinsing pad.
  • the loading pad is a porous carrier to which a blood group antibody is immobilized.
  • the method of fixing the blood group antibody may be a method such as physical adsorption or chemical crosslinking.
  • said blood sample comprises a blood sample having a certain degree of hemolysis or a higher blood lipid content.
  • the determination of the detection result can be accomplished by the naked eye or by an optical signal or an electrical signal acquisition and interpretation instrument.
  • detecting a person can be accomplished by the naked eye or by an optical signal or an electrical signal acquisition and interpretation instrument.
  • an optical signal or an electrical signal acquisition and interpretation instrument can be accomplished by the naked eye or by an optical signal or an electrical signal acquisition and interpretation instrument.
  • the steps of the ABO/Rh/MN blood type include:
  • Fig. 2 Fix the blood group antibody on a glass fiber mat with a certain pore diameter by physical adsorption or chemical crosslinking.
  • the physical or chemical immobilization method used has little effect on the biological activity of the antibody, and the immobilized antibody is added with an antibody protein preservative, and the protective agent may be selected from the group consisting of bovine serum albumin, calf serum, peptone, sucrose, polyethylene. Pyrrolidone and its compositions.
  • High titer human ABO/Rh/MN blood group monoclonal antibody diluted to appropriate potency in 5 mM phosphate buffer, and added antibody immobilization protectant to facilitate immobilization and preservation of antibody on glass fiber mat, diluted antibody A reaction enhancer may be added to the solution to facilitate the subsequent immune response.
  • the antibody was spread on a pre-activated glass fiber mat at a ratio of 5 cm x 5 cm of fiber mat per 1.5 ml of diluted antibody, dried at 37 ° C, and sealed for storage.
  • the blood type of the blood sample to be tested can be determined based on whether there is red residue on the sample pad.
  • the agglutination reaction after rinsing, there is no significant red substance residue on the loading pad containing the anti-B antibody, indicating that the blood sample to be tested does not undergo agglutination reaction with the anti-B antibody. Therefore, it can be determined from the test results that the blood type blood type of the agglutination reaction with the anti-A anti-B antibody is AB type, and the blood type of the blood sample only agglutinating with the anti-A antibody is type A; only agglutination reaction with the anti-B antibody occurs.
  • the blood type of the blood sample is type B.
  • the blood type of the blood sample which did not undergo agglutination reaction with the anti-A 3 ⁇ 4 B antibody was type 0. '
  • Rh blood group detection reagent strip when there is a significant red substance residue on the loading pad containing the anti-D antibody after the reaction is rinsed, it indicates that the blood sample to be tested does not undergo agglutination reaction with the anti-D antibody; when the reaction is rinsed, the anti-D is contained. When there is no significant red substance residue on the sample pad of the antibody, it indicates that the blood sample to be tested does not undergo agglutination reaction with the anti-D antibody.
  • the blood type of the blood sample which is agglutinated with the anti-D antibody is a Rh-positive blood type
  • the blood type of the blood sample which does not agglutinate with the anti-D antibody is a Rh-negative blood type.
  • the MN blood group detection reagent strip when the reactants are washed, there is a significant red substance residue on the loading pad containing the anti-M antibody, indicating that the blood test to be tested is captured by the corresponding anti-M antibody, thereby occurring with the anti-M antibody. Agglutination reaction; After rinsing, there is no significant red substance residue on the pad containing anti-M antibody, indicating that the blood sample to be tested has no agglutination reaction with anti-M antibody; when the reaction is rinsed, the anti-N antibody-containing sample pad has significant When the red substance remains, it indicates that the blood sample to be tested has an agglutination reaction with the anti-N antibody.
  • the blood type blood type which is agglutinated with the anti-M anti-N antibody is MN type
  • the blood type of the blood sample which only agglutinates with the anti-M antibody is M type; only agglutination reaction with the anti-N antibody occurs.
  • the blood type of the blood sample is N type.
  • the rapid test reagent strip is composed of a backing, a rinse pad, a sample pad, a cushion, an absorbent pad, and a waterproof tape.
  • a rinsing pad, a loading pad, a cushion, an absorbent pad, and a waterproof tape are sequentially attached to the backing.
  • the edge of the pad is pressed against the edge of the pad, the edge of the pad is pressed against the edge of the pad, the edge of the pad is pressed against the edge of the pad, and the tape is pasted on the overlapping edge of the pad and the pad to prevent flushing.
  • the liquid spreads from the surface of the loading pad.
  • the backing can be made of, but not limited to, a polyester material.
  • said antibody immobilization means immobilizing an antibody against A or anti-B or anti-D or anti-M or anti-N to a glass fiber having a certain pore diameter by physical or chemical crosslinking or the like. Pad or other carrier of similar pore size.
  • the antibody Prior to fixation, the antibody may be diluted to a certain extent depending on the potency of the antibody to a suitable concentration that can be used.
  • the antibody may be immobilized on a glass fiber mat (sampling pad) by soaking or evenly applying or spraying, and then lyophilized or dried.
  • the antibody referred to therein may be previously treated with a glutaraldehyde crosslinking agent.
  • the blood sample to be tested may be red blood cells or whole blood or a red blood cell suspension diluted with a solution of 10 mM phosphate buffer (PBS), physiological saline or the like (the dilution is usually 2). -50%). Blood samples having a certain degree of hemolysis or having a higher blood lipid content are also suitable for use in the kit of the present invention.
  • PBS phosphate buffer
  • physiological saline physiological saline
  • the appropriately-prepared or undiluted whole blood or blood cell sample to be tested may be added to the sample pad to which the blood group antibody is immobilized, and the sample pad is rinsed with the rinse liquid after the immunological binding reaction occurs.
  • the rinse solution can be 10 mM phosphate buffer (PBS) or other solution.
  • PBS phosphate buffer
  • the presence of red blood cells agglutinated after the immune response can be easily observed with the naked eye, or the blood type detection results of the batch samples can be read using an optical signal or an electrical signal acquisition and interpretation instrument.
  • the actual operation can be performed on two test reagent strips which are separated and juxtaposed on the same polyester sheet backing and fixed with anti-A or anti-B blood type antibodies on the sample pad.
  • the actual operation can be performed on two test reagent strips which are separated and juxtaposed on the same polyester sheet backing and fixed with anti-M or anti-N blood type antibodies on the sample pad.
  • the human ABO/Rh/MN blood type rapid test kit of the present invention is suitable for blood group blood type screening, street blood sampling, and home ABO/Rh/MN blood type self-test. Quick and easy identification of blood types is achieved through rapid immune response and simple and convenient results interpretation.
  • the method is simple and easy to operate, and no additional reagents such as anti-human globulin are required.
  • the amount of the blood sample to be tested may not exceed 10 ⁇ l, and the entire detection process does not exceed 2 minutes.
  • the method and the detection kit for rapidly detecting the human ABO/Rh/MN blood type according to the present invention are based on the principle of immunochromatography, and whether the blood type is determined by using a red substance remaining after red blood cell agglutination after the blood type substance antigen antibody reaction remains. .
  • the method of the present invention is simple in operation and can avoid various defects of the conventional blood type detecting method.
  • the method of the invention is suitable for single-person or multi-person rapid detection. It is especially suitable for rapid detection of blood type ABO/Rh/MN blood samples and family self-test of blood type. If used in conjunction with the appropriate instrument, this method can be used for rapid screening of blood bank systems for populations of blood samples with ABO/Rh/MN. Specific implementation
  • Example 1 Preparation and detection steps of detection reagent strips in human ABO blood group kit (1) Fix the blood group antibody (anti-A, anti-B antibody) on the loading pad:
  • a high-value blood group monoclonal anti-A anti-B antibody (produced by Changchun Bode Biotechnology Co., Ltd.) was diluted to 5 ml with 5 mM phosphate buffer solution, and the final titer was 64. Adding an antibody immobilization protectant (composed of 5% bovine serum albumin, 5% sucrose, 5% PVP, 5% calf serum phosphate buffer) to the diluted antibody solution to promote immobilization of the antibody on the glass fiber mat And save.
  • the antibody was spread on a pre-activated glass fiber mat in a ratio of 5 cm X 5 cm glass fiber mat per 1.5 ml of diluted antibody, then dried and sealed in a 37 ⁇ solution.
  • a 20 mm wide rinsing fiberglass mat is placed on the center of a rectangular polyester sheet having a width of about 5-10 cm, and then 10 mm wide loading pads, cushions and 30 mm wide absorbent pads are applied to both sides, and finally rinsed.
  • the pad and the pad are pasted with a waterproof tape to prevent the pad from turning up and the application and rinsing fluid to spread.
  • the polyester sheets adhered to each layer were cut into strips of 5 mm width to obtain test strips having a length of about 5 to 10 cm and a width of about 5 mm (see Figs. 1 and 2).
  • Example 2 Preparation and detection steps of a test reagent strip in a human Rh blood test kit
  • a high-value blood group monoclonal anti-D antibody (manufactured by MILLIPORE, USA) was diluted to 1 ml with a 5 mM phosphate buffer solution, and the final titer was 32. Adding an antibody immobilization protectant (composed of 5% albumin, 5% sucrose, 5% sucrose, 5% calf serum in PBS) to the diluted antibody solution to promote immobilization and preservation of the antibody on the glass fiber mat, and A reaction enhancer (phosphate buffer containing 2% Tween 20 and 2% Triton X-100) was added to facilitate the subsequent immune response.
  • an antibody immobilization protectant composed of 5% albumin, 5% sucrose, 5% sucrose, 5% calf serum in PBS
  • a reaction enhancer phosphate buffer containing 2% Tween 20 and 2% Triton X-100
  • the antibody was spread on a pre-activated glass fiber mat at a ratio of 5 cm x 5 cm of fiber mat per 1.5 ml of diluted antibody, then dried at 37 ° C, and then sealed for storage (see Figure 3).
  • the other steps are similar to the preparation and detection steps of the ABO blood group kit rapid test reagent strip.
  • Example 3 Preparation and detection steps of detection reagent strips in human MN blood group detection kit
  • the preparation and detection steps of the rapid detection reagent strip of the human MN blood type detection kit are the same as the preparation and detection steps of the rapid detection reagent strip of the human Rh blood type detection kit, and the only difference is that the antibody immobilized on the detection reagent strip is replaced with anti-M and Anti-N antibody (see Figure 4).
  • Example 4 Comparison of blood typing detection method of the present invention with existing conventional methods
  • the invention can detect a wide range of samples, and can also detect samples such as partial hemolysis and hyperlipidemia.
  • the kit detection method of the invention avoids the defects that the commonly used clinical blood type identification methods have a long verification time, a large manual labor intensity, and a large influence of the operator's subjective deviation and the possibility of contamination.

Description

快速检测人 ABO/Rh/MN血型的方法及试剂盒
发明所属领域
本发明涉及快速检测人类 ABO/Rh/MN血型的方法及相应的试剂盒。 发明背景
血液分型方法是将人血液红细胞分成 、 B、 AB和 0四种不同类型以及 R 血型 系统和 MN血型系统的常规方法。 鉴定血型的一般方法是将受检红细胞与含有血型抗 体的试剂混合， 两者反应后， 通过观察红细胞是否发生细胞凝集来判断被检红细胞的 血型。 此类方法是首先从病人血清中分离红细胞。 将适量的红细胞与引入试管中或玻 片上的试验溶液（含有抗 A血清、抗 B血清或者抗 D血清或者抗 M血清、抗 N血清） 混和， 可以另外加入利于免疫反应进行的其他试剂并保温、 离心和搅拌。 然后肉眼观 察， 确定细胞是否发生了聚集。
如果样品在抗 A试剂存在下发生细胞聚集，而在抗 B存在下不发生聚集即鉴定为 A型血。 样品在抗 B试剂存在下发生细胞聚集， 但在抗 A存在下不发生聚集则鉴定为 B型血。样品与抗 A和抗 B试剂均有聚集， 样品为 AB型血。样品与抗 A和抗 B试剂 均不发生反应则定为 0型血。 样品与抗 D试剂发生聚集的为 Rh阳性血型， 样品与抗 D试剂不发生凝集的血样为 Rh阴性血型。 MN血型系统根据是否发生相应的凝集反应 可以分为1^1、 N和 MN三种血型。
目前检测人 ABO/Rh/MN血型的技术可以分为两类， 一类是检测细胞膜上结合的 血型抗原， 包括红细胞盐水凝集法、 中和实验、 吸收实验、 解离实验和混合凝集反应 等。 另一类是直接检测游离血型抗原， 包括协同凝集反应、 放射免疫反应等。
目前常用的红细胞盐水凝集法是通过已知的 IgM或 IgG型抗体结合新鲜红细胞表 面的血型抗原， 导致红细胞凝集， 进而判断被检血液的血型。 此方法被广泛用于临床 输血前的血型检验（配型）。 然而， 这种方法需要相应的配套试剂， 而且试剂需要低温 保存， 结果也难以判断。
其他血型检测方法也都存在各自的缺点， 例如中和实验方法费时较长， 而且灵敏 度比较低。 解离实验和混合凝集反应法操作要求高， 每次实验需要反复摸索条件， 其 灵敏度也不高；协同凝结反应方法是用 IgG型抗体致敏金黄色葡萄球菌 A蛋白（SPA)， 通过观察葡萄球菌是否凝集来判断被检样品的血型， 但该方法对试剂保存要求较高; 放射免疫和酶联免疫方法虽然灵敏度高， 但是费时较长、 不适 ¾革人份检测 也无法 进行现场检测。
总之， 目前常用的临床血型鉴定方法存在检定时间长、 手工劳动强度大， 以及结 果受主观偏差影响较大和存在污染的可能等问题。 因此， 有必要对传统的血型检测方 法做出实质性的改进。 发明概要
本方法的一个目的是提供一种检测人 ABO Rh/MN血型的方法， 该方法包括将被 测血样滴加在预先包被固定了抗 A或抗 B或抗 D或者抗 M或者抗 N血型抗体的测试 条上， 在测试条的一端滴加冲洗液后， 使未与抗体发生凝集反应的红细胞在冲洗液的 泳动下远离加样点处， 而与抗体发生免疫凝集的红细胞被残留在加样点处， 然后即可 根据反应位点是否有凝集的红细胞残留来判定被测样品的血型。
根据本发明的一个优选实施方案， 其中测试条上滴加的待测血样的体积可以小至 1-10μ1。 所说的冲洗液是 10mM磷酸盐缓冲液 （PBS )、 生理盐水或者其他等张溶液。 所说的待检血样是滴加在检测试剂条的加样垫上。
根据本发明的一个优选实施方案， 其中所说的固定血型抗体的方法可以是物理吸 附或者化学交联等方法。
根据本发明的一个优选实施方案， 其中所说的待测血样可以是红细胞或者全血或 者是用生理盐水、 磷酸盐缓冲液等等张溶液稀释的红细胞或全血悬浮液。 并且所说的 血样也可以包括部分溶血或者高血脂含量的血样。
根据本发明的一个优选实施方案， 其中检测结果的判断可以是肉眼观察或者利用 光学信号或者电信号采集仪器进行判断。
简单地说， 当血液样品与结合了相应的血型抗体的加样垫接触并反应后， 即在加 样垫上与相应的抗体发生免疫结合反应。 用冲洗液体冲洗之后， 发生了免疫结合反应 的样品即在加样垫上残留显著的红色物质， 而没有发生免疫结合反应的血样则被冲洗 液冲洗而离幵加样垫， 加样点处便没有红色物质残留。 从而可根琚红细胞与相应抗体 发生免疫反应后， 加样垫上是否有红色物质的残留来判断红细胞是否和血型抗体发生 了免疫反应， 进而判定血液样品的 ABO/Rh/MN血型。
本发明的另一个目的是提供完成上述检测方法的试剂盒，该试剂盒由检测试剂条、 冲洗液和采血工具组成， 其中检测试剂条由冲洗垫、 加样垫、 缓冲垫、 吸水垫、 防水 胶布以及用来做背衬的聚酯板组成。 发明的附图说明
图 1显示本发明血型检测试剂盒中 ABO血型快速检测试剂条的模式图。
图 2显示本发明血型检测试剂盒中 ABO血型快速检测试剂条的进一步模式图结构 分解。 其中 1为冲洗垫， 2为防水胶布， 3为含有血型抗体的加样垫， 4为缓冲垫， 5 为吸水垫， 6为聚酯板背衬。
图 3显示本发明血型检测试剂盒中 Rh血型快速检测试剂条的模式图。 其中
1为冲洗垫， 2为防水胶布， 3为含有抗 D抗体的加样垫， 4为缓冲垫， 5为吸水垫。
图 4显示本发明血型检测试剂盒中 MN血型快速检测试剂条的模式图。
图 5显示为固定有血型抗体的玻璃纤维垫上滴加阳性血样后的反应示意图。
其中 1为红细胞， 2为血型抗体， 3为玻璃纤维加样垫。 发明的详细说明
本发明涉及快速检测人类待测血样的 ABO/R /MN血型的方法及相应的试剂盒。 本发明首先提供了检测人类待测血样的 ABO/Rh/MN血型的方法。 所说的方法包 括将被测血样滴加在预先包被固定了抗 A或抗 B或抗 D或者抗 M或者抗 N血型抗体 的测试条上， 在测试条的一端滴加冲洗液后， 使未与抗体发生凝集反应的红细胞在冲 洗液的泳动下远离加样点处， 而与抗体发生免疫凝集的红细胞被残留在加样点处， 然 后即可根据反应位点是否有凝集的红细胞残留来判定被测样品的血型。 根据本发明的一个优选实施方案， 其中所说的待测血样可以是红细胞或者全血或 者是用生理盐水、 磷酸盐缓冲液 （PBS ) 等等张溶液稀释的红细胞悬浮液或者全血稀 释液。 并且所说的血样也可以包括溶血样品或者高血脂含量的血样。
根据本发明的一个优选实施方案，其中所说的滴加的待测血样的体积不大于 10μ1。 所说的冲洗液是 10mM磷酸盐缓冲液、 生理盐水或者其他溶液。
根据本发明的一个优选实施方案, 其中所说的固定血型抗体的方法可以是物理吸 附或者化学交联等方法。
本发明进一步提供完成上述检测方法的试剂盒， 该试剂盒由检测试剂条、 冲洗液 和釆血工具组成， 其中检测试剂条由冲洗垫、 加样垫、 缓冲垫、 吸水垫、 防水胶布以 及用来做背衬的聚酯板组成 （参见图 1 )。
根据本发明的一个优选实施方案， 其中所说的冲洗垫、 加样垫、 缓冲垫、 吸水垫 是由玻璃纤维等具有一定孔径的多孔高分子材料制成的， 并且其孔径大小至少允许单 个红细胞通过。 根据其材质的选择， 其中冲洗垫、 加样垫和缓冲垫有时又统称为玻璃 纤维垫。
根据本发明的一个优选实施方案， 其中所说的待测血样是滴加在加样垫上， 并且 所说的冲洗液体是滴加在冲洗垫上。 加样垫是固定有血型抗体的多孔载体。
并且固定血型抗体的方法可以是物理吸附或者化学交联等方法。
根据本发明的一个优选实施方案，其中所说的血样包括有一定程度溶血或者血脂含 量较高的血样。
根据本发明的一个优选实施方案， 其中检测结果的判断可以是用肉眼或者利用光 学信号或者电信号采集和判读仪器来完成。 具体地说， 使用本发明的试剂盒， 检测人
ABO/Rh/MN血型的操作步骤包括：
1) 将人 ABO/Rh/MN血型抗体直接或者间接固定在加样垫 3 (图 2)上： 通过物理 吸附或者化学交联手段将血型抗体固定在具有一定孔径的玻璃纤维垫上。 其中所采用 的物理或者化学固定方法对抗体的生物活性影响不大， 同时固定化抗体内添加有抗体 蛋白保存剂， 保护剂可选自牛血清白蛋白、 小牛血清、 蛋白胨、 蔗糖、 聚乙烯吡咯烷 酮及其组合物。
取高效价的人 ABO/Rh/MN血型单克隆抗体，以 5mM磷酸盐缓冲液稀释到适当的 效价， 并加入抗体固定保护剂以利于抗体在玻璃纤维垫上的固定和保存， 稀释后的抗 体溶液中可加入反应增强剂， 以促进后续免疫反应的进行。 按照每 1.5ml稀释后的抗 体铺展 5cm X 5cm的纤维垫的比例， 将抗体铺敷在预先活化的玻璃纤维垫上， 37°C烘 干后密封保存。
2) 在加样垫 3上滴加待测样品， 然后在冲洗垫 2 (图 3 )上滴加冲洗液体。 加样并 滴加冲洗液后， 即发生免疫结合反应， 同时显现结果。
3) 反应完成并冲洗后， 可根据加样垫上是否有红色残留物来判定被检血样的血 型。
在 ABO血型检测试剂条上， 当反应物被冲洗后， 含有抗 A抗体的加样垫上有显 著的红色物质残留时， 说明待测血样被相应抗 A抗体捕获， 从而与抗 A抗体发生了凝 集反应 （即阳性反应）； 冲洗后， 含有抗 A抗体的加样垫上没有显著的红色物质残留 时， 说明待测血样与抗 A抗体未发生凝集反应 （即阴性反应）； 当反应冲洗完毕后含 有抗 B抗体的加样垫上有显著的红色物质残留时， 说明待测血样与抗 B抗体发生了凝 集反应； 冲洗后， 含有抗 B抗体的加样垫上没有显著的红色物质残留时， 说明待测血 样与抗 B抗体未发生凝集反应。 因此， 由检测结果可以判定， 与抗 A抗 B抗体均发生 凝集反应的血样血型为 AB型， 仅与抗 A抗体发生凝集反应的血样的血型为 A型； 仅 与抗 B抗体发生凝集反应的血样的血型为 B型。 与抗 A ¾ B抗体均未发生凝集反应 的血样的血型为 0型。 '
在 Rh血型检测试剂条上， 当反应冲洗完毕后含有抗 D抗体的加样垫上有 显著的红色物质残留时， 说明待测血样与抗 D抗体未发生凝集反应； 当反应冲洗完毕 后含有抗 D抗体的加样垫上没有显著的红色物质残留时，说明待测血样与抗 D抗体未 发生凝集反应。 因此， 由检测结果可以判定， 与抗 D抗体发生凝集反应的血样的血型 为 Rh阳性血型， 与抗 D抗体不发生凝集反应的血样的血型为 Rh阴性血型。
在 MN血型检测试剂条上， 当反应物被冲洗后， 含有抗 M抗体的加样垫上有显 著的红色物质残留时， 说明待测血杼被相应抗 M抗体捕获， 从而与抗 M抗体发生了 凝集反应； 冲洗后， 含有抗 M抗体的加 垫上没有显著的红色物质残留时， 说明待测 血样与抗 M抗体未发生凝集反应；当反应冲洗完毕后含有抗 N抗体的加样垫上有显著 的红色物质残留时， 说明待测血样与抗 N抗体发生了凝集反应。 因此， 由检测结果可 以判定， 与抗 M抗 N抗体均发生凝集反应的血样血型为 MN型， 仅与抗 M抗体发生 凝集反应的血样的血型为 M型； 仅与抗 N抗体发生凝集反应的血样的血型为 N型。
根据本发明的一个优选实施方案， .其中所说的快速检测试剂条由背衬、 冲洗 垫、 加样垫、 缓冲垫、 吸水垫以及防水胶布组成。 在背衬上依次贴上冲洗垫、 加样垫、 缓冲垫、 吸水垫以及防水胶布。 加样垫边缘压在冲洗垫边缘上， 缓冲垫边缘压在加样 垫边缘上， 吸水垫边缘压在缓冲垫边缘上， 防水胶布粘贴在冲洗垫和加样垫重叠的边 缘上， 以防止冲洗液体从加样垫表面漫延。 背衬可以由但是不限于聚酯材料制成。
根据本发明的一个优选实施方案， 其中所说的抗体固定是指利用物理或者化 学交联等方法将抗 A或抗 B或抗 D或抗 M或抗 N等抗体固定在具有一定孔径的玻璃 纤维垫或者其他相似孔径的载体上。 固定前， 可根据抗体的效价不同对抗体进行一定 比例的稀释， 使其达到可以使用的适当浓 。 可以采用浸泡或者均匀涂抹或者喷雾的 方法将抗体固定在玻璃纤维垫 （加样垫） 上， 然后再行冻干或者烘干处理。 其中所说 的抗体可以是预先用戊二醛交联剂处理的。 根据本发明的一个优选实施方案， 其中所说的待测血样可以是红细胞或者全 血或者用 10mM磷酸盐缓冲液 (PBS)、 生理盐水等等张溶液稀释的红细胞悬浮液 (稀释 度通常为 2-50%)。 有一定程度的溶血或者有较高血脂含量的血样也适用于本发明的试 剂盒。
可以将经过适当稀释或未稀释的待检全血或者血细胞样品加至固定有血型抗体的 加样垫上， 待发生免疫结合反应后用冲洗液冲洗加样垫.。 冲洗液可以是 10mM磷酸 盐缓冲液 （PBS )或者其他溶液。 免疫反应后凝集的红细胞的存在可以很容易用肉眼 观察到，或者也可利用光信号或者电信号采集和判读仪器读出批量样品的血型检测结 果。
如进行 ABO血型的检测， 实际操作可以在分隔并列制作于同一块聚酯板背衬上、 分别在加样垫上固定有抗 A或抗 B血型抗体的两条检测试剂条上完成。
如进行 MN血型的检测， 实际操作可以在分隔并列制作于同一块聚酯板背衬上、 分别在加样垫上固定有抗 M或抗 N血型抗体的两条检测试剂条上完成。
本发明的人 ABO/Rh/MN血型快速检测试剂盒适合血站血型筛査、 街头采血以及 家庭 ABO/Rh/MN血型自检使用。 通过快速免疫反应及简单方便的结果判读， 达到对 血型快速简单准确的鉴定。该方法操作简单易行， 不需要额外添加抗人球蛋白等试剂。 本发明的方法中，被检血样的加样量可以不超过 10微升，全部检测过程不超过 2分钟。
本发明快速检测人 ABO/Rh/MN血型的方法和检测试剂盒是根据免疫层析法的原 理，利用血型物质抗原抗体反应后是否残留有因为红细胞凝集而残留的红色物质来实 现对血型的判断。本发明的方法操作简单、可避免现有普通血型检测方法的各种缺陷。 本发明方法适合单人份或者多人份快速检测。特别适合采血现场 ABO/Rh/MN血型的 快速检测以及血型的家庭自检。如结合使用相应的仪器， 该方法可用于血站系统对批 量血液样品 ABO/Rh/MN血型的人群快速筛査。 具体实施方案
以下实施例旨在进一步举例描述本发明， 这些实施例并不以任何方式限制本发明。 在不背离本发明技术解决方案的前提下， 对本发明所作的本领域普通技术人员容易实 现的任何改动或者改变都将落入本发明的权利要求范围之内。 实施例 1： 人 ABO血型试剂盒中检测试剂条的制备及检测步骤 ( 1 ) 在加样垫上固定血型抗体 （抗 A、 抗 B抗体）：
取高效价的血型单克隆抗 A抗 B抗体 （由长春博德生物技术有限公司生产）， 以 5mM磷酸盐缓冲溶液稀释到 lml， 终效价为 64。在稀释后的抗体液中加入抗体固定保 护剂 （由 5%牛血清白蛋白、 5%蔗糖、 5 %PVP、 5%小牛血清的磷酸盐缓冲液组成） 以便促进抗体在玻璃纤维垫上的固定和保存。 按照每 1.5ml稀释后的抗体铺展 5cm X 5cm玻璃纤维垫的比例在预先活化的玻璃纤维垫上铺展抗体， 然后 37Ό烘干处理并密 封保存。
(2) 快速检测试剂条的组装：
在一宽度约 5-10cm的长方形的聚酯板背衬中央贴上 20mm宽的冲洗玻璃纤维垫， 然后两边依次贴上 10mm宽的加样垫、 缓冲垫和 30mm宽的吸水垫， 最后在冲洗垫和 加样垫交接处用防水胶布粘贴， 防止贴垫翻起以及加样和冲洗液的漫延。 然后将各层 均粘贴好的聚酯板切成 5mm宽的条，即得到长约 5-lOcm, 宽约 5mm的检测试剂条（参 见图 1和图 2 )。
( 3 ) 样品检测：
在试剂条的加样垫上滴加 5μ1的全血样品。 滴加待检测样品后， 在冲洗垫 2上缓 慢匀速滴加反应冲洗液 5滴。 待冲洗液层析后观察加样垫的颜色。 加样垫 3上有显著 的红色物质残留为阳性反应，加样垫 3上没有红色物质残留为阴性反应。结合 ΑΒΟ血 型抗体和红细胞反应的关系， 可以很容易得知待检测样品的血型。 或者， 也可以利用 光信号或者电信号采集系统采集加样垫上残留的红色物质， 然后可以对结果自动化判 断， 从而实现对批量血液样品的 ΑΒΟ血型的快速筛査。 实施例 2: 人 Rh血型检测试剂盒中检测试剂条的制备及检测步骤
( 1 ) 在加样垫上固定血型抗体 （抗 D抗体）：
取高效价的血型单克隆抗 D抗体 （美国 MILLIPORE公司生产）， 以 5mM磷酸 盐缓冲溶液稀释到 lml， 终效价为 32。 在稀释后的抗体液中加入抗体固定保护剂 （由 5%白蛋白、 5%蔗糖、 5 %PVP、 5%小牛血清的 PBS溶液组成） 以便促进抗体在玻璃 纤维垫上的固定和保存， 并且加入反应增强剂 （含有 2%Tween20和 2%Triton X-100 的磷酸盐缓冲液),用以促进后续免疫反应的进行。按照每 1.5ml稀释后的抗体铺展 5cm X 5cm的纤维垫的比例在预先活化的玻璃纤维垫上铺展抗体， 然后 37°C烘干处理， 然 后密封保存 （参见附图 3 )。 其他步骤与 ABO血型试剂盒快速检测试剂条的制备及检测步骤类似。 实施例 3: 人 MN血型检测试剂盒中检测试剂条的制备及检测步骤
人 MN血型检测试剂盒快速检测试剂条的制备及检测步骤与人 Rh血型检测试剂 盒快速检测试剂条的制备及检测步骤相同， 不同的只是将其中检测试剂条上固定的抗 体换成抗 M和抗 N抗体 （参见附图 4)。 实施例 4: 本发明血型检测方法与现有常规方法的比较
我们使用传统的血型检测方法和本发明的方法与试剂盒，对普通人群 1500例个体 (献血员） 同时进行血型双盲法检测。 结果显示， 本方法的检测结果和与已知的血球 液体凝集方法相比较， 符合率达到 100%。 而且本发明可检测样本范围比较广， 对于 部分溶血、 高血脂等样本也可以检测。 同时， 本发明的试剂盒检测方法避免了目前常 用的临床血型鉴定方法存在检定时间长、 手工劳动强度大， 以及结果受操作者主观偏 差影响较大和存在污染的可能等缺陷。

Claims

权 利 要 求
1、 一种检测人 ABO/Rh/MN血型的方法， 该方法包括将被测血样滴加在预先包被 固定了抗 A或抗 B或抗 D或抗 M或抗 N血型抗体的测试条上， 在测试条的一端滴加 冲洗液后， 使未与抗体发生凝集反应的红细胞在冲洗液的泳动下远离加样点处， 而与 抗体发生免疫凝集的红细胞被残留在加样点处， 然后即可根据反应位点是否有凝集的 红细胞残留来判定被测样品的血型。
2、根据权利要求 1的方法， 其中所说的待测血样可以是红细胞或者全血或者是用 生理盐水或等渗磷酸盐缓冲液稀释的红细胞或全血。
3、根据权利要求 1的方法， 其中所说的血样可以包括部分溶血或者高血脂含量的 血样
4、 根据权利要求 1 的方法， 其中所说的抗体是以物理吸附或者化学交联法固定 在测试条上。
5、根据权利要求 1的方法， 其中反应位点是否有凝集可以是以肉眼观察或者利用 光学信号或者电信号采集仪器进行判断。
6、用于完成根据权利要求 1的方法的试剂盒， 该试剂盒由检测试剂条和冲洗液及 采血工具组成， 其中检测试剂条由冲洗垫、 加样垫、 缓冲垫、 吸水垫、 透明防水胶布 以及用来做被衬的聚酯板组成。
7、 根据权利要求 7的试剂盒， 其中构成检测试剂条的所说的冲洗垫、 加样垫、 缓 冲垫、 吸水垫是由具有一定孔径的多孔 ¾分子材料制成的， 并且其孔径大小至少允许 单个红细胞通过。
8、 根据权利要求 7的试剂盒， 其中在固定有血型抗体的加样垫上滴加待测血样， 并且在冲洗垫上滴加等渗的冲洗液， 然后观察加样垫上是否有凝集的红细胞残留来判 断被测血样的血型。
PCT/CN2009/000322 2008-06-10 2009-03-26 快速检测人abo/rh/mn血型的方法及试剂盒 WO2009149615A1 (zh)

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MX2010013460A MX2010013460A (es) 2008-06-10 2009-03-26 Un metodo para determinar rapidamente el tipo sanguineo humano abo/rh/mn y su kit de prueba.
JP2011510804A JP5181058B2 (ja) 2008-06-10 2009-03-26 ヒトａｂｏ／ｒｈ／ｍｎ血液型を迅速に判定する方法及びキット
KR1020107028483A KR101333654B1 (ko) 2008-06-10 2009-03-26 인간 ＡＢＯ／Ｒｈ／ＭＮ 혈액형을 신속하게 결정하는 방법 및 그 테스트 키트
US12/995,288 US20110076698A1 (en) 2008-06-10 2009-03-26 method of quickly determining human abo/rh/mn blood type and a test kit thereof
RU2010150683/15A RU2475758C2 (ru) 2008-06-10 2009-03-26 СПОСОБ БЫСТРОГО ОПРЕДЕЛЕНИЯ ТИПА КРОВИ ЧЕЛОВЕКА АВ0/Rh/MN И ТЕСТ-НАБОР ДЛЯ НЕГО
BRPI0914801A BRPI0914801A2 (pt) 2008-06-10 2009-03-26 método para detectar o grupo sanguíneo abo/rh/mn humano, e, kit
ES09761240.2T ES2444430T3 (es) 2008-06-10 2009-03-26 Un procedimiento de determinación rápida del grupo sanguíneo ABO/Rh/MN y un kit de prueba del mismo

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