WO2017045397A1 - 用于血型检测的微管、一体式ABO/RhD血型定型检测卡及制备 - Google Patents

用于血型检测的微管、一体式ABO/RhD血型定型检测卡及制备 Download PDF

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WO2017045397A1
WO2017045397A1 PCT/CN2016/078941 CN2016078941W WO2017045397A1 WO 2017045397 A1 WO2017045397 A1 WO 2017045397A1 CN 2016078941 W CN2016078941 W CN 2016078941W WO 2017045397 A1 WO2017045397 A1 WO 2017045397A1
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antibody
gel
blood
microtube
monoclonal
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PCT/CN2016/078941
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French (fr)
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钟志荣
周翔
吴庆军
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深圳市达科为生物工程有限公司
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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

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  • the invention relates to blood type detection, in particular to a micro tube for blood type detection, an integrated ABO/RhD blood type styling test card and a preparation method thereof.
  • Rh blood group system is an important blood type system after the ABO blood group system, and is also the most complicated system in the red blood cell type system.
  • the antigens in the Rh blood type system include D, C, E, etc., and the D antigen is the most antigenic.
  • the surface of red blood cells contains D antigen positive for RhD, and D antigen is negative for RhD. After RhD negative patients receive RhD positive blood, at least 20% of patients will have an immune response to D antigen, which will occur after the second exposure to the antigen in vivo. Hemolysis reaction, so it is also important to shape RhD.
  • the current ABO/RhD blood type stereotype test card needs to be added with diluted red blood cells for detection, and the operation steps are cumbersome and inefficient.
  • the present invention provides a microtube for blood type detection, an integrated ABO/RhD blood type styling test card, and a preparation method.
  • the microtubule for blood group detection comprises: the microtube comprises a gel, an antibody working solution and a serum separating gel in order from the bottom to the top; the antibody working solution contains a blood group antibody and makes the blood type The antibody-preserving antibody dilution; the serum separation gel has a specific gravity of 1.075 to 1.077, and can undergo thixotropic reaction under the centrifugal force of 150 G to 300 G, and changes from a solid to a fluid; the antibody diluent has a specific gravity greater than that of the serum.
  • the gel is separated and smaller than the red blood cells.
  • the integrated ABO/RhD blood type stereotype test card provided by the invention has: the test card has 8 micro tubes, and the 8 micro tubes are:
  • a first microtubule the microtubule, and the blood group antibody contained in the working fluid of the antibody is anti-A monogram Antibody
  • a second microtubule the microtubule, and the blood group antibody contained in the working fluid of the antibody is an anti-B monoclonal antibody;
  • a third microtubule the microtubule, and the blood group antibody contained in the working fluid of the antibody is an anti-D monoclonal antibody;
  • a fourth microtube comprising a gel, an antibody diluent and a serum separating gel in order from the bottom of the fourth microtube;
  • the fifth micro tube to the eighth micro tube the first to fourth micro tubes are respectively repeated.
  • the preparation method of the integrated ABO/RhD blood type stereotype test card provided by the invention comprises the following steps:
  • the anti-A monoclonal antibody, the anti-B monoclonal antibody and the anti-D monoclonal antibody are respectively formulated into 7 parts by volume of the antibody dilution solution per 1 part by volume of the antibody to prepare the antibody.
  • an anti-monoclonal anti-A gel having IgM properties and having IgM properties are prepared according to the ratio of adding 1.2-1.7 ml of the antibody working solution per 1 g of the swollen gel.
  • the anti-monoclonal anti-A gel, anti-monoclonal anti-B gel, anti-monoclonal anti-D gel and neutral gel are respectively added to Among the first micro tube, the second micro tube, the third micro tube and the fourth micro tube, the fifth to eighth micro tubes respectively repeat the first to fourth micro tubes;
  • 0.8g-2g of the serum separating gel is added to the first micro tube to the eighth micro tube, and 150G-300G is centrifuged for a certain time, so that the serum separation gel is thixotropically changed from solid to fluid and then captured. At the interface of the antibody working fluid;
  • the integrated ABO/RhD blood type stereotype test card of the present invention can directly add peripheral whole blood for detection, and does not need to centrifuge the peripheral whole blood to remove red blood cells for dilution and then detect.
  • the operation steps of the traditional ABO/RhD blood type stereotype test card can be reduced, and the efficiency is improved.
  • the serum separation gel used in the present invention has a specific gravity greater than that of the serum than the red blood cells, and the specific gravity is between 1.075 and 1.077.
  • the thixotropic deformation can be changed from solid to fluid, and the good thixotropy of the blood separation gel is prolonging the red blood cells.
  • the necessary conditions for the agglutination reaction with the blood group antibody, the thixotropy of the serum separation gel should be sufficient for the red blood cells to stay in the antibody working solution for a sufficient time, so that the red blood cells can not sink with the blood group antibody and have already sunk at the bottom of the tube, resulting in a false negative test result.
  • FIG. 1 is a schematic view of a microtube for blood group detection in a specific embodiment of the present invention.
  • the microtube 1 comprises a gel 11 , an antibody working solution 12 and a serum separating gel 13 in order from the bottom to the top, and the antibody works.
  • the liquid contains a blood type antibody and an antibody dilution for keeping the blood type antibody active;
  • the serum separation gel 13 has a specific gravity of 1.075 to 1.077, and can undergo thixotropic reaction under the centrifugal force of 150 G to 2000 G, and turns from a solid to a fluid;
  • the specific gravity of the liquid is greater than the serum separation gel and less than red blood cells.
  • the specific gravity relationship of each substance is as follows: serum ⁇ serum separation gel ⁇ antibody specific gravity liquid ⁇ red blood cells, in a preferred embodiment:
  • the antibody dilution has a specific gravity between 1.078 and 1.082 and a pH between 6.6 and 6.8.
  • the serum separating gel comprises the following components by weight: 35-45 parts of cyclopentadiene oligomer, 12-25 parts of organic gelling agent, 15-30 parts of organic gelling agent dispersing agent, 8-12 of viscosity reducing agent Parts and specific gravity adjusting agent 8-12 parts.
  • the serum separating gel comprises the following components by weight: 40 parts of cyclopentadiene oligomer, 20 parts of organic gelling agent, 20 parts of organic gelling agent dispersing agent, viscosity reducing agent 10 10 parts by weight and specific gravity regulator.
  • the gel was a dextran gel having a particle size of 40-60 ⁇ m.
  • the centrifugal separation force of the serum separation gel was 200 G, and the centrifugation time was 2 min.
  • the centrifugal force gradients were designed to be 80G, 100G, 120G, 150G, 180G, 200G, 250G, 300G, 400G.
  • serum separation gel can be very good under centrifugal force greater than 150G. Separation of red blood cells and serum, the effect is poor; if the centrifugal force is higher than 300G, although the serum separation gel can separate the red blood cells and serum, but because of the excessive centrifugal force, the red blood cells do not have enough time and the antibody working fluid The blood group antibody reaction has already sunk into the bottom of the tube, which is not conducive to correctly distinguish the blood type.
  • the organic gelling agent is dibenzylidene sorbitol; the organic gelling agent dispersing agent is polyoxyethylene-polyoxypropylene block copolymer, 1-methyl-2-pyrrolidone, DMSO, DMA and DMF At least one of; the viscosity reducing agent is at least one of di(2-ethylhexyl) phthalate and an epoxy resin; and the specific gravity adjusting agent is silica having a particle diameter of 5 ⁇ m to 10 ⁇ m. , titanium dioxide, chlorinated paraffin with a chlorination degree of 40%, and chlorinated paraffin with a chlorination degree of 70%.
  • the ratio of the volume of the antibody working solution to the weight of the gel is between 1:3 and 1:5; the serum separating gel is added in an amount of from 0.8 to 2 g, more preferably 1 g.
  • the invention also provides an integrated ABO/RhD blood type stereotype detection card.
  • the detection card has 8 micro tubes, and the 8 micro tubes are:
  • the first microtube the microtube in the above embodiment, and the blood group antibody contained in the antibody working solution is an anti-A monoclonal antibody;
  • a second microtube the microtube in the above embodiment, and the blood group antibody contained in the antibody working solution is an anti-B monoclonal antibody;
  • a third microtubule the microtubule in the above embodiment, and the blood group antibody contained in the antibody working solution is an anti-D monoclonal antibody;
  • the fourth microtube from the bottom of the fourth microtube, the gel, the antibody dilution solution and the serum separation gel are sequentially arranged, that is, the fourth microtube does not contain the blood group antibody;
  • the fifth micro tube to the eighth micro tube the first to fourth micro tubes are respectively repeated.
  • the invention also provides a method for preparing an integrated ABO/RhD blood type stereotype detection card.
  • the method comprises the following steps:
  • the anti-A monoclonal antibody, the anti-B monoclonal antibody and the anti-D monoclonal antibody are respectively formulated into 7 parts by volume of the antibody dilution solution per 1 part by volume of the antibody to prepare the antibody.
  • an anti-monoclonal anti-A gel having IgM properties and having IgM properties are prepared according to the ratio of adding 1.2-1.7 ml of the antibody working solution per 1 g of the swollen gel.
  • the anti-monoclonal anti-A gel, anti-monoclonal anti-B gel, anti-monoclonal anti-D gel and neutral gel are respectively added to Among the first micro tube, the second micro tube, the third micro tube and the fourth micro tube, the fifth to eighth micro tubes respectively repeat the first to fourth micro tubes;
  • 0.8g-2g of the serum separating gel is added to the first micro tube to the eighth micro tube, and 150G-300G is centrifuged for a certain time, so that the serum separation gel is thixotropically changed from solid to fluid and then captured. At the interface of the antibody working fluid;
  • the step (6) specifically includes the following steps:
  • the process of formulating the antibody dilution in the step (1) is as follows: 1.2-1.7 g of sodium chloride, 12-30 g of glycine, 0.2-0.3 g of disodium hydrogen phosphate, 0.46-0.56 g are added per 1 L of purified water. Sodium dihydrogen phosphate, 10-20 g bovine serum albumin, 12-25 g glycerol, 0.45-0.68 g methyl p-hydroxybenzoate and 0.1-0.16 g Propyl p-hydroxybenzoate.
  • the method for preparing the integrated ABO/RhD blood type test card comprises the following steps:
  • the antibody dilution liquid has a specific gravity of 1.078-1.082 and a pH of 6.6, and the formula is as follows: 1.5 g of chlorine is added to 1 L of purified water. Sodium, 20 g glycine, 0.26 g disodium hydrogen phosphate, 0.52 g sodium dihydrogen phosphate, 10 g of bovine serum albumin, 20 g of glycerol, 0.6 g of methyl p-hydroxybenzoate and 0.13 g of propyl p-hydroxybenzoate.
  • the gel after the sterilization in the step (21) is allowed to stand for a certain period of time (6 hours in this example), the gel is allowed to settle naturally, the supernatant is decanted, and then the volume ratio is 1:1.2 (the sedimentation is set.
  • the antibody dilution of the ratio of the gel to the antibody dilution was resuspended, and this was repeated twice to remove the gel broken fragments, and the swollen gel having a uniform particle size (in the present example, the particle size was in the range of 40-60 ⁇ m) was collected.
  • anti-A monoclonal antibody, anti-B monoclonal antibody and anti-D monoclonal antibody in this example, all three antibodies were purchased from Merck Millipore), respectively, per 1 part by volume
  • the antibody was added to a ratio of 7 parts by volume of the antibody diluent (in this example, specifically, the volume of each antibody was 5 ml, and the volume of the antibody dilution for diluting each antibody was 35 ml, respectively) to prepare an antibody working solution.
  • anti-monoclonal anti-A gel, anti-monoclonal anti-B gel with IgM properties and anti-monoclonal anti-D gel with IgM properties and formulation of neutral gel: 1.2 per 1g of swollen gel -1.7ml antibody dilution ratio (in this case, 1g swollen gel is added to 1.5ml of antibody dilution) to prepare neutral gel; wherein: anti-A monoclonal antibody titer ⁇ 128, anti-B monoclonal antibody effect The price is ⁇ 128, and the anti-D monoclonal antibody titer is ⁇ 128.
  • sealing, labeling and encapsulation wherein the sealing means: the nozzle of the sub-packed micro-tube is thermally plastic-sealed; the labeling means: after the sealing is completed, the micro-tubes are labeled, Forming an integrated ABO/RhD blood type stereotype test card; encapsulation means: aseptic plastic sealing and cartoning of the integrated ABO/RhD blood type stereotype test card.
  • the conventional card to which the red blood cell suspension was added was centrifuged at 70 G for 1 minute in a centrifuge, and then centrifuged at 100 G for 4 minutes.
  • the conventional card to which the red blood cell suspension was added was centrifuged at 70 G for 1 minute in a centrifuge, and then centrifuged at 100 G for 4 minutes.
  • the integrated ABO/RhD blood type stereotype test card of the present embodiment can directly add peripheral whole blood for detection without removing the red blood cells by centrifugation of the peripheral whole blood. After dilution, the test can reduce the operation steps of the traditional ABO/RhD blood type test card and improve the efficiency.

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Abstract

一种用于血型检测的微管（1）、一体式ABO/RhD血型定型检测卡及制备方法，该血型检测的微管（1）从底部往上依次包含凝胶（11）、抗体工作液（12）和血清分离胶（13），所述抗体工作液（12）中含有血型抗体和使所述血型抗体保持活性的抗体稀释液，所述血清分离胶（13）的比重在1.075～1.077之间，在150G～300G的离心力作用下能够发生触变，由固体变成流体；所述抗体稀释液的比重大于血清分离胶（13）小于红细胞。采用微管制成的一体式ABO/RhD血型定型检测卡可以直接添加外周全血进行检测，而不需要对外周全血进行离心取出红细胞进行稀释后再进行检测，在保证血型检测卡的灵敏度和特异性的前提下，可以减少传统的ABO/RhD血型定型检测卡的操作步骤，提高效率。

Description

用于血型检测的微管、一体式ABO/RhD血型定型检测卡及制备 技术领域
本发明涉及血型检测，特别是涉及一种用于血型检测的微管、一体式ABO/RhD血型定型检测卡及制备方法。
背景技术
自发现ABO血型以来，ABO血型在临床中应用广泛，ABO血型的准确定型非常重要，是安全输血的基础和保障。Rh血型系统是仅次于ABO血型系统的重要血型系统，也是红细胞血型系统中最为复杂的一个系统，Rh血型系统中的抗原包括D、C、E等，其中D抗原抗原性最强。红细胞表面含有D抗原为RhD阳性，不含有D抗原为RhD阴性，RhD阴性患者接受RhD阳性血液后，至少有20％患者会对D抗原产生免疫应答，在第二次体内接触该抗原后会发生溶血反应，因此对RhD进行定型也十分重要。
目前的ABO/RhD血型定型检测卡需要添加稀释后的红细胞才能进行检测，操作步骤繁琐，效率低下。
发明内容
为了弥补上述现有技术的不足，本发明提出一种用于血型检测的微管、一体式ABO/RhD血型定型检测卡及制备方法。
本发明的技术问题通过以下的技术方案予以解决：
本发明提供的一种用于血型检测的微管为：所述微管从底部往上依次包含凝胶、抗体工作液和血清分离胶；所述抗体工作液中含有血型抗体和使所述血型抗体保持活性的抗体稀释液；所述血清分离胶的比重在1.075～1.077之间，在150G～300G的离心力作用下能够发生触变，由固体变成流体；所述抗体稀释液的比重大于血清分离胶且小于红细胞。
本发明提供的一体式ABO/RhD血型定型检测卡为：所述检测卡上具有8个微管，所述8个微管分别是：
第一微管：为所述微管，且所述抗体工作液中含有的血型抗体为抗A单克 隆抗体；
第二微管：为所述微管，且所述抗体工作液中含有的血型抗体为抗B单克隆抗体；
第三微管：为所述微管，且所述抗体工作液中含有的血型抗体为抗D单克隆抗体；
第四微管：从所述第四微管的底部往上依次包含凝胶、抗体稀释液和血清分离胶；
第五微管～第八微管：分别重复第一微管～第四微管。
本发明提供的一体式ABO/RhD血型定型检测卡的制备方法，包含如下步骤：
(1)配制抗体稀释液，所述抗体稀释液的比重大于血清分离胶且小于红细胞；
(2)凝胶溶胀：采用生理盐水或者所述抗体稀释液对凝胶进行溶胀；
(3)配制抗体工作液：分别将抗A单克隆抗体、抗B单克隆抗体和抗D单克隆抗体按每1体积份的抗体加入7体积份的所述抗体稀释液比例配制成所述抗体工作液；
(4)配制特异性凝胶：按照每1g所述已溶胀的凝胶加入1.2-1.7ml所述抗体工作液的比例分别配制成具有IgM性质的抗单克隆抗A凝胶、具有IgM性质的抗单克隆抗B凝胶和具有IgM性质的抗单克隆抗D凝胶；以及配制中性胶：按照每1g所述已溶胀的凝胶加入1.2-1.7ml所述抗体稀释液的比例配制成中性胶；其中：抗A单克隆抗体效价≥128，抗B单克隆抗体效价≥128，抗D单克隆抗体效价≥128；
(5)分装：按照每管45-55μl的灌装量，将所述抗单克隆抗A凝胶、抗单克隆抗B凝胶、抗单克隆抗D凝胶、中性胶分别加入到第一微管、第二微管、第三微管和第四微管中，第五微管～第八微管分别重复第一微管～第四微管；
(6)配制血清分离胶，所述血清分离胶的比重在1.075～1.077之间，在150G～2000G的离心力作用下能够发生触变，由固体变成流体；
(7)分别在第一微管～第八微管中加入0.8g-2g的所述血清分离胶，150G-300G离心一定时间，使所述血清分离胶发生触变由固体变成流体后捕捉在所述抗体工作液的界面上；
(8)封口、贴签以及封装。
本发明与现有技术对比的有益效果是：本发明的一体式ABO/RhD血型定型检测卡可以直接添加外周全血进行检测，而不需要对外周全血进行离心取出红细胞进行稀释后再进行检测，在保证血型检测卡的灵敏度和特异性的前提下，可以减少传统的ABO/RhD血型定型检测卡的操作步骤，提高效率。具体的，本发明采用的血清分离胶比重大于血清小于红细胞，比重在1.075-1.077之间，在一定的离心力条件下能够发生触变由固体变成流体，血型分离胶良好的触变性是延长红细胞与血型抗体发生凝集反应的必要条件，血清分离胶的触变性要满足红细胞停留在抗体工作液中的时间充足，以免红细胞不能与血型抗体反应就已经沉于管底，造成假阴性的检测结果。
附图说明
图1是本发明具体实施方式中的用于血型检测的微管的示意图。
具体实施方式
下面对照附图并结合优选的实施方式对本发明作进一步说明。
本发明提供一种用于血型检测的微管，在具体实施方式中，如图1所示，微管1从底部往上依次包含凝胶11、抗体工作液12和血清分离胶13，抗体工作液中含有血型抗体和使血型抗体保持活性的抗体稀释液；血清分离胶13的比重在1.075～1.077之间，在150G～2000G的离心力作用下能够发生触变，由固体变成流体；抗体稀释液的比重大于血清分离胶且小于红细胞。
各物质的比重关系如下：血清＜血清分离胶＜抗体比重液＜红细胞，在优选的实施例中：
抗体稀释液的比重在1.078-1.082之间，pH值在6.6-6.8之间。
血清分离胶包括如下重量份的各组分：环戊二烯低聚物35-45份，有机胶凝剂12-25份，有机胶凝剂分散剂15-30份，减粘剂8-12份和比重调节剂8-12份。在更优选的实施例中，血清分离胶包括如下重量份的各组分：环戊二烯低聚物40份，有机胶凝剂20份，有机胶凝剂分散剂20份，减粘剂10份和比重调节剂10份。
凝胶为葡聚糖凝胶，粒径为40-60μm。
血清分离胶发生触变的离心力为200G，离心时间为2min。
在相同时间(本实验采用2分钟)下，设计离心力梯度80G、100G、120G、150G、180G、200G、250G、300G、400G，实验证明，在大于150G的离心力下，血清分离胶能很好的把红细胞和血清分离开，低于则效果较差；而若离心力高于300G，虽然血清分离胶能将红细胞和血清分离开，但由于离心力过大，红细胞没有足够的时间与抗体工作液中的血型抗体反应就已经沉入管底，不利于正确分辨血型。
在相同离心力下进行三组实验，分别是150G、180G和200G，设计一个时间梯度30s、1min、1.5min、2min，实验证明，在200G时离心2分钟效果较佳。
所述有机胶凝剂为二亚苄基山梨糖醇；所述有机胶凝剂分散剂为聚氧乙烯-聚氧丙烯嵌段共聚物、1-甲基-2-吡咯烷酮、DMSO、DMA和DMF中的至少一种；所述减粘剂为邻苯二甲酸二(2-乙基己基)酯和环氧树脂中的至少一种；所述比重调节剂为粒径5μm-10μm的二氧化硅、二氧化钛、氯化度40％的氯化石蜡、氯化度70％的氯化石蜡。
所述抗体工作液的体积与所述凝胶的重量之比在1∶3至1∶5之间；所述血清分离胶的加入量为0.8-2g，更优的是加入1g。本发明还提供一种一体式ABO/RhD血型定型检测卡，在具体实施方式中，检测卡上具有8个微管，8个微管分别是：
第一微管：为上述实施方式中的微管，且抗体工作液中含有的血型抗体为抗A单克隆抗体；
第二微管：为上述实施方式中的微管，且所述抗体工作液中含有的血型抗体为抗B单克隆抗体；
第三微管：为上述实施方式中的微管，且所述抗体工作液中含有的血型抗体为抗D单克隆抗体；
第四微管：从第四微管的底部往上依次包含凝胶、抗体稀释液和血清分离胶，也即第四微管中不含有血型抗体；
第五微管～第八微管：分别重复第一微管～第四微管。
本发明还提供一种一体式ABO/RhD血型定型检测卡的制备方法，在具体实施方式中，包含如下步骤：
(1)配制抗体稀释液，所述抗体稀释液的比重大于血清分离胶且小于红细胞；
(2)凝胶溶胀：采用生理盐水或者所述抗体稀释液对凝胶进行溶胀；
(3)配制抗体工作液：分别将抗A单克隆抗体、抗B单克隆抗体和抗D单克隆抗体按每1体积份的抗体加入7体积份的所述抗体稀释液比例配制成所述抗体工作液；
(4)配制特异性凝胶：按照每1g所述已溶胀的凝胶加入1.2-1.7ml所述抗体工作液的比例分别配制成具有IgM性质的抗单克隆抗A凝胶、具有IgM性质的抗单克隆抗B凝胶和具有IgM性质的抗单克隆抗D凝胶；以及配制中性胶：按照每1g所述已溶胀的凝胶加入1.2-1.7ml所述抗体稀释液的比例配制成中性胶；其中：抗A单克隆抗体效价≥128，抗B单克隆抗体效价≥128，抗D单克隆抗体效价≥128；
(5)分装：按照每管45-55μl的灌装量，将所述抗单克隆抗A凝胶、抗单克隆抗B凝胶、抗单克隆抗D凝胶、中性胶分别加入到第一微管、第二微管、第三微管和第四微管中，第五微管～第八微管分别重复第一微管～第四微管；
(6)配制血清分离胶，所述血清分离胶的比重在1.075～1.077之间，在150G～2000G的离心力作用下能够发生触变，由固体变成流体；
(7)分别在第一微管～第八微管中加入0.8g-2g的所述血清分离胶，150G-300G离心一定时间，使所述血清分离胶发生触变由固体变成流体后捕捉在所述抗体工作液的界面上；
(8)封口、贴签以及封装。
在优选的实施例中：
所述步骤(6)具体包括如下步骤：
(61)将35-45份环戊二烯低聚物加热融化；
(62)在融化的环戊二烯低聚物中加入有机胶凝剂12-25份，有机胶凝剂分散剂15-30份和减粘剂8-12份，捏合混合物20-40分钟；
(63)冷却至常温(即20-30℃)，加入比重调节剂8-12份，在0.1bar-0.5bar的真空度下真空捏合混合物20-40分钟。
所述步骤(1)中的配制所述抗体稀释液的过程如下：在每1L纯净水中加入1.2-1.7g氯化钠，12-30g甘氨酸，0.2-0.3g磷酸氢二钠，0.46-0.56g磷酸二氢钠，10-20g牛血清白蛋白，12-25g甘油，0.45-0.68g对羟基苯甲酸甲酯和0.1-0.16g 对羟基苯甲酸丙酯。
在一个优选的实施例中，该一体式ABO/RhD血型定型检测卡的制备方法包含如下步骤：
(1)配制抗体稀释液，所述抗体稀释液的比重大于血清分离胶小于红细胞，本例中，抗体稀释液比重为1.078-1.082，pH为6.6，配方如下：在1L纯净水中加入1.5g氯化钠，20g甘氨酸，0.26g磷酸氢二钠，0.52g磷酸二氢钠，牛血清白蛋白10g，甘油20g，对羟基苯甲酸甲酯0.6g和对羟基苯甲酸丙酯0.13g。
(2)凝胶溶胀，本例中直接采用抗体稀释液对凝胶进行溶胀，具体包括如下步骤：
(21)在每1g凝胶(本例中采用葡聚糖凝胶)中加入2-10ml抗体稀释液(本例中为1g凝胶加入2ml抗体稀释液)，悬浮凝胶，进行湿热灭菌，灭菌后备用。
(22)将步骤(21)灭菌后的凝胶放置一定时间(本例中为6个小时)，使凝胶自然沉降，倒去上清液，再加入体积比1∶1.2(沉降的凝胶与抗体稀释液的比)的抗体稀释液重悬，如此重复两次，除去凝胶破损碎片，收集颗粒大小均匀(本例中粒径在40-60μm范围内)的已溶胀的凝胶。
(3)配制抗体工作液：分别将抗A单克隆抗体、抗B单克隆抗体和抗D单克隆抗体(本例中，三种抗体均购自默克密理博公司)按每1体积份的抗体加入7体积份的抗体稀释液比例(本例中，具体的，每一种抗体的体积为5ml，用于稀释每一种抗体的抗体稀释液的体积分别为35ml)配制成抗体工作液。
(4)配制特异性凝胶：按照每1g已溶胀的凝胶加入1.2-1.7ml抗体工作液的比例(本例中是1g已溶胀凝胶加入1.5ml的抗体稀释液)分别配制成具有IgM性质的抗单克隆抗A凝胶、具有IgM性质的抗单克隆抗B凝胶和具有IgM性质的抗单克隆抗D凝胶；以及配制中性胶：按照每1g已溶胀的凝胶加入1.2-1.7ml抗体稀释液的比例(本例中是1g已溶胀凝胶加入1.5ml的抗体稀释液)配制成中性胶；其中：抗A单克隆抗体效价≥128，抗B单克隆抗体效价≥128，抗D单克隆抗体效价≥128。
(5)分装：按照每管50μl的灌装量，将所述抗单克隆抗A凝胶、抗单克隆抗B凝胶、抗单克隆抗D凝胶、中性胶分别加入到第一微管、第二微管、第 三微管和第四微管中，第五微管～第八微管分别重复第一微管～第四微管。
(6)配制血清分离胶，具体如下：
(61)将40份环戊二烯低聚物在130℃下加热融化。
(62)在融化的环戊二烯低聚物中加入有机胶凝剂(本例中为二亚苄基山梨糖醇)20份，有机胶凝剂分散剂(本例中为聚氧乙烯-聚氧丙烯嵌段共聚物)20份和减粘剂(本例中为邻苯二甲酸二(2-乙基己基)酯)10份，捏合混合物30分钟。
(63)冷却至常温，加入比重调节剂(本例中采用二氧化钛)10份，真空捏合混合物30分钟。
(7)分别在第一微管～第八微管中加入1g血清分离胶，200G离心3分钟，使血清分离胶发生触变由固体变成流体后捕捉在抗体工作液的界面上。
(8)封口、贴签以及封装，其中，封口是指：将分装好的微管的管口用铝箔进行热力塑封；贴签是指：在封口完成后，在各个微管上贴签，形成一体式ABO/RhD血型定型检测卡；封装是指：对一体式ABO/RhD血型定型检测卡进行无菌塑封和装盒。
以下分别设计两组实验，采用以上实施例的一体式ABO/RhD血型定型检测卡(以下简称一体卡)与长春博迅公司的ABO血型检测卡(以下简称常规卡)进行血型检测对比如下：
第一组实验：
1、抽取未知血型供血者10人，每人抽取外周血10ml(样本1～样本10)。
2、取上述外周血2.5ml加入含促凝剂的试管中，300G离心2分钟，取16μl压积红细胞，加入1984μl生理盐水，配制成0.8％红细胞悬液。
3、取一体卡和常规卡各50张，200G离心2分钟，撕掉铝膜。
4、往一体卡中直接加入2μl外周血，同时往常规卡中加入50μl的红细胞悬液。
5、将加了红细胞悬液的常规卡放入离心机中70G离心1分钟，然后100G离心4分钟。
6、将加了外周血的一体卡放入离心机中200G离心2分钟，然后100G离心 4分钟。
7、取卡判定结果，结果如下表一。
表一 未知血型样本检测结果
样本 1 2 3 4 5 6 7 8 9 10
一体卡 A O B A B B AB B O A
常规卡 A O B A B B AB B O A
备注：上述10个样本的RhD检测结果均为阴性。
第二组实验：
1、准备A、B、O和AB血型外周血10份，其中5份为RhD阴性，另外5份为RhD阳性，每份10ml。
2、取上述外周血2.5ml加入含促凝剂的试管中，300G离心2分钟，取16μl压积红细胞，加入1984μl生理盐水，配制成0.8％红细胞悬液。
3、取一体卡和常规卡各50张，200G离心2分钟，撕掉铝膜。
4、往一体卡中直接加入2μl外周血，同时往常规卡中加入50μl的红细胞悬液。
5、将加了红细胞悬液的常规卡放入离心机中70G离心1分钟，然后100G离心4分钟。
6、将加了外周血的一体卡放入离心机中200G离心2分钟，然后100G离心4分钟。
7、取卡判定结果，结果如下表二。
表二 已知血型样本检测结果
Figure PCTCN2016078941-appb-000001
Figure PCTCN2016078941-appb-000002
从以上实验结果可知，在保证血型检测卡的准确性前提下，本实施例的一体式ABO/RhD血型定型检测卡可以直接添加外周全血进行检测，而不需要对外周全血进行离心取出红细胞进行稀释后再进行检测，可以减少传统的ABO/RhD血型定型检测卡的操作步骤，提高效率。
以上内容是结合具体的优选实施方式对本发明所作的进一步详细说明，不能认定本发明的具体实施只局限于这些说明。对于本发明所属技术领域的技术人员来说，在不脱离本发明构思的前提下，还可以做出若干等同替代或明显变型，而且性能或用途相同，都应当视为属于本发明的保护范围。

Claims (10)

  1. 一种用于血型检测的微管，其特征在于：所述微管从底部往上依次包含凝胶、抗体工作液和血清分离胶；所述抗体工作液中含有血型抗体和使所述血型抗体保持活性的抗体稀释液；所述血清分离胶的比重在1.075～1.077之间，在150G～300G的离心力作用下能够发生触变，由固体变成流体；所述抗体稀释液的比重大于血清分离胶且小于红细胞。
  2. 如权利要求1所述的微管，其特征在于：所述抗体稀释液的比重在1.078-1.082之间，pH值在6.6-6.8之间。
  3. 如权利要求1所述的微管，其特征在于：所述血清分离胶包括如下重量份的各组分：环戊二烯低聚物35-45份，有机胶凝剂12-25份，有机胶凝剂分散剂15-30份，减粘剂8-12份和比重调节剂8-12份。
  4. 如权利要求1所述的微管，其特征在于：所述凝胶为葡聚糖凝胶，粒径为40-60μm。
  5. 如权利要求3所述的微管，其特征在于：所述有机胶凝剂为二亚苄基山梨糖醇；所述有机胶凝剂分散剂为聚氧乙烯-聚氧丙烯嵌段共聚物、1-甲基-2-吡咯烷酮、DMSO、DMA和DMF中的至少一种；所述减粘剂为邻苯二甲酸二(2-乙基己基)酯和环氧树脂中的至少一种；所述比重调节剂为粒径5μm-10μm的二氧化硅、二氧化钛、氯化度40％的氯化石蜡、氯化度70％的氯化石蜡。
  6. 如权利要求1所述的微管，其特征在于：在所述微管中，所述抗体工作液的体积与所述凝胶的重量之比在1∶3至1∶5之间；所述血清分离胶的加入量为0.8g-2g。
  7. 一种一体式ABO/RhD血型定型检测卡，其特征在于：所述检测卡上具有8个微管，所述8个微管分别是：
    第一微管：为权利要求1-6任意一项所述的微管，且所述抗体工作液中含有的血型抗体为抗A单克隆抗体；
    第二微管：为权利要求1-6任意一项所述的微管，且所述抗体工作液中含有的血型抗体为抗B单克隆抗体；
    第三微管：为权利要求1-6任意一项所述的微管，且所述抗体工作液中含有的血型抗体为抗D单克隆抗体；
    第四微管：从所述第四微管的底部往上依次包含凝胶、抗体稀释液和血清分离胶；
    第五微管～第八微管：分别重复第一微管～第四微管。
  8. 一种一体式ABO/RhD血型定型检测卡的制备方法，其特征在于，包含如下步骤：
    (1)配制抗体稀释液，所述抗体稀释液的比重大于血清分离胶且小于红细胞；
    (2)凝胶溶胀：采用生理盐水或者所述抗体稀释液对凝胶进行溶胀；
    (3)配制抗体工作液：分别将抗A单克隆抗体、抗B单克隆抗体和抗D单克隆抗体按每1体积份的抗体加入7体积份的所述抗体稀释液比例配制成所述抗体工作液；
    (4)配制特异性凝胶：按照每1g所述已溶胀的凝胶加入1.2-1.7ml所述抗体工作液的比例分别配制成具有IgM性质的抗单克隆抗A凝胶、具有IgM性质的抗单克隆抗B凝胶和具有IgM性质的抗单克隆抗D凝胶；以及配制中性胶：按照每1g所述已溶胀的凝胶加入1.2-1.7ml所述抗体稀释液的比例配制成中性胶；其中：抗A单克隆抗体效价≥128，抗B单克隆抗体效价≥128，抗D单克隆抗体效价≥128；
    (5)分装：按照每管45-55μl的灌装量，将所述抗单克隆抗A凝胶、抗单克隆抗B凝胶、抗单克隆抗D凝胶、中性胶分别加入到第一微管、第二微管、第三微管和第四微管中，第五微管～第八微管分别重复第一微管～第四微管；
    (6)配制血清分离胶，所述血清分离胶的比重在1.075～1.077之间，在150G～2000G的离心力作用下能够发生触变，由固体变成流体；
    (7)分别在第一微管～第八微管中加入0.8g-2g的所述血清分离胶，150G-300G离心一定时间，使所述血清分离胶发生触变由固体变成流体后捕捉在所述抗体工作液的界面上；
    (8)封口、贴签以及封装。
  9. 如权利要求8所述的制备方法，其特征在于，所述步骤(6)具体包括如下步骤：
    (61)将35-45份环戊二烯低聚物加热融化；
    (62)在融化的环戊二烯低聚物中加入有机胶凝剂12-25份，有机胶凝剂分散剂15-30份和减粘剂8-12份，捏合混合物20-40分钟；
    (63)冷却至常温，加入比重调节剂8-12份，在0.1bar-0.5bar的真空度下真空捏合混合物20-40分钟。
  10. 如权利要求8所述的制备方法，其特征在于，所述步骤(1)中的配制所述抗体稀释液的过程如下：在每1L纯净水中加入1.2-1.7g氯化钠，12-30g甘氨酸，0.2-0.3g磷酸氢二钠，0.46-0.56g磷酸二氢钠，10-20g牛血清白蛋白，12-25g甘油，0.45-0.68g对羟基苯甲酸甲酯和0.1-0.16g对羟基苯甲酸丙酯。
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CN112444518A (zh) * 2020-11-18 2021-03-05 天津德祥生物技术有限公司 一种血型试剂卡离心检测装置
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CN111337693A (zh) * 2020-03-30 2020-06-26 江苏中济万泰生物医药有限公司 Rh血型检测卡的制备方法
CN112444518A (zh) * 2020-11-18 2021-03-05 天津德祥生物技术有限公司 一种血型试剂卡离心检测装置
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CN113189354A (zh) * 2021-04-30 2021-07-30 重庆国际旅行卫生保健中心(重庆海关口岸门诊部) 一种便携式快速血型正定型检测组件
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