CN103760333B - A method for separating erythrocytes in a blood sample and the use of - Google Patents

A method for separating erythrocytes in a blood sample and the use of Download PDF

Info

Publication number
CN103760333B
CN103760333B CN 201410006059 CN201410006059A CN103760333B CN 103760333 B CN103760333 B CN 103760333B CN 201410006059 CN201410006059 CN 201410006059 CN 201410006059 A CN201410006059 A CN 201410006059A CN 103760333 B CN103760333 B CN 103760333B
Authority
CN
Grant status
Grant
Patent type
Application number
CN 201410006059
Other languages
Chinese (zh)
Other versions
CN103760333A (en )
Inventor
刘毅
刘杰
马天涯
吴银飞
高飞
Original Assignee
艾博生物医药(杭州)有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Grant date

Links

Abstract

本发明涉及一种从血液样品中分离红细胞的装置和方法,以及检测血液样本中被分析物质的检测装置和方法。 The present invention relates to an apparatus and method for separating red blood cells from a blood sample, and a detection apparatus and method for detecting a substance to be analyzed in a blood sample. 使用这些装置和方法可以有效分离血液样品中的红细胞,从而能够减少红细胞对液体流动的阻碍作用和对反应背景的干扰。 Using these apparatus and methods may effectively separate the red blood cells in a blood sample, it is possible to reduce the impediment to fluid flow of red blood cells and the background interference with the reaction. 并且使用该方法和装置不会对检测的结果带来不利影响。 And does not adversely affect the result of the detection using the method and apparatus.

Description

-种分离血液样本中红细胞的方法w及运用 - Method isolates erythrocytes in a blood sample and use of w

[0001] 本申请是申请号为200910206522. 6、申请日2009年10月13日的中国专利申请的分案申请。 [0001] This application is a number 200,910,206,522.6, a divisional application filing date of the patent application of China October 13, 2009 of.

技术领域 FIELD

[0002] 本发明是关于分离全血样本中红细胞的装置和方法,更具体的讲,是关于一种检测装置,他应用分离全血中纤维蛋白来分离红细胞的方法W及装置来减少红细胞对检测结果的干扰。 [0002] The present invention relates to apparatus and method for whole blood sample erythrocytes separated, say more particularly, relates to a detection apparatus, a method and apparatus for his application W whole blood to separate red blood cell separating fibrin to reduce erythrocyte interference detection result.

背景技术 Background technique

[0003] 现代临床诊断方法通常是对血液样品进行的。 [0003] Modern clinical diagnostic methods are usually carried out on blood samples. 不幸的,红细胞对许多诊断检测有干扰作用。 Unfortunately, red blood cells have a disruptive effect on many diagnostic tests. 运就需要先把红细胞从血液样品中分离出去,然后再进一步对样品进行检测或化验。 Yun need to first be separated from the red blood cells in the blood sample, then further test the samples or testing. 另一方面,在层析检测装置中,特别是免疫检测装置中,红细胞可能阻止液体流动,液体流动对于运种检测装置上发生反应是必不可少的。 On the other hand, the chromatographic detection device, in particular immunoassay device, red blood cells may prevent the flow of liquid is essential for the reaction to occur on the liquid flow transported species detection means. 基于运种原因和其他一些原因,许多检测都用血浆或血清进行,运就需要从全血中分离出血浆或血清。 And transport a variety of reasons based on other reasons, many detection were carried out using blood plasma or serum, the operation will need to separate plasma or serum from whole blood.

[0004] 有许多现有技术可用在全血样品中使红细胞与血浆分离。 [0004] There are many available prior art plasma separating erythrocytes in a whole blood sample manipulation. 离屯、是一种惯用的手段。 From the village, it is a means of conventional. 另外,在专利和公开的专利申请中也披露过一些方法,例如美国专利593933U6673629、 4594327、4678757、5558834、6818180,美国公开专利申请2006/0029923、2004/0202783。 In addition, patents and published patent applications also disclosed some methods, for example, U.S. Patent No. 593933U6673629, 4594327,4678757,5558834,6818180, U.S. Patent Application Publication 2006 / 0029923,2004 / 0202783. 总结起来,运些分离的方法主要包括物理和化学方法来分离全血样品中的红细胞。 To sum up, separating operation such methods include physical and chemical methods for separating red blood cells in a whole blood sample. 运些用于使红细胞与血浆分离的技术许多是昂贵的、复杂的,可能导致红细胞不完全分离。 These operation for separating erythrocytes and plasma in the art many are expensive, complicated, may result in incomplete separation of red blood cells. 特别是对那些被放置了很久时间,并产生了凝血的全血样本来讲,现有技术不能很好的进行分离。 Especially those that are placed in the separation of long time, and generating a whole blood sample in terms of clotting, the prior art is not well. 特别地,在层析免疫检测装置中,运种凝血样本会引起非特异性的结合或很高的背景干扰,导致丧失检测的灵敏度,而且还能够堵塞层析载体从而阻碍血清或血浆在上流动,导致检测无效。 In particular, in the chromatography immunoassay device, blood coagulation sample transported species cause nonspecific binding or high background interference, resulting in loss of detection sensitivity, but also blocked thus preventing the chromatography carrier in the serum or plasma flow, caused an invalid test. 特别对一些需要在很短时间内就可W得出检测结果的装置来讲,运一缺点更为致命。 Some devices require special W can be obtained the test results in a very short period of time in terms of transport disadvantage is more deadly. 运就需要一种试剂和包括有该试剂的装置和方法,用于分离全血样本中的红细胞,而运种装置和方法对检测系统没有不利的影响。 Transport agents and a need for a device and a method comprising the agent for separating red blood cells in a whole blood sample, the transport apparatus and method species has no adverse effect on the detection system.

发明内容 SUMMARY

[0005] 为了解决W上问题,本发明提供一种从血液样品中分离红细胞的试剂W及该试剂的一些运用,使用该试剂可W有效地从血液样品中分离红细胞。 [0005] In order to solve the problem W, the present invention provides an isolated using a number of reagents W, and erythrocytes from a blood sample of the reagent in the reagent can be used effectively W from the red cells from a blood sample.

[0006] 一方面,本发明设及一种从血液样本中分离红细胞的一种方法。 [0006] In one aspect, the present invention is provided a method of red blood cells, and one isolated from the blood sample. 具体的讲,包括, 让血液样本样本和一种结合血液中纤维蛋白的受体反应,除去血液样本中的纤维蛋白受体。 To be specific, including, so that a blood sample and one sample of receptor binding reaction fibrin blood to remove fibers receptor blood sample. 在另一个实施方式中,该方法包括,提供一种载体,载体上包括一种结合纤维蛋白的受体,让血液样本流过该载体。 In another embodiment, the method includes providing a carrier, the carrier comprising one binding receptor fibrin, so that the blood sample to flow through the carrier.

[0007] 另一方面,本发明还设及一种检测血液样品中被分析物质的检测装置,运种装置包括一个载体,载体上包括一个检测区域,在检测区域上游处理有结合血液样本中纤维蛋白的受体。 [0007] In another aspect, the present invention is also provided, and detection means for detecting an analyte in a blood sample substance, comprising a carrier transport device types, comprising a detection zone on the carrier, upstream of the detection region in a blood sample treated with a binding fiber receptor protein. 优选地,载体为层析载体。 Preferably, the carrier of the chromatography carrier. 更优选的,在检测区域的上游包括样本接受区域,在样本接受区域上处理有结合血液样本中纤维蛋白的受体。 More preferably, upstream from the detection zone includes a sample receiving zone, the sample receiving treatment with a blood sample fibrin binding receptor region.

[0008] 另一方面,本发明还提供一种用于检测全血样本中被分析物质的一种方法。 [0008] In another aspect, the present invention further provides a method for detecting a substance to be analyzed in whole blood samples. 此方法优选地应用本发明的层析载体装置中。 This method is preferably applied to the chromatography carrier device of the present invention. 具体的讲,此方法包括,提供一种层析载体,层析载体上包括一个样本接受区域,其中,在样本接受区域上处理有结合血液样本中合纤维蛋白的受体;使血液样品流过样本接受区域;检测血液样本中是否存在被分析物质。 To be specific, this method comprises, there is provided a chromatography carrier, including a sample receiving zone on the chromatographic carrier, wherein the processing in the sample receiving region on the receptor binding bonded fibrin blood sample; blood sample to flow through sample receiving region; detecting whether there is analyte in a blood sample.

[0009] 在W上所有实施方式中,结合纤维蛋白的受体包括纤维蛋白的抗体,结合红细胞的受体包括抗红细胞的抗体,抗体可W是单抗或多抗,也可W是抗体片段;载体可W是层析载体或非吸水性载体等;结合纤维蛋白的受体可W被固定在载体上。 [0009] W in all embodiments, the fibrin binding of fibrin receptors include antibodies, receptor binding erythrocytes include anti-erythrocyte antibodies, antibody is monoclonal or polyclonal may be W, W is an antibody fragment may be ; W is a chromatography carrier may be a carrier or the like water absorbent carrier; fibrin binding receptor W may be immobilized on a support. .

[0010] 有益效果:利用本发明的技术,可W有效的去处血液样本里的红细胞,而不管血液样本是新鲜的还是已经产生凝血的样本,增长了被检测样品的使用时间。 [0010] The beneficial effects: the use of technology according to the present invention, W effectively place the blood sample in the red blood cells, regardless of whether the blood sample is fresh or has clotting sample, increase the time the sample is detected. 另外,使用了能够结合纤维蛋白的受体的红细胞分离装置或检测血液样本中被分析物质的检测装置可W延长使用寿命。 Further, the use of red blood cell separation apparatus capable of binding to a receptor or detection of fibrin in the blood sample being analyzed substance detecting means W can extend the service life.

附图说明 BRIEF DESCRIPTION

[0011] 图1为本发明的一个具体实施方式,在检测装置10中,包括一个过滤层11来接受样本并让样本通过该层W及一个反应层12,在反应层上处理有还原反应底物,和手持柄13,其中在过滤层11上处理有结合血液样本中纤维蛋白的多克隆抗体和结合红细胞的多克隆抗体。 A specific embodiment [0011] FIG. 1 of the present invention, in the detection means 10, 11 comprises a filter layer to receive a sample and allow the sample through the layer 12 W, and a reaction layer, the reaction layer in a reducing reaction substrate processing was held and the shank 13, wherein the filter layer 11 on the blood samples treated with a fibrin binding polyclonal antibodies and polyclonal antibodies binding erythrocytes. 当把全血样本01施加到样本过滤层11上后,处理在过滤层11上的纤维蛋白抗体和红细胞抗体一起把红细胞拦截在过滤层上,让血浆通过过滤层到达反应层12上,如果样本中存在被分析物质,例如血糖,在反应层12的下表面直接可W观察到颜色变化。 When the whole blood sample 01 is applied to the sample filter layer was 11, the processing in the filter layer 11 fibrin antibody and red cell antibodies with the erythrocyte intercept on the filter layer, so that the plasma through the filter layer to the reaction layer 12, if the sample in the presence of analyte, e.g. glucose, W may be a direct color change is observed in the surface of the reaction layer 12.

[0012] 如图2描绘了本发明一个优选的具体实施方式,在检测装置30中,构成样本接受区域35的载体材料为玻璃纤维,构成检测区域31的材料为硝酸纤维素膜并位于样本接受区域的下游,含有被标记物质的区域34位于样本接受区域35和检测区域31的之间。 [0012] FIG. 2 depicts a preferred embodiment of the present invention DETAILED embodiments, the detection device 30, the carrier material constituting the sample receiving region 35 is a glass fiber, a material constituting the detection region 31 is located a nitrocellulose membrane and sample receiving the downstream region, a region containing the labeled substance 34 is located between the sample receiving region 35 and the detection region 31. 在检测区域上包括固定有被固定的特异结合分子带33和检测结果控制分子带32 ;在玻璃纤维载体上包括红细胞表面抗原的多克隆抗体和纤维蛋白的多克隆抗体,其中样本接受区域35,标记区域34和检测区域31之间相互连接可W让液体从样本接受区域35流到检测区域31上进行反应。 On the detection area comprises a fixed specific binding molecules immobilized belt 33 and belt 32 controlling the molecular detection result; on glass fiber support comprises a red blood cell surface antigen polyclonal antibody polyclonal antibody and fibrin, wherein the sample receiving region 35, marker area 34 and the detection region may be interconnected between 31 W 35 allow liquid to flow from the sample receiving zone 31 is detected on the reaction region.

[0013] 标记说明:检测装置10, 30;血液样本01,02;过滤层11;反应层12;手持柄13;样本接受垫35,标记垫34,检测区域(检测线)36,控制线32,硝酸纤维膜36,吸水层31。 [0013] Symbols: detecting means 10, 30; 01, 02, the blood sample; filtration layer 11; reaction layer 12; hand shank 13; sample receiving pad 35, marking pad 34, the detection zone (test line) 36, the control line 32 , nitrocellulose membrane 36, the water-absorbing layer 31.

具体实施方式 detailed description

[0014] 下面对本发明设及的结构或运些所使用的技术术语做进一步的说明。 [0014] Next, the structure of the present invention is provided or transported and technical terms used in some further explanation.

[001引检测 [001 detects primer

[0016] 检测表示化验或测试一种物质或材料是否存在,比如,但并不限于此,化学物质、 有机化合物、无机化合物、新陈代谢产物、药物或者药物代谢物、有机组织或有机组织的代谢物、核酸、蛋白质或聚合物。 [0016] detecting a laboratory or testing a substance or presence or absence of material, such as, but not limited to this metabolites, chemicals, organic compounds, inorganic compounds, metabolites, drugs or drug metabolites, organic tissue, or organism of , nucleic acid, protein, or polymer. 另外,检测表示测试物质或材料的数量。 Further, the number of detecting a test substance or material. 进一步说,化验还表示免疫检测,化学检测、酶检测等。 Furthermore, laboratory tests also indicate immunoassay, chemical detection, detection of enzyme.

[0017] 纤维蛋白和纤维蛋白的受体 [0017] Fibrin and fibrin receptor

[0018] 纤维蛋白原是发现最早的一种凝血因子。 [0018] Fibrinogen is a clotting factor found in the earliest. 呈伸长的楠球体,是由Ξ对多肤链(一对α链、一对β链、一对丫链)W二硫键连接而成的二聚物,其分子量约为34万道尔顿。 Nan was elongated spheres, the skin is multi-chain Ξ (a pair of α chain, a pair of β chain, a pair of chain Ah) W formed disulfide-linked dimer having a molecular weight of about 340,000 Seoul pause. 在肝脏中合成后进入血浆,W溶解形式存在。 After synthesis in the liver into the plasma, the presence of W in dissolved form. 每100ml人血浆中含量约0.3g。 Per 100ml human plasma content of about 0.3g. 纤维蛋白是一种高度不溶的蛋白质多聚体,是像细针一样的晶状物。 Fibrin is a highly insoluble protein multimers, needle like crystals of the same substance. 纤维蛋白原转变为纤维蛋白是整个血凝过程中最基本的变化,要经过3个环节:①纤维蛋白原的水解在凝血酶作用下,纤维蛋白原分子中的两条α链和两条β链每条都有一个肤键断裂,结果形成纤维蛋白单体,并同时释放出两对小分子的纤维蛋白多肤(即纤维蛋白多肤A和Β,分子量合计约为9000道尔顿)。 Fibrinogen into fibrin during blood clotting whole basic change, to go through three links: ① Hydrolysis under fibrinogen thrombin, fibrinogen molecule two α chains and two β each strand has a skin bond breaking, resulting in the formation of fibrin monomers, and fibrinogen with release of two pairs of small molecules plurality skin (i.e., skin a fibrin and Beta, the total molecular weight of about 9000 daltons). 因此,最后形成的纤维蛋白的分子量比纤维蛋白原小些。 Thus, the molecular weight of fibrin is finally formed smaller than fibrinogen. ②纤维蛋白单体的聚集。 ② aggregated fibrin monomers. 在Ca2+的参与下,若干纤维蛋白单体聚合成可溶性的纤维蛋白多聚体。 Involvement in Ca2 + in a number of fibrin monomers into soluble fibrin polymer. ③血凝块的形成在纤维蛋白酶和Ca2+的作用下,不同的纤维蛋白分子的α链之间形成交键,使纤维蛋白转变为最终的不溶性的纤维蛋白多聚体。 ③ clot formation in the fibers and the role of Ca2 + protease, to form a cross bond between the α chains of different molecules of fibrin, fibrin into the final insoluble fibrin polymer. 形成中的纤维蛋白相互交错重叠并网罗血细胞,使原来呈溶胶状的血液转变成凝胶状的血凝块。 Fibrin formation in the interleaved and overlapped recruited blood cells, so that the original form of the sol into gel form blood clots.

[0019] 在我们的实验中,我们惊讶地发现,利用结合纤维蛋白的受体去除血液样本中的纤维蛋白可W有效的分离出红细胞。 [0019] In our experiments, we have surprisingly found that the use of the fibrin binding receptor remove fibrin blood sample W can be effectively separated from the red blood cells. 特别的,当运种结合纤维蛋白的受体和结合红细胞的受体配合使用情况下,可W更好的使血液中的红细胞得到分离。 In particular, when the kind of transport receptor binding and fibrin binding erythrocytes with the case of using the receptor, W may be better to make the red blood cells be isolated. 并且,运种去除红细胞的技术不管对新鲜的血液还是对已经产生凝血的血液样本同样有效。 Further, the transported species to remove red cells or equally effective regardless of the art for blood sample clotting already to the fresh blood.

[0020] 分离方法 [0020] Separation

[0021] 一方面,本发明设及一种从全血样本中分离红细胞的一种方法。 [0021] In one aspect, the present invention is provided a method of red blood cells, and one isolated from whole blood samples. 具体的讲,包括, 让全血样本和一种结合血液中纤维蛋白的受体反应,除去血液样本中的纤维蛋白受体。 To be specific, including, so that the whole blood sample and one binding receptor responses fibrin in the blood, removing fibrin blood sample receptor. 在一个具体的实施例子里,可W用抗纤维蛋白的抗体作为受体来结合或捕获血液样本里的纤维蛋白单体或纤维蛋白多聚体,抗纤维蛋白的抗体可W是多克隆抗体、单克隆抗体或抗体片段。 In a specific embodiment example, the antibody may be an anti-fibrin with W as a receptor to bind or capture a blood sample in the fibrin monomer or fibrin polymer, an anti-fibrin antibody is a polyclonal antibody may be W, The monoclonal antibody or antibody fragment. 运种受体还可W是自然合成或人工合成的其他可W结合纤维蛋白的受体,运里所说的结合可W是"特异"的结合,也可W是非"特异"的结合。 W may be transported receptors synthetic natural or synthetic fibers other W binding protein receptor, said binding may be transported in conjunction with W is "specific", but also in conjunction with W "specific" non. 运种结合纤维蛋白的受体只要可W结合血液样本中的纤维蛋白就可W了。 Fibrin binding species transport receptor binding as long as W fibrin in the blood sample can be a W. 一个比较简单的例子,先让血液样本和抗纤维蛋白的抗体接触,然后通过分离血液样本中抗纤维蛋白的抗体来分离血液样本中红细胞。 A relatively simple example, let blood sample and an anti-fibrin antibody and then contacting the blood sample to separate the red blood cells by antibodies against fibrin separating a blood sample. 在一个更优先的实施方式中,让血液样本和抗红细胞的抗体接触,再让该血液样本和抗纤维蛋白的抗体接触,然后通过分离血液样本中抗纤维蛋白的抗体来分离样本中的红细胞。 In a more preferred embodiment, the blood sample and make contact with antibodies against red blood cells, and let the blood sample in contact with anti-fibrin antibody, and then separating the sample red blood cells in the blood sample by antibody antifibrin separation. 在另一个实施方式中,也可W让血液样本同时和抗纤维蛋白的抗体和抗红细胞的抗体接触, 然后通过同时分离样本中的抗纤维蛋白的抗体和抗红细胞的抗体来分离样本中的红细胞。 In another embodiment, W may be a blood sample at the same time and allow antibodies against fibrin and red blood cells in contact with antibodies, antibody against fibrin sample to separate the sample and then separating red blood cells and anti-erythrocyte antibodies by simultaneously . 运里所说的"分离"是指通过物理或化学的方式来直接或间接地让抗纤维蛋白的抗体从血液样本中分离开来。 Operation in said "isolated" means to make an anti-fibrin antibody directly or indirectly, by physical or chemical means from the blood sample to carve away. 例如,可W直接用一种抗纤维蛋白受体的抗体来分离,也可W是其它的方式,例如在一些载体上固定一些结合纤维蛋白的抗体或抗红细胞的抗体,然后让全血样本流过该载体。 For example, W can be directly used an anti-fibrin antibody receptors isolated, W may be other ways, such as a fixed number of antibody or an anti-erythrocyte antibody binding in a number of fibrin the support, and then let the whole blood sample stream through the carrier.

[0022] 载体、含有载体的分离装置 [0022] The vector containing the vector of the separation device

[0023] 在另一个实施方式中,该方法包括,提供一种载体,载体上包括一种结合纤维蛋白的受体,让血液样本流过该载体。 [0023] In another embodiment, the method includes providing a carrier, the carrier comprising one receptor binding fibrin, so that the blood sample to flow through the carrier. 另外,本发明还设及一种分离全血样本中红细胞的装置, 该装置包括一个载体,其中在载体上处理有结合纤维蛋白受体。 Further, the present invention is also provided an apparatus and a whole blood sample in the separation of red blood cells, the apparatus comprising a carrier, wherein the carrier process with a fibrin binding receptor. 运种载体可W由吸水性,又叫"层析载体"或非吸水性材料构成或组成。 W may be transported by a water-absorbing carrier species, also known as "chromatographic carrier" or non-absorbent material or composition.

[0024] 非吸水性的材料包括,但不限于,塑料、玻璃、陶瓷和其他金属材料。 [0024] non-bibulous materials include, but are not limited to, plastics, glass, ceramics, metals, and other materials. 例如,可先让结合纤维蛋白的受体,例如抗体,固定在塑料表面上,然后让血液样本和塑料表面上的受体接触,运样血液里的纤维蛋白就被固定在塑料表面的受体捕获,从而导致血液里的纤维蛋白从血液样本中分离,运样红细胞也被分离出来。 For example, let fibrin binding receptors, such as antibodies, immobilized on a plastic surface, and then let the blood sample contacting the receptor on the surface of the plastic and the sample transport blood of fibrin is fixed on the plastic surface receptor capture, resulting in fibrin blood isolated from a blood sample, the sample transport erythrocytes are also isolated. 在一个优选的实施方式中,提供运样一种非吸水性的材料,其中结合红细胞的抗体和结合纤维蛋白的抗体同时被固定处理在运些材料上,让血液样本流过非吸水性的材料表面。 In a preferred embodiment, there is provided a non-water-absorbing sample transport material, wherein the fibrin binding antibodies and antibody binding erythrocytes treated while being fixed on the transport of these materials, so that the blood sample to flow through the non-bibulous material surface.

[00巧]另一方面,运种载体也可W是层析载体,运里所说的层析载体是指任何适合的多孔性,有吸收能力的材料或毛细材料。 [Qiao 00] On the other hand, the carrier may also be transported species and W is the chromatography carrier, the chromatography carrier transport in said refers to any suitable porous, absorbent material or capillary with a material capacity. 运种材料本身有一定的吸收能力,液体通过毛细作用可W在上流动,例如一些滤纸,玻璃纤维素,聚脂膜,尼龙膜,硝酸纤维素膜,醋酸纤维素, 天然原料(例如棉花)织物和合成原料(尼龙)织物;W及多孔凝胶等等。 Transport materials itself has a certain absorption capacity, the liquid may be by capillary action W in the flow, such as some paper, cellulose glass, polyester film, nylon film, cellulose nitrate film, cellulose acetate, natural materials (such as cotton) fabrics and synthetic materials (nylon) fabric; W is a porous gel and the like. 在一个优先的实施方式中,当用运些支持液体流动的载体时候,可W先把结合纤维蛋白的受体固定处理在运些载体上,让其不可因为液体的流动而被溶解在液体里。 In one preferred embodiment, when the carrier when transported by liquid flow of some support, can bind to the receptor W first fixing treatment fibrin in some operation on the vehicle, because the flow of the liquid is not allowed to be dissolved in the liquid . 当让血液样本被施加到运些载体上的时候,血液样本中的纤维蛋白分子被它的受体结合而被聚集在载体上,随着血液样本的继续流动,样本中的纤维蛋白就被分离开来。 When the blood sample is applied to allow the transport vehicle some time, fibrin blood sample molecule bound to its receptor is aggregated on the support, with the continued flow of the blood sample, the sample was separated fibrin off. 在另一个优选的实施方式中,在运些载体上还可W处理有一种或几种结合红细胞的受体,运些受体可W聚集血液样本中的红细胞,运些红细胞的受体可W被处理在纤维蛋白受体的上游,例如固定处理在载体上。 In another preferred embodiment, the carrier may also in some transport W to bind erythrocytes with one or several receptors, these receptors may be transported in the blood sample W aggregate red blood cells, some red blood cells transport receptors W the fibers to be treated upstream of the receptor, for example on a carrier fixing process. 处理结合纤维蛋白的受体和结合红细胞的受体在载体上的位置可W和纤维蛋白受体位于同一位置,还可W分别位于上下或前后,或者是其它的位置;当血液样本流过载体的时候,血液样本可W 同时和纤维蛋白的受体和红细胞的受体同时接触;也可W是血液样本先和纤维蛋白的受体接触然后再和红细胞的受体接触,或者先和红细胞的受体接触然后再和纤维蛋白的受体接触。 Processing receptor binding and fibrin binding erythrocytes receptor sites on the carrier available fibrin receptor and W in the same position, which are located further back and up, or W, or other position; when the blood sample to flow through the carrier when the blood sample can be simultaneously and W erythrocyte receptors and receptor while contacting fibrin; W is a blood sample can be first contacted and fibrin receptor and then contacting the receptor erythrocytes, or red blood cells and the first receptor and then contacting the fibrin receptor contacts. 如何把运些受体处理并让其固定在载体上是本领域的公知常识。 How to process and allowed to transport these receptors immobilized on the carrier are common knowledge in the art. 并且,本领域的技术人员应该理解到,载体可W是单一材料构成或者由一种W上的材料构成(例如,可W由不同的材料构成不同的部位,区或层),只要运些多层的材料处于液流相互接触的状态,从而使液体能够在运些材料间通过。 Further, those skilled in the art will appreciate that the vector may be W is a single material or a material (e.g., W may be made of different materials in different parts of the configuration, zone or layer) on a W, as long as the operation of such multiple- the material layer in a state of mutual contact of the liquid flow, so that the liquid can be transported through between these materials. 例如,运种载体接受样本后可W让液体样本垂直通过,如图1 所示,或水平通过载体,如图2所示,也可W是其他的形式。 For example, after receiving the sample carrier transported species may allow W vertically through the liquid sample, shown in Figure 1, or by the level of the carrier 2, W may be other forms.

[0026] 运种通过分离血液样本中纤维蛋白能够分离或除去红细胞的原因可能是,纤维蛋白的受体,例如抗纤维蛋白的抗体,通过结合纤维蛋白分子让单个纤维蛋白分子聚集起来, 形成一个聚集网,该网相互交错并网络住样本中的红细胞,通过分离纤维蛋白的受体从而间接的分离样本中的红细胞。 [0026] By separating species transported in the blood sample can be separated from fibrin, or erythrocytes may be removed reason, fibrin receptors such as anti-fibrin antibodies, the fibrin formed by binding a fibrin molecule so that a single molecule come together, a aggregation network, the network and the network interlace erythrocytes live samples each, separated by a receptor indirectly fibrin separating red blood cells in the sample. 在另一个优选的实施方式中,当有结合红细胞的受体,例如红细胞表面抗原的抗体,存在的情况下,红细胞受体可W使单个的红细胞聚集在一起形成"红细胞小团",同时在有纤维蛋白分子形成的网络状织物的时候,可W网络住运些"红细胞小团"而不容易被血液样本溶解或带走,运样可W更好地分离血液样本中的红细胞。 In another preferred embodiment, when there is a receptor binding erythrocytes, erythrocyte surface antigens such as antibodies, in the presence of red blood cells so that a single receptor can erythrocyte W together form a "small RBC group", while have a network-like structure formed of fibrous protein molecules when W can be transported more live network "RBC pellet was" not easily dissolved or blood samples taken, sample transport W may be better separation of red blood cells in a blood sample. 在一个更优先的实施方式中,运种结合纤维蛋白的受体为多克隆抗体并且被固定处理在层析载体上,另外,在层析载体上还固定处理有结合红细胞表面抗原的多克隆抗体,当把血液样本施加到运种层析载体上时,处理在载体上的红细胞抗体结合或捕获样品中的红细胞形成"红细胞小团",同时载体上的纤维蛋白抗体结合或捕获样品中的纤维蛋白形成纤维蛋白分子网络,运种蛋白分子网络可W网住运些比单个红细胞体积更大的"红细胞小团"从而可W更有效的阻碍红细胞继续向前流动,运样可W有效的从血液样本中分离出红细胞。 In a more preferred embodiment, the fibrin binding species transport receptor is a polyclonal antibody and is fixed to the chromatography carrier in the process, additionally, on a further fixed to the chromatography carrier processing polyclonal antibodies bound red blood cell surface antigen , when the blood sample is applied to the transported species chromatographic support, on a carrier treated erythrocytes or an antibody binding in a sample of red blood cell formation capture "RBC small group", while fibers bound or captured sample antibodies fibers on the carrier fibrin network formed protein molecules, transport proteins molecule transport network netted W is larger than those of a single red blood cell volume "small group of red blood cells" such that W can be more effectively impede flow further erythrocytes, the sample can be transported efficiently from W separating erythrocytes in a blood sample. 运种利用处理在载体上纤维蛋白的受体分离红细胞的方法可W通过让受体直接固定在载体上得W 实现,也可W间接地来分离纤维蛋白。 Species transported using the method of treatment on the carrier erythrocytes fibrin receptor may be isolated by allowing the W receptor immobilized on a carrier obtained directly implemented W, W may be isolated indirectly fibrin. 例如,把抗纤维蛋白抗体的抗体(简称纤维蛋白二抗) 处理并固定在载体上,先让血液样本和抗纤维蛋白的抗体混合反应,然后把该混合溶液施加到处理有二抗的载体上,该二抗抗体捕获纤维蛋白抗体,从而间接的捕获纤维蛋白。 For example, the antibodies against fibrin antibody (referred to fibrin secondary antibodies) immobilized on a carrier and treatment, blood samples and let the anti-fibrin antibody reaction mix, the mixed solution is then applied to the antibody treatment for two carriers the two anti-fibrin antibody capture antibody, and indirectly to capture the fibrin. 同样道理,也可W把抗红细胞抗体的抗体(简称红细胞二抗)处理并固定在载体上来间接捕获血液样品中的红细胞。 Similarly, W may be the anti-erythrocyte antibody (abbreviated RBC secondary antibody) fixed in the support and processing the captured indirectly onto erythrocytes in the blood sample. 在一个优选的方式中,把抗纤维蛋白的抗体和抗红细胞的抗体直接固定处理在载体上。 In a preferred embodiment, the anti-fibrin antibody and anti-erythrocyte antibodies process is directly fixed on the support.

[0027] 检测装置 [0027] detecting means

[0028]另一方面,本发明还设及一种检测全血样品中被分析物质的检测装置,运种装置包括一个载体,载体上包括一个检测区域,在检测区域上游处理有结合血液样本中纤维蛋白的受体。 [0028] another aspect, the present invention is also provided, and detection means for detecting analyte substance whole blood sample, the transport means comprises a seed carrier, the carrier comprising a detection region, the detection region upstream of a blood sample processed with a binding receptor fibrin. 优选地,载体为层析载体;更优选的,在该载体上还包括一个位于检测区域上游的样本接受区域;在样本接受区域上处理有结合血液样本中纤维蛋白的受体。 Preferably, the carrier of the chromatography carrier; more preferably, on the carrier further comprises a sample receiving zone located upstream of the detection region; sample receiving process in conjunction with a fibrin blood sample receptor region. 任何检测系统都可W用于本发明的目的。 Any detection system can be used for purposes of the present invention W. 优选的是层析免疫检测系统,包括但不局限于水平流动系统、 垂直液流系统、和检测棒。 Preferably the chromatography immunoassay system, including but not limited horizontal flow systems, vertical flow systems, and the detection rod. 下面将描述一些已知的检测系统。 Here are some known detection system will be described.

[0029] -般来讲,对于层析载体试剂条上至少安排一个样本接受区域和一个检测区域, 检测区域上包括一些特异分子或化学物质,通过他们可W检测到样本中是否存在被分析物质或数量。 [0029] - generally speaking, for at least the chromatography carrier arrangements reagent strip and a sample receiving area of ​​a detection area, including some specific molecules or chemicals on the detection area, they may be W is detected by the presence or absence of analyte in the sample material or number. 当将被检测的样品液施加到样本接受区域的时候,借助毛细作用它将沿着层析载体移动,并且在检测区域通过结合反应巧日免疫学反应)的作用,积累预先安排在层析载体上的带有标记的被标记物质,运种积累作用与样本中被分析物质的存在或含量成正比(例如,双抗体夹屯、法)或反比(竞争法),运种通过测定带有标记物质的存在或含量,就可W 检测出样品液中是否存在或含量多少。 When the sample solution to be detected is applied to the sample receiving region, it will move by capillary action along the chromatographic support, and a binding reaction in the detection region by Qiao day immunological response) effects, in the chromatography carrier accumulation prearranged is marked on the labeling substance, the analyte presence or proportional to the concentration effect of substance accumulation transported species in the sample (e.g., diabodies clip Tun, France) or inversely (competitive method), labeled with a measuring operation by species the presence or amount of substance, can detect the presence or W content in many liquid samples. 当然,检测区域上也可W是纯化学性质低物的颜色反应,例如氧化还原反应等等,通过检测区域上颜色的深浅来测量被分析物质有无或数量, 利用运一原理的检测装置常见的有血糖检测试剂条等,例如在美国专利6818180中所描述的那样。 Of course, the detection area may be W is a pure chemical nature of the color of the reaction was low, for example, redox reactions, etc., to measure the presence or absence or amount of the analyte by the color depth of the detection area, the detecting means using the operation principle of a common there blood glucose test strip and the like, for example, as in U.S. Patent No. 6,818,180 described. 在一个具体的实施例子中,样本接受区域与被标记物质处于相同的位置,优选地是在被标记物质的上游(把通过毛细作用引起液体流动的方向称为"上游",相反的方向称为"下游")。 In a specific embodiment example, the sample receiving region and the labeled substance in the same position, preferably upstream of the labeling substance (the direction of flow of the liquid caused by capillary action is called "upstream", the opposite direction is called "downstream"). 当让样品液体(怀疑含有被分析物质)和样品接受区域接触时,样品液借助毛细作用,沿层析载体连同被分析物质一同向下游流动。 When the sample liquid so that (a substance suspected of containing an analyte) and the sample receiving region of the contact time, the sample liquid by capillary action, along the chromatography carrier together with the analyte flows downstream. 通常被分析物质为一种化合物,它W特定的方式结合于固定在检测区域上的检测分子。 Typically the analyte is a compound which binds to a specific W immobilized on the detection region of the detection molecules. 例如,被分析物质为一种抗原,被标记的物质为该抗原的第一抗体,固定在检测区域的分子为该抗原的另一个抗体分子。 For example, the analyte is an antigen, the first antibody being labeled substance for an antigen, antibody molecules fixed to the molecule another detection region for the antigen. 利用运些原理来检测样本中是否存在被分析物质的装置和方法为现有技术,不是本发明的重点。 To detect the presence of the prior art, the present invention is not the focus of the analysis method and apparatus utilizing the sample material is transported these principles. 本发明可W用于任何血液样品,包括血清和血浆,但是优先的是用含有红细胞的全血样本。 The present invention W may be used in any blood sample, including serum and plasma, but preferred is a whole blood sample containing red blood cells.

[0030] 如果希望W理想的灵敏度进行检测,优选地是对血液样本中所需被分析物进行检测前将红细胞除去。 [0030] If desired W ideal detection sensitivity, it is preferably removed prior to detection of the red blood cells in a blood sample the desired analyte. 因此,根据本发明的检测装置,让结合纤维蛋白的受体被固定处理在层析载体上,优选地处理在样本接受区域上。 Accordingly, the detection device according to the invention, so that the fibrin binding of receptor was immobilized on the chromatography carrier process, preferably in process sample receiving area. 之所W是优选,是因为血液样本一旦施加到样本接受区域上,运种处理可W有效将血液样品中的红细胞除去,而减少血清或血浆在W后沿着载体流动的最少干扰,从而使血清或血浆在W后的流动速度不受影响,另外降低了背景干扰。 Of W is preferably is because, once a blood sample is applied to the sample-receiving area, W transported treatments can be effective to remove red blood cells in a blood sample, serum or plasma to reduce flow along the least interference carrier after W, so that serum or plasma flow rate after W unaffected, further reducing background interference. 如图2描绘了本发明一个优选的具体实施方式,在检测装置30中,构成样本接受区域35的载体材料为玻璃纤维,构成检测区域31的材料为硝酸纤维素膜并位于样本接受区域的下游,含有被标记物质的区域34位于样本接受区域35和检测区域31的之间。 2 depicts a preferred embodiment of the present invention DETAILED embodiments, the detection device 30, the carrier material constituting the sample receiving region 35 is a glass fiber, a material constituting the detection region 31 is a nitrocellulose membrane and located downstream of the sample receiving region , the region containing the labeled substance 34 is located between the sample receiving region 35 and the detection region 31. 在检测区域上包括固定有被固定的特异结合分子带33和检测结果控制分子带32 ;在玻璃纤维载体上包括红细胞表面抗原的多克隆抗体和纤维蛋白的多克隆抗体,其中样本接受区域35, 标记区域34和检测区域31之间相互连接可W让液体从样本接受区域35流到检测区域31 上进行反应。 On the detection area comprises a fixed specific binding molecules immobilized belt 33 and belt 32 controlling the molecular detection result; on glass fiber support comprises a red blood cell surface antigen polyclonal antibody polyclonal antibody and fibrin, wherein the sample receiving region 35, marker area 34 and the detection region may be interconnected between 31 W 35 allow liquid to flow from the sample receiving zone 31 is detected on the reaction region.

[0031] 图1描绘了本发明的另一个优选的具体实施方式,在检测装置10中,包括一个过滤层11来接受样本并让样本通过该层和反应层12,在反应层上处理有还原反应底物,和手持柄13,其中在过滤层上处理有结合血液样本中纤维蛋白的多克隆抗体和结合红细胞的多克隆抗体。 [0031] Figure 1 depicts a further preferred embodiment of the present invention are described in the detecting means 10, comprising a filter layer 11 to receive a sample and allow the sample 12, in the process layer and the reaction layer by layer reduction reaction the reaction substrate, and holding the shank 13, wherein the filter layer on the treated blood sample with a binding fibrin binding polyclonal antibodies and polyclonal antibodies erythrocytes. 当把全血样本01施加到样本接受层11上后,处理在过滤层11上的纤维蛋白抗体和红细胞抗体一起把红细胞拦截在过滤层上,让血浆通过过滤层到达反应层12上,如果样本中存在被分析物质,例如血糖,在反应层12的下表面直接可W观察到颜色变化。 After When a whole blood sample 01 is applied to the sample-receiving layer 11, the processing in the filter layer 11 fibrin antibody and red cell antibodies with the erythrocyte intercept on the filter layer, so that the plasma through the filter layer to the reaction layer 12, if the sample in the presence of analyte, e.g. glucose, W may be a direct color change is observed in the surface of the reaction layer 12. 关于本发明设及去除血液样本中红细胞方法的应用不仅局限与W上具体实施方式的列举,还可W被运用到其他任何检测系统中去,例如可W被运用到美国专利6818180图1所描绘的检测装置中去,具体的讲,可W被处理到多孔性膜1的表层5上来分离通过孔21施加的全血样本。 Application and removal of a blood sample provided on erythrocytes in the methods of the invention include not only confined to the particular embodiments of the W, W may be applied to any other detection system to, for example, W can be applied to U.S. Patent 6,818,180 depicted in FIG. 1 detecting means go, specifically speaking, W can be processed into the porous surface of the membrane 51 is separated from a whole blood sample is applied up through the hole 21. 该领域的一般技术人员可W理解,本发明所描述的方法或装置中都可W和现有技术中其它分离红细胞的方法,结构或试剂结合使用,例如与美国专利6818180总所描述的那样的结构和试剂结合使用。 One skilled in the art may be appreciated that W, method or apparatus of the present invention as described in the prior art and W can be other methods of separating red blood cells, structures or binding agents, for example U.S. Patent No. 6,818,180 and as described in the total structure and reagents used in combination.

[0032] 检测方法 [0032] Detection

[0033] 另一方面,本发明还提供一种用于检测全血样本中被分析物质的一种方法。 [0033] another aspect, the present invention further provides a method for detecting a substance to be analyzed in whole blood samples. 此方法优选地应用本发明的层析载体装置中。 This method is preferably applied to the chromatography carrier device of the present invention. 具体的讲,此方法包括,提供一种载体,载体上包括一个样本接受区域,其中,在样本接受区域上处理有结合血液样本中纤维蛋白的受体;使血液样品流过样本接受区域;检测血液样本中是否存在被分析物质。 To be specific, the method comprising providing a vector comprising a sample application zone on a carrier, wherein the sample receiving process in the blood sample with a binding receptor region fibrin; the blood sample to flow through sample receiving region; detecting blood samples for the presence analyte. 优选的,在样本接受区域上还处理有结合红细胞的抗体。 Preferably, the sample-receiving region further processing antibody binding erythrocytes. 优选的,在样本接受区域的下游还包括一个检测区域, 该检测区域包括一个被固定处理的特异结合分子,让流过样本接受区域的样本流过检测区域。 Preferably, downstream of the sample receiving region further comprises a detection region, the detection region comprises a fixed handle specific binding molecule, so that the sample flows through sample receiving area flows through the detection region. 载体可W是层析载体或非吸水性载体。 W is a chromatography carrier may support or water-absorbing support.

[0034] 下面的实施例将进一步说明本发明,但无论如何不应理解为对本发明范围的限制。 [0034] The following examples will further illustrate the present invention but should in no way be construed as limiting the scope of the present invention.

[0035] 实验 [0035] Experiment

[0036]连輪1,利用杭纤维帝白杭体夹分离血液样本奸细胸在檢测CTI(屯、肌钩帝白)中的麻用 [0036] The wheel 1 is connected by fiber Hang Tai Hang sandwiched white blood sample isolated thoracic spies hemp (Tun, white muscle hook Di) is detected by CTI

[0037] 本实验,包括W下其他实验中所用的抗纤维蛋白的多抗购买于北京Ξ博远志生物技术有限责任公司,地址北京市海淀区东北旺南路26号,批号为G11135796J162 ;使用的抗红细胞的单抗买于Genclonn公司,地址为中国浙江杭州天目山路198号,古荡经济园区,批号为ME060822-1-0419 ;使用的抗红细胞的多抗购买于Genclonn公司,地址为杭州天目山路198号,古荡经济园区,批号为Q05920-1113.。 [0037] In this experiment, including W under anti-fibrin polyclonal antibody used in other experiments were purchased from Beijing Ξ Sunbiotech limited liability company, address Dongbeiwang South Road, Haidian District, Beijing, No. 26, lot number G11135796J162; use of anti red blood cells in Genclonn monoclonal antibody to buy the company, address, 198 Mount Road, Hangzhou, Zhejiang, China, Gudang economic zone, lot number ME060822-1-0419; polyclonal anti-erythrocyte used to purchase Genclonn company, address of Hangzhou Tian Mu Shan Road 198 No, Gudang economic zone, lot Q05920-1113 .. 除了额外标出的地方,可W通过现有技术所描述的方法制造CTI检测试剂条。 Except where otherwise indicated, the CTI can be manufactured test strip W by the process described in the prior art. 本实验参照图2来说明如何制备和组装试剂条。 This experiment will be described with reference to FIG. 2 how to make and assemble reagent strip.

[0038] 1.抗纤维蛋白的抗体的配置和处理样本接受垫35 [0038] The configuration and processing of the sample 1. The anti-fibrin antibodies of the receiving pad 35

[0039] 样本接受垫为玻璃纤维,先在上面处理Tris缓冲溶液,对玻璃纤维做如下几个不同的处理。 [0039] Sample receiving pad is a glass fiber, the first process in the above Tris buffer solution, glass fiber several different treatment as follows.

[0040] 1),只在玻璃纤维上处理抗纤维蛋白多抗(100个,每个处理20个)。 [0040] 1), only the anti-fibrin polyclonal antibody treatment (100, 20 per treatment) on glass fiber. 抗纤维蛋白的多克隆抗体按照比例1 :50,1 :100,1 :200,1 :300,1 :400稀释,并均匀地处理到玻璃纤维上,分别为处理A,B,C,D,E。 Polyclonal antibodies against fibrin in accordance with the ratio of 1: 50, 1: 100, 1: 200, 1: 300, 1: 400 dilution, and uniform treatment to glass fibers, respectively, the processing A, B, C, D, E.

[0041] 2),同时在玻璃纤维上处理抗红细胞和纤维蛋白的多抗(50个,每个处理10个)。 [0041] 2), while processing multiple anti-RBC and anti-fibrin (on glass fiber 50, each processing 10). 把(1)中的A,B,C,化E分为2组,每组10个,再在其中一组的每个玻璃纤维上处理抗红细胞的多克隆抗体,抗红细胞抗体的稀释比例为1 :1〇,分别为G=1 :50(抗纤维蛋白多抗)+1 : 1〇(抗红细胞多抗);,H=1 :100(抗纤维蛋白多抗)+1 :10(抗红细胞多抗),1=1 :200(抗纤维蛋白多抗)+1 :1〇(抗红细胞多抗)J=1 :300(抗纤维蛋白多抗)+1 :10(抗红细胞多抗), K=1 :400(抗纤维蛋白多抗)+1 :10(抗红细胞多抗)。 The (1) A, B, C, E of the divided two groups of 10 each, and then a polyclonal antibody dilution ratio on the glass fibers which each set of anti-treated erythrocytes, is an anti-red cell antibodies 1: 1〇, respectively, G = 1: 50 (anti-fibrin antibodies) +1: 1〇 (polyclonal anti-erythrocyte) ;, H = 1: 100 (anti-fibrin antibodies) +1: 10 (anti multi-erythrocyte antibodies), 1 = 1: 200 (anti-fibrin antibodies) +1: 1〇 (polyclonal anti-erythrocyte) J = 1: 300 (anti-fibrin antibodies) +1: 10 (multiple anti-RBC antibodies) , K = 1: 400 (anti-fibrin antibodies) +1: 10 (multiple anti-RBC antibodies).

[0042] 3),只在玻璃纤维上处理1 :10的红细胞多抗,处理为F(10个)。 [0042] 3) process only on glass fiber 1: 10 polyclonal antibody erythrocytes, is treated F (10 unit).

[0043] 最后在37°C烘箱里烘干处理好的玻璃纤维备用。 [0043] Finally, glass fibers good alternate drying process at 37 ° C oven.

[0044] 2.标记垫的准备34 [0044] 2. Preparation of the marking pad 34

[0045] 用常用的胶体金乳胶,平均粒径为20-40nm钢米.标记兔抗CTI的单抗和鼠抗人IgG多抗,并用机器均匀的喷洒在聚脂膜上,并在37Γ的烘箱下烘干处理好的标记垫。 [0045] Colloidal gold with a conventional latex, average particle size 20-40nm steel meters. CTI labeled rabbit anti-mouse anti-human antibody and the polyclonal antibody IgG, and sprayed uniformly with the machine in the polyester film, and the 37Γ deal marks drying oven pad.

[0046] 3.硝酸纤维膜的准备36 [0046] 3. The nitrocellulose membranes Preparation 36

[0047] 首先由一个微处理器控制的微量调节注射器把羊抗鼠IgG多抗(1. 3mg/ml,作为检测结果控制线32)处理到硝酸纤维素膜上,然后再把鼠抗CTI单抗(浓度为4.Omg/ml)处理到硝酸纤维素膜作为检测线33,作为被分析物结合区域。 [0047] First, regulated by a microprocessor-controlled micro-syringe to the sheep anti-mouse IgG polyclonal antibody (1. 3mg / ml, as a detection result of the control line 32) to process a nitrocellulose membrane, and then a single mouse anti CTI anti (concentration 4.Omg / ml) to a nitrocellulose membrane treated as a detection line 33, as the analyte binding region. 喷量都为1.1μ1/cm。 Ejection amount are 1.1μ1 / cm. 完成后, 硝酸纤维素膜立即在45°C干燥(2小时)来固定抗体试剂。 Upon completion, the nitrocellulose membrane was immediately dried at 45 ° C (2 hours) the immobilized antibody reagent.

[004引4.试剂条的组装 [004 primer reagent strip 4. The assembly

[0049] 按照图2所描绘的式样组装检测CTI的试剂条,让玻璃纤维作为样品吸收垫35并叠加在标记垫34上,同时让标记垫叠加在硝酸纤维膜36上;W及吸水滤纸31叠加在硝酸纤维膜上组成如图2所示的试剂条。 [0049] According to FIG. 2 depicted style assembly detected CTI reagent strip, so that the glass fiber as a sample absorbent pad 35 and superimposed marking pad 34, while allowing marking pad is superimposed on the nitrocellulose membrane 36; W and water absorbing filter paper 31 nitrocellulose membranes superimposed components shown in Figure 2 in the reagent strip shown in FIG.

[0050] 5.血液样本的准备 [0050] The blood sample prepared

[0051] 准备20个全血样本(储存在2-8°C,采集时间都超过7天) [0051] Preparation 20 whole blood samples (stored at 2-8 ° C, the acquisition time is more than 7 days)

[005引6.检测过程 [005 6 lead detection process

[0053] 在每个处理的样本接受垫上滴加lOOul的全血样本,并记录控制线条(C线)出现的时间,控制线出现时间的快慢可W说明液体在试剂条上流动的速度。 [0053] In the samples of each receiving whole blood sample pad treated dropwise lOOul and recording the time control line (C line) occurs, the control lines may appear speed W times described in the velocity of the liquid flowing through the reagent strip. 按照预先设置的标准,在5分钟之内要出现控制线为合格,在10分钟内需要完成检测,即血液样本需要流过硝酸纤维膜然后到达吸水滤纸;否则为不合格产品。 In accordance with pre-set standard, within 5 minutes of the control line to appear to be acceptable, in 10 minutes to complete testing, i.e. it requires a blood sample to flow through a nitrocellulose membrane filter and then reaches the absorbent; otherwise substandard products. 选用F和J处理来进行灵敏度和特异性检测。 F and J are selected to process the detection sensitivity and specificity. 用阴性样本配置成阳性标本,让待检测的CT1的浓度为0. 5ng/ml,记录检测结果。 Concentration of the positive samples arranged with the negative sample, so that CT1 is detected to be 0. 5ng / ml, recording the detection result. 特异性检测,用标准的阴性样本20个来检测运些试剂条。 Specific detection, using standard negative samples 20 to detect the operation of these reagent strips. 另外,如果出现红色背景也被判为不合格产品。 In addition, if a red background appears also convicted of substandard products.

[0054] 实验结果 [0054] The results

[00巧]表1,C线出现的时间测定 [Qiao 00] Table 1, the time measurement lines appear C

[0056] [0056]

Figure CN103760333BD00101

[0057] 注意:在表1的结果中,在F,H,I处理中带有下划线的数字中虽然控制线出现的时间都小于5分钟,但是他们都没有在10分钟内完成反应,也被判断为不合格产品。 [0057] Note: The results in Table 1, the F, H, I underlined number processing time, although a control line appearing less than five minutes, but they are not the reaction was completed within 10 minutes, is also judged as substandard products.

[00则表2 :不合格率[0059] [00 Table 2: failure rate [0059]

Figure CN103760333BD00102

[0064] [0064]

Figure CN103760333BD00111

[0065] 7.分析和结论 [0065] 7. The analysis and conclusions

[0066] 由上面的实验结果可W看出,利用抗纤维蛋白的抗体可W分离血液样本中的红细胞,大大降低血液对层析载体阻塞的几率,提高血液分离的效率,并且对检测本身并不带来不利的影响。 [0066] W can be seen from the above experimental results, the use of an anti-fibrin antibodies may be isolated from blood samples W red blood cells, to substantially reduce the risk of blood clogging of the chromatographic carrier to improve the efficiency of the separation of blood, and detection itself not adversely affected. 另外,如果与抗红细胞的抗体结合使用,作用更为明显。 Further, if used in conjunction with an anti-erythrocyte antibody, it was more effective.

[0067]连輪2,利用杭纤维帝白杭体夹分离血液样本奸细胸在檢郷IPSA(前列腺特择杭原) 中的麻用 [0067] The wheel 2 is connected by fiber Hang Tai Hang sandwiched separated white blood sample in the sample spies chest Hongo IPSA (Optional prostate Laid Hang original) with Ma

[006引同样参照附图2来说明如何组装该实验的试剂条。 [006 Primer 2 to illustrate how to assemble the same reagent strip test reference to the drawings. 和实验1相比,结构没有太大的差异,所用的红细胞抗体和纤维蛋白的抗体批号和来源相同。 Experiment 1 compared to not much difference in the structure, the same source and lot number used antibodies erythrocytes and fibrin antibody. 只是所检测的被分析物质不同和在样本接受垫上做不同的处理。 Except that different substances detected analyte in a sample receiving pad and processed differently.

[0069] 1.抗纤维蛋白的抗体的配置和处理样本接受垫 [0069] The configuration and processing of the sample 1. The anti-fibrin antibodies of receiving pad

[0070] 样本接受垫为玻璃纤维,先在上面处理Tris缓冲溶液,对玻璃纤维做如下几个不同的处理。 [0070] Sample receiving pad is a glass fiber, the first process in the above Tris buffer solution, glass fiber several different treatment as follows.

[0071] 1),处理1。 [0071] 1), for 1. 在玻璃纤维处理抗纤维蛋白和抗红细胞的多克隆抗体。 Glass fiber treatment and the anti-fibrin polyclonal anti-erythrocyte antibody. 抗纤维蛋白的抗体按照比例1 :300,稀释,并均匀地处理到玻璃纤维上。 Anti-fibrin antibody according to Comparative Example 1: 300 dilution, and uniform treatment to glass fibers.

[0072] 2),处理2。 [0072] 2), 2 treated. 在玻璃纤维上处理抗红细胞的多克隆抗体(购买于Genclonn公司,与实验1中所用的为同一批号)。 Anti treated erythrocytes on glass fiber polyclonal antibodies (purchased from companies Genclonn, in Experiment 1 was used for the same lot). 红细胞多克隆抗体的稀释比例为1:10最后在37°C烘箱里烘干处理好的玻璃纤维备用。 RBC polyclonal antibody dilution ratio 1:10 at 37 ° C oven for final drying process in good spare glass fiber.

[0073] 3),对照处理CK。 [0073] 3), the control process CK. 在玻璃纤维上处理抗红细胞的单克隆抗体(购买于Genclonn公司,批号为:与实验1中所用的为同一批号),稀释比例为1:75。 Anti treated erythrocytes on glass fiber monoclonal antibody (purchased from Genclonn company, lot number: as in Experiment 1 was used in the same batch), 1:75 dilution ratio.

[0074] 2.标记垫的准备34 [0074] 2. Preparation of the marking pad 34

[00巧]用常用的胶体金乳胶,粒径为20-40nm钢米.标记兔抗PSA的单抗体和鼠抗人IgG 多抗用机器均匀的喷洒在聚脂膜上,并在37°C的烘箱下烘干处理好的标记垫。 [Qiao 00] by a conventional latex colloidal gold particle size of 20-40nm steel meters. Single labeled rabbit anti-PSA antibody and the mouse anti-human IgG polyclonal antibody sprayed uniformly with the machine in a polyester film, and at 37 ° C deal marks drying pad under the oven.

[0076] 3.硝酸纤维膜的准备36 [0076] 3. The nitrocellulose membranes Preparation 36

[0077] 首先由一个微处理器控制的微量调节注射器把羊抗鼠IgG多抗(1. 3mg/ml,作为检测结果控制线32)处理到硝酸纤维素膜上,然后再把鼠抗PSA单抗(浓度为4.Omg/ml)处理到硝酸纤维素膜作为检测线31,作为被分析物结合区域。 [0077] First, regulated by a microprocessor-controlled micro-syringe to the sheep anti-mouse IgG polyclonal antibody (1. 3mg / ml, as a detection result of the control line 32) to process a nitrocellulose membrane, and then a single mouse anti-PSA anti (concentration 4.Omg / ml) to a nitrocellulose membrane treated as a detection line 31, as the analyte binding region. 喷量都为1.1μ1/cm。 Ejection amount are 1.1μ1 / cm. 完成后, 硝酸纤维素膜立即在45°C干燥(2小时)来固定抗体试剂。 Upon completion, the nitrocellulose membrane was immediately dried at 45 ° C (2 hours) the immobilized antibody reagent.

[007引4.试剂条的组装 [007 primer reagent strip 4. The assembly

[0079] 按照图2所描绘的式样组装检测PSA的试剂条,让玻璃纤维作为样品吸收垫并与标记垫叠加,同时让标记垫和硝酸纤维膜W及吸水滤纸叠加组成如图2所示的试剂条。 [0079] As depicted in Figure 2 assembled style detecting PSA reagent strip, so that the glass fiber as a sample pad and the absorbent pad is superimposed with the labeled, while allowing marking pad and nitrocellulose membrane and water absorbing filter paper superposition of W as shown in FIG. 2 reagent strips.

[0080] 5.血液样本的准备 [0080] The blood sample prepared

[0081] 准备20个7全血样本(储存在2-8°C,采集时间超过7天)。 [0081] Preparation 20 7 whole blood samples (stored at 2-8 ° C, acquisition time over seven days).

[00的]6.检测过程 6. The detection process [00]

[0083] 在每个处理的样本接受垫上滴加40ul的全血样本并滴加40ul的全血憐酸缓冲溶液帮助跑板,并记录控制线条(C线)出现的时间,控制线出现时间的快慢可W说明液体在试剂条上流动的速度。 [0083] each sample receiving pad dropwise treated whole blood sample was added dropwise and the whole blood 40ul 40ul of acid buffer solution pity help running board, and the recording time of the control line (C line) occurs, the control line appearance time W speed can be described in the speed of the liquid flowing through the reagent strip. 按照标准,在3分钟之内要出现控制线为合格,在5分钟内要完成检巧。 As standard, within 3 minutes in the control line to appear to be acceptable in the subject within 5 minutes to complete coincidence. ,即血液样本需要流过硝酸纤维膜然后到达吸水滤纸,否则为不合格产品。 , I.e. it requires a blood sample to flow through a nitrocellulose membrane filter and then reaches the absorbent, an unqualified products. 选用对照和处理1来进行灵敏度和特异性检测。 Selection of control and treated to a sensitivity and specificity of detection. 用阴性样本配置成阳性标本,让待检测的PSA的浓度为2ng/ml,4ng/ml,lOng/ml,20ng/ml,记录检测结果。 Concentration of the positive samples arranged with the negative sample, so as to be detected PSA 2ng / ml, 4ng / ml, lOng / ml, 20ng / ml, the detection result record. 特异性检测,用标准的阴性样本20个来检测运些试剂条。 Specific detection, using standard negative samples 20 to detect the operation of these reagent strips.

[0084] 7.检测结果 [0084] 7. The detection result

[00财表1对照和处理2的控制线出现的时间比较(秒) [Table 1 Control 00 fiscal control line 2 and a processing time of occurrence of the comparison (s)

[0086] [0086]

Figure CN103760333BD00121

[0089] 注表示在3分钟内样本不能到达控制线,*表示在5分钟内仍然不能完成检测。 [0089] Note the sample does not reach the control line represented in 3 minutes, * represents still can not detect completion within 5 minutes.

[0090] 表3 :处理1和对照的灵敏度比较 [0090] Table 3: Sensitivity 1 treated and control comparison

[0091] [0091]

Figure CN103760333BD00131

[009引"G"表示Τ线/检测线的颜色,后面的数字越大,表示颜色越深。 [009 primer "G" indicates the color Τ line / lines, behind the larger the number, the deeper the color represented.

[009引表4 :处理1和对照的特异性比较 [Table 009 Primer 4: 1 treated and control-specific comparison

[0094] [0094]

Figure CN103760333BD00132

[009引结论 [009 cited conclusions

[0096] 由上面的实验结果可W看出,利用抗纤维蛋白的抗体可W分离血液样本中的红细胞,大大降低血液对层析载体阻塞的几率,提高血液分离的效率,并且对检测本身并不带来不利的影响。 [0096] W can be seen from the above experimental results, the use of an anti-fibrin antibodies may be isolated from blood samples W red blood cells, to substantially reduce the risk of blood clogging of the chromatographic carrier to improve the efficiency of the separation of blood, and detection itself not adversely affected. 另外,如果与抗红细胞的抗体结合使用,作用更为明显。 Further, if used in conjunction with an anti-erythrocyte antibody, it was more effective.

[0097]连輪3,加巧稳定忡连輪。 [0097] Even wheel 3, wheel plus Qiao stable even sad.

[0098] 用实验1中的处理F和J来做加速稳定性实验,用来检验产品的最长有效时间。 [0098] do accelerated stability experiment F Experiment 1 processing and J, the maximum effective time for product testing. 方法:把处理F和J的样品放在55°C下处理,并按照下表1所列的天数进行灵敏度,特异性, 每次用10个产品进行,在每个处理的样本接受垫上滴加lOOul的全血样本,并记录控制线条(C线)出现的时间。 Method: The sample handling F and J on treatment at 55 ° C, the sensitivity and specificity of the number of days set forth in Table 1 below, each time with 10 products, each sample receiving pad treated dropwise lOOul of a whole blood sample, and recording the time control line (C line) appears.

[0099] 表1,稳定性试验天数和检测时间对照表 [0099] Table 1, the number of days and the detection time stability test table

[0100] [0100]

Figure CN103760333BD00133

阳101]"X"表示在55°C的情况下需要进行实验进行验证。 Male 101] "X" represents a need to experiment to verify in the case of 55 ° C.

[0102] 实验结果[010引0天 [0102] Experimental results [0 days 010 primer

[0104] 表2-1处理F的结果 [0104] Table 2-1 in the processing result F

[0105] [0105]

Figure CN103760333BD00134

Figure CN103760333BD00141

[0120] [0120]

Figure CN103760333BD00151

阳~注表示阴性结果;数字加"+"表示结果为阳性,数字越大,表示检测线条的颜色越深。 Note indicates a negative result - male; Digital Plus "+" indicates a positive result, the larger the number, the more color depth detecting lines.

[0心]结论 [0 Heart] Conclusions

[0123] 从W上实验结果可W明显看出,当在样本接受垫上添加了抗纤维蛋白的抗体的时候,产品的有效使用时间可W达到2年,同时减少了样本到达C线的时间。 [0123] From the experimental results W W apparent, when added in a sample receiving pad antibodies against fibrin time, effective use time of the product W can be up to 2 years, while reducing the sample time line C is reached.

Claims (13)

  1. 1. 一种从血液样品中分离红细胞的方法,包括:让血液样品和一种结合血液样品中纤维蛋白的受体接触;从血液样品中分离出纤维蛋白受体来分离血液样品中的红细胞。 CLAIMS 1. A method of separating red blood cells from a blood sample, comprising: a blood sample so that one binding and fibrin blood sample contacting the receptor; isolated from the blood sample to separate the fibrinogen receptor blood sample erythrocytes.
  2. 2. 根据权利要求1所述的方法,其特征在于:该方法还包括让血液样品和一种结合血液样品中红细胞的受体接触。 2. The method according to claim 1, wherein: the method further comprises a blood sample and make contact with one binding receptor erythrocytes in the blood sample.
  3. 3. 根据权利要求2所述的方法,其特征在于:所述的结合纤维蛋白的受体和结合红细胞的受体被固定在一个支持液体流动的载体上。 3. The method according to claim 2, wherein: said receptor binding and fibrin binding erythrocytes receptor is immobilized on a carrier to support a flow of liquid.
  4. 4. 根据权利要求1所述的方法,其特征在于:所述的纤维蛋白受体包括纤维蛋白的单克隆或多克隆抗体或抗体片段。 4. The method according to claim 1, wherein: said receptor comprising fibrin fibrin monoclonal or polyclonal antibody or antibody fragment.
  5. 5. 根据权利要求3所述的方法,其特征在于:所述的载体为层析载体。 5. The method according to claim 3, wherein: said carrier is a chromatography carrier.
  6. 6. -种从血液样品中分离红细胞的方法,包括:提供一种载体,让血液样品从该载体上流过,其中,该载体上处理有结合血液样本中纤维蛋白的受体;通过分离出纤维蛋白受体来分离血液样品中的红细胞。 6. - methods of separating red blood cells from a blood sample, comprising: providing a carrier, so that a blood sample flowing through from the vector, wherein the vector processing on a blood sample with a binding fibrin receptor; by separating the fibers receptor isolated red blood cells in a blood sample.
  7. 7. 根据权利要求6所述的方法,其特征在于:该载体上还包括结合红细胞的受体。 7. The method according to claim 6, wherein: the carrier further comprises a receptor binding erythrocytes.
  8. 8. 根据权利要求6所述的方法,所述的受体包括单克隆、多克隆抗体或抗体片段。 8. The method according to claim 6, said receptor includes monoclonal, polyclonal antibody or antibody fragment.
  9. 9. 根据权利要求6-8之一所述的方法,其特征在于:所述的载体为层析载体。 9. The method according to any one of claims 6-8, wherein: said carrier is a chromatography carrier.
  10. 10. -种检测血液样品中被分析物质的方法,包括:提供一种层析载体,让血液样品从层析载体上流过;测量血液样品中被分析物质的含量,其中该层析载体上固定一种结合血液样品中纤维蛋白的受体,通过分离出纤维蛋白受体来分离血液样品中的红细胞。 10. - Method analyte substance and seed detecting blood sample, comprising: providing a chromatography carrier so that a blood sample flowing from the chromatography carrier; substance content measuring analyte in a blood sample, which is fixed on the chromatographic support one binding receptor fibrin blood sample, the blood sample to separate the red blood cells by separating the fibrinogen receptor.
  11. 11. 根据权利要求10所述的方法,其特征在于,所述的载体上还包括结合红细胞的受体。 11. The method according to claim 10, wherein said carrier further comprises a receptor binding erythrocytes.
  12. 12. 根据权利要求10所述的方法,其特征在于,所述的受体包括抗体或抗体片段。 12. The method according to claim 10, wherein said receptor comprises an antibody or antibody fragment.
  13. 13. 根据权利要求10所述的方法,其特征在于,在该层析载体的下游还包括一个检测样本中被分析物质的检测区域。 13. The method according to claim 10, wherein the chromatography carrier in the downstream of the detector further comprises a detection zone analyte substance in the sample.
CN 201410006059 2008-10-13 2009-10-13 A method for separating erythrocytes in a blood sample and the use of CN103760333B (en)

Priority Applications (3)

Application Number Priority Date Filing Date Title
CN 201410006059 CN103760333B (en) 2009-10-13 2009-10-13 A method for separating erythrocytes in a blood sample and the use of
CN 200910206522 CN101750244B (en) 2008-10-13 2009-10-13 Method for separating red cells from blood sample and application
CN200910206522.62009.10.13 2009-10-13

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN 201410006059 CN103760333B (en) 2009-10-13 2009-10-13 A method for separating erythrocytes in a blood sample and the use of

Publications (2)

Publication Number Publication Date
CN103760333A true CN103760333A (en) 2014-04-30
CN103760333B true CN103760333B (en) 2016-03-16

Family

ID=50527606

Family Applications (1)

Application Number Title Priority Date Filing Date
CN 201410006059 CN103760333B (en) 2008-10-13 2009-10-13 A method for separating erythrocytes in a blood sample and the use of

Country Status (1)

Country Link
CN (1) CN103760333B (en)

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5939331A (en) * 1992-03-10 1999-08-17 Quidel Corporation Red blood cell separation means for specific binding assays
CN1499978A (en) * 2001-02-28 2004-05-26 柏尔纯公司 Manufacture of hemoglobin-based oxygen carrier
CN101275942A (en) * 2007-03-30 2008-10-01 希森美康株式会社 Chromatographic test device
CN101402671A (en) * 2008-10-21 2009-04-08 浙江大学 Method simultaneously separating fibrinogen and immunoglobulin from livestock and poultry blood

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5939331A (en) * 1992-03-10 1999-08-17 Quidel Corporation Red blood cell separation means for specific binding assays
CN1499978A (en) * 2001-02-28 2004-05-26 柏尔纯公司 Manufacture of hemoglobin-based oxygen carrier
CN101275942A (en) * 2007-03-30 2008-10-01 希森美康株式会社 Chromatographic test device
CN101402671A (en) * 2008-10-21 2009-04-08 浙江大学 Method simultaneously separating fibrinogen and immunoglobulin from livestock and poultry blood

Also Published As

Publication number Publication date Type
CN103760333A (en) 2014-04-30 application

Similar Documents

Publication Publication Date Title
US5616467A (en) Method and kit for analyte detection employing gold-sol bound antibodies
US5766552A (en) Apparatus for red blood cell separation
US7393697B2 (en) Diagnostic test for analytes in a sample
US20040018576A1 (en) Bence Jones protein testing cassette
US5160486A (en) Test carrier utilizing reaction of two bioaffine binding partners
US5652148A (en) Method and apparatus for red blood cell separation
US5079142A (en) Orthogonal flow immunoassays and devices
US20070224701A1 (en) Combination vertical and lateral flow immunoassay device
US20020132370A1 (en) Detection of a blood coagulation activity marker in a body fluid sample
WO2010001598A1 (en) Porous solid phase for binding assay, and binding assay method using the same
US20040023412A1 (en) Ligand binding assay and kit with a separation zone for disturbing analytes
US5118428A (en) Method to remove red blood cells from whole blood samples
WO1984002193A1 (en) Chromogenic support immunoassay
US5215886A (en) HDL determination in whole blood
US5660798A (en) Apparatus for red blood cell separation
CN101059513A (en) Method and apparatus for detecting drug residue of food
Campbell et al. Immunoadsorbents-Preparation and use of cellulose derivatives
US20100143941A1 (en) Devices and methods for detecting analytes
JPH10253632A (en) Method, kit and device for analysis
US20100297611A1 (en) Method and Device For Combined Detection Of Viral And Bacterial Infections
JP2006189317A (en) Testing element for immunochromatographic method
EP0291194B2 (en) Immunoassays and devices therefor
Kimura et al. Rapid quantitation of immunoglobulin G antibodies specific for blood group antigens A and B by surface plasmon resonance
WO1989003044A1 (en) Colloidal gold particle concentration immunoassay
JPH10123137A (en) Highly sensitive immunoassay method

Legal Events

Date Code Title Description
C06 Publication
C10 Entry into substantive examination
C14 Grant of patent or utility model