WO2016176859A1 - 体外诊断装置和试剂盒 - Google Patents

体外诊断装置和试剂盒 Download PDF

Info

Publication number
WO2016176859A1
WO2016176859A1 PCT/CN2015/078470 CN2015078470W WO2016176859A1 WO 2016176859 A1 WO2016176859 A1 WO 2016176859A1 CN 2015078470 W CN2015078470 W CN 2015078470W WO 2016176859 A1 WO2016176859 A1 WO 2016176859A1
Authority
WO
WIPO (PCT)
Prior art keywords
layer
diafiltration
eclampsia
detecting device
percolation
Prior art date
Application number
PCT/CN2015/078470
Other languages
English (en)
French (fr)
Inventor
谭卓
刘术金
林洁
吴飞宏
Original Assignee
浙江数问生物技术有限公司
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
Application filed by 浙江数问生物技术有限公司 filed Critical 浙江数问生物技术有限公司
Priority to PCT/CN2015/078470 priority Critical patent/WO2016176859A1/zh
Publication of WO2016176859A1 publication Critical patent/WO2016176859A1/zh

Links

Images

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids

Definitions

  • the present invention relates to the diagnosis of diseases, in particular to the kit for diagnosing pre-eclampsia.
  • Eclampsia is a complex and dangerous disease that occurs in pregnant women 20 weeks after pregnancy, especially in the third trimester of pregnancy. It is characterized by convulsions and coma, accompanied by major symptoms such as edema, hypertension and proteinuria. Can be complicated by renal failure, heart failure, pulmonary edema, cerebral hemorrhage, early exfoliation of the placenta.
  • Pre-eclampsia also known as "pre-eclampsia” (pre-eclampsia) is preceded by eclampsia, headache, dizziness, upper abdominal discomfort, nausea and vomiting, chest tightness, blurred vision, golden eyes, anxiety, Symptoms such as irritability, predicting the stage of eclampsia, often accompanied by major symptoms such as hypertension and proteinuria. If blood pressure and proteinuria continue to increase or symptoms of terminal organ damage (including fetal growth restriction) are considered, it is considered It is severe pre-eclampsia (sPE). Most of the symptoms of pre-eclampsia before convulsions are pre-eclampsia, but some patients have no obvious pre-existing symptoms, sudden seizures or coma.
  • sPE severe pre-eclampsia
  • intravenous magnesium sulfate is currently used clinically to prevent convulsions. If blood pressure is particularly high, an antihypertensive drug is added.
  • the labor is scheduled to be delivered or the caesarean section is delivered, that is, there is medical indication for childbirth. According to statistics, approximately 76,000 pregnant women worldwide die every year from pre-eclampsia/eclampsia.
  • the present invention provides a percolation detecting device for predicting or detecting pre-eclampsia or eclampsia, comprising a closed casing formed by the upper and lower grooves being mutually cemented.
  • the upper cover opens a number of holes for adding the sample to be tested as well as other reagents such as stains, eluents, and the like.
  • At least a percolating layer and a water absorbing layer are stacked in the casing from top to bottom.
  • the diafiltration layer comprises a microporous membrane that allows normal proteins and other small molecule compounds to pass, but does not allow misfolded protein aggregates to pass.
  • Such a microporous membrane may be a cellulose membrane such as a nitrocellulose membrane or a cellulose acetate membrane.
  • the microporous membrane of the diafiltration layer comprises a labeling reagent that specifically binds to the misfolded protein aggregate, or the misfolded protein aggregate itself, or both.
  • a support layer is placed between the percolating layer and the water absorbing layer for supporting the diafiltration layer so as not to deform after wetting.
  • a coloring layer is placed between the percolating layer and the water absorbing layer as a background to set off the diafiltration layer to make color development more apparent.
  • Another aspect of the present invention provides the use of the above diafiltration detecting device in the preparation of a kit for detecting and predicting pre-eclampsia.
  • kits for detecting and predicting pre-eclampsia including the above-described diafiltration detecting device, eluent or color chart or all three.
  • the present invention relates to the above diafiltration detecting device, a method of preparing the kit, and a method for detecting and predicting pre-eclampsia using the above-described diafiltration detecting device and kit.
  • Figure 1 is a cross-sectional view of one embodiment of a percolating device of the present invention
  • Fig. 2 is a photograph showing the results of urine test for detecting pre-eclampsia pregnant women and normal pregnant women using the percolating device of the present invention.
  • the present invention relates to a diafiltration method for detecting the presence of misfolded protein aggregates in the urine of a pregnant woman to determine whether the pregnant woman tested has pre-eclampsia (or eclampsia) or has a pre-eclampsia (or eclampsia) risk.
  • the present invention provides a percolation detecting device for predicting or detecting pre-eclampsia (or eclampsia), comprising a closed casing formed by the upper and lower grooves being mutually cemented.
  • the upper cover opens a number of holes for adding the sample to be tested as well as other reagents such as stains, eluents, and the like.
  • the closed casing may be of any material (plastic, metal, etc.), and the shape is preferably a flat casing, such as a circle, a square, a rectangle, an ellipse, a triangle, etc., and the thickness from top to bottom may be 2-10 mm.
  • the diafiltration layer comprises a microporous membrane that allows normal proteins and other small molecule compounds to pass, but does not allow misfolded protein aggregates to pass.
  • a microporous membrane may be a cellulose membrane such as a nitrocellulose membrane or a cellulose acetate membrane.
  • the microporous membrane of the diafiltration layer comprises a specific binding misfolded protein aggregate
  • the labeling reagent that specifically binds to the misfolded protein aggregates can be any agent capable of binding to misfolded protein aggregates, all of which are well known to those skilled in the art, including dyes (e.g., heterocyclic dyes, azo dyes, and Thioflavin), antibodies (eg, A11 antibody, Officcr antibody, OC antibody, M118 antibody, M204 antibody, M205 antibody, M89 antibody, and M09 antibody) and the like.
  • the method of coating the above-mentioned misfolded protein aggregate labeling reagent onto the microporous membrane of the diafiltration layer may be carried out by any conventional method, for example, by dropping a dye solution or an antibody-containing solution onto the membrane, followed by drying, air drying or baking. dry.
  • the membrane may also be immersed in a dye solution or an antibody-containing solution to adsorb the dye or antibody onto the membrane and then air dried, air dried or dried.
  • the purpose of the water absorbing layer is to suck the liquid from the top of the diafiltration layer.
  • the material must generally be a highly water absorbing material such as sponge, absorbent paper, cotton, filter paper, etc., which may be a single layer or multiple layers.
  • a support layer is further included between the water absorbing layer and the diafiltration layer for supporting the diafiltration layer to maintain the microporous membrane flat without deforming or pleating, but the liquid in the sample and reagent should still Free to pass through the support layer. Therefore, the support layer can be made of a filter paper material having a slightly hard texture, a relatively smooth surface, and particularly no deformation after wetting, such as fiberglass paper.
  • a coloring layer may be included between the water absorbing layer and the diafiltration layer, and the color thereof is a color having a high contrast with the coloring agent of the labeling reagent.
  • the color of the lining layer as a background makes the color of the diafiltration layer more apparent.
  • the microporous membrane of the diafiltration layer can be white, and the color-imparting layer is blue or other color that has a high contrast with the marking reagent.
  • the Congo red binding sample misfolds protein aggregates, it appears red, while the blue coloring layer acts as a background to make the red color appearing in the percolation layer more pronounced.
  • the material of the color-imparting layer should be such that the liquid can pass through and is not dyed, and it can be a colored chemical fiber cloth, a colored filter film such as a colored cellulose film, or the like.
  • a colored chemical fiber cloth such as a colored cellulose film, or the like.
  • the microporous membrane of the percolation layer is red after staining the positive sample.
  • the color layer may be light blue, sky blue or blue.
  • Colored chemical fiber cloth or filter nitrocellulose membrane, cellulose acetate membrane).
  • the percolating apparatus 100 includes a closed housing formed by the upper cover 102 and the bottom groove 104 being secured to each other. In the closed housing, the upper cover opens a sample hole 112 for adding the sample to be tested and other reagents. At least a percolating layer 106 and a water absorbing layer 110 are stacked in the casing from top to bottom. The diafiltration layer 106 is coated with a Congo red dye. The support layer 108 may be between the percolation layer 106 and the water absorbing layer 110, and the support layer may be replaced with the color layer 108.
  • a coloring layer and a support layer can be included between the percolating layer and the water absorbing layer.
  • a percolating layer from top to bottom are a percolating layer, a coloring layer, a supporting layer, and a water absorbing layer.
  • the microporous membrane of the diafiltration layer comprises a labeling reagent (eg, Congo red or antibody) that specifically binds to the misfolded protein aggregate, or the misfolded protein aggregate itself, or both.
  • the general method of preparing the percolation detecting device of the present invention may comprise the following steps (in any order): (1) providing an upper cover and a bottom groove, a closed casing which can be mutually formed; (2) providing a microporous membrane (diafiltration layer) coated with a misfolded protein aggregate labeling reagent, and the method may be any conventional method, for example, dropping a solution containing a labeling reagent (such as a dye, an antibody) onto the membrane, or The membrane is immersed in a solution containing a labeling reagent (such as a dye, an antibody), and the labeling reagent (such as a dye, an antibody) is adsorbed onto the membrane, and then air-dried, air-dried or dried; (3) the water-absorbing layer and the support layer are If necessary, a color-imprinting layer (if needed), and a microporous membrane (diafiltration layer) coated with a misfolded protein aggregate labeling reagent (or a percolation layer
  • kits for detecting and predicting pre-eclampsia including the above-described diafiltration detecting device, and optionally further comprising an eluent, a reference or a color chart or two of them Or three at the same time.
  • the use of the above diafiltration detecting device in preparing a kit for detecting and predicting pre-eclampsia is provided, that is, assembling the diafiltration detecting device with other components such as an eluent, a reference or a color chart in one In the box.
  • the present invention relates to a method for detecting and predicting pre-eclampsia using the above-described diafiltration detecting device and kit.
  • the diafiltration detecting device and kit of the present invention can be conveniently used to detect a urine sample of a pregnant woman to predict or detect pre-eclampsia.
  • a method is the above diafiltration detecting device, wherein the microporous membrane of the diafiltration layer comprises a labeling reagent such as Congo red which specifically binds to the misfolded protein aggregate, and the pregnant woman urine sample is added from the spotting hole (may be 1-500 ⁇ l volume), after all the urine has permeated the percolation layer, add the eluent (may be 1-500 ⁇ l volume), and the eluent is completely permeated through the percolation layer and observed in the sample well.
  • a labeling reagent such as Congo red which specifically binds to the misfolded protein aggregate
  • the pregnant woman urine sample is added from the spotting hole (may be 1-500 ⁇ l volume)
  • the eluent may be 1-500 ⁇ l volume
  • the eluent can be ethanol or other solvent that can dissolve Congo red.
  • the color is red, it is positive, that is, the pregnant woman has preeclampsia or has Pre-eclampsia risk, if the color is still the primary color of the microporous membrane, the test is negative, and the pregnant women in the test have no preeclampsia.
  • a pregnant woman's urine sample is added from the spotting hole, and after the urine is completely permeated through the diafiltration layer, the pregnant woman suffering from pre-eclampsia or having pre-eclampsia risk has misfolded protein aggregation in the urine. And the misfolded protein aggregates are adsorbed on the percolation layer and cannot pass through the percolation layer. Especially after elution by the eluent, these misfolded protein aggregates still adsorb to the percolation layer.
  • a diafiltration test provided by the present invention is formed One of the embodiments of the device, wherein the upper cover is provided with a sample hole for adding a sample to be tested and other reagents, and the casing is provided with a percolation layer and a water absorption layer from top to bottom, and the percolation layer comprises A labeling reagent that specifically binds to a misfolded protein aggregate and a misfolded protein aggregate that binds to the labeling reagent.
  • the nitrocellulose membrane was dip-coated in Congo red working solution at 0.1 mg/ml overnight, and the impregnated nitrocellulose membrane was dried and cut into 5 mm x 5 mm squares. Cut other materials such as filter paper and absorbent paper into 5mm x 5mm squares.
  • absorbent paper > Whatman filter paper > nitrocellulose membrane, sealed plastic shell. Apply 250 ul of pregnant women's urine to the plastic shell and dry for 20 minutes. 250 ul of 50% ethanol was added to the spotting hole. After the ethanol was absorbed, 250 ul of 50% ethanol was added again. After the absorption was dried, the color in the sampled well was observed, and the red color was positive and the white color was negative. The results are shown in Figure 2.
  • the nitrocellulose membrane was dip-dyed in a Thioflavin S working solution at 0.1 mg/ml overnight, and the impregnated nitrocellulose membrane was dried and cut into 5 mm x 5 mm squares. Cut other materials such as filter paper and absorbent paper into 5mm x 5mm squares.
  • absorbent paper > Whatman filter paper > nitrocellulose membrane, sealed plastic shell. Apply 250 ul of pregnant women's urine to the plastic shell and dry for 20 minutes. 250 ul of 50% ethanol was added to the wells. After the ethanol was absorbed, 250 ul of 50% ethanol was added again. After absorption, the color in the sample was observed. The yellow color was positive and the white color was negative.
  • the nitrocellulose membrane was dip-coated in a mouse IgG antibody SW11 working solution against A? oligomer at 4 ° C overnight, and the impregnated nitrocellulose membrane was dried and cut into 5 mm x 5 mm squares. Cut other materials such as filter paper and absorbent paper into 5mm x 5mm squares.
  • plastic shell sample well add 250 ul of pregnant women's urine. When the urine is completely infiltrated, add horseradish peroxidase-labeled goat anti-mouse IgG antibody to the spotted well. After infiltration, add 250 ul of washing solution, then add 5 - The aminosalicylic acid substrate developed color and the brown color was positive.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Chemical & Material Sciences (AREA)
  • Biomedical Technology (AREA)
  • Urology & Nephrology (AREA)
  • Hematology (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Cell Biology (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Investigating Or Analysing Biological Materials (AREA)

Abstract

一种用于检测子痫前期或子痫的检测装置和体外诊断试剂盒，包括一个由上盖和底槽相互卡固形成的一个封闭壳体。在封闭壳体上盖开有点样孔，壳体中自上而下叠有至少渗滤层和吸水层，其中渗滤层包含有特异性结合错误折叠蛋白聚集物的标记试剂。

Description

体外诊断装置和试剂盒
相关申请
本申请案主张2014年5月7日递交的题为“体外诊断装置和试剂盒”的中国专利申请案201410190153.7以及2014年5月8日递交的题为“渗滤检测装置和试剂盒”的中国专利申请案201410192368.2之权益及优先权，其整体揭示内容全文以引用的方式并入本文中。
技术领域
本发明涉及疾病诊断，尤其是诊断子痫前期的试剂盒。
技术背景
子痫(eclampsia)是怀孕妇女在怀孕后20周以后、特别是妊娠晚期发生的一种复杂和危险的疾病，以抽搐及昏迷为特点，并伴有水肿、高血压和蛋白尿等主要症状，可并发肾功能衰竭、心力衰竭、肺水肿、大脑出血、胎盘早期剥离等。先兆子痫(亦称为“子痫前期”)(pre-eclampsia)则是于子痫抽搐前，有头痛、头晕、上腹不适、恶心呕吐、胸闷、视力模糊、眼冒金花、忧虑、易激动等症状，预示子痫即将发生的阶段，常伴有高血压和蛋白尿等主要症状，如果血压和蛋白尿持续增加或者出现末端器官损坏(包括胎儿生长限制)的症状，就会被认为是重度先兆子痫(sPE)。子痫抽搐前多数有先兆子痫症状，但也有些患者前驱症状不明显，突然发作抽搐或进入昏迷。
对于重度子痫前期，目前临床上采用静脉点滴硫酸镁，防止发生抽搐，如果血压特别高，另外加用降压药。当怀孕在34周以上时，安排引产或进行剖腹产分娩，即有医学指征分娩。据统计，全球每年有大约76,000孕妇因先兆子痫/子痫而死亡。
目前，临床上还没有任何能够预测或者诊断先兆子痫的检测试剂，只能通过临床观察、根据血压升高和尿蛋白质的出现来主观地判断，具体地说，根据孕前无高血压、肾小球肾炎病史，孕20周后出现高血压、蛋白尿、水肿及血尿酸升高等症状。这种诊断方法存在几方面的缺陷：一是诊断时机太晚，有些患者在诊断时或诊断后不久就出现严重而危险的子痫症状，诊断晚了，不能有效地治疗先兆子痫而预防子痫；二是不准确，据统计有38％的子痫患者没有高血压或蛋白尿症状；三是对一大部分孕妇无法应用：孕前即患有慢性高血压、蛋白尿、或肾病的孕妇，以及孕20周后才来第一次检查而对其之前是否有高血压、或蛋白尿无从知晓的孕妇。因此临床急需一种更早期、更准确的不需要医师进行主观判断的临床检验和诊断先兆子痫的试剂盒和方法。
发明概述
本发明提供了一种用于预测或检测子痫前期或子痫的渗滤检测装置，包括一个由上盖和底槽相互卡固形成的一封闭壳体。在封闭壳体中，上盖开有点样孔用于添加待测样本以及其它试剂诸如染色剂、洗脱液等等。所述壳体中自上而下叠有至少渗滤层和吸水层。所述渗滤层包括一种微孔膜，它能让正常蛋白和其它小分子化合物通过，但却不能让错误折叠蛋白聚集物通过。这种微孔膜可以是纤维素膜，例如硝酸纤维素膜或醋酸纤维素膜。优选地，渗滤层的微孔膜包含有特异性结合错误折叠蛋白聚集物的标记试剂、或者错误折叠蛋白聚集物本身、或者二者兼具。在一些实施例中，渗滤层和吸水层之间置有支撑层用于支持渗滤层使其在浸湿后不变形。在一些实施例中，渗滤层和吸水层之间置有衬色层作为背景以衬托渗滤层使其显色更加明显。
本发明的另一方面提供了上述渗滤检测装置在制备用于检测和预测子痫前期的试剂盒中的应用。
另一方面，还提供了用于检测和预测子痫前期的试剂盒，其中包括上述渗滤检测装置、洗脱液或比色卡或者三者同时。
另外，本发明还涉及上述渗滤检测装置、试剂盒的制备方法，以及使用上述渗滤检测装置、试剂盒用于检测和预测子痫前期的方法。
附图说明
图1为本发明的渗滤装置的一个实施方案的剖面图；
图2是显示应用本发明渗滤装置检测子痫前期孕妇和正常孕妇尿液试验结果的照片。
发明详述
本发明涉及一种渗滤法检测孕妇尿液中是否存在错误折叠蛋白聚集物，以判定所检测的孕妇是否患有子痫前期(或子痫)或者具有子痫前期(或子痫)风险。
特别地，本发明提供了一种用于预测或检测子痫前期(或子痫)的渗滤检测装置，包括一个由上盖和底槽相互卡固形成的一封闭壳体。在封闭壳体中，上盖开有点样孔用于添加待测样本以及其它试剂诸如染色剂、洗脱液等等。封闭壳体可以采用任何材质(塑料、金属等等)，形状以扁壳体较好，例如圆形、正方形、长方形、椭圆形、三角形等等，从上到下的厚度可以是2-10mm。所述壳体中自上而下叠有至少渗滤层和吸水层。所述渗滤层包括一种微孔膜，它能让正常蛋白和其它小分子化合物通过，但却不能让错误折叠蛋白聚集物通过。这种微孔膜可以是纤维素膜，例如硝酸纤维素膜或醋酸纤维素膜。
优选地，渗滤层的微孔膜包含有特异性结合错误折叠蛋白聚集物 的标记试剂、或者错误折叠蛋白聚集物本身、或者二者兼具。特异性结合错误折叠蛋白聚集物的标记试剂可以是任何能够和错误折叠蛋白聚集物结合的试剂，这些试剂都已为领域内技术人员所熟知，包括染料(例如杂环染料、偶氮类染料和硫磺素)、抗体(例如A11抗体、Officcr抗体、OC抗体、M118抗体、M204抗体、M205抗体、M89抗体和M09抗体)等等。将上述错误折叠蛋白聚集物的标记试剂包被到渗滤层的微孔膜上方法可以采用任何常规方法，例如，将染料溶液或者含抗体的溶液点滴到膜上，然后晾干、风干或烘干。也可以将膜浸泡于染料溶液或者含抗体的溶液中，使染料或抗体吸附到膜上，然后晾干、风干或烘干。
吸水层的目的是将液体从渗滤层上面吸下来，其材质一般来说必须是吸水性较强的材料，如海绵、吸水纸、棉花、滤纸等等，可以是单层或多层。
在一些实施方案中，在吸水层和渗滤层之间还包含一支撑层，用于支撑渗滤层使微孔膜能够保持平整而不变形或出褶，但是样品和试剂中的液体应该仍能自由透过支撑层。因此支撑层可以采用质地稍微坚硬、表面比较平滑、尤其浸湿之后不变形的滤纸材料，例如玻璃纤维纸。
在一些实施方案中，在吸水层和渗滤层之间可以包含一衬色层，其颜色是一种与标记试剂显色对比度高的颜色，在实际结果颜色较浅、不易判断的情况时，衬色层作为背景使渗滤层所显现的颜色更为明显。例如，渗滤层的微孔膜可以是白色的，衬色层是蓝色或其它与标记试剂显色对比度高的颜色。例如，当刚果红结合样品中错误折叠蛋白聚集物后，显现红色，而蓝色的衬色层作为背景使渗滤层所显现的红色更为明显。
衬色层的材质应该是能够让液体透过，并且不被染色，可以是有色化纤布料、有色滤膜例如有色纤维素膜等等。举个例子，当刚果红用作错误折叠蛋白聚集物的标记试剂时，阳性样品染色后渗滤层的微孔膜上显红色，此时，衬色层可以是浅蓝色、天蓝色或蓝色的化纤布料或滤膜(硝酸纤维素膜、醋酸纤维素膜)。
图1是本发明的装置的一个实施方案的示意图，具体地说，渗滤装置100包括一个由上盖102和底槽104相互卡固形成的一封闭壳体。在封闭壳体中，上盖开有点样孔112用于添加待测样本以及其它试剂。壳体中自上而下叠有至少渗滤层106和吸水层110。渗滤层106包被有刚果红染料。在渗滤层106和吸水层110之间可以是支撑层108，也可以将支撑层换成衬色层108。
在一些实施方案中，在渗滤层和吸水层之间可以同时包含衬色层和支撑层。优选地，从上至下为渗滤层、衬色层、支撑层、和吸水层。 优选地，渗滤层的微孔膜包含有特异性结合错误折叠蛋白聚集物的标记试剂(例如刚果红或抗体)、或者错误折叠蛋白聚集物本身、或者二者兼具。
制备本发明的渗滤检测装置的一般方法可以包括以下步骤(顺序不限)：(1)提供一个上盖和一个底槽，两者能够相互卡固形成的一封闭壳体；(2)提供一个包被有错误折叠蛋白聚集物标记试剂的微孔膜(渗滤层)，具体方法可以采用任何常规方法，例如，将含有标记试剂(例如染料、抗体)的溶液点滴到膜上，或者将膜浸泡在将含有标记试剂(例如染料、抗体)的溶液中，使标记试剂(例如染料、抗体)吸附到膜上，然后晾干、风干或烘干；(3)将吸水层、支撑层(如果需要的话)、衬色层(如果需要的话)、以及包被有错误折叠蛋白聚集物标记试剂的微孔膜(渗滤层)(或者不含有错误折叠蛋白聚集物标记试剂的渗滤层)按一定的顺序叠放在扁平壳体里；(4)然后将上盖和底槽相互卡固形成一封闭壳体，并且使得上盖在点样孔处与渗滤层紧密接触以至于液体在渗滤层上基本不从侧面横向流出点样孔以外的区域。
本发明的另一方面还提供了用于检测和预测子痫前期的试剂盒，其中包括上述渗滤检测装置，另外还可以可选地包括洗脱液、对照品或比色卡或者其中两个或三者同时。另一方面提供了上述渗滤检测装置在制备用于检测和预测子痫前期的试剂盒中的应用，即将上述渗滤检测装置与其它成分例如洗脱液、对照品或比色卡组装在一个包装盒中。
另外，本发明还涉及使用上述渗滤检测装置、试剂盒用于检测和预测子痫前期的方法。本发明的渗滤检测装置和试剂盒可以很方便地用于检测孕妇尿样以预测或检测子痫前期。具体地说，一种方法是取上述渗滤检测装置其中渗滤层的微孔膜包含有特异性结合错误折叠蛋白聚集物的标记试剂如刚果红，从点样孔加入孕妇尿样(可以是1-500微升体积)，待尿液全部渗透过渗滤层后，加入洗脱液(可以是1-500微升体积)，待洗脱液完全渗透过渗滤层后观察点样孔内微孔膜颜色，以刚果红为例，洗脱液可以是乙醇或其它可以溶解刚果红的溶剂，洗脱后，如果颜色是红色则为检测阳性，即受检孕妇患有子痫前期或者有子痫前期风险，若颜色仍是微孔膜原色，则检测为阴性，受检孕妇没有子痫前期。
特别地，在上述检测过程中，从点样孔加入孕妇尿样、待尿液全部渗透过渗滤层后，患有子痫前期或者有子痫前期风险的孕妇尿液中有错误折叠蛋白聚集物，而这些错误折叠蛋白聚集物会吸附在渗滤层之上而不能透过渗滤层，尤其在经洗脱液洗脱后，这些错误折叠蛋白聚集物仍会吸附在渗滤层，此时便形成了本发明提供的一种渗滤检测 装置的实施方案之一，其中所述上盖开有点样孔用于添加待测样本以及其它试剂，所述壳体中自上而下设有渗滤层和吸水层，所述渗滤层包含有特异性结合错误折叠蛋白聚集物的标记试剂以及与所述标记试剂结合的错误折叠蛋白聚集物。
实施例一
将硝酸纤维素膜浸染在刚果红工作液0.1mg/ml过夜，将浸染好的硝酸纤维素膜干燥，将其裁剪成5mmx5mm方块。将滤纸、吸水纸等其它材料裁剪成5mmx 5mm方块。在渗滤塑料壳中从下至上按下列顺序组装：吸水纸〉Whatman滤纸〉硝酸纤维素膜，密封塑料壳。在塑料壳加样孔加样250ul孕妇尿液，干燥20min。在点样孔加入50％乙醇250ul，待乙醇吸收干后再次加入50％乙醇250ul，待吸收干后观察加样孔内颜色，红色为阳性，白色为阴性。结果见附图2。
共计试验了12个确诊为子痫前期的孕妇的尿样，6个临产或产后但没有子痫前期症状的孕妇的尿样，灵敏度75％，特异性100％，PPV＝100％，NPV＝67％。
实施例二
将硝酸纤维素膜浸染在硫磺素S工作液0.1mg/ml过夜，将浸染好的硝酸纤维素膜干燥，将其裁剪成5mmx5mm方块。将滤纸、吸水纸等其它材料裁剪成5mmx 5mm方块。在渗滤塑料壳中从下至上按下列顺序组装：吸水纸〉Whatman滤纸〉硝酸纤维素膜，密封塑料壳。在塑料壳加样孔加样250ul孕妇尿液，干燥20min。在点样孔加入50％乙醇250ul，待乙醇吸收干后再次加入50％乙醇250ul，待吸收干后观察加样空内颜色，黄色为阳性，白色为阴性。
实施例三
将硝酸纤维素膜浸染在针对Aβ寡聚物的小鼠IgG抗体SW11工作液中4℃过夜，将浸染好的硝酸纤维素膜干燥，将其裁剪成5mmx5mm方块。将滤纸、吸水纸等其它材料裁剪成5mmx 5mm方块。在渗滤塑料壳中从下至上按下列顺序组装：吸水纸〉Whatman滤纸〉硝酸纤维素膜，密封塑料壳。在塑料壳加样孔加样250ul孕妇尿液，待尿液完全渗入，在点样孔加入辣根过氧化物酶标记的羊抗鼠IgG抗体，待渗入后，加250ul洗涤液，然后加入5-氨基水杨酸底物显色，棕色为阳性。
实施例四
将硝酸纤维素膜浸染在刚果红工作液0.1mg/ml过夜，将浸染好 的硝酸纤维素膜干燥，将其裁剪成5mmx5mm方块。将滤纸、蓝色化纤布、吸水纸等其它材料裁剪成5mmx 5mm方块。在渗滤塑料壳中从下至上按下列顺序组装：吸水纸〉Whatman滤纸〉蓝色化纤布〉硝酸纤维素膜，密封塑料壳。在塑料壳加样孔加样250ul孕妇尿液，干燥20min。在点样孔加入50％乙醇250ul，待乙醇吸收干后再次加入50％乙醇250ul，待吸收干后观察加样空内颜色，红色为阳性，白色为阴性。

Claims (9)

  1. 一种渗滤检测装置，包括
    上盖和底槽相互卡固形成一封闭壳体，其中
    所述上盖开有点样孔用于添加待测样本以及其它试剂；
    所述壳体中自上而下设有渗滤层和吸水层，所述渗滤层包含有特异性结合错误折叠蛋白聚集物的标记试剂。
  2. 权利要求1所述渗滤检测装置，其中所述渗滤层还包括与所述标记试剂结合的错误折叠蛋白聚集物。
  3. 权利要求1或2中所述渗滤检测装置，还包括支撑层位于所述渗滤层和吸水层之间。
  4. 权利要求1或2中所述渗滤检测装置，还包括衬色层位于所述渗滤层和吸水层之间。
  5. 一种渗滤检测装置，包括
    上盖和底槽相互卡固形成一封闭壳体，其中
    所述上盖开有点样孔用于添加待测样本以及其它试剂；
    所述壳体中自上而下设有渗滤层、衬色层和吸水层，所述渗滤层包含有特异性结合错误折叠蛋白聚集物的标记试剂。
  6. 权利要求5所述渗滤检测装置，其中所述渗滤层还包括与所述标记试剂结合的错误折叠蛋白聚集物。
  7. 权利要求5所述渗滤检测装置，其中所述渗滤层为白色，所述标记试剂为刚果红，所述衬色层为蓝色。
  8. 权利要求1-7中任一所述渗滤检测装置，在制备用于检测和预测子痫前期的试剂盒中的应用。
  9. 用于检测和预测子痫前期的试剂盒，包括权利要求1-7中所述任一渗滤检测装置，以及洗脱液和比色卡。
PCT/CN2015/078470 2015-05-07 2015-05-07 体外诊断装置和试剂盒 WO2016176859A1 (zh)

Priority Applications (1)

Application Number Priority Date Filing Date Title
PCT/CN2015/078470 WO2016176859A1 (zh) 2015-05-07 2015-05-07 体外诊断装置和试剂盒

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/CN2015/078470 WO2016176859A1 (zh) 2015-05-07 2015-05-07 体外诊断装置和试剂盒

Publications (1)

Publication Number Publication Date
WO2016176859A1 true WO2016176859A1 (zh) 2016-11-10

Family

ID=57217563

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2015/078470 WO2016176859A1 (zh) 2015-05-07 2015-05-07 体外诊断装置和试剂盒

Country Status (1)

Country Link
WO (1) WO2016176859A1 (zh)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110702902A (zh) * 2019-11-04 2020-01-17 珠海丽珠试剂股份有限公司 试剂条及其使用方法

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6461830B1 (en) * 2000-06-01 2002-10-08 Atairgin Technologies, Inc. Determining existence of preeclampsia in pregnancies by measuring levels of glycerophosphatidyl compounds, glycerophosphatidycholine, lysophospholipids and lysophosphatidylcholine
CN101819209A (zh) * 2010-05-15 2010-09-01 厦门出入境检验检疫局检验检疫技术中心 一种利用斑点免疫金渗滤法检测口蹄疫非结构蛋白3ab抗体的检测方法
CN102483418A (zh) * 2008-10-31 2012-05-30 耶鲁大学 先兆子痫检测和治疗的方法和组合物
CN102608321A (zh) * 2012-02-29 2012-07-25 厦门大学 多房棘球绦虫循环抗原斑点金免疫渗滤试剂盒及制备方法
CN103760340A (zh) * 2014-01-28 2014-04-30 成都创宜生物科技有限公司 以flrg作为标志物制备检测工具的应用及检测工具
CN105092854A (zh) * 2014-05-07 2015-11-25 湖州数问生物技术有限公司 体外诊断装置和试剂盒

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6461830B1 (en) * 2000-06-01 2002-10-08 Atairgin Technologies, Inc. Determining existence of preeclampsia in pregnancies by measuring levels of glycerophosphatidyl compounds, glycerophosphatidycholine, lysophospholipids and lysophosphatidylcholine
CN102483418A (zh) * 2008-10-31 2012-05-30 耶鲁大学 先兆子痫检测和治疗的方法和组合物
CN101819209A (zh) * 2010-05-15 2010-09-01 厦门出入境检验检疫局检验检疫技术中心 一种利用斑点免疫金渗滤法检测口蹄疫非结构蛋白3ab抗体的检测方法
CN102608321A (zh) * 2012-02-29 2012-07-25 厦门大学 多房棘球绦虫循环抗原斑点金免疫渗滤试剂盒及制备方法
CN103760340A (zh) * 2014-01-28 2014-04-30 成都创宜生物科技有限公司 以flrg作为标志物制备检测工具的应用及检测工具
CN105092854A (zh) * 2014-05-07 2015-11-25 湖州数问生物技术有限公司 体外诊断装置和试剂盒

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110702902A (zh) * 2019-11-04 2020-01-17 珠海丽珠试剂股份有限公司 试剂条及其使用方法
CN110702902B (zh) * 2019-11-04 2022-12-09 珠海丽珠试剂股份有限公司 试剂条及其使用方法

Similar Documents

Publication Publication Date Title
CN105445449B (zh) 唾液尿酸快速半定量检测装置及其制备方法
US10329335B2 (en) Composition and system for separating and detecting alpha-fetoprotein variant and use thereof
JP6697394B2 (ja) 異常折り畳みタンパク質を検出するための方法および組成物
WO2021000949A1 (zh) 一种肿瘤细胞表面标志分子pd-l1的检测方法
WO2008031288A1 (fr) Colonne centrifuge préremplie servant à dépister un variant d'alpha-fétoprotéine spécifique de l'hépatocarcinome, et trousse d'analyse contenant ladite colonne
CN110157801B (zh) 一种组合标志物及其在制备胃癌发生风险预测试剂盒中的应用及其测定系统和方法
EP2579041B1 (en) Rapid detection tool and kit for premature rupture of fetal membrane using ICAM-1 as marker.
JP2023027284A (ja) ミスフォールドタンパク質を検出するためのデバイスおよびその使用方法
CN105793707A (zh) 免疫层析辅助的检测方法
CN106353511A (zh) 一种尿微量白蛋白/尿肌酐一体化检测二联条及其制备方法
CN107515307A (zh) 一种干式免疫心血管八指标检测试纸条
CN105092854A (zh) 体外诊断装置和试剂盒
WO2016176859A1 (zh) 体外诊断装置和试剂盒
CN104714031A (zh) 一种心肌梗死快速检测试剂盒
CN104237504A (zh) 一种病理尸检过敏性休克的免疫荧光诊断方法及试剂盒
TW201602576A (zh) 檢測套組及檢測方法
JP2007163151A (ja) 対象生物の生理状態を判定する方法
CA2813497A1 (en) A new method for diagnosing hypertension as well as cardiomyopathies
CN204832205U (zh) 一种快速定量检测sFlt-1和Endoglin试纸
CN104849455B (zh) 一种基于梅毒血清学检测蛋白芯片制备与应用
JP6457119B2 (ja) 生化学検査と免疫反応検査を行うマルチユニット、及びこれを用いた検査方法
CN216160652U (zh) 错误折叠蛋白检测试纸条及试剂盒
WO2023065609A1 (zh) 确定胃癌极早期发生风险及评估胃癌前病变进展风险的分子标志及其在诊断试剂盒中的应用
CN206362809U (zh) 人生长分化因子‑15的检测试纸组件
CN206146947U (zh) 胱抑素C和β2微球蛋白半定量金标联合检测试剂盒

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 15891111

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 15891111

Country of ref document: EP

Kind code of ref document: A1