WO2020224344A1 - 一种点免疫印迹检测的检测装置及检测方法 - Google Patents

一种点免疫印迹检测的检测装置及检测方法 Download PDF

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WO2020224344A1
WO2020224344A1 PCT/CN2020/080798 CN2020080798W WO2020224344A1 WO 2020224344 A1 WO2020224344 A1 WO 2020224344A1 CN 2020080798 W CN2020080798 W CN 2020080798W WO 2020224344 A1 WO2020224344 A1 WO 2020224344A1
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detection
nitrocellulose membrane
sample
negative pressure
suction device
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PCT/CN2020/080798
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English (en)
French (fr)
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肖明兵
刘肇修
施炜
顾志峰
许敏雪
谭忠华
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南通大学附属医院
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Priority to CA3137436A priority Critical patent/CA3137436A1/en
Priority to US17/593,787 priority patent/US20220187302A1/en
Priority to ZA2020/06356A priority patent/ZA202006356B/en
Publication of WO2020224344A1 publication Critical patent/WO2020224344A1/zh

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54306—Solid-phase reaction mechanisms
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5023—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures with a sample being transported to, and subsequently stored in an absorbent for analysis
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L9/00—Supporting devices; Holding devices
    • B01L9/52—Supports specially adapted for flat sample carriers, e.g. for plates, slides, chips
    • 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
    • G01N33/5302—Apparatus specially adapted for immunological test procedures
    • G01N33/5304—Reaction vessels, e.g. agglutination plates
    • 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
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
    • 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
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/02—Adapting objects or devices to another
    • B01L2200/026—Fluid interfacing between devices or objects, e.g. connectors, inlet details
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/16—Reagents, handling or storing thereof
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/04—Moving fluids with specific forces or mechanical means
    • B01L2400/0475—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure
    • B01L2400/0487—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure fluid pressure, pneumatics
    • B01L2400/049—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure fluid pressure, pneumatics vacuum
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/745—Assays involving non-enzymic blood coagulation factors
    • G01N2333/75—Fibrin; Fibrinogen
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2474/00—Immunochemical assays or immunoassays characterised by detection mode or means of detection
    • G01N2474/10—Immunoblots, e.g. Western blot or Dot blot
    • 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
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57438—Specifically defined cancers of liver, pancreas or kidney
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the invention belongs to the technical field of immunodetection, and specifically relates to a detection device and a detection method for dot immunoblotting detection.
  • Western blot or ELISA methods are often used to detect the target protein.
  • Western blot method transfer the protein sample separated by PAGE (polyacrylamide gel electrophoresis) to a PVDF membrane (polyvinylidene fluoride membrane), bind the protein antigen with the antibody corresponding to the target protein, and then bind to enzyme or isotope labeling Used secondary antibody to detect the expression of the target protein by chemiluminescence.
  • ELISA method The test sample (to determine the antibody or antigen in it) reacts with the antigen or antibody on the surface of the solid carrier. Separate the formed antigen-antibody complex from other substances by washing.
  • the enzyme-labeled antigen or antibody adds the enzyme-labeled antigen or antibody, and bind to the solid-phase carrier through a specific reaction.
  • the amount of enzyme on the solid phase carrier is in a certain ratio to the amount of the tested substance in the specimen, so qualitative or quantitative analysis can be performed according to the intensity of the color.
  • the two methods are complicated to operate, require a large amount of samples, and cannot simultaneously take into account the advantages of sensitivity, specificity, and multi-sample detection.
  • the technical problem to be solved by the present invention is to provide a detection device and a detection method for dot immunoblotting detection to solve the problems raised in the background art.
  • an embodiment of the present invention provides a detection device for dot immunoblotting detection, which is characterized by including a negative pressure suction device, an orifice plate, a hose connecting the orifice plate and the negative pressure suction device, and a tight fit
  • the nitrocellulose membrane on the end surface of the orifice plate, the orifice plate includes an upper pallet, a lower pallet sealed and connected under the upper pallet, and a negative pressure cavity arranged between the upper pallet and the lower pallet, the lower pallet One side is provided with a connecting pipe for the hose to be sleeved, the upper supporting plate has 70 through holes, and the nitrocellulose membrane has the same size as the end surface of the orifice plate.
  • the size of the upper pallet is 8 cm ⁇ 6 cm, and the diameter of the through hole is 2 mm.
  • a portable sputum suction device is selected as the negative pressure suction device, and the negative pressure value of the portable sputum suction device is set to 0.06 MPa.
  • the 70 through holes are arranged in a 10 ⁇ 7 configuration, and the distance between every two through holes is equal.
  • a pressure plate is detachably connected to the upper part of the orifice plate.
  • the pressing plate includes a rectangular frame and a buckle rotatably connected around the rectangular frame, and a clamping block for the buckle is provided around the lower tray.
  • the present invention also provides a detection method for dot immunoblotting detection, which is characterized in that it comprises the following steps:
  • Sample addition Divide the 70 sample addition grooves into the reference sample hole and the sample hole, and repeat 3 times for each. Add 1 ⁇ L of the reference substance and each sample to be tested into the reference sample hole and sample hole respectively; Dry the fiber membrane naturally for 30 minutes;
  • the TBS-T solution includes Tris, NaCl, Tween-20 and ddH2O, wherein the mass of Tris is 2.42g, the mass of NaCl is 8.0g, the volume of Tween-20 is 1mL, and the TBS-T solution Use ddH2O to make up the volume to 1000ml.
  • sample holes can be set as multiple groups of sample holes according to the number of groups of samples to be tested.
  • the detection device for dot immunoblotting detection of the present invention makes the nitrocellulose membrane closely adhere to the 70-well plate and form 70 sample loading grooves on the surface.
  • the negative pressure suction device is used for negative pressure suction, which is beneficial for adding The concentration of samples during the sample avoids cross-contamination after sample diffusion and affects the experimental results, and the experimental results are more accurate.
  • the detection device and detection method for dot immunoblotting detection of the present invention perform dot immunoblotting detection. Compared with Western blot and ELISA methods, the immunoblotting detection performed by the dot immunoblotting device is not only sensitive, specific, and requires less sample, It has the advantages of simplicity, economy and speed, and it can perform multi-sample testing at one time, which is suitable for testing a large number of people.
  • Figure 1 is a schematic structural diagram of a detection device for midpoint western blot detection of the present invention
  • Figure 2 is a top view of the orifice plate of the present invention.
  • Figure 3 is a graph showing the detection results of the relative concentration of serum FGB in the pancreatic cancer mouse group, pancreatitis mouse group and normal control group by the detection method of the midpoint western blot detection of the present invention
  • Fig. 4 is a comparison diagram of the relative concentration of serum FGB in the pancreatic cancer mouse group, pancreatitis mouse group and normal control group by the detection method of the midpoint western blot detection of the present invention.
  • Negative pressure suction device 2. Orifice plate; 21, upper pallet; 22, lower tray; 23, connecting pipe; 24, through hole; 25, negative pressure cavity; 3. hose; 4. Nitrocellulose membrane; 5. Negative pressure cavity.
  • the terms “installed”, “connected”, and “connected” should be understood in a broad sense unless otherwise clearly specified and limited.
  • it can be a fixed connection or a detachable connection.
  • Connected or integrally connected it can be a mechanical connection or an electrical connection; it can be directly connected or indirectly connected through an intermediate medium, and it can be the internal communication between two components.
  • the specific meaning of the above-mentioned terms in the present invention can be understood in specific situations.
  • a detection device for dot immunoblotting detection includes a negative pressure suction device 1, an orifice plate 2, a hose 3 connected to the orifice plate 2 and the negative pressure suction device 1, and a hose 3 that is closely attached to the hole
  • the nitrocellulose membrane 4 on the upper end of the plate 2 is feasible.
  • the hose 3 can be a rubber tube or a plastic hose.
  • the orifice plate 2 includes an upper pallet 21 and a lower pallet 22 that is sealed and connected under the upper pallet 21 And a negative pressure cavity 25 arranged between the upper pallet 21 and the lower pallet 22.
  • One side of the lower pallet 22 is provided with a connecting pipe 23 for the hose 3 to be sleeved, and the upper pallet 21 has 70 Through holes 24, the size of the nitrocellulose membrane 4 and the upper end surface of the orifice plate 2 are the same.
  • the hose 3 When in use, use the hose 3 to tightly connect the connecting pipe 23 of the 70-well plate 2 and the interface of the negative pressure suction device 1; place the nitrocellulose membrane 4 on the 70-well plate 2 to form a closed cavity, and then turn on the negative pressure
  • the suction device 1 pumps air to provide a negative pressure environment, so that the nitrocellulose membrane 4 is closely attached to the 70-well plate 2 and 70 sample loading grooves are formed on the surface.
  • the negative pressure suction device 1 is used for negative pressure suction.
  • the method is conducive to the concentration of samples when adding samples, avoiding cross-contamination of sample diffusion and affecting the experimental results.
  • the size of the upper support plate 21 is 8 cm ⁇ 6 cm, and the diameter of the through hole 24 is 2 mm.
  • the negative pressure suction device 1 is a portable sputum suction device, and the negative pressure value of the portable sputum suction device is set to 0.06 MPa.
  • the 70 through holes 24 are arranged in a 10 ⁇ 7 arrangement, and the distance between every two through holes 24 is equal. If possible, the distance between the through holes is 5.5 cm-7 cm.
  • a pressure plate is detachably connected to the upper part of the orifice plate 2.
  • the pressing plate includes a rectangular frame and buckles rotatably connected around the rectangular frame, and the lower tray 22 is provided with clamping blocks for clamping the buckles around the periphery.
  • the connection method of the clamping blocks and the buckles please refer to The connection method of the lunch box is enough to compress the nitrocellulose membrane 4.
  • the orifice plate of the utility model is detachably connected with a pressing plate, and the nitrocellulose membrane 4 is pressed above the orifice plate through the pressing plate to ensure that the nitrocellulose membrane 4 is tightly attached to the orifice plate.
  • the principle of dot immunoblotting detection is to adsorb the sample to be tested on the nitrocellulose membrane 4 (NC membrane), and the solid-phase carrier adsorbs proteins in the form of non-covalent bonds, and can maintain the type of polypeptide and its biological activity constant.
  • the protein or polypeptide on the nitrocellulose membrane 4 as the antigen, react with the antibody corresponding to the target protein, and react with the horseradish peroxidase (HRP)-labeled secondary antibody, after autoradiography to detect the expression of the target protein Level.
  • HRP horseradish peroxidase
  • the present invention also provides a detection method for dot immunoblotting detection, which includes the following steps:
  • the primary antibody used in the detection of FGB in serum in this patent is FGB mouse anti-human monoclonal antibody, purchased from Santa Cruz Company, item number: sc-271035; secondary antibody is HRP-labeled mouse anti-human monoclonal antibody, purchased from Santa Cruz company, article number: sc-516102.
  • the TBS-T solution includes Tris, NaCl, Tween-20 and ddH2O, wherein the mass of Tris is 2.42g, the mass of NaCl is 8.0g, the volume of Tween-20 is 1mL, and the Use ddH2O to make up the volume of TBS-T solution to 1000ml, as shown in Table 1 below;
  • sample holes may be set as multiple groups of sample holes according to the number of groups of samples to be tested.
  • mass spectrometry analysis is used to find that the abundance of FGB in the serum of pancreatic cancer patients is higher than that of pancreatitis patients and normal people; then, a dot immunoblotting detection device and its detection method for detecting serum FGB are used to compare the previous mass spectrometry The analysis results are verified.
  • two groups of mice (7 mice in each group) were injected subcutaneously with pancreatic cancer cells and intraperitoneally injected with caerulein to construct a mouse model of pancreatic cancer and a mouse model of pancreatitis.
  • the sera of the mouse model and 7 pancreatitis mouse models were prepared into a pancreatic cancer mouse group and a pancreatitis mouse group.
  • mice (7) were left untreated, and the serum of the control group mice was drawn to prepare a normal control group; then the relative concentration of serum FGB in the three groups of mice was detected by the detection method of dot western blotting.
  • the results are shown in 3; among them, columns a2 to 4 and columns a5 to 7 are the control substances with different concentration gradients, respectively, and columns b, c, and d are the pancreatic cancer mouse group (b1, c1, d1 are the same mouse , B2, c2, and d2 are the same mouse, and so on, the serum of 7 pancreatic cancer mice is dripped into the sample hole), e, f, g are listed as pancreatitis mouse group (e1, f1, g1 It is the same mouse, e2, f2, and g2 are the same mouse, and so on, the serum of 7 pancreatitis mice is dripped into the sample well), h, i, j are listed as the normal control group (h1, i1 and
  • Table 2 The detection of three groups of serum FGB levels by a western blot device

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Abstract

一种点免疫印迹检测的检测装置和检测方法。该检测装置包括负压吸引装置（1）、孔板（2）、连接孔板（2）和负压吸引装置（1）的软管（3）及紧密贴合在孔板（2）上端面的硝酸纤维膜（4）；孔板（2）包括上层托板（21）、密封连接在上层托板（21）下方的下层托盘（22）及设置于上层托板（21）和下层托盘（22）之间的负压空腔（25）；下层托盘22一侧设置有一供软管（3）套接的连接管（23）；上层托板（21）上具有70个通孔（24）。硝酸纤维膜（4）与孔板（2）上端面的大小相同。该检测方法包括准备、加样、封闭、孵育一抗、孵育二抗、显影、分析的过程。通过负压吸引装置（1）进行负压吸引，有利于加样时样品的集中，避免样品扩散后交叉污染影响实验结果，实验结果更准确。

Description

一种点免疫印迹检测的检测装置及检测方法 技术领域
本发明属于免疫检测技术领域，具体涉及一种点免疫印迹检测的检测装置及检测方法。
背景技术
目前多用Western blot或者ELISA的方法来检测目的蛋白。Western blot法：将经过PAGE(聚丙烯酰胺凝胶电泳)分离出的蛋白质样品转移到PVDF膜(聚偏二氟乙烯膜)上，用目的蛋白对应的抗体结合蛋白抗原，再结合酶或者同位素标记过的二抗，以化学发光的方式检测目的蛋白的表达。ELISA法：受检样品(测定其中的抗体或抗原)与固相载体表面的抗原或抗体起反应。用洗涤的方法把形成的抗原抗体复合物和其他物质分开。再加入酶标记的抗原或抗体，通过特异性反应而结合在固相载体上。此时固相载体上的酶量与标本中受检物质的量呈一定的比例，所以可根据呈色的深浅进行定性或定量分析。但两种方法操作复杂、样品需求量大，并且不能同时兼顾灵敏、特异、多样本检测等优点。
发明内容
本发明要解决的技术问题是提供一种点免疫印迹检测的检测装置及检测方法，以解决背景技术中所提出的问题。
为解决上述技术问题，本发明的实施例提供一种点免疫印迹检测的检测装置，其特征在于，包括负压吸引装置、孔板、连接孔板和负压吸引装置的软管及紧密贴合在孔板上端面的硝酸纤维膜，所述孔板包括上层托板、密封连接在上层托板下方的下层托盘及设置于上层托板、下层托盘之间的负压空腔，所述下层托盘一侧设置有一供软管套接的连接管，所述上层托板上具有70个通孔，所述硝酸纤维膜与孔板上端面的大小相同。
进一步的，所述上层托板的尺寸为8cm×6cm，所述通孔直径为2mm。
进一步的，所述负压吸引装置选用手提式吸痰器，所述手提式吸痰器的负压值设定为0.06MPa。
进一步的，70个所述通孔呈10×7设置，每两个通孔之间距离相等。
进一步的，所述孔板的上方可拆卸连接有一压板。
其中，所述压板包括矩形边框及转动连接在矩形边框四周的卡扣，所述下层托盘的四周设置有供卡扣卡接的卡块。
本发明还提供一种点免疫印迹检测的检测方法，其特征在于，包括以下步骤：
S1、准备：将手提式吸痰器通过软管和孔板相连接；将硝酸纤维膜剪至孔板上端面的上层托板大小，置于具有70个通孔的孔板上，打开手提式吸痰器，将手提式吸痰器的负压值设置为0.06MPa，在硝酸纤维膜表面形成70个加样凹槽；
S2、加样：将70个加样凹槽分为对照品孔和样本孔，每个重复3次，对照品孔和样本孔分别加入对照品和各待测样本1μL；加样结束，将硝酸纤维膜自然晾干30min；
S3、封闭：将硝酸纤维膜轻置于5％的脱脂奶粉溶液中，置于摇床上室温下封闭1.5h；然后，弃去封闭液，用TBS-T漂洗1-2次；其中，5％脱脂奶粉溶液使用TBS-T溶液稀释至溶度为5％；
S4、孵育一抗：用5％牛血清白蛋白溶液按1:500比例稀释一抗，将硝酸纤维膜均匀的完全浸入其中，置于摇床上，37℃恒温孵育4h；结束后将硝酸纤维膜放入TBS-T中摇床上洗涤3次，每次5min；其中，5％牛血清白蛋白溶液采用TBS-T溶液稀释至溶度为5％；
S5、孵育二抗：用1％脱脂奶粉溶液按1:5000比例稀释二抗，将硝酸纤维膜均匀的完全浸入其中，置于摇床上室温孵育1.5h；结束后将硝酸纤维膜放入TBS-T中洗涤3次，每次15min；1％脱脂奶粉溶液使用TBS-T溶液稀释至溶度为1％；
S6、显影：将洗涤完毕的硝酸纤维膜平铺于显影仪的适当位置，将显影剂均匀滴加于硝酸纤维膜上，采用凝胶成像系统曝光显影并保存；
S7、分析：显影所得图像用Image J软件进行灰度值扫描分析；各样本目的蛋白的相对浓度用待测样本灰度值/对照样本灰度值表示。
进一步的，所述TBS-T溶液包括Tris、NaCl、Tween-20及ddH2O，其中，Tris的质量为2.42g，NaCl的质量为8.0g，Tween-20的体积为1mL，所述 TBS-T溶液使用ddH2O将体积补充至1000ml。
进一步的，所述样本孔可根据待测样本的组数设置为多组样本孔。
本发明的上述技术方案的有益效果如下：
(1)本发明的点免疫印迹检测的检测装置，使硝酸纤维膜紧密贴合70孔板并在表面形成70个加样凹槽，利用这种负压吸引装置进行负压吸引，有利于加样时样品的集中，避免样品扩散后交叉污染影响实验结果，实验结果更准确。
(2)本发明点免疫印迹检测的检测装置及检测方法，进行点免疫印迹检测相比Western blot和ELISA的方法，点免疫印迹装置进行的免疫印迹检测不仅具有灵敏、特异、样品需要量少、简单、经济和快速的优点，同时还能一次进行多样本检测，适合大量人群的检测。
附图说明
图1为本发明中点免疫印迹检测的检测装置的结构示意图；
图2为本发明中孔板的俯视图；
图3为本发明中点免疫印迹检测的检测方法对胰腺癌小鼠组、胰腺炎小鼠组和正常对照组的血清FGB的相对浓度的检测结果图；
图4为本发明中点免疫印迹检测的检测方法检测胰腺癌小鼠组、胰腺炎小鼠组和正常对照组的血清FGB的相对浓度对比图。
附图标记说明：1、负压吸引装置；2、孔板；21、上层托板；22、下层托盘；23、连接管；24、通孔；25、负压空腔；3、软管；4、硝酸纤维膜；5、负压空腔。
具体实施方式
为使本发明要解决的技术问题、技术方案和优点更加清楚，下面将结合附图及具体实施例进行详细描述。
在本发明的描述中，需要说明的是，术语“中心”、“上”“下”、“左”、“右”、“竖直”、“水平”、“内”、“外”、“前”、“后”等指示的方位或位置关系为基于附图所示的方位或位置关系，仅是为了便于描述本发明和简化描述，而不是指示或暗示所指的装置或元件必须具有特定的方位、以特定的方位构造和操作，因此不能理解为对本发明的限制。此外，术语“第一”、“第二”、“第三”仅用 于描述目的，而不能理解为指示或暗示相对重要性。
在本发明的描述中，需要说明的是，除非另有明确的规定和限定，术语“安装”、“相连”、“连接”应作为广义理解，例如，可以是固定连接，也可以是可拆卸连接，或一体地连接；可以是机械连接，也可以是电连接；可以是直接相连，也可以通过中间媒介间接相连，可以是两个元件内部的连通。对于本领域的普通技术人员而言，可以具体情况理解上述术语在本发明中的具体含义。
如图1和图2所示，一种点免疫印迹检测的检测装置，包括负压吸引装置1、孔板2、连接孔板2和负压吸引装置1的软管3及紧密贴合在孔板2上端面的硝酸纤维膜4，可行的，所述软管3可采用橡胶管或塑料软管，所述孔板2包括上层托板21、密封连接在上层托板21下方的下层托盘22及设置于上层托板21、下层托盘22之间的负压空腔25，所述下层托盘22一侧设置有一供软管3套接的连接管23，所述上层托板21上具有70个通孔24，所述硝酸纤维膜4与孔板2上端面的大小相同。使用时，用软管3紧密连接70孔板2的连接管23处和负压吸引装置1的接口；将硝酸纤维膜4置于70孔板2上，形成封闭的内腔，再打开负压吸引装置1进行抽气提供负压环境，使硝酸纤维膜紧4密贴合70孔板2并在表面形成70个加样凹槽，利用这种负压吸引装置1进行负压吸引，这种方式有利于加样时样品的集中，避免样品扩散交叉污染，影响实验结果。
在进一步的实施例中，所述上层托板21的尺寸为8cm×6cm，所述通孔24直径为2mm。
在进一步的实施例中，所述负压吸引装置1选用手提式吸痰器，所述手提式吸痰器的负压值设定为0.06MPa。
在进一步的实施例中，70个所述通孔24呈10×7设置，每两个通孔24之间距离相等，可行的，所述通孔之间的距离为5.5cm-7cm。
在进一步的实施例中，所述孔板2的上方可拆卸连接有一压板。
其中，所述压板包括矩形边框及转动连接在矩形边框四周的卡扣，所述下层托盘22的四周设置有供卡扣卡接的卡块，这种卡块与卡扣的连接方式，可参照饭盒的连接方式，起到压紧硝酸纤维膜4即可。本实用新型的孔板上可拆卸连接有压板，通过压板将硝酸纤维膜4压在孔板上方，以保证硝酸纤维膜4与孔板紧密贴合。
在本发明中，点免疫印迹检测的原理是将待测样品吸附至硝酸纤维膜4(NC膜)上，固相载体以非共价键形式吸附蛋白质，且能保持多肽类型及其生物学活性不变。以硝酸纤维膜4上的蛋白质或多肽作为抗原，与目的蛋白相应的抗体发生免疫反应，再与辣根过氧化物酶(HRP)标记的二抗反应，经过放射自显影以检测目的蛋白的表达水平。
本发明还提供一种点免疫印迹检测的检测方法，包括以下步骤：
S1、准备：将手提式吸痰器通过软管3和孔板2相连接；将硝酸纤维膜4剪至具有70个通孔24的孔板3大小，置于具有70个通孔24的孔板2上，打开手提式吸痰器，将手提式吸痰器的负压值设置为0.06MPa，在硝酸纤维膜4表面形成70个加样凹槽。
S2、加样：将70个加样凹槽分为对照品孔和样本孔，每个重复3次(即对照品孔和样本孔均具有3个复孔)，对照品孔和样本孔分别加入对照品和各待测样本1μL；加样结束，将硝酸纤维膜自然晾干30min。
S3、封闭：将硝酸纤维膜4轻置于5％的脱脂奶粉溶液中，置于摇床上室温下封闭1.5h；然后，弃去封闭液，用TBS-T漂洗1-2次；其中，5％脱脂奶粉溶液使用TBS-T溶液稀释至溶度为5％。
S4、孵育一抗：用5％牛血清白蛋白溶液按1:500比例稀释一抗，将硝酸纤维膜均匀的完全浸入其中，置于摇床上，37℃恒温孵育4h；结束后将硝酸纤维膜放入TBS-T中摇床上洗涤3次，每次5min；其中，5％牛血清白蛋白溶液采用TBS-T溶液稀释至溶度为5％。
S5、孵育二抗：用1％脱脂奶粉溶液按1:5000比例稀释二抗，将硝酸纤维膜均匀的完全浸入其中，置于摇床上室温孵育1.5h；结束后将硝酸纤维膜放入TBS-T中洗涤3次，每次15min；1％脱脂奶粉溶液使用TBS-T溶液稀释至溶度为1％。在本专利中检测血清中FGB中使用的一抗为FGB鼠抗人单克隆抗体，购自Santa Cruz公司，货号：sc-271035；二抗为HRP标记的鼠抗人单克隆抗体，购自Santa Cruz公司，货号：sc-516102。
S6、显影：将洗涤完毕的硝酸纤维膜平铺于显影仪的适当位置，将显影剂均匀滴加于硝酸纤维膜上，采用凝胶成像系统曝光显影并保存；
S7、分析：显影所得图像用Image J软件进行灰度值扫描分析；各样本目 的蛋白的相对浓度用待测样本灰度值/对照样本灰度值表示。
在进一步的实施例中，所述TBS-T溶液包括Tris、NaCl、Tween-20及ddH2O，其中，Tris的质量为2.42g，NaCl的质量为8.0g，Tween-20的体积为1mL，所述TBS-T溶液使用ddH2O将体积补充至1000ml，如下表1所示；
表1 TBS-T溶液的配方表
Figure PCTCN2020080798-appb-000001
在进一步的实施例中，所述样本孔可根据待测样本的组数设置为多组样本孔。
在本实施例中，先采用质谱分析发现胰腺癌患者血清中FGB的丰度高于胰腺炎患者和正常人；然后，采用了点免疫印迹检测装置及其检测血清FGB的检测方法对前期的质谱分析结果进行验证。在本实施例中，分别对两组小鼠(每组7只)进行皮下注射胰腺癌细胞和腹腔注射雨蛙素，构建胰腺癌小鼠模型和胰腺炎小鼠模型，分别提取7只胰腺癌小鼠模型和7只胰腺炎小鼠模型的血清，制备成胰腺癌小鼠组和胰腺炎小鼠组。对照组小鼠(7只)不予处理，抽取对照组小鼠的血清，制备成正常对照组；然后采用点免疫印迹检测的检测方法对三组小鼠的血清FGB的相对浓度进行检测，检测结果如3所示；其中，a2～4列及a5～7列分别为3个不同浓度梯度的对照品，b、c、d列为胰腺癌小鼠组(b1、c1、d1为同一小鼠，b2、c2、d2为同一小鼠，以此类推，将7只胰腺癌小鼠的血清都滴入样品孔内)，e、f、g列为胰腺炎小鼠组(e1、f1、g1为同一小鼠，e2、f2、g2为同一小鼠，以此类推，将7只胰腺炎小鼠的血清都滴入样品孔内)，h、i、j列为正常对照品组(h1、i1、j1为同一小鼠，h2、i2、j2为同一小鼠，以此类推，将7只正常小鼠的血清均滴入样品孔内)。用Image J软件将图像反向后，统计各血清标本灰度值。各血清样本FGB相对浓度用待测样本灰度值/对照样本灰度值表示。各组FGB相对浓度统计见表2及图4所示。
表2 点免疫印迹装置检测三组血清FGB水平
Figure PCTCN2020080798-appb-000002
注：*与胰腺炎组、正常对照组比较，P<0.05。QL-QU：四分位间距；Max-Min：最大值-最小值。
表2及图4的结果显示：FGB在胰腺癌小鼠血清中的相对浓度明显高于胰腺炎小鼠及正常对照组(P均<0.05)，而胰腺炎组与正常对照组之间的相对浓度差异尚无统计学意义(P>0.05)，结果与前期的质谱结果相一致，证实了点免疫印迹检测的检测装置及检测方法进行血清FGB检测的可行性。
以上所述是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明所述原理的前提下，还可以作出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。

Claims (9)

  1. 一种点免疫印迹检测的检测装置，其特征在于，包括负压吸引装置、孔板、连接孔板和负压吸引装置的软管及紧密贴合在孔板上端面的硝酸纤维膜，所述孔板包括上层托板、密封连接在上层托板下方的下层托盘及设置于上层托板、下层托盘之间的负压空腔，所述下层托盘一侧设置有一供软管套接的连接管，所述上层托板上具有70个通孔，所述硝酸纤维膜与孔板上端面的大小相同。
  2. 根据权利要求1所述的一种点免疫印迹检测的检测装置，其特征在于，所述上层托板的尺寸为8cm×6cm，所述通孔直径为2mm。
  3. 根据权利要求1所述的一种点免疫印迹检测的检测装置，其特征在于，所述负压吸引装置选用手提式吸痰器，所述手提式吸痰器的负压值设定为0.06MPa。
  4. 根据权利要求1所述的一种点免疫印迹检测的检测装置，其特征在于，70个所述通孔呈10×7设置，每两个通孔之间距离相等。
  5. 根据权利要求1所述的一种用于免疫印迹检测的新型检测装置，其特征在于，所述孔板的上方可拆卸连接有一压板。
  6. 根据权利要求5所述的一种用于免疫印迹检测的新型检测装置，其特征在于，所述压板包括矩形边框及转动连接在矩形边框四周的卡扣，所述下层托盘的四周设置有供卡扣卡接的卡块。
  7. 一种使用权利要求1所述的点免疫印迹检测的检测装置进行点免疫印迹检测的检测方法，其特征在于，包括以下步骤：
    S1、准备：将手提式吸痰器通过软管和孔板相连接；将硝酸纤维膜剪至孔板上端面的上层托板大小，置于具有70个通孔的孔板上，打开手提式吸痰器，将手提式吸痰器的负压值设置为0.06MPa，在硝酸纤维膜表面形成70个加样凹槽；
    S2、加样：将70个加样凹槽分为对照品孔和样本孔，每个重复3次，对照品孔和样本孔分别加入对照品和各待测样本1μL；加样结束，将硝酸纤维膜自然晾干30min；
    S3、封闭：将硝酸纤维膜轻置于5％的脱脂奶粉溶液中，置于摇床上室温下封闭1.5h；然后，弃去封闭液，用TBS-T漂洗1-2次；其中，5％脱脂奶粉溶液使用TBS-T溶液稀释至溶度为5％；
    S4、孵育一抗：用5％牛血清白蛋白溶液按1:500比例稀释一抗，将硝酸纤维膜均匀的完全浸入其中，置于摇床上，37℃恒温孵育4h；结束后将硝酸纤维膜放入TBS-T中摇床上洗涤3次，每次5min；其中，5％牛血清白蛋白溶液采用TBS-T溶液稀释至溶度为5％；
    S5、孵育二抗：用1％脱脂奶粉溶液按1:5000比例稀释二抗，将硝酸纤维膜均匀的完全浸入其中，置于摇床上室温孵育1.5h；结束后将硝酸纤维膜放入TBS-T中洗涤3次，每次15min；1％脱脂奶粉溶液使用TBS-T溶液稀释至溶度为1％；
    S6、显影：将洗涤完毕的硝酸纤维膜平铺于显影仪的适当位置，将显影剂均匀滴加于硝酸纤维膜上，采用凝胶成像系统曝光显影并保存；
    S7、分析：显影所得图像用Image J软件进行灰度值扫描分析；各样本目的蛋白的相对浓度用待测样本灰度值/对照样本灰度值表示。
  8. 根据权利要求7所述的一种点免疫印迹检测的检测方法，其特征在于，所述TBS-T溶液包括Tris、NaCl、Tween-20及ddH2O，其中，Tris的质量为2.42g，NaCl的质量为8.0g，Tween-20的体积为1mL，所述TBS-T溶液使用ddH2O将体积补充至1000ml。
  9. 根据权利要求7所述的一种点免疫印迹检测的检测装置检测血清FGB的检测方法，其特征在于，所述样本孔可根据待测样本的组数设置为多组样本孔。
PCT/CN2020/080798 2019-05-05 2020-03-24 一种点免疫印迹检测的检测装置及检测方法 WO2020224344A1 (zh)

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