WO2023284476A1 - 一种新型冠状病毒SARS-CoV-2抗体检测试纸条 - Google Patents
一种新型冠状病毒SARS-CoV-2抗体检测试纸条 Download PDFInfo
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- WO2023284476A1 WO2023284476A1 PCT/CN2022/098998 CN2022098998W WO2023284476A1 WO 2023284476 A1 WO2023284476 A1 WO 2023284476A1 CN 2022098998 W CN2022098998 W CN 2022098998W WO 2023284476 A1 WO2023284476 A1 WO 2023284476A1
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Images
Classifications
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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
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
-
- 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
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
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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
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
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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
- G01N33/68—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 utility model relates to a novel coronavirus SARS-CoV-2 antibody detection test strip, belonging to the novel coronavirus SARS-CoV-2 detection technical field.
- the N protein is a protein with a high content and strong immunogenicity of the new coronavirus, which is wrapped around the core component of the viral RNA to form a helical protein nucleocapsid. Due to the strong immunogenicity of the N protein, it is usually used as one of the immunogen components for the detection of the new crown antibody. Obviously, the antibody against the N antigen does not have protective immunity and can only show that there has been a natural infection with the new crown or a full vaccination. Inactivated virus vaccine.
- the new crown inactivated vaccine produced in China should produce antibodies against many proteins of the new crown virus, including N protein, S protein, M protein, and E protein, but only a small part of the antibodies against S-RBD have protective immunity.
- the subunit vaccines developed by other countries in the world and my country including mRNA vaccines, adenovirus vaccines and recombinant protein vaccines, all use the S-RBD immunogen.
- S-RBD antibodies For this part of the vaccinators, only S-RBD antibodies can be detected, while Other antigens such as N protein were negative.
- antibody levels can fluctuate over time in both those who have recovered from infection and those who have been vaccinated.
- a test strip for analyzing the immune status of novel coronavirus detects the antibodies produced by the human body against various protein antigens of the new coronavirus, including but not limited to the S protein, RBD protein, N protein, M protein and E protein of the new coronavirus. , and then analyze the immune status of the human body, such as whether to produce antibodies, and distinguish between virus infection immunity and subunit vaccine immunity according to the type of antibody produced, and analyze the type of vaccine inoculated.
- the base plate is a polyvinyl chloride plate.
- the detection line is immobilized with a protein
- the protein is an antigen of the novel coronavirus.
- the antigen can be S protein, RBD protein, N protein, M protein or E protein.
- the gold label pad has a protein marked by a signal molecule, which is an antigen of the novel coronavirus or a secondary antibody of an anti-human antibody.
- the antigen can be S protein, RBD protein, N protein and E protein.
- the label includes but is not limited to colloidal gold particles, and any molecule that plays a signaling role can be used as a label.
- the number of detection lines is 2, 3, 4, 5, or more than 5.
- Each detection line is immobilized with different novel coronavirus antigens.
- the order of the detection lines immobilizing different novel coronavirus antigens can be arranged arbitrarily.
- detection lines which are respectively immobilized with the S protein, RBD protein, N protein and E protein of the novel coronavirus.
- the quality control line is immobilized with antibodies against the secondary antibody or novel coronavirus antibodies or other determined immune response combinations including human or non-human.
- the detection test strips of the utility model aim at the detection lines of different antibodies, visually display the types of antibodies against different antigenic proteins contained in the human body samples, and can conveniently analyze different immune conditions.
- the integrated test strip structure makes the detection of the required components simple and convenient.
- the one-time immunochromatographic reaction reduces the risk of non-specific reaction, and the color development is more stable. Mature lateral flow technology requires shorter detection time.
- Fig. 1 is the structure of an embodiment of novel coronavirus SARS-CoV-2 antibody detection test strip of the present utility model
- Fig. 2 is the structure of another embodiment of the novel coronavirus SARS-CoV-2 antibody detection test strip of the present invention.
- Embodiment 1 Determination of spray film concentration on detection line
- Coated RBD protein test strips were tested using serum from 3 healthy volunteers who had been vaccinated with Anhui Zhifeilong Koma recombinant subunit vaccine, and 3 healthy volunteers who had not been vaccinated.
- the test strips coated with N protein used the sera of 3 healthy volunteers who had been vaccinated with the Beijing Kexing inactivated vaccine, and the sera of 3 healthy volunteers who had not been vaccinated.
- the test results are as follows:
- the antibody results in the coated RBD protein test sample are as follows:
- the antibody results in the coated N protein test sample are as follows:
- the optimal concentration of coated RBD protein should be 1.00mg/mL-1.25mg/mL, and the optimal concentration of coated N protein should be 0.75mg/mL-1.00mg/mL.
- Embodiment 2 Preparation of 2 kinds of antibody detection test strips
- T-line (detection line) colloidal gold particle modification Take 20mL colloidal gold solution, add 100 ⁇ L 0.1mol/L potassium carbonate solution, then add 200 ⁇ L N protein antigen and RBD protein antigen with a concentration of 1mg/mL, mix evenly, and place at room temperature 30 minutes. Then add 200 ⁇ L of 20% casein solution and mix well, let stand at room temperature for 30 minutes, and then centrifuge at 12000rpm for 20 minutes. After centrifugation, discard the supernatant, then add 10 mL of 50 mmol/L PBS buffer with pH 7.4 to resuspend, and centrifuge at 12,000 rpm for 20 minutes.
- colloidal gold buffer Discard the supernatant after centrifugation, resuspend the pellet in 2.0 mL of colloidal gold buffer, and store at 4°C in the dark.
- the components of the colloidal gold buffer are 10 mmol/L pH9.0 Tris-HCl, 0.3% sodium citrate, 3% sucrose, 6% trehalose, and 0.15% casein.
- 2C line (quality control line) colloidal gold particle modification Take 20mL colloidal gold solution, add 100 ⁇ L 0.2mol/L potassium carbonate solution, then add 200 ⁇ L rabbit anti-chicken IgY antibody with a concentration of 1mg/mL, mix well, and place at room temperature for 30 minutes . Then add 200 ⁇ L of 20% casein solution and mix well, let stand at room temperature for 30 minutes, and then centrifuge at 12000rpm for 20 minutes. After centrifugation, discard the supernatant, then add 10 mL of 50 mmol/L PBS buffer with pH 7.4 to resuspend, and centrifuge at 12,000 rpm for 20 minutes.
- colloidal gold buffer Discard the supernatant after centrifugation, resuspend the pellet in 2.0 mL of colloidal gold buffer, and store at 4°C in the dark.
- the components of the colloidal gold buffer are 10 mmol/L pH9.0 Tris-HCl, 0.3% sodium citrate, 3% sucrose, 6% trehalose, and 0.15% casein.
- 1Preparation of spray solution prepare 0.1M PBS.
- Use 0.1M PBS to prepare the T1 line prepare the mixed recombinant N protein to a concentration of 1.00mg/mL, and the final concentration of PBS is 0.01M
- use 0.1M PBS to prepare the T2 line and prepare the recombinant RBD protein to a concentration of 1.25mg/mL, The final concentration of PBS is 0.01M
- use 0.1M PBS to prepare C-line the C-line system is coated with chicken IgY antibody, the corresponding colloidal gold particles are labeled with rabbit anti-chicken IgY antibody, chicken IgY is prepared to a concentration of 1.50mg/mL, PBS The final concentration was 0.01M.
- the structures of the two kinds of antibody detection test strips are shown in FIG. 1 .
- the test strips include: a sample pad 10 , a gold standard pad 20 , a detection membrane 50 , an absorbent pad 60 and a bottom plate 70 .
- the sample pad 10 , the gold standard pad 20 , the detection membrane 50 and the water-absorbing pad 60 are all placed above the bottom plate 70 .
- the gold standard pad 20 and the water absorption pad 60 are arranged on both sides of the detection film 50, and a part of the gold standard pad 20 and a part of the water absorption pad 60 overlap the ends of the detection film 50 respectively.
- a part of the sample pad 10 overlaps the end of the gold standard pad 20 .
- the detection film 50 is provided with a detection line 30 and a quality control line 40 , and the detection line 30 and the quality control line 40 are arranged at intervals along the extending direction of the detection film 50 .
- the detection line 30 includes: a first detection sub-wire 31 and a second detection sub-wire 32 , and along the extending direction of the detection film 50 , the first detection sub-wire 31 and the second detection sub-wire 32 are arranged at intervals.
- Embodiment 3 the test of different kinds of antibodies
- Test objects RBD antibody, N protein antibody, SARS antibody, influenza A antibody, hepatitis B antibody, dengue fever antibody, etc. of the new coronavirus, and 3 test strips were tested in parallel for each antibody test.
- test strips responded well to the RBD antibody and N protein antibody of the new coronavirus, but had no cross-reaction to other types of antibodies.
- Embodiment 4 Sample test of 2 kinds of antibody detection test strips
- test strip test steps :
- test strip described in the utility model and the new coronavirus IgM/IgG antibody test strip of a certain brand all use the following steps to test.
- the Sanxiang Novel Coronavirus Nucleic Acid Detection Kit detects the samples according to the kit instructions.
- test strips are better at distinguishing between different vaccinated populations.
- Embodiment 5 Preparation of 4 kinds of antibody detection test strips
- T line (detection line) colloidal gold particle modification take 40mL colloidal gold solution, add 200 ⁇ L 0.1mol/L potassium carbonate solution, and then add 200 ⁇ L of N protein antigen, RBD protein antigen, M protein antigen and E protein at a concentration of 1mg/mL The antigens were mixed evenly and left at room temperature for 30 minutes. Then add 400 ⁇ L 20% casein solution and mix well, let stand at room temperature for 30 minutes, and then centrifuge at 12000rpm for 20 minutes. After centrifugation, discard the supernatant, add 20 mL of 50 mmol/L PBS buffer with pH 7.4 to resuspend, and centrifuge at 12,000 rpm for 20 minutes.
- colloidal gold buffer Discard the supernatant after centrifugation, resuspend the pellet in 2.0 mL of colloidal gold buffer, and store at 4°C in the dark.
- the components of the colloidal gold buffer are 10 mmol/L pH9.0 Tris-HCl, 0.3% sodium citrate, 3% sucrose, 6% trehalose, and 0.15% casein.
- 2C line (quality control line) colloidal gold particle modification Take 20mL colloidal gold solution, add 100 ⁇ L 0.2mol/L potassium carbonate solution, then add 200 ⁇ L rabbit anti-chicken IgY antibody with a concentration of 1mg/mL, mix well, and place at room temperature for 30 minutes . Then add 200 ⁇ L of 20% casein solution and mix well, let stand at room temperature for 30 minutes, and then centrifuge at 12000rpm for 20 minutes. After centrifugation, discard the supernatant, then add 10 mL of 50 mmol/L PBS buffer with pH 7.4 to resuspend, and centrifuge at 12,000 rpm for 20 minutes.
- colloidal gold buffer Discard the supernatant after centrifugation, resuspend the pellet in 2.0 mL of colloidal gold buffer, and store at 4°C in the dark.
- the components of the colloidal gold buffer are 10 mmol/L pH9.0 Tris-HCl, 0.3% sodium citrate, 3% sucrose, 6% trehalose, and 0.15% casein.
- 1Preparation of spray solution prepare 0.1M PBS.
- Use 0.1M PBS to prepare the T1 line prepare the mixed recombinant N protein to a concentration of 1.00mg/mL, and the final concentration of PBS is 0.01M
- use 0.1M PBS to prepare the T2 line and prepare the recombinant M protein to a concentration of 1.50mg/mL, The final concentration of PBS is 0.01M
- use 0.1M PBS to prepare the T3 line prepare the mixed recombinant RBD protein to a concentration of 1.25mg/mL, and the final concentration of PBS is 0.01M
- use 0.1M PBS to prepare the T4 line and prepare the recombinant E protein
- the final concentration of PBS is 0.01M
- use 0.1M PBS to prepare C-line the C-line system is coated with chicken IgY antibody, and the corresponding colloidal gold particles are labeled with rabbit anti-chicken IgY antibody, and chicken IgY is prepared into 1.50
- the test strips include: a sample pad 10 , a gold standard pad 20 , a detection membrane 50 , an absorbent pad 60 and a bottom plate 70 .
- the sample pad 10 , the gold standard pad 20 , the detection membrane 50 and the water-absorbing pad 60 are all placed above the bottom plate 70 .
- the gold standard pad 20 and the water absorption pad 60 are arranged on both sides of the detection film 50, and a part of the gold standard pad 20 and a part of the water absorption pad 60 overlap the ends of the detection film 50 respectively.
- a part of the sample pad 10 overlaps the end of the gold standard pad 20 .
- the detection film 50 is provided with a detection line 30 and a quality control line 40 , and the detection line 30 and the quality control line 40 are arranged at intervals along the extending direction of the detection film 50 .
- the detection line 30 includes: a first detection sub-wire 31, a second detection sub-wire 32, a third detection sub-wire 33, and a fourth detection sub-wire 34.
- the first detection sub-wire 31 It is spaced apart from the second detection sub-line 32 , the third detection sub-line 33 , and the fourth detection sub-line 34 .
- Embodiment 6 Sample test of 4 kinds of antibody detection test strips
- the sera of 10 healthy volunteers who had been vaccinated with the Anhui Zhifeilong Koma recombinant subunit vaccine, the sera of 10 healthy volunteers who had been vaccinated with the Beijing Kexing inactivated vaccine, and the sera of 10 healthy volunteers who had not been vaccinated were used for testing.
- test strip test steps :
- the 4 kinds of antibody test strips and a certain brand of new coronavirus IgM/IgG antibody test strips are tested using the following steps.
- the Sanxiang Novel Coronavirus Nucleic Acid Detection Kit detects the samples according to the kit instructions.
- the four antibody test strips can better distinguish different vaccinated populations.
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Abstract
一种新型冠状病毒SARS-CoV-2抗体检测试纸条,包括:底板(70)、检测膜(50)、样品垫(10)、金标垫(20)及吸水垫(60),检测膜(50)上设有相互平行的检测线(30)和质控线(40);检测线(30)的数量至少为2,并且检测线(30)与金标垫(20)固定有联合检测新型冠状病毒SARS-CoV-2的抗体的蛋白。抗体检测试纸条直观显示人体样本含有的针对不同抗原蛋白的抗体种类,可方便分析不同的免疫情况。
Description
相关申请的交叉引用
本申请要求申请日为2021年07月12日,申请号为202121573774.5,题名为“一种新型冠状病毒SARS-CoV-2抗体检测试纸条”的优先权,以上中国专利申请的全部内容在此以引入方式并入本申请。
本实用新型涉及一种新型冠状病毒SARS-CoV-2抗体检测试纸条,属于新型冠状病毒SARS-CoV-2检测技术领域。
新型冠状病毒(SARS-CoV-2)属于冠状病毒家族,含有棘突状蛋白(Spike protein,简称S蛋白),膜蛋白(membrane protein,简称M蛋白),包膜蛋白(Envelope protein,简称E蛋白),核衣壳蛋白(Nucleocapsid protein,简称N蛋白)。新冠病毒的致病过程是病毒S蛋白中的受体结合结构域(Receptor Binding Domain,简称RBD)和人体呼吸道上皮细胞表面的ACE2受体进行结合,启动和介导了病毒对人体细胞的入侵和致病。因此S-RBD蛋白是机体中和抗体阻断新冠病毒入侵的主要靶点。当机体受到新冠病毒感染后,会产生许多种抗体,包括针对N、S、E、M蛋白中多种抗原决定族的抗体。其中大部分抗体不具备中和病毒入侵的作用,甚至具有抗体加强效应(Antibody Dependant Enhancement,简称ADE)。大量的研究表明,针对S-RBD蛋白的部分抗体,可以阻断S-RBD和人体ACE2受体的结合,从而阻断新冠病毒的入侵和致病。因此S-RBD蛋白已成为大多数新冠亚单位疫苗使用的免疫原,检测抗S-RBD抗体可以反映机体对新冠病毒的免疫状态。而N蛋白是新冠病毒含量较多、免疫原性较强的一种蛋白,包裹在病毒RNA核心组分外面形成螺旋状的蛋白核衣壳。由于N蛋白的免疫原性较强,因此通常被用作新冠抗体检测的免疫原组分之一,显然针对N抗原的抗体不具备保护性免疫,只能显示曾经有新冠自然感染或接种过全病毒灭活疫苗。
目前上市的绝大多数新冠抗体试剂采用新冠混合组分抗原(通常为混合N和S蛋白),检测IgM和IgG总抗体,也就是一条检测线固定有多种新冠抗原。其目的是仅区分过去感染(IgG阳性)还是新近感染(IgM阳性),而不能区分抗体具体针对的新冠病毒抗原的类型。如果IgM阳性,表明新 近感染新冠肺炎,具有辅助诊断意义。该类抗体检测试剂大多为免疫层析法,也有酶联免疫或化学发光法。
目前各国大规模接种新冠疫苗,也使人们产生了不同种类的抗体。新型冠状病毒疫苗是指用新型冠状病毒相关组分制作的用于预防接种的生物制品,根据其特性可分为核酸mRNA疫苗,灭活疫苗,减毒流感病毒载体疫苗,腺病毒载体疫苗,重组亚单位多肽疫苗。不同的疫苗虽然最终都是使人体对病原体产生保护性的抗体,但是由于其不同的特性,使人体产生的抗体种类和数量也并不相同。譬如中国产的新冠灭活疫苗,应该对新冠病毒的许多蛋白包括N蛋白、S蛋白、M蛋白、E蛋白产生抗体,但其中只有一小部分针对S-RBD的抗体具有保护性免疫。而世界上其他国家以及我国研发的亚单位疫苗,包括mRNA疫苗,腺病毒疫苗和重组蛋白疫苗均采用了S-RBD的免疫原,对这部分接种者,只能测到S-RBD抗体,而N蛋白等其他抗原阴性。此外,无论是感染康复者,还是疫苗接种者,其抗体的水平均可以随着时间而又所波动。
因此,通过区别不同的新型冠状病毒的抗体,来分析个体接种疫苗情况,对于区分不同个体的免疫状态有重要作用,尤其是在世界大流行的病原体传播中。而区分不同个体的免疫状态,对于新型冠状病毒传播中的防疫策略的制定,防疫政策的执行,防疫情况的分析等等都有非常重要的意义。而目前为止,并未存在相关产品。
实用新型内容
针对现有技术中存在的问题,提供了一种分析针对新型冠状病毒免疫状况的试纸条。本实用新型中的检测试纸条通过检测人体针对新型冠状病毒的多种蛋白抗原所产生的抗体,包括但不局限于新型冠状病毒的S蛋白、RBD蛋白、N蛋白、M蛋白和E蛋白等,进而分析人体的免疫情况,例如是否产生抗体,并且依据产生抗体的种类区分病毒感染免疫与亚单元疫苗免疫,分析其接种的疫苗种类。
本实用新型提供了一种新型冠状病毒SARS-CoV-2抗体检测试纸条,包括:底板、检测膜、样品垫、金标垫及吸水垫,其中,所述检测膜上设有相互平行的检测线和质控线;其中,所述检测线的数量至少为2,并且所述检测线与金标垫固定有联合检测新型冠状病毒SARS-CoV-2的抗体的蛋白。
在一个具体的实施方案中,所述底板为聚氯乙烯板。
在一个具体的实施方案中,所述检测膜为硝酸纤维素膜、或纤维素膜、或羧化纤维素膜、或聚偏二氟乙烯纤维素膜。
进一步地,新型冠状病毒SARS-CoV-2的抗体包括:针对新型冠状病毒的S蛋白、RBD蛋白、N蛋白和E蛋白产生的抗体。
进一步地,所述检测线固定有蛋白,所述蛋白为新型冠状病毒的抗原。在一些具体的实施方案中,所述抗原可以为S蛋白、RBD蛋白、N蛋白、M蛋白或E蛋白。
进一步地,金标垫上具有信号分子标记的蛋白,所述蛋白为新型冠状病毒的抗原或者抗人抗体的二抗。在一些具体的实施方案中,所述抗原可以为S蛋白、RBD蛋白、N蛋白和E蛋白。所述标记包括但不限于胶体金颗粒,任何起到信号指示作用的分子均可以用作标记。
本实用新型中任意所述蛋白可以是该蛋白天然或重组的蛋白片段,可以是天然或重组的全长蛋白,可以是天然或重组的蛋白片段与全长蛋白的组合。
在一些具体的实施方案中,所述检测线的数量为2条、3条、4条、5条,或者大于5条。每条检测线固定有不同新型冠状病毒抗原。固定不同新型冠状病毒抗原的检测线的顺序可以任意进行排列。
在一些具体的实施方案中,所述检测线的数量为2~5条。每条检测线可以可以分别固定有S蛋白、RBD蛋白、N蛋白、M蛋白,以及E蛋白抗原中的任意一种。
在一个具体的实施方案中,所述检测线为2条,分别固定有新型冠状病毒的RBD蛋白和N蛋白。
在一个具体的实施方案中,所述检测线为2条,分别固定有新型冠状病毒的RBD蛋白和S蛋白。
在一个具体的实施方案中,所述检测线为2条,分别固定有新型冠状病毒的RBD蛋白和E蛋白。
在一个具体的实施方案中,所述检测线为3条,分别固定有新型冠状病毒的RBD蛋白、S蛋白和N蛋白。
在一个具体的实施方案中,所述检测线为4条,分别固定有新型冠状病毒的S蛋白、RBD蛋白、N蛋白和E蛋白。
在一个具体的实施方案中,所述检测线为5条,分别固定有新型冠状病毒的S蛋白、RBD蛋白、N蛋白、M蛋白和E蛋白。
在一些具体的实施方案中,质控线固定有针对二抗的抗体或新型冠状病毒抗体或者包括人源或非人源的其他确定免疫反应组合。
在一个具体的实施方案中,本实用新型所述检测试纸条的作用原理为:样本中的新型冠状病毒抗体加入到样品垫后,由于毛细作用进入到金标垫,不同蛋白的抗体会与金标垫上胶体金标记的对应抗原或二抗反应结合,同时由于毛细作用继续进入NC膜(硝酸纤维素膜),在NC膜上开始层析。当结合后的复合分子经过对应的检测线时,会与该检测线上的抗原发生免疫反应实现捕获,而非对应的抗体则不会发生免疫反应,复合分子则继续在NC膜上层析。当检测线上捕获的对应复合分子数量累积到一定数量时,则会显示胶体金颜色。试纸条上的C线,则通过捕获未反应的胶体金颗粒, 或者捕获其专门标记的胶体金颗粒显色。
本实用新型所述检测试纸条针对不同抗体的检测线,直观的显示了人体样本含有的针对不同抗原蛋白抗体种类,可方便分析不同的免疫情况。一体化的试纸条结构,使得检测所需组分简单、便捷。一次性的免疫层析反应,降低了非特异性反应的风险,显色更稳定。成熟的侧向层析技术,需要的检测时间更短。
图1为本实用新型的新型冠状病毒SARS-CoV-2抗体检测试纸条的一个实施方案的结构;
图2为本实用新型的新型冠状病毒SARS-CoV-2抗体检测试纸条的另一个实施方案的结构。
实施例1:检测线喷膜浓度确定
1)、喷膜液配制:
①配制0.1M的PBS,备用。
②使用0.1M的PBS,将重组RBD蛋白配制成0.50mg/mL、0.75mg/mL、1.00mg/mL、1.25mg/mL、1.50mg/mL、2.00mg/mL浓度,PBS终浓度为0.01M。
③使用0.1M的PBS,将混合重组N蛋白配制成0.50mg/mL、0.75mg/mL、1.00mg/mL、1.25mg/mL、1.50mg/mL、2.00mg/mL浓度,PBS终浓度为0.01M。
2)、重组蛋白喷膜:
设定喷膜量为0.1μL/mm,移动速度为60mm/s,开始喷膜,每个浓度分别制备一种试纸条以供优化实验。
3)、各浓度测试:
包被RBD蛋白试纸条测试使用3例接种过安徽智飞龙科马重组亚单位疫苗的健康志愿者血清,以及3例未接种疫苗健康志愿者血清。包被N蛋白试纸条测试使用3例接种过北京科兴灭活疫苗的健康志愿者血清,以及3例未接种疫苗健康志愿者血清。观察反应条带的显色程度,显色深度以“+”表示;“1+”代表有显色但显色较弱,“2+”代表显色明显但显色不深,“3+”代表有显色很深,“-”表示无显色。测试结果如下:
包被RBD蛋白测试样本中抗体结果如下:
表1、RBD蛋白试纸条测试结果
包被N蛋白测试样本中抗体结果如下:
表2、N蛋白试纸条测试结果
4)、结果分析:
根据测试结果,包被RBD蛋白的最优浓度应为1.00mg/mL-1.25mg/mL,包被N蛋白的最优浓度应为0.75mg/mL-1.00mg/mL。
实施例2:2种抗体检测试纸条的制备
1)、胶体金颗粒修饰:
①T线(检测线)胶体金颗粒修饰:取20mL胶体金溶液,加入100μL0.1mol/L碳酸钾溶液,再分别加入200μL浓度1mg/mL的N蛋白抗原和RBD蛋白抗原混和均匀,在室温下放置30分钟。再加入200μL 20%酪蛋白溶液混合均匀,在室温放置30分钟,然后12000rpm离心20分钟。离心完成后弃去上清,再加入pH7.4的50mmol/L PBS缓冲液10mL重悬,12000rpm离心20分钟。离心完成后弃去上清,将沉淀重悬于2.0mL胶体金缓冲液中,4℃避光保存。胶体金缓冲液成分为10mmol/L的pH9.0Tris-HCl,0.3%柠檬酸钠,3%蔗糖,6%海藻糖,0.15%酪蛋白。
②C线(质控线)胶体金颗粒修饰:取20mL胶体金溶液,加入100μL0.2mol/L碳酸钾溶液,再加入200μL浓度1mg/mL的兔抗鸡IgY抗体混和均匀,在室温下放置30分钟。再加入200μL 20%酪蛋白溶液混合均匀,在室温放置30分钟,然后12000rpm离心20分钟。离心完成后弃去上清,再加入pH7.4的50mmol/L PBS缓冲液10mL重悬,12000rpm离心20分钟。离心完成后弃去上清,将沉淀重悬于2.0mL胶体金缓冲液中,4℃避光保存。胶体金缓冲液成分为10mmol/L的pH9.0Tris-HCl、0.3%柠檬酸钠、3%蔗糖、6%海藻糖、0.15%酪蛋白。
③将修饰好的T线胶体金颗粒与C线胶体金颗粒按照8:2比例混合备用。
2)、胶体金溶液喷涂:
①设定喷涂量为3μL/mm,移动速度为60mm/s,将胶体金溶液喷涂到金标垫上;
②喷涂完成后,将金标垫转移到37℃恒温箱烘干过夜;
③将干燥好的金标垫收集到干燥箱中保存备用。
3)、喷膜:
①配制喷膜液:配制0.1M的PBS。使用0.1M的PBS配制T1线,将混合重组N蛋白配制成1.00mg/mL浓度,PBS终浓度为0.01M;使用0.1M的PBS配制T2线,将重组RBD蛋白配制成1.25mg/mL浓度,PBS终浓度为0.01M;使用0.1M的PBS配制C线,C线系统采用鸡IgY抗体包被,对应胶体金颗粒使用兔抗鸡IgY抗体标记,将鸡IgY配制成1.50mg/mL浓度,PBS终浓度为0.01M。
②喷涂:设定喷膜量为0.1μL/mm,移动速度为60mm/s,进行喷膜。
③将喷涂完成的NC膜大板转移到37℃恒温箱烘干过夜,完成后收集到干燥箱中保存备用。
4)、样品垫处理:
配制0.01M的PBST溶液,将样品垫浸入溶液中,浸透之后将样品垫放入纱网架,置于37℃烘箱烘干过夜,完成后收集到干燥箱中保存备用。
5)、大板的组装:
将喷涂好NC膜的大板、样品垫、金标垫、吸水垫按照图1的顺序依次粘贴好。
6)、切条
使用切条机将大板切成宽度为3.5-4.5mm宽的试纸条。
2种抗体检测试纸条的结构如图1所示,试纸条包括:样品垫10、金标垫20、检测膜50、吸水垫60和底板70。样品垫10、金标垫20、检测膜50和吸水垫60均放置在底板70的上方。其中,沿检测膜50的延伸方向,金标垫20和吸水垫60设置于检测膜50的两侧,且金标垫20的一部分和吸水垫60的一部分分别搭接于检测膜50的端部。样品垫10的一部分搭接在金标垫20的端部。
检测膜50设置有检测线30和质控线40,沿检测膜50的延伸方向,检测线30和质控线40间隔设置。其中,检测线30包括:第一检测子线31和第二检测子线32,沿检测膜50的延伸方向,第一检测子线31和第二检测子线32间隔设置。
实施例3:不同种类抗体的测试
1)、测试对象:新型冠状病毒的RBD抗体、N蛋白抗体、SARS抗体、甲流抗体、乙肝抗体、登革热抗体等,每种抗体测试平行测试3支试纸条。
2)、测试步骤:
①测试样本处理,使用pH7.4 0.01M PBS将各待测抗体稀释到0.1mg/mL(或等同于0.1mg/mL)浓度。
②吸样,吸取各样本各75μL,将各试纸条平放,分别加入检测试纸条的样品垫区。
③反应读数,反应10分钟后,肉眼读取试纸条上的显色结果。
3)、结果判读规则:
观察反应条带的显色程度,显色深度以“+”表示;“1+”代表有显色但显色较弱,“2+”代表显色明显但显色不深,“3+”代表有显色很深,“-”表示无显色。3支平行试纸条,新型冠状病毒的RBD抗体、N蛋白抗体以每条检测线中最弱的结果作为判定结果,SARS抗体、甲流抗体、乙肝抗体、登革热抗体以每条检测线中最强的结果作为判定结果。
4)、各种抗体测试结果:
表3、抗体测试结果
5)、结果分析:
从测试结果来看,试纸条对新型冠状病毒的RBD抗体、N蛋白抗体响应良好,而对其他种类的抗体并无交叉反应。
实施例4:2种抗体检测试纸条的样本测试
1)、测试方法:
分别使用本实用新型所述试纸条、某品牌新型冠状病毒IgM/IgG抗体测试试纸条、圣湘新型冠状病毒核酸检测试剂盒进行检测。
2)、测试样本:
分别使用10例接种过安徽智飞龙科马重组亚单位疫苗的健康志愿者血清,10例接种过北京科兴灭活疫苗的健康志愿者血清,以及10例未接种疫苗健康志愿者血清进行测试。
3)、试纸条测试步骤:
本实用新型所述试纸条、某品牌新型冠状病毒IgM/IgG抗体测试试纸条均使用如下步骤测试。
①吸样,吸取各样本各75μL,将各试纸条平放,分别加入检测试纸条 的样品垫区。
②反应读数,反应15分钟后,肉眼读取试纸条上的显色结果。
圣湘新型冠状病毒核酸检测试剂盒按照试剂盒说明书对样本进行检测。
4)、结果判读规则:
观察反应条带的显色程度,显色深度以“+”表示;“1+”代表有显色但显色较弱,“2+”代表显色明显但显色不深,“3+”代表有显色很深,“-”表示无显色。
5)、样本测试结果:
表4、安徽智飞龙科马重组亚单位疫苗志愿者测试结果
表5、北京科兴灭活疫苗志愿者测试结果
表6、未接种疫苗志愿者测试结果
6)、结果分析:
从测试结果来看,试纸条对不同的疫苗接种人群的区分度较好。
实施例5:4种抗体检测试纸条的制备
1)、胶体金颗粒修饰:
①T线(检测线)胶体金颗粒修饰:取40mL胶体金溶液,加入200μL0.1mol/L碳酸钾溶液,再分别加入200μL浓度1mg/mL的N蛋白抗原、RBD蛋白抗原、M蛋白抗原和E蛋白抗原混和均匀,在室温下放置30分钟。再加入400μL 20%酪蛋白溶液混合均匀,在室温放置30分钟,然后12000rpm离心20分钟。离心完成后弃去上清,再加入pH7.4的50mmol/L PBS缓冲液20mL重悬,12000rpm离心20分钟。离心完成后弃去上清,将沉淀重悬于2.0mL胶体金缓冲液中,4℃避光保存。胶体金缓冲液成分为10mmol/L的pH9.0Tris-HCl,0.3%柠檬酸钠,3%蔗糖,6%海藻糖,0.15%酪蛋白。
②C线(质控线)胶体金颗粒修饰:取20mL胶体金溶液,加入100μL0.2mol/L碳酸钾溶液,再加入200μL浓度1mg/mL的兔抗鸡IgY抗体混和均匀,在室温下放置30分钟。再加入200μL 20%酪蛋白溶液混合均匀,在室温放置30分钟,然后12000rpm离心20分钟。离心完成后弃去上清,再加入pH7.4的50mmol/L PBS缓冲液10mL重悬,12000rpm离心20分钟。离心完成后弃去上清,将沉淀重悬于2.0mL胶体金缓冲液中,4℃避光保存。胶体金缓冲液成分为10mmol/L的pH9.0Tris-HCl、0.3%柠檬酸钠、3%蔗糖、6%海藻糖、0.15%酪蛋白。
③将修饰好的T线胶体金颗粒与C线胶体金颗粒按照8:2比例混合备用。
2)、胶体金溶液喷涂:
①设定喷涂量为3μL/mm,移动速度为60mm/s,将胶体金溶液喷涂到金标垫上;
②喷涂完成后,将金标垫转移到37℃恒温箱烘干过夜;
③将干燥好的金标垫收集到干燥箱中保存备用。
3)、喷膜:
①配制喷膜液:配制0.1M的PBS。使用0.1M的PBS配制T1线,将混合重组N蛋白配制成1.00mg/mL浓度,PBS终浓度为0.01M;使用0.1M的PBS配制T2线,将重组M蛋白配制成1.50mg/mL浓度,PBS终浓度为0.01M;使用0.1M的PBS配制T3线,将混合重组RBD蛋白配制成1.25mg/mL浓度,PBS终浓度为0.01M;使用0.1M的PBS配制T4线,将重组E蛋白配制成1.50mg/mL浓度,PBS终浓度为0.01M;使用0.1M的PBS配制C线,C线系统采用鸡IgY抗体包被,对应胶体金颗粒使用兔抗鸡IgY抗体标记,将鸡IgY配制成1.50mg/mL浓度,PBS终浓度为0.01M。
②喷涂:设定喷膜量为0.1μL/mm,移动速度为60mm/s,进行喷膜。
③将喷涂完成的NC膜大板转移到37℃恒温箱烘干过夜,完成后收集到干燥箱中保存备用。
4)、样品垫处理:
配制0.01M的PBST溶液,将样品垫浸入溶液中,浸透之后将样品垫放入纱网架,置于37℃烘箱烘干过夜,完成后收集到干燥箱中保存备用。
5)、大板的组装:
将喷涂好NC膜的大板、样品垫、金标垫、吸水垫按照图1的顺序依次粘贴好。
6)、切条
使用切条机将大板切成宽度为3.5-4.5mm宽的试纸条。
4种抗体检测试纸条的结构如图2所示,试纸条包括:样品垫10、金标垫20、检测膜50、吸水垫60和底板70。样品垫10、金标垫20、检测膜50和吸水垫60均放置在底板70的上方。其中,沿检测膜50的延伸方向,金标垫20和吸水垫60设置于检测膜50的两侧,且金标垫20的一部分和吸水垫60的一部分分别搭接于检测膜50的端部。样品垫10的一部分搭接在金标垫20的端部。
检测膜50设置有检测线30和质控线40,沿检测膜50的延伸方向,检测线30和质控线40间隔设置。其中,检测线30包括:第一检测子线31和第二检测子线32、第三检测子线33,和第四检测子线34,沿检测膜50的延伸方向,第一检测子线31和第二检测子线32、第三检测子线33,和第四检测子线34间隔设置。
实施例6:4种抗体检测试纸条的样本测试
1)、测试方法:
分别使用4种抗体检测试纸条、某品牌新型冠状病毒IgM/IgG抗体测试试纸条、圣湘新型冠状病毒核酸检测试剂盒进行检测。
2)、测试样本:
分别使用10例接种过安徽智飞龙科马重组亚单位疫苗的健康志愿者血清,10例接种过北京科兴灭活疫苗的健康志愿者血清,以及10例未接种疫苗健康志愿者血清进行测试。
3)、试纸条测试步骤:
4种抗体检测试纸条、某品牌新型冠状病毒IgM/IgG抗体测试试纸条均使用如下步骤测试。
①吸样,吸取各样本各75μL,将各试纸条平放,分别加入检测试纸条的样品垫区。
②反应读数,反应15分钟后,肉眼读取试纸条上的显色结果。
圣湘新型冠状病毒核酸检测试剂盒按照试剂盒说明书对样本进行检测。
4)、结果判读规则:
观察反应条带的显色程度,显色深度以“+”表示;“1+”代表有显色但显色较弱,“2+”代表显色明显但显色不深,“3+”代表有显色很深,“-”表示无显色。
5)、样本测试结果:
表7、安徽智飞龙科马重组亚单位疫苗志愿者测试结果
表8、北京科兴灭活疫苗志愿者测试结果
表9、未接种疫苗志愿者测试结果
6)、结果分析:
从测试结果来看,4种抗体检测试纸条对不同的疫苗接种人群的区分度较好。
Claims (10)
- 一种新型冠状病毒SARS-CoV-2抗体检测试纸条,其中,包括:底板、检测膜、样品垫、金标垫及吸水垫,其中,所述检测膜上设有相互平行的检测线和质控线;其中,所述检测线的数量至少为2,并且所述检测线与金标垫固定有联合检测新型冠状病毒SARS-CoV-2的抗体的蛋白。
- 根据权利要求1所述的新型冠状病毒SARS-CoV-2抗体检测试纸条,其中,所述底板为聚氯乙烯板。
- 根据权利要求1所述的新型冠状病毒SARS-CoV-2抗体检测试纸条,其中,所述检测膜为硝酸纤维素膜、或纤维素膜、或羧化纤维素膜,或聚偏二氟乙烯纤维素膜。
- 根据权利要求1所述的新型冠状病毒SARS-CoV-2抗体检测试纸条,其中,所述蛋白为S蛋白、RBD蛋白、N蛋白、M蛋白,或E蛋白。
- 根据权利要求1所述的新型冠状病毒SARS-CoV-2抗体检测试纸条,其中,所述金标垫上具有信号分子标记的蛋白,所述蛋白为新型冠状病毒的抗原或者抗人抗体的二抗。
- 根据权利要求5所述的新型冠状病毒SARS-CoV-2抗体检测试纸条,其中,所述抗原为S蛋白、RBD蛋白、N蛋白、M蛋白,或E蛋白。
- 根据权利要求5或6所述的新型冠状病毒SARS-CoV-2抗体检测试纸条,其中,所述信号分子为胶体金颗粒。
- 根据权利要求1所述的新型冠状病毒SARS-CoV-2抗体检测试纸条,其中,所述检测线的数量为2~5条。
- 根据权利要求1所述的新型冠状病毒SARS-CoV-2抗体检测试纸条,其中,所述检测线的数量为2条。
- 根据权利要求9所述的新型冠状病毒SARS-CoV-2抗体检测试纸条,其中,所述检测线分别固定有RBD蛋白和N蛋白。
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