WO2022205629A1 - 一种高灵敏SARS-CoV-2中和抗体的检测方法、检测试剂盒 - Google Patents

一种高灵敏SARS-CoV-2中和抗体的检测方法、检测试剂盒 Download PDF

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WO2022205629A1
WO2022205629A1 PCT/CN2021/101904 CN2021101904W WO2022205629A1 WO 2022205629 A1 WO2022205629 A1 WO 2022205629A1 CN 2021101904 W CN2021101904 W CN 2021101904W WO 2022205629 A1 WO2022205629 A1 WO 2022205629A1
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detection
line
rbd
neutralizing antibody
capture
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张玉基
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南京立顶医疗科技有限公司
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6854—Immunoglobulins
    • 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
    • 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
    • G01N33/56983—Viruses
    • 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
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/08—RNA viruses
    • G01N2333/165—Coronaviridae, e.g. avian infectious bronchitis virus
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2469/00—Immunoassays for the detection of microorganisms
    • G01N2469/20—Detection of antibodies in sample from host which are directed against antigens from microorganisms

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  • the invention belongs to the technical field of biomedical detection, and in particular relates to a detection method and detection kit for a highly sensitive SARS-CoV-2 neutralizing antibody.
  • the pathogen of new coronavirus pneumonia (new coronary pneumonia, COVID-19) is a new type of coronavirus, which belongs to the ⁇ genus. It has 5 essential genes, respectively targeting 4 structural proteins, nucleoprotein (N), viral envelope (E), matrix protein (M) and spike protein (S), and RNA-dependent RNA polymerase (RdRp).
  • the nucleoprotein (N) wraps the RNA genome to form the nucleocapsid, which is surrounded by the viral envelope (E), which is embedded with proteins such as matrix protein (M) and spike protein (S).
  • the spike protein (S) enters the cell by binding to the host's angiotensin-converting enzyme 2 (ACE-2) through the receptor binding site (RBD).
  • ACE-2 angiotensin-converting enzyme 2
  • the S protein is the key for the virus to enter the human body and is the main target of vaccines and therapeutic neutralizing antibodies.
  • the receptor-binding (RBD) domain of the S protein can bind to the host ACE2 receptor, mediate viral invasion into host cells, and is also the binding site for most potent neutralizing antibodies.
  • the neutralizing antibody After the neutralizing antibody binds to the RBD domain, it can make the virus lose its ability to adsorb and invade host cells, thereby inhibiting the replication and spread of the virus in the host.
  • the virus neutralizing antibody plays an important role in eliminating the virus in the host.
  • the "gold standard" of traditional neutralizing antibody detection is infection inhibition test, which is mainly to mix the virus with the antibody to be detected first, and then inoculate the mixture into sensitive animals, embryos or cells, and detect the disease by the lesions of the sensitive animals, embryos or cells.
  • the residual virus infectivity is determined to indirectly determine the neutralizing effect of the antibody to be detected on the virus.
  • PRNT plaque reduction neutralization test
  • CPE cytopathic effct
  • colloidal gold-labeled RBD binds to ACE2 to form a detection line.
  • the neutralizing antibodies block the binding of RBD and ACE2, thereby reducing the color intensity of the detection line.
  • the presence of neutralizing antibodies can be judged by machine interpretation or by the change in the color intensity of the detection line. When interpreting with the naked eye, a large change in the depth of the detection line is required to make a judgment. The sensitivity of this form of detection card needs to be improved.
  • the purpose of the present invention is to solve the technical problems of cumbersome, time-consuming and low-sensitivity detection of SARS-CoV-2 neutralizing antibodies in the prior art, and to provide a simple, fast and highly sensitive method for SARS-CoV-2 neutralizing antibodies. Detection methods and detection kits.
  • the technical scheme of the present invention is as follows:
  • a highly sensitive SARS-CoV-2 neutralizing antibody detection method characterized in that the detection of SARS-CoV-2 neutralizing antibody is realized based on a sandwich method combined with a blocking method.
  • the highly sensitive SARS-CoV-2 neutralizing antibody detection method enhances the specificity of the subsequent detection of neutralizing antibodies by sandwich method by capturing non-neutralizing antibodies in advance.
  • the highly sensitive SARS-CoV-2 neutralizing antibody detection method uses ACE2 protein as a capture material, blocks non-neutralizing antibodies, and forms a coated RBD protein-neutralizing antibody-RBD protein-labeled carrier complex to achieve Detection of SARS-CoV-2 neutralizing antibodies.
  • a high-sensitivity SARS-CoV-2 neutralizing antibody detection kit based on the above-mentioned detection method includes a capture line, and the capture line is coated with ACE2 protein.
  • a detection line, a quality control line, and a water absorbing pad are sequentially arranged on one side of the capture line, and a binding pad and a sample pad are arranged on the other side of the capture line in sequence;
  • the capture line, detection line and quality control line are all set on the NC membrane;
  • the detection line is coated with RBD protein, and the binding pad is provided with an RBD protein marker, and the RBD protein marker includes a marker carrier and the RBD protein bound to the marker carrier.
  • the labeling carrier comprises one or more of colloidal gold, latex microspheres, nanocarbon, magnetic microspheres or fluorescent microspheres.
  • the sample pad is further provided with a blood filter pad or an anti-RBC antibody.
  • the detection result determination method determines the SARS-CoV-2 neutralizing antibody content by directly observing the reaction results of the capture line, the detection line and the quality control line.
  • the detection result determination method uses an instrument to read the reaction results of the capture line, the detection line and the quality control line, and judges the neutralization of SARS-CoV-2 by the ratio of the detection line and the quality control line antibody content.
  • the detection principle of the present invention is as follows: the present invention detects the neutralizing antibody by adopting the blocking method and the sandwich method, and by adding a capture line, the non-neutralizing antibody targeting RBD is captured in advance, so as to ensure the subsequent detection of neutralization by the sandwich method.
  • Specificity of the antibody Through the design of capture line, detection line and RBD protein label, the RBD protein label is used to capture the non-neutralizing antibody targeting RBD in the sample, and the antibody-RBD protein-labeled carrier complex is formed.
  • the ACE2 protein in the wire captures the RBD protein, thereby immobilizing a non-neutralizing antibody targeting RBD on the capture wire.
  • the neutralizing antibody-RBD protein-labeled carrier complex formed after the neutralizing antibody is captured by the RBD protein marker cannot be captured by the capture line. As the chromatography reaches the detection line, it is captured by the RBD at the detection line to form a packet.
  • the RBD protein-neutralizing antibody-RBD protein-labeled carrier complex causes a corresponding signal to appear on the detection line, and the neutralizing antibody concentration in the sample is judged by this signal.
  • the present invention captures non-neutralizing antibodies targeting RBD in advance by adding a capture line to ensure subsequent detection of the specificity of neutralizing antibodies by the sandwich method, and determines whether there is neutralizing antibody in the sample by the presence or absence of the detection line. and antibody, the sensitivity is better than that by blocking competition method.
  • a second concentration determination point can be formed by comparing the color depth of the detection line and the capture line: when the neutralizing antibody exceeds a certain concentration, the detection line is lighter than the capture line. At higher concentrations of neutralizing antibody, the capture line disappeared.
  • the present invention adopts the method of immunochromatography, which has simple operation and fast detection speed.
  • the concentration of neutralizing antibody in the sample when the concentration of neutralizing antibody in the sample is extremely low, the system design of capture line and detection line increases the screening of ACE2 and ensures the specificity of detection; the concentration of neutralizing antibody in the sample is relatively high When , the concentration of neutralizing antibody can also be judged by the inhibition signal of the capture line, which greatly improves the detection range.
  • Fig. 1 is the structural representation of the kit of the present invention
  • the detection principle of the present invention is as follows: the present invention detects the neutralizing antibody by adopting the blocking method and the sandwich method, and by adding a capture line, the non-neutralizing antibody targeting RBD is captured in advance, so as to ensure the subsequent detection of neutralization by the sandwich method.
  • Specificity of the antibody Through the design of capture line, detection line and RBD protein label, the RBD protein label is used to capture the non-neutralizing antibody targeting RBD in the sample, and the antibody-RBD protein-labeled carrier complex is formed.
  • the ACE2 protein in the wire captures the RBD protein, thereby immobilizing a non-neutralizing antibody targeting RBD on the capture wire.
  • the neutralizing antibody-RBD protein-labeled carrier complex formed after the neutralizing antibody is captured by the RBD protein marker cannot be captured by the capture line. As the chromatography reaches the detection line, it is captured by the RBD at the detection line to form a packet.
  • the RBD protein-neutralizing antibody-RBD protein-labeled carrier complex causes a corresponding signal to appear on the detection line, and the neutralizing antibody concentration in the sample is judged by this signal.
  • a SARS-CoV-2 neutralizing antibody detection kit comprising a chromatography test paper, a cartridge and a sample diluent, the chromatography test paper comprising a bottom plate, a sample pad 1, a binding pad 2, an NC membrane 3 and a water-absorbing pad 4, The sample pad 1, the binding pad 2, the NC film 3 and the water absorbing pad 4 are sequentially attached to one side of the bottom plate; the NC film 3 is provided with a capture line 5, a detection line 6 and a quality control line 7 in sequence, and the capture line 5 is arranged on the NC film 3 in sequence.
  • Line 5 is close to the binding pad 2
  • the quality control line 7 is close to the water absorption pad 4
  • the capture line 5 is coated with ACE2 protein
  • the detection line 6 is coated with RBD protein
  • the binding pad is provided with RBD protein markers and anti-chicken IgY marker
  • the marker carrier is colloidal gold
  • the quality control line is coated with a quality control test substance
  • the quality control test substance is chicken IgY.
  • NC membrane coating using 1XPBS + 4% sucrose as solvent, respectively prepare the capture line coating solution with ACE2 protein concentration of 0.2 mg/mL, the detection line coating solution with RBD protein concentration of 0.2 mg/mL, and 0.5 mg/mL of RBD protein concentration.
  • mg/mL chicken IgY protein concentration quality control line coating solution the coating solution prepared above was scratched on the NC membrane using a gold-spraying membrane device, dried at 35°C for 12 hours, and coated with the NC membrane;
  • the bottom plate is made of PVC material
  • the sample pad 1 is glass fiber cut into a width of 18mm
  • the absorbent pad is absorbent paper cut into a width of 25mm.
  • the pads 4 are overlapped in sequence and pasted on one side of the bottom plate to obtain a large plate;
  • sample diluent of the present invention is prepared by ultrapure water, and the formula is 0.02moL/L PB+0.3% Triton 100.
  • a detection kit for SARS-CoV-2 neutralizing antibody comprising a chromatography test paper, a cartridge and a sample diluent, the chromatography test paper comprising a bottom plate, a sample pad, a binding pad, an NC membrane and a water-absorbing pad, the sample pad , the binding pad, the NC film and the water absorbing pad are sequentially attached to one side of the bottom plate;
  • the NC film is sequentially provided with a capture line, a detection line and a quality control line, the capture line is close to the binding pad, and the quality control line is close to the water absorption line pad, the capture line is coated with ACE2 protein, and the detection line is coated with RBD protein;
  • the binding pad is provided with an RBD protein marker, and the RBD protein marker includes a marker carrier and an RBD protein bound to the marker carrier , the labeling carrier is red latex microspheres.
  • the quality control line is coated with a quality control detection substance, and the quality control detection substance is an anti-RBD antibody.
  • the labeled carrier is red latex microspheres
  • the labeled protein is only RBD protein
  • the coating solution of the quality control line is 0.5 mg/mL anti-RBD monoclonal antibody
  • a sample addition hole and a diluent hole are set on the cartridge, which are used to add sample and diluent respectively.
  • a detection kit for SARS-CoV-2 neutralizing antibody comprising a chromatography test paper, a cartridge and a sample diluent, the chromatography test paper comprising a bottom plate, a sample pad, a binding pad, an NC membrane and a water-absorbing pad, the sample pad , the binding pad, the NC film and the water absorbing pad are sequentially attached to one side of the bottom plate;
  • the NC film is sequentially provided with a capture line, a detection line and a quality control line, the capture line is close to the binding pad, and the quality control line is close to the water absorption line pad, the capture line is coated with ACE2 protein, and the detection line is coated with RBD protein;
  • the binding pad is provided with an RBD protein marker, and the RBD protein marker includes a marker carrier and an RBD protein bound to the marker carrier , the labeled carrier time-resolved fluorescent microspheres.
  • the quality control line is coated with a quality control detection substance, and the quality control detection substance is an anti-RBD
  • the labeled carrier is a time-resolved fluorescent microsphere
  • the labeled protein is only RBD protein
  • the coating solution of the quality control line is 0.5 mg/mL anti-RBD monoclonal Antibody.
  • Example 2 The difference between this example and Example 1, Example 2, and Example 3 is that the sample pad includes a blood filter pad or an anti-RBC antibody is added to the sample pad, which can directly detect fingertip blood and whole blood samples.
  • the sample pad includes a blood filter pad or an anti-RBC antibody is added to the sample pad, which can directly detect fingertip blood and whole blood samples.
  • Example 3 Collect the serum samples of the subjects before the injection of the vaccine and the serum samples of the 1st, 2nd, 3rd, 4th, 5th and 6th weeks after the vaccine injection, and use the test kit of Example 3 for detection.
  • the collected serum is added dropwise to the sample addition hole of the detection card of the present invention, and then the diluent is added dropwise to the diluent hole, and the reaction is carried out for 15 minutes; then the matching fluorescence detector is used to read the reaction results of the capture line, the detection line and the quality control line. , and the SARS-CoV-2 neutralizing antibody content was judged by the ratio of the detection line and the capture line.
  • the detection kit of the present invention has high sensitivity, strong specificity, simple detection operation and fast detection speed; it is suitable for the detection of SARS-CoV-2 neutralizing antibodies.

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Abstract

一种高灵敏SARS-CoV-2中和抗体的检测方法和检测试剂盒，其中试剂盒包括层析试纸、卡壳和样本稀释液，层析试纸包括底板、样品垫（1）、结合垫（2）、NC膜（3）和吸水垫（4）， NC膜（3）上依次设置有捕获线（5）、检测线（6）和质控线（7），捕获线（5）包被有ACE2蛋白，检测线（6）包被有RBD蛋白，结合垫（2）设置有RBD蛋白标记物；采用阻断法加夹心法原理提高检测中和抗体的灵敏度，通过添加捕获线的方式，将靶向RBD的非中和抗体提前捕获，保证后续通过夹心法检测中和抗体的特异性。

Description

一种高灵敏SARS-CoV-2中和抗体的检测方法、检测试剂盒 技术领域
本发明属于生物医学检测技术领域，尤其涉及一种高灵敏SARS-CoV-2中和抗体的检测方法、检测试剂盒。
背景技术
新型冠状病毒肺炎(新冠肺炎，COVID-19)的病原为新型冠状病毒，属于β属的冠状病毒，有包膜，颗粒呈圆形或椭圆形，直径60～140nm。具有5个必需基因，分别针对核蛋白(N)、病毒包膜(E)、基质蛋白(M)和刺突蛋白(S)4种结构蛋白及RNA依赖性的RNA聚合酶(RdRp)。核蛋白(N)包裹RNA基因组构成核衣壳，外面围绕着病毒包膜(E)，病毒包膜包埋有基质蛋白(M)和刺突蛋白(S)等蛋白。刺突蛋白(S)通过受体结合位点(RBD)结合宿主的血管紧张素转化酶2(ACE-2)进入细胞。S蛋白是病毒进入人体的关键，是疫苗和治疗性中和抗体的主要靶点。S蛋白的受体结合(RBD)结构域可与宿主ACE2受体结合，介导病毒入侵宿主细胞，也是大多数有效的中和抗体的结合部位。
中和抗体与RBD结构域结合后，可使病毒失去吸附、入侵宿主细胞能力，从而抑制病毒在宿主体内复制传播的抗体，病毒中和抗体在清除宿主体内的病毒中起着重要作用。
传统中和抗体检测的“金标准”是感染抑制试验，主要是先将病毒与待检测抗体进行混合，再将混合物接种到敏感动物、胚胎或细胞，通过敏感动物、胚胎或细胞的病变情况来测定残存的病毒感染力，从而间接确定待检测抗体对病毒的中和效果。其中在体外进行利用活病毒进行蚀斑减少中和试验(plaque reduction neutralizationtest，PRNT)和通过检测细胞病变(cytopathic effct，CPE)量来进行分析的微量细胞中和试验是常用方法。
对于新型冠状病毒烈性病毒性传染病来讲，即使是体外实验也面临活病毒株来源和需要在高等级生物安全实验室操作的限制，杜克-新加坡国立大学医学院新发传染病重点研究项目主任王林发教授带领的研究团队，基于中和抗体可阻断RBD和ACE2结合的原理开发了阻断ELISA的cPass血清检测盒，由金斯瑞生物进行商业化推广，并获FDA授权用于恢复期血浆筛查。但仍存在检测操作繁 琐、耗时长的缺点。之后，多家公司基于此原理开发了侧向层析试纸条，主要方法是将ACE2划到膜上，并用胶体金标记RBD蛋白。当样本中无中和抗体时候，胶体金标记的RBD和ACE2结合形成检测线。样本中有中和抗体时，中和抗体阻断RBD和ACE2的结合，从而降低检测线的显色强度。通过机器判读，或者检测线显色强度的深浅变化来判断中和抗体存在情况。肉眼判读时候需要检测线深浅有较大变化才能做判定，此形式检测卡的灵敏度有待提高。
发明内容
本发明的目的在于解决现有技术中SARS-CoV-2中和抗体检测操作繁琐、耗时长，灵敏度低的技术问题，提供一种简便、快速、灵敏度高的SARS-CoV-2中和抗体的检测方法、检测试剂盒。本发明的技术方案如下：
一种高灵敏SARS-CoV-2中和抗体的检测方法，其特征在于，基于夹心法结合阻断法实现SARS-CoV-2中和抗体的检测。
优选的，高灵敏SARS-CoV-2中和抗体的检测方法通过提前捕获非中和抗体，增强后续通过夹心法检测中和抗体的特异性。
优选的，高灵敏SARS-CoV-2中和抗体的检测方法以ACE2蛋白为捕获物，阻断非中和抗体，通过形成包被RBD蛋白-中和抗体-RBD蛋白-标记载体复合物，实现SARS-CoV-2中和抗体的检测。
一种基于上述检测方法的高灵敏SARS-CoV-2中和抗体检测试剂盒，包括捕获线，所述捕获线包被有ACE2蛋白。
优选的，在所述捕获线的一侧依次设有检测线、质控线、吸水垫，在所述捕获线的另一侧依次设有结合垫、样品垫；
所述捕获线、检测线、质控线均设置在NC膜上；
所述检测线上包被有RBD蛋白，所述结合垫设置有RBD蛋白标记物，所述RBD蛋白标记物包括标记载体和结合于所述标记载体上的RBD蛋白。
优选的，所述标记载体包括胶体金、乳胶微球、纳米碳、磁微球或荧光微球中的一种或多种。
优选的，所述样品垫上还设有滤血垫或抗RBC抗体。
优选的，所述检测试剂盒，其检测结果判定方法通过直接观测捕获线、检测线和质控线的反应结果，来判断SARS-CoV-2中和抗体含量。
优选的，所述检测试剂盒，其检测结果判定方法通过采用仪器读取捕获线、检测线和质控线的反应结果，并通过检测线和质控线的比值判断SARS-CoV-2中和抗体含量。
本发明的检测原理为：本发明通过采用阻断法加夹心法原理检测中和抗体，通过添加捕获线的方式，将靶向RBD的非中和抗体提前捕获，保证后续通过夹心法检测中和抗体的特异性：通过捕获线、检测线和RBD蛋白标记物的设计，利用RBD蛋白标记物捕获样品中靶向RBD的非中和抗体，形成抗体-RBD蛋白-标记载体复合物，并通过捕获线中的ACE2蛋白捕捉RBD蛋白，从而将靶向RBD的非中和抗体固定在捕获线上。而中和抗体被RBD蛋白标记物捕获后形成的中和抗体-RBD蛋白-标记载体复合物不能被捕获线被捕获，随着层析到达检测线处，被检测线处的RBD捕获，形成包被RBD蛋白-中和抗体-RBD蛋白-标记载体复合物，使检测线出现相应的信号，并通过此信号判断样本中的中和抗体浓度。
通过采用上述技术方案，达到的技术效果如下：
(1)本发明通过添加捕获线的方式，将靶向RBD的非中和抗体提前捕获，保证后续通过夹心法检测中和抗体的特异性，通过检测线的有无来判定样本中是否有中和抗体，灵敏度较通过阻断式竞争法的更好。
(2)条带有无的判读方式，较常规通过对比条带显色深浅的方式，判读方法更加直观、容易一些。
(3)除了通过检测线出现与否来判定样品中和抗体存在情况，还能通过检测线和捕获线颜色深浅对比形成第二个浓度判定点：在中和抗体超过特定浓度的时候，检测线的颜色较捕获线浅。当中和抗体浓度更高时，捕获线消失。
(4)本发明采用免疫层析的方法，操作简单，检测速度快。
(5)本发明在样本中的中和抗体浓度极低时，通过捕获线、检测线的系统设计，增加了ACE2的筛选，保证了检测的特异性；在样本中的中和抗体浓度较高时，还可以通过捕获线的抑制信号判断中和抗体的浓度，极大的提升了检测范围。
附图说明
图1是本发明试剂盒的结构示意图；
图中：1、样品垫；2、结合垫；3、NC膜；4、吸水垫；5、捕获线；6、检测线；7、质控线。
具体实施方式
下面结合附图对本发明作进一步的说明。
下述的实施例是为了进一步说明本发明的一些优选实施例，并非全部实施例。本领域专业人员在没有进行创造性劳动的前提下做出的基于本发明的其他实施例，都属于本发明的权利保护范围。
本发明的检测原理为：本发明通过采用阻断法加夹心法原理检测中和抗体，通过添加捕获线的方式，将靶向RBD的非中和抗体提前捕获，保证后续通过夹心法检测中和抗体的特异性：通过捕获线、检测线和RBD蛋白标记物的设计，利用RBD蛋白标记物捕获样品中靶向RBD的非中和抗体，形成抗体-RBD蛋白-标记载体复合物，并通过捕获线中的ACE2蛋白捕捉RBD蛋白，从而将靶向RBD的非中和抗体固定在捕获线上。而中和抗体被RBD蛋白标记物捕获后形成的中和抗体-RBD蛋白-标记载体复合物不能被捕获线被捕获，随着层析到达检测线处，被检测线处的RBD捕获，形成包被RBD蛋白-中和抗体-RBD蛋白-标记载体复合物，使检测线出现相应的信号，并通过此信号判断样本中的中和抗体浓度。
实施例1胶体金检测试剂盒
一种SARS-CoV-2中和抗体的检测试剂盒，包括层析试纸、卡壳和样本稀释液，所述层析试纸包括底板、样品垫1、结合垫2、NC膜3和吸水垫4，所述样品垫1、结合垫2、NC膜3和吸水垫4依次黏贴与底板一侧；所述NC膜3上依次设置有捕获线5、检测线6和质控线7，所述捕获线5靠近结合垫2，所述质控线7靠近吸水垫4，所述捕获线5包被有ACE2蛋白，所述检测线6包被有RBD蛋白；所述结合垫设置有RBD蛋白标记物和抗鸡IgY标记物，所述标记载体为胶体金，所述质控线包被有质控检测物，所述质控检测物为鸡IgY。
本实施例胶体金测试试剂盒的制备方法：
(1)胶体金标记，采用万分之一浓度的胶体金分别标记RBD蛋白和抗鸡IgY，离心去上清，浓缩十倍得到RBD蛋白标记物和抗鸡IgY标记物；
(2)结合垫制备，将(1)得到的RBD蛋白标记物和抗鸡IgY标记物按照1： 1比例混合后，再稀释3倍后均匀涂抹于玻璃纤维垫片上，35℃烘干30分钟，然后采用斩切机，切割成5mm宽，得到结合垫；
(3)NC膜包被，采用1XPBS+4％蔗糖作为溶剂，分别配制0.2mg/mL的ACE2蛋白浓度的捕获线包被液、0.2mg/mL的RBD蛋白浓度的检测线包被液、0.5mg/mL的鸡IgY蛋白浓度质控线包被液，采用喷金划膜仪器将上述配制的包被液划在NC膜上，35℃烘干12小时，包被NC膜；
(4)大板组装，底板采用PVC材质底板，样品垫1为切割成宽度18mm的玻璃纤维，吸水垫为切割成宽度25mm的吸水纸，将样品垫1、结合垫2、NC膜3和吸水垫4依次重叠，黏贴于底板一侧，得到大板；
(5)样本稀释液配制，本发明样本稀释液采用超纯水配制，配方为0.02moL/L PB+0.3％曲拉通100。
(6)切条、组装，封装，采用切条机将步骤(4)得到的大板切成3mm宽度的试纸条，放置于所述卡壳的内部完成组装，然后同干燥剂一起封装于铝箔袋内，再同配套的样本稀释液一起放置于包装盒内，完成本发明试剂盒的制备。
使用时，收集待检测的血清并滴加在所述卡壳的加样孔，然后向加样孔滴加稀释液，反应15分钟；通过直接观测捕获线、检测线和质控线的反应结果，并与配套使用的浓度显色卡进行比对，来判断SARS-CoV-2中和抗体含量。
实施例2乳胶微球检测试剂盒
一种SARS-CoV-2中和抗体的检测试剂盒，包括层析试纸、卡壳和样本稀释液，所述层析试纸包括底板、样品垫、结合垫、NC膜和吸水垫，所述样品垫、结合垫、NC膜和吸水垫依次黏贴与底板一侧；所述NC膜上依次设置有捕获线、检测线和质控线，所述捕获线靠近结合垫，所述质控线靠近吸水垫，所述捕获线包被有ACE2蛋白，所述检测线包被有RBD蛋白；所述结合垫设置有RBD蛋白标记物，所述RBD蛋白标记物包括标记载体和结合于标记载体的RBD蛋白，所述标记载体为红色乳胶微球。所述质控线包被有质控检测物，所述质控检测物为抗RBD抗体。
本发明试剂盒的制备方法与实施例1的不同之处在于，所述标记载体为红色乳胶微球，标记蛋白只有RBD蛋白，质控线包被液为0.5mg/mL的抗RBD单克隆抗体，另外，卡壳上设置加样孔和稀释液孔，分别用于添加样本和稀释液。
使用时，收集待检测的血清并滴加在所述卡壳的加样孔，然后向稀释液孔滴加稀释液，反应15分钟；通过直接观测捕获线、检测线和质控线的反应结果，并与配套使用的浓度显色卡进行比对，来判断SARS-CoV-2中和抗体含量。
实施例3荧光检测试剂盒
一种SARS-CoV-2中和抗体的检测试剂盒，包括层析试纸、卡壳和样本稀释液，所述层析试纸包括底板、样品垫、结合垫、NC膜和吸水垫，所述样品垫、结合垫、NC膜和吸水垫依次黏贴与底板一侧；所述NC膜上依次设置有捕获线、检测线和质控线，所述捕获线靠近结合垫，所述质控线靠近吸水垫，所述捕获线包被有ACE2蛋白，所述检测线包被有RBD蛋白；所述结合垫设置有RBD蛋白标记物，所述RBD蛋白标记物包括标记载体和结合于标记载体的RBD蛋白，所述标记载体时间分辨荧光微球。所述质控线包被有质控检测物，所述质控检测物为抗RBD抗体。
本发明试剂盒的制备方法与实施例1的不同之处在于，所述标记载体为时间分辨荧光微球，标记蛋白只有RBD蛋白，质控线包被液为0.5mg/mL的抗RBD单克隆抗体。
使用时，收集待检测的血清并滴加在所述卡壳的加样孔，然后向加样孔滴加稀释液，反应15分钟；然后采用仪器读取捕获线、检测线和质控线的反应结果，并通过检测线和质控线的比值判断SARS-CoV-2中和抗体含量。
实施例4
本实施例与实施例1、实施例2、实施例3不同之处在于所述样品垫包括滤血垫或所述样品垫上添加有抗RBC抗体，能够直接检测指尖血和全血样本。
实施例5中和抗体检测
收集受测试者注射疫苗前血清及注射疫苗后第1周、第2周、第3周、第4周、第5周和第6周的血清样本，采用实施例3检测试剂盒进行检测，将收集的血清并滴加在本发明检测卡的加样孔，然后向稀释液孔滴加稀释液，反应15分钟；然后采用配套荧光检测仪读取捕获线、检测线和质控线的反应结果，并通过检测线和捕获线的比值判断SARS-CoV-2中和抗体含量。
检测结果如下表：
Figure PCTCN2021101904-appb-000001
从以实施例可以看出，本发明检测试剂盒灵敏度高，特异性强，检测操作简单，检测速度快；适合于SARS-CoV-2中和抗体的检测。
以上所述仅为本发明的优选实施例，并非因此限制本发明的专利范围，凡是利用本发明说明书内容所作的简单修改或变换，或直接或间接运用在其他相关的技术领域，均同理包括在本发明的专利保护范围内。以上实施例仅用以说明本发明而并非限制本发明所描述的技术方案；因此，尽管本说明书参照上述的实施例对本发明已进行了详细的说明，但是，本领域的普通技术人员应当理解，仍然可以对本发明进行修改或等同替换；而一切不脱离本发明的精神和范围的技术方案及其改进，其均应涵盖在本发明的权利要求范围中。

Claims (7)

  1. 一种高灵敏SARS-CoV-2中和抗体的检测方法，其特征在于，基于夹心法结合阻断法实现SARS-CoV-2中和抗体的检测，通过添加捕获线的方式，将靶向RBD的非中和抗体提前捕获，增强后续通过夹心法检测中和抗体的特异性；通过捕获线、检测线和RBD蛋白标记物的设计，利用RBD蛋白标记物捕获样品中靶向RBD的非中和抗体，形成抗体-RBD蛋白-标记载体复合物，并通过捕获线中的ACE2蛋白捕捉RBD蛋白，从而将靶向RBD的非中和抗体固定在捕获线上；而中和抗体被RBD蛋白标记物捕获后形成的中和抗体-RBD蛋白-标记载体复合物不能被捕获线被捕获，随着层析到达检测线处，被检测线处的RBD捕获，形成包被RBD蛋白-中和抗体-RBD蛋白-标记载体复合物，使检测线出现相应的信号，并通过此信号判断样本中的中和抗体浓度。
  2. 一种基于权利要求1所述的检测方法的高灵敏SARS-CoV-2中和抗体检测试剂盒，其特征在于，包括捕获线，所述捕获线包被有ACE2蛋白。
  3. 根据权利要求2所述的检测试剂盒，其特征在于，在所述捕获线的一侧依次设有检测线、质控线、吸水垫，在所述捕获线的另一侧依次设有结合垫、样品垫；
    所述捕获线、检测线、质控线均设置在NC膜上；
    所述检测线上包被有RBD蛋白，所述结合垫设置有RBD蛋白标记物，所述RBD蛋白标记物包括标记载体和结合于所述标记载体上的RBD蛋白。
  4. 根据权利要求3所述的检测试剂盒，其特征在于，所述标记载体包括胶体金、乳胶微球、纳米碳、磁微球或荧光微球中的一种或多种。
  5. 根据权利要求3所述的检测试剂盒，其特征在于，所述样品垫上还设有滤血垫或抗RBC抗体。
  6. 根据权利要求3所述的检测试剂盒，其特征在于，其检测结果判定方法通过直接观测捕获线、检测线和质控线的反应结果，来判断SARS-CoV-2中和抗体含量。
  7. 根据权利要求3所述的检测试剂盒，其特征在于，其检测结果判定方法通过采用仪器读取捕获线、检测线和质控线的反应结果，并通过检测线和质控线的比值判断SARS-CoV-2中和抗体含量。
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