WO2022165982A1 - 一种抗体检测方法 - Google Patents

一种抗体检测方法 Download PDF

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WO2022165982A1
WO2022165982A1 PCT/CN2021/086008 CN2021086008W WO2022165982A1 WO 2022165982 A1 WO2022165982 A1 WO 2022165982A1 CN 2021086008 W CN2021086008 W CN 2021086008W WO 2022165982 A1 WO2022165982 A1 WO 2022165982A1
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ace2
rbd
detection
sample
cov
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French (fr)
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夏良雨
俞先
吕伟华
赵艾钧
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广东菲鹏生物有限公司
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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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • 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
    • 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
    • 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
    • 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
    • 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
    • G01N33/585—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with a particulate label, e.g. coloured latex
    • 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
    • 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

Definitions

  • the present disclosure relates to the field of biotechnology, and in particular, to an antibody detection method.
  • viral surface receptor-binding proteins are the key “keys” for pathogenic microorganisms to enter cells, which can open the “lock” of cellular receptor proteins, thereby entering cells and initiating the replication process of pathogenic microorganisms.
  • Neutralizing antibodies are certain antibodies produced by B lymphocytes, which can bind to antigens (ligands) on the surface of pathogenic microorganisms, thereby preventing the pathogenic microorganisms from adhering to target cell receptors and preventing pathogenic microorganisms from invading host cells.
  • BSL3 biosafety level 3 laboratories
  • BSL2 biosafety secondary laboratory
  • the spike protein on the surface of SARS-CoV-2 is an important receptor binding site on the surface of the coronavirus, which can bind to virus-specific receptors on the host cell surface, mediate the fusion of the viral outer membrane with the host cell, Adsorption and membrane penetration; the receptor binding domain (RBM) of the coronavirus S protein can bind the virus to angiotensin-converting enzyme 2 (ACE2) on the surface of the host cell and allow the virus to enter the host cell .
  • ACE2 angiotensin-converting enzyme 2
  • SARS-CoV-2 invades the body, it stimulates the body to produce protective neutralizing antibodies, which specifically prevent pathogenic microorganisms from entering host cells and prevent infection.
  • the amount of neutralizing antibodies is an important indicator of the protective effect of vaccine immunity and an important basis for vaccine evaluation and quality control.
  • protective antibodies namely neutralizing antibodies
  • the titer of the neutralizing antibodies can be determined to determine the clinical efficacy of the vaccine. Therefore, the detection of neutralizing antibodies can be applied to the development and evaluation of vaccines, as well as the evaluation of individual autoimmune effects after vaccination. In addition, the detection of neutralizing antibodies in cured patients with novel coronavirus can determine whether there is a risk of re-infection.
  • the present disclosure relates to a method for realizing anti-coronavirus antibody detection on an immunoturbidimetric platform, the method comprising:
  • the detection method is used to detect the presence or absence of neutralizing antibodies in a sample.
  • the coronavirus is selected from SARS-CoV-2 or SARS-CoV.
  • the sample is selected from any sample that may contain antibodies.
  • the sample is selected from one or more of serum, plasma, whole blood, lymph, cerebrospinal fluid, tissue fluid, saliva, urine, or lymphocytes.
  • RBD aggregates or polyploids can be obtained by expressing two or more RBD sequences in tandem or by polymerizing two or more RBD proteins by chemical coupling.
  • a detection assembly comprises: a reagent comprising an RBD polymer or polyploid and a reagent of latex-conjugated ACE2 or a fragment thereof.
  • the detection component contains reagents for RBD aggregates or polyploids, and RBD aggregates or polyploids can be obtained by expressing two or more RBD sequences in tandem or by chemical coupling It is obtained by polymerizing more than two RBD proteins.
  • a detection assembly is used to detect the presence of neutralizing antibodies in a sample.
  • the detection component is for the detection of neutralizing antibodies against coronavirus, in one or more embodiments, the detection of neutralizing antibodies against SARS-CoV-2, in a In one or more embodiments, neutralizing antibodies against SARS-CoV are detected.
  • SARS-CoV-2 includes its wild type and variants
  • SARS-CoV includes its wild type and variants
  • ACE2 includes ACE2 isoforms, fragments, variants (including mutants), or homologs from any species.
  • the ACE2 is mammalian ACE2. Mammals include therathera, eumammalia, eutheria, protothera, monothera, or primates.
  • the ACE2 is human, bat, pangolin, civet, or porcine ACE2.
  • the method described in any of the above embodiments and the detection assembly described in any of the above embodiments are used in antibody detection or preparation of antibody detection reagents.
  • Figure 1 shows an SDS-PAGE profile of an RBD polymer or polyploid, 1: RBD monomer; 2: RBD dimer.
  • Figure 2 shows a schematic representation of the reaction process when neutralizing antibodies are present in the sample, resulting in no effective turbidity being generated.
  • Figure 3 shows a schematic representation of the reaction process when bound antibody is present in the sample, resulting in effective turbidity.
  • Figure 4 shows a schematic representation of the reaction process when no antibody is present in the sample, resulting in effective turbidity.
  • One or more embodiments of the present disclosure enable anti-coronavirus antibody detection on an immunoturbidimetric platform.
  • the term "immunoturbidimetric platform” refers to a kinetic assay method for antigen-antibody binding.
  • the basic principle is: when the antigen and antibody react specifically in a special dilution system and the ratio is appropriate (generally stipulates that the antibody is in excess), the formation of the soluble immune complex in the dilution system
  • the accelerator polyethylene glycol, etc.
  • the amount of the formed immune complex increases with the increase in the amount of antigen in the test sample, and the turbidity of the reaction solution also increases.
  • the content of the antigen in the test sample can be calculated.
  • neutralizing antibodies refers to certain antibodies produced by B lymphocytes that bind to antigens on the surface of pathogenic microorganisms, thereby preventing the pathogenic microorganisms from adhering to target cell receptors and preventing cell invasion.
  • RBD polymer or polyploid refers to a protein with multiple RBD domains, which can be a multi-copy RBD vector constructed by gene recombination technology and expressed to obtain a recombinant protein with multiple RBD domains in series; A single RBD protein can be obtained by recombinant technology, and then a multimeric protein can be obtained by chemical coupling; its characteristic is that the newly formed molecular structure has more than two RBD domains.
  • sample can be understood as any sample that may contain antibodies, in one or more embodiments, the sample is from a sample after infection or active immunization; in one or more embodiments, the sample is selected from the group consisting of Body fluids, excreta, cells; such as serum, plasma, whole blood, lymph, cerebrospinal fluid, tissue fluid, saliva, urine, lymphocytes, etc., but not limited thereto.
  • fragment refers to an amino acid fragment comprising at least a binding segment.
  • contact is understood to mean allowing it to bind.
  • the contact time is not specifically limited, and the contact time varies with different embodiments and detection platforms, but belongs to the category that can be understood by those skilled in the art.
  • ligand can be understood as any protein or polypeptide capable of binding to a receptor.
  • a "receptor” can be understood as a molecule capable of recognizing a ligand and specifically binding the ligand, the ligand-receptor binding can achieve signal transduction, and the receptor may include a co-receptor.
  • Common cell surface receptors include, but are not limited to, G protein-coupled receptors, receptor tyrosine kinases, guanylate cyclase-coupled receptors, ion channels, adhesion receptors, and the like.
  • affinity The easier the two are combined, the stronger the binding after binding, the stronger the affinity, and vice versa.
  • reagent can be understood as a substance or product, etc., which is not limited to its form or state, and can be either liquid or solid.
  • ACE2 is not limited to species, and ACE2 of different species can be selected according to the species source of the sample to be tested, such as mammals, non-cold-blooded flying animals, bats and birds, etc.
  • human ACE2 is used.
  • ACE2 for coronavirus neutralizing antibody detection in human samples.
  • the present disclosure provides a method for realizing coronavirus antibody detection on an immunoturbidimetric platform, comprising:
  • the above method detects neutralizing antibodies, that is, the above method is used to detect the presence or absence of neutralizing antibodies in a sample.
  • the coronavirus refers to SARS-CoV-2 or SARS-CoV.
  • SARS-CoV-2 includes its wild type and variant.
  • SARS-CoV includes its wild type and variant.
  • RBD aggregates or polyploids can be obtained by expressing two or more RBD sequences in tandem or by polymerizing two or more RBD proteins by chemical coupling.
  • the sequence of the RBM can be referred to Wrapp Daniel, Wang Nianshuang, Corbett Kizzmekia S, et al. Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation, and its sequence identity is at least 70%. %, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the amino acid sequences that still have Ligand binding to receptor and/or antibody function.
  • ACE2 refers to ACE2 of any species, and includes ACE2 isoforms, fragments, variants (including mutants), or homologs from any species.
  • the ACE2 is a mammal (eg, Thera, eutheria, eutheria, protothera, monarch, primate (monkey, non-human primate). , or human)) ACE2.
  • the ACE2 is human, bat, pangolin, civet, or porcine ACE2.
  • the ACE2 is human ACE2, the sequence can be referred to Gene ID: 59272, and its sequence identity is at least 70%, 75%, 80%, 85%, 90%, 91%, 92% , 93%, 94%, 95%, 96%, 97%, 98% or 99% of the amino acid sequence, and these amino acid sequences still have the function of receptor binding ligand.
  • the present disclosure also relates to a detection assembly comprising: an RBD-mer or polyploid-containing reagent and a latex-conjugated ACE2 or fragment thereof.
  • the RBD-mer or polyploid-containing agent or the latex-conjugated ACE2 or fragment thereof further independently contains a buffer (or buffer).
  • Buffers include: mixed solutions of weak acids and their salts; or, mixed solutions of weak bases and their salts.
  • the buffer (or buffer) can be Tris buffer, PBS buffer, carbonate buffer, borate buffer, and the like.
  • the RBD-mer or polyploid-containing agent or the latex-conjugated ACE2 or fragment thereof may also independently include one or more stabilizers, typically in vitro Stabilizers commonly used in detection reagents (helping reagent preservation or detection), such as amino acid substances, such as glycine, etc.; protein substances, such as BSA (bovine serum albumin), casein, etc.; carbohydrate substances, such as sucrose, glucose etc.; surfactants, such as Tween, TRITON, etc.
  • the agent may also independently include a preservative, such as sodium azide.
  • the RBD polymer or polyploid in the detection component can be obtained by tandem expression of two or more RBD sequences or by chemical coupling of two or more RBD proteins.
  • a detection assembly is used to detect the presence of neutralizing antibodies in a sample.
  • the detection component is for the detection of neutralizing antibodies against coronavirus, in one or more embodiments, the detection of neutralizing antibodies against SARS-CoV-2, in a In one or more embodiments, neutralizing antibodies against SARS-CoV are detected.
  • the present disclosure also relates to the detection method described in any of the above embodiments, or the application of the detection component described in any of the above embodiments in antibody detection or preparation of antibody detection reagents.
  • the sample is contacted with a reagent containing RBD aggregates or polyploids, and the neutralizing antibody and total antibody in the sample are combined with RBD to form a neutralizing antibody-RBD complex and a total antibody-RBD complex, respectively;
  • ACE2 By adding the latex-conjugated ACE2 reagent, ACE2 can further bind to the total antibody-RBD complex to form a complex, but not to the neutralizing antibody-RBD complex.
  • RBD multimers or polyploids bound with neutralizing antibodies cannot re-associate with latex-conjugated ACE2 and thus cannot undergo turbidity reactions.
  • the RBD multimer or polyploid can bind to the latex-conjugated ACE2, resulting in a turbidity reaction.
  • the content of the neutralizing antibody in the sample is inversely proportional to the signal value of the turbidity reaction.
  • Neutralizing antibodies in a sample can be quantified.
  • Recombinant human ACE2, recombinant RBD antigen (RBD multimeric fragment), recombinant RBD antigen (RBD monomer fragment), neutralizing antibody standard against novel coronavirus (G3), and latex microspheres used in the following examples were all purchased From Guangdong Feipeng Biological Co., Ltd.
  • the instrument used is a fully automatic biochemical analyzer, which is any conventional biochemical analyzer on the market.
  • Dilute neutralizing antibody standards with matrix serum to the following concentrations: 0ng/mL, 50ng/mL, 100ng/mL, 200ng/mL, 500ng/mL, 1000ng/mL, 2000ng/mL, 4000ng/mL, 8000ng/mL .
  • Both the RBD polymer fragment and the RBD monomer fragment were purchased from Guangdong Feipeng Biological Co., Ltd.
  • the results of SDS-PAGE electrophoresis are shown in Figure 1. From the molecular weight, it can be determined that lane 2 is the RBD polymer fragment.
  • Diluent 1 Diluent 2 Diluent 3 Antigen concentration 40 ⁇ g/mL 25 ⁇ g/mL 10 ⁇ g/mL
  • Diluent 4 Diluent 5 Diluent 6
  • Antigen concentration 40 ⁇ g/mL 25 ⁇ g/mL 10 ⁇ g/mL
  • Detection process Aspirate 5 ⁇ L of the sample on the sample plate, and at the same time draw 135 ⁇ L of R1, add it to the cuvette, stir mechanically, and incubate at 37°C for 5 minutes; add 15 ⁇ L of R2 to the cuvette, stir mechanically, and incubate at 37°C for 8 minutes ; Add 50 ⁇ L of R3 to the cuvette, stir mechanically, and incubate at 37°C for 10 minutes.
  • the instrument records the absorbance values at 15 minutes and 30 seconds and 25 minutes and 30 seconds after adding the sample. The difference is the reaction absorbance of the sample. The absorbance and the neutralizing antibody concentration in the sample are negatively correlated under specific conditions.
  • the dilution 1-diluent 3 prepared with RBD polymer can produce obvious turbidity response, and the turbidity response is related to the amount of neutralizing antibody, while the RBD monomer configuration Diluent 4 - Diluent 6 failed to produce a turbidity response.
  • the applicant has further studied the detection principle of the method of the present disclosure.
  • the RBD multimeric fragment contains at least 2 or more sites that can bind to ACE2. When there is no neutralizing antibody in the detection sample, the RBD multimeric fragment binds to ACE2.
  • Turbidity reaction Figure 4; when there is only binding antibody but no neutralizing antibody in the test sample, the RBD multimeric fragment binds to ACE2 in a turbidity reaction ( Figure 3); when neutralizing antibody is present in the test sample, neutralizing antibody And the antibody binds to the RBD multimeric fragment, blocking the binding of the RBD multimeric fragment to ACE2, thereby inhibiting the occurrence of the turbidity reaction ( Figure 2).
  • the method for realizing anti-coronavirus antibody detection on the immunoturbidimetric platform provided by the present disclosure can be implemented in industry, and the detection components provided by the present disclosure can also be mass-produced in industry, and they are used in antibody detection or preparation of antibody detection reagents.
  • the application has the advantages of universal application, low cost and fast detection.

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Abstract

本申请提供了免疫比浊检测冠状病毒抗体的方法和试剂盒，其中使用多聚体RBD或多倍体RBD在胶乳平台实现中和抗体的检测。

Description

一种抗体检测方法
相关申请的交叉引用
本公开要求于2021年02月05日提交中国专利局的申请号为202110160942.6、名称为“一种抗体检测方法”的中国专利申请的优先权，其全部内容通过引用结合在本公开中。
技术领域
本公开涉及生物技术领域，具体而言，涉及一种抗体检测方法。
背景技术
在病原微生物侵染宿主细胞的过程中，病毒表面受体结合蛋白是病原微生物进入细胞的关键“钥匙”，可以打开细胞受体蛋白的“锁”，从而进入细胞并启动病原微生物的复制过程。
中和抗体是B淋巴细胞产生的某些抗体，能够与病原微生物表面的抗原(配体)结合，从而阻止该病原微生物黏附于靶细胞受体，防止病原微生物侵入宿主细胞。
目前，中和抗体检测主要在生物安全三级实验室(BSL3)内进行。利用活病毒细胞培养的中和试验检测，对实验室等级要求高，严重限制了临床大规模的推广使用。开发出可在生物安全二级实验室(BSL2)或普通实验室开展的快速中和抗体检测方法，具有重要意义。
SARS-CoV-2表面棘突S蛋白(spike protein)是冠状病毒表面重要的受体结合位点，其可与宿主细胞表面病毒特异性受体结合、介导病毒外膜与宿主细胞融合、病毒吸附及穿膜；冠状病毒S蛋白受体结合域(receptor binding domain，RBM)可使病毒与宿主细胞表面的血管紧张素转化酶2(angiotensin-converting enzyme2，ACE2)结合，并使病毒进入宿主细胞。当SARS-CoV-2在侵入机体时，会刺激机体产生具有保护作用的中和抗体，专门阻止病原微生物进入宿主细胞，防止感染。中和抗体的多少是疫苗免疫保护效果的一项重要指标，是疫苗评估和质控的重要依据。
个体接种疫苗后可通过免疫应答产生保护性抗体，即中和抗体，对中和抗体进行滴度测定，可以判断疫苗的临床疗效。因此，对中和抗体的检测，可应用于疫苗的研发评价，以及疫苗接种后，对个体自身免疫效果的评价。另外，对于治愈后的新型冠状病毒患者的中和抗体的检测，可以判断是否存在再次感染的风险。
随着新型冠状病毒疫苗的推广接种，现在急需开发方便、快捷且无需在BSL3实验室条件下进行总抗体和中和抗体检测的试剂，现有开发的产品都是在ELISA和发光平台上检 测的，但是ELISA和发光平台对设备要求高，大部分三甲以下的医院或者社康诊所都没有配套仪器，而生化比浊检测有应用普遍、成本低、检测快的优势，开发能够在免疫比浊平台实现中和抗体检测的方法和产品具有重要意义。
发明内容
本公开提供了以下至少一种实施方式：
在一种或多种实施方式中，本公开涉及一种在免疫比浊平台实现抗冠状病毒抗体检测的方法，方法包括：
1)样品与含有RBD聚体或多倍体的试剂接触；
2)再与偶联有胶乳的ACE2或其片段的试剂接触。
在一种或多种实施方式中，检测方法用于检测样品中是否存在中和抗体。
在一种或多种实施方式中，冠状病毒选自SARS-CoV-2或SARS-CoV。
在一种或多种实施方式中，样品选自可能含有抗体的任何样品。
在一种或多种实施方式中，样品选自血清、血浆、全血、淋巴液、脑脊液、组织液、唾液、尿液或淋巴细胞中的一种或多种。
在一种或多种实施方式中，RBD聚体或多倍体可以是对RBD序列进行两个以上串联表达得到或通过化学偶联的方式将两个以上的RBD蛋白聚合得到。
在一种或多种实施方式中，检测组件，该检测组件包括：含有RBD聚体或多倍体的试剂和偶联有胶乳的ACE2或其片段的试剂。
在一种或多种实施方式中，检测组件中含有RBD聚体或多倍体的试剂，RBD聚体或多倍体可以是对RBD序列进行两个以上串联表达得到或通过化学偶联的方式将两个以上的RBD蛋白聚合得到。
在一种或多种实施方式中，检测组件用于检测样品中是否存在中和抗体。
在一种或多种实施方式中，检测组件是用于检测抗冠状病毒的中和抗体，在一种或多种实施方式中，检测的是抗SARS-CoV-2的中和抗体，在一种或多种实施方式中，检测的是抗SARS-CoV的中和抗体。
在一种或多种实施方式中，SARS-CoV-2包括其野生型及变异型，SARS-CoV包括其野生型及变异型。
在一种或多种实施方式中，ACE2包括来自任何物种的ACE2亚型、片段、变体(包括突变体)、或同系物。
在一种或多种实施方式中，ACE2是哺乳动物的ACE2。哺乳动物包括兽亚纲、真哺乳亚纲、真兽亚纲、原兽亚纲、统兽总目、或灵长目。
在一种或多种实施方式中，ACE2是人、蝙蝠、穿山甲、麝猫、或猪的ACE2。
在一种或多种实施方式中，以上任一实施方式所述的方法、以上任一实施方式所述的检测组件在抗体检测或制备抗体检测试剂中的应用。
附图说明
图1示出了RBD聚体或多倍体的SDS-PAGE图，1：RBD单体；2：RBD二聚体。
图2示出了当样品中存在中和抗体时的反应过程示意图，结果是不能产生有效浊度。
图3示出了当样品中存在结合抗体时的反应过程示意图，结果是产生了有效的浊度。
图4示出了当样品中不存在抗体时的反应过程示意图，结果是产生了有效的浊度。
具体实施方式
现将详细地提供本公开实施方式的参考，其一个或多个实例描述于下文。提供每一实例作为解释而非限制本公开。实际上，对本领域技术人员而言，显而易见的是，可以对本公开进行多种修改和变化而不背离本公开的范围或精神。例如，作为一个实施方式的部分而说明或描述的特征可以用于另一实施方式中，来产生更进一步的实施方式。
本公开的一种或多种实施例能够在免疫比浊平台实现抗冠状病毒抗体检测。
本文中，术语“免疫比浊平台”是指是抗原抗体结合动态测定方法。其基本原理是:当抗原与抗体在特殊稀释系统中特异性反应而且比例合适(一般规定抗体过量)时，形成的可溶性免疫复合物在稀释系统中的促聚剂(聚乙二醇等)的作用下，自液相析出，形成微粒，使反应液出现浊度。当抗体浓度固定时，形成的免疫复合物的量随着检样中抗原量的增加而增加，反应液的浊度也随之增加。通过测定反应液的浊度与一系列标准品对照，即可计算出检样中抗原的含量。
本文中，术语“中和抗体”指的是B淋巴细胞产生的某些抗体，这些抗体能够与病原微生物表面的抗原结合，从而阻止该病原微生物黏附靶细胞受体，防止侵入细胞。
本文中，“RBD聚体或多倍体”指的是具有多个RBD结构域的蛋白，可以是通过基因重组技术构建多拷贝RBD载体，表达得到串联有多个RBD结构域的重组蛋白；也可以通过重组技术得到单个RBD蛋白再通过化学偶联的方式得到多聚体形式的蛋白；其特点是新形成的分子结构中具有两个以上的RBD结构域。
本文中，“样品”可理解为可能含有抗体的任何样品，在一种或多种实施方式中，样品来自于感染或主动免疫后的样品；在一种或多种实施方式中，样品选自体液、排泄物、细胞；例如血清、血浆、全血、淋巴液、脑脊液、组织液、唾液、尿液、淋巴细胞等，但不限于此。
本文中，术语“片段”指的是至少含有结合区段的氨基酸片段。
本文中，“接触”可理解为允许其发生结合。对接触时间不作具体限定，不同的实施方 式和检测平台，接触时间有所差异，但属于本领域技术人员可以理解的范畴。
本文中，“配体”可理解为能够与受体结合的任何蛋白或多肽。“受体”可理解为能够识别配体并特异性结合配体的分子，配体受体结合能够实现信号传导，受体可以包括辅助性受体。常见的细胞表面受体包括但不限于G蛋白偶联受体、受体酪氨酸激酶、鸟苷酸环化酶偶联受体、离子通道、黏附受体等。配体与受体结合的难易度与结合后的强度叫做亲和力。两者越容易结合，结合后结合的强度越大，则亲和力越强，反之亦然。
本文中，“试剂”可以理解为物质或产品等，不限其形式或状态，可以是液态也可以是固态。
本文中，ACE2不限定物种，根据待检样品的物种来源可以选择不同物种的ACE2，例如哺乳动物、非冷血飞行动物、蝙蝠和鸟类等，在一种或多种实施方式中，使用人的ACE2，进行人样品的冠状病毒中和抗体检测。
在一种或多种实施方式中，本公开提供在免疫比浊平台实现冠状病毒抗体检测的方法，包括：
1)样品与含有RBD聚体或多倍体的试剂接触；
2)再与偶联有胶乳的ACE2或其片段的试剂接触。
在一种或多种实施方式中，上述方法检测的是中和抗体，即上述方法用于检测样品中是否存在中和抗体。
在一种或多种实施方式中，冠状病毒是指SARS-CoV-2或SARS-CoV。SARS-CoV-2包括其野生型及变异型。SARS-CoV包括其野生型及变异型。
在一种或多种实施方式中，RBD聚体或多倍体可以是对RBD序列进行两个以上串联表达得到或通过化学偶联的方式将两个以上的RBD蛋白聚合得到。
在一种或多种实施方式中，RBM的序列可参考文献Wrapp Daniel,Wang Nianshuang,Corbett Kizzmekia S,等人Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation，以及与其序列一致性至少70％、75％、80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的氨基酸序列，且这些氨基酸序列仍具有配体结合受体和/或抗体的功能。
在一种或多种实施方式中，“ACE2”是指任何物种的ACE2，且包括得自任何物种的ACE2亚型、片段、变体(包括突变体)、或同系物。在一种或多种实施方式中，ACE2是哺乳动物(例如，兽亚纲、真哺乳亚纲、真兽亚纲、原兽亚纲、统兽总目、灵长目(猴、非人灵长类、或人))的ACE2。在一种或多种实施方式中，ACE2是人、蝙蝠、穿山甲、麝猫、或猪的ACE2。在一种或多种实施方式中，ACE2是人的ACE2，序列可参考Gene ID:59272，以及与其序列一致性至少70％、75％、80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的氨基酸序列，且这些氨基酸序列仍具有受体结合配体的功 能。
在一种或多种实施方式中，本公开还涉及检测组件，该检测组件包括：含有RBD聚体或多倍体的试剂和偶联有胶乳的ACE2或其片段的试剂。
在一种或多种实施方式中，含有RBD聚体或多倍体的试剂或偶联有胶乳的ACE2或其片段的试剂还独立地含有缓冲剂(或缓冲液)。缓冲剂(或缓冲液)包括：弱酸及其盐的混合溶液；或，弱碱及其盐的混合溶液。例如，缓冲剂(或缓冲液)可以是Tris缓冲液、PBS缓冲液、碳酸盐缓冲液、硼酸盐缓冲液等等。在一种或多种实施方式中，含有RBD聚体或多倍体的试剂或偶联有胶乳的ACE2或其片段的试剂还可以独立地包括一种或多种稳定剂，稳定剂通常为体外检测试剂常用的稳定剂(助于试剂保存或检测)，诸如，氨基酸类物质，例如甘氨酸等；蛋白类物质，例如BSA(牛血清白蛋白)、酪蛋白等；糖类物质，例如蔗糖、葡萄糖等；表面活性剂，例如吐温、TRITON等。在一种或多种实施方式中，所述试剂还可以独立地包括防腐剂，例如叠氮钠。
在一种或多种实施方式中，检测组件中RBD聚体或多倍体可以是对RBD序列进行两个以上串联表达得到或通过化学偶联的方式将两个以上的RBD蛋白聚合得到。
在一种或多种实施方式中，检测组件用于检测样品中是否存在中和抗体。
在一种或多种实施方式中，检测组件是用于检测抗冠状病毒的中和抗体，在一种或多种实施方式中，检测的是抗SARS-CoV-2的中和抗体，在一种或多种实施方式中，检测的是抗SARS-CoV的中和抗体。
本公开还涉及以上任一实施方式所述的检测方法，或以上任一实施方式所述的检测组件在抗体检测或制备抗体检测试剂中的应用。
以检测抗SARS-COV-2的中和抗体为例说明本公开的原理：
(1)样品与含有RBD聚体或多倍体的试剂接触，样品中的中和抗体和总抗体分别与RBD结合形成中和抗体-RBD复合物以及总抗体-RBD复合物；
(2)加入偶联有胶乳的ACE2的试剂，ACE2能够与总抗体-RBD复合物进一步结合形成复合物，而不与中和抗体-RBD复合物结合。
结合有中和抗体的RBD多聚体或多倍体不能够再结合偶联有胶乳的ACE2，从而不能够发生浊度反应。当样品中没有中和抗体时，RBD多聚体或多倍体能够与偶联有胶乳的ACE2结合，发生浊度反应，样品中的中和抗体含量与浊度反应信号值成反比关系，从而可以定量样品中的中和抗体。
本申请的各方面和实施方式将参照附图和以下实施例进行讨论。其他方面和实施方式对于本领域技术人员是清楚的。尽管本申请已经结合示例性实施方式进行了描述，很多等同修改和变化在给出本申请时对于本领域技术人员是清楚的。因而，本申请的示例性实施方式是示例性的，非限制性的。可以对所述实施方式做出多种变化，而不脱离本申请的宗 旨和范围。
以下实施例中使用到的重组人ACE2、重组RBD抗原(RBD多聚片段)、重组RBD抗原(RBD单体片段)、抗新型冠状病毒的中和抗体标准品(G3)、胶乳微球均购自广东菲鹏生物有限公司。所用仪器为全自动生化分析仪，是市面上任意的常规生化分析仪。
实施例1 ACE2偶联胶乳微球
1)390μL交联液(50mM HEPES，pH6.1)，加入100μL胶乳微球(JSR)，在摇床上震荡混匀，为胶乳微球液；
2)胶乳微球液中加入13μL的EDAC溶液，37℃震荡活化1小时；
3)加入50μL的ACE2/RBD(2mg/mL)到胶乳微球液中，37℃震荡交联4小时；
4)加入50μL的TW-20溶液，37℃震荡封闭1小时；
5)将交联液体加入到离心管中，离心后，小心用移液枪弃上清；
6)加入2mL清洗液(50mM甘氨酸+0.1％NaN 3，pH8.0)，旋涡震荡，再离心一次，小心弃上清；
7)加入5mL胶乳微球悬浮液(50mM甘氨酸+0.1％BSA+10％蔗糖+0.1％NaN 3，pH8.0)，超声分散处理。
实施例2标准品制备
用基质血清将中和抗体标准品稀释成以下不同浓度：0ng/mL、50ng/mL、100ng/mL、200ng/mL、500ng/mL、1000ng/mL、2000ng/mL、4000ng/mL、8000ng/mL。
实施例3样品稀释液制备
RBD多聚片段和RBD单体片段均购置广东菲鹏生物有限公司，其SDS-PAGE电泳结果如图1所示，从分子量上可以确定泳道2是RBD多聚体片段。
3.1用重组RBD抗原(RBD多聚片段)配置如下不同浓度的样品稀释液，稀释液基本配方是：Tris缓冲液
  稀释液1 稀释液2 稀释液3
抗原浓度 40μg/mL 25μg/mL 10μg/mL
3.2用重组RBD抗原(RBD单体片段)配置如下不同浓度的样品稀释液，稀释液基本配方是：Tris缓冲液
  稀释液4 稀释液5 稀释液6
抗原浓度 40μg/mL 25μg/mL 10μg/mL
实施例4上机测试
仪器：全自动生化分析仪
检测流程：吸取样品盘上的样品5μL，同时吸取R1 135μL，加入比色杯中，机械搅拌，37℃下孵育5分钟；在比色杯中加入R2 15μL，机械搅拌，37℃下孵育8分钟；在比色杯中加入R3 50μL，机械搅拌，37℃下孵育10分钟。仪器记录下从加入样品开始算起的15分30秒和25分钟30秒的吸光度值，差值就是样品的反应吸光度，吸光度和样品中中和抗体浓度在特定条件下负相关。
加样比S:R1:R2:R3＝5:135:15:50
波长：570nm
空白时间：104-104
反应时间：172-172
R1：Tris缓冲液
R2：样品稀释液
R3：ACE2偶联胶乳微球试剂
表1检测样品中的中和抗体
Figure PCTCN2021086008-appb-000001
表2中和抗体抑制率计算结果
Figure PCTCN2021086008-appb-000002
Figure PCTCN2021086008-appb-000003
分析表1和表2数据，使用RBD聚体配置的稀释液1-稀释液3可以产生明显的浊度反应，且浊度反应与中和抗体的量成相关关系，而用RBD单体配置的稀释液4-稀释液6不能产生浊度反应。
本申请人进一步研究了采用本公开方法的检测原理，RBD多聚片段至少包含有2个以上的位点可以结合ACE2，当检测样品中不存在中和抗体时，RBD多聚片段和ACE2结合发生浊度反应(图4)；当检测样品中仅有结合抗体但是没有中和抗体时，RBD多聚片段和ACE2结合发生浊度反应(图3)；当检测样品中存在中和抗体时，中和抗体与RBD多聚片段结合，阻断了RBD多聚片段与ACE2的结合，从而抑制浊度反应的发生(图2)。
以上所述仅为本公开的典型实施例而已，并不用于限制本公开，对于本领域的技术人员来说，本公开可以有各种更改和变化。凡在本公开的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本公开的保护范围。
工业实用性
本公开提供的在免疫比浊平台实现抗冠状病毒抗体检测的方法能够在工业上实施，且本公开提供的检测组件也能够在工业上批量生产，并且它们在抗体检测或制备抗体检测试剂中的应用具有应用普遍、成本低、检测快的优势。

Claims (20)

  1. 一种在免疫比浊平台实现抗冠状病毒抗体检测的方法，所述方法包括：
    1)样品与含有RBD聚体或多倍体的试剂接触；
    2)再与偶联有胶乳的ACE2或其片段的试剂接触。
  2. 根据权利要求1所述的方法，其中，所述方法用于检测样品中是否存在中和抗体。
  3. 根据权利要求1或2所述的方法，其中，所述冠状病毒选自SARS-CoV-2或SARS-CoV。
  4. 根据权利要求3所述的方法，其中，所述SARS-CoV-2包括其野生型及变异型，所述SARS-CoV包括其野生型及变异型。
  5. 根据权利要求1-4中任一项所述的方法，其中，样品选自可能含有抗体的任何样品；
    可选的，所述样品选自血清、血浆、全血、淋巴液、脑脊液、组织液、唾液、尿液或淋巴细胞。
  6. 根据权利要求1-5中任一项所述的方法，其中，RBD聚体或多倍体可以是对RBD序列进行两个以上串联表达得到或通过化学偶联的方式将两个以上的RBD蛋白聚合得到。
  7. 根据权利要求1-6中任一项所述的方法，其中，ACE2包括来自任何物种的ACE2亚型、片段、变体或同系物。
  8. 根据权利要求1-7中任一项所述的方法，其中，ACE2是哺乳动物的ACE2。
  9. 根据权利要求8所述的方法，其中，哺乳动物包括兽亚纲、真哺乳亚纲、真兽亚纲、原兽亚纲、统兽总目、或灵长目。
  10. 根据权利要求1-7中任一项所述的方法，其中，ACE2是人、蝙蝠、穿山甲、麝猫、或猪的ACE2。
  11. 检测组件，所述检测组件包括：含有RBD聚体或多倍体的试剂和偶联有胶乳的ACE2或其片段的试剂。
  12. 根据权利要求11所述的检测组件，其中，RBD聚体或多倍体可以是对RBD序列进行两个以上串联表达得到或通过化学偶联的方式将两个以上的RBD蛋白聚合得到。
  13. 根据权利要求11或12所述的检测组件，其中，检测组件用于检测样品中是否存在中和抗体。
  14. 根据权利要求11-13中任一项所述的检测组件，其中，所述检测组件用于检测 抗冠状病毒的中和抗体；
    可选的，检测组件用于检测抗SARS-CoV-2的中和抗体或抗SARS-CoV的中和抗体。
  15. 根据权利要求14所述的检测组件，其中，所述SARS-CoV-2包括其野生型及变异型，所述SARS-CoV包括其野生型及变异型。
  16. 根据权利要求11-15中任一项所述的检测组件，其中，ACE2包括来自任何物种的ACE2亚型、片段、变体、或同系物。
  17. 根据权利要求11-16中任一项所述的检测组件，其中，ACE2是哺乳动物的ACE2。
  18. 根据权利要求17所述的检测组件，其中，哺乳动物包括兽亚纲、真哺乳亚纲、真兽亚纲、原兽亚纲、统兽总目、或灵长目。
  19. 根据权利要求1-16中任一项所述的检测组件，其中，ACE2是人、蝙蝠、穿山甲、麝猫、或猪的ACE2。
  20. 权利要求1-10中任一项所述的方法、权利要求11-19中任一项所述的检测组件在抗体检测或制备抗体检测试剂中的应用。
PCT/CN2021/086008 2021-02-05 2021-04-08 一种抗体检测方法 WO2022165982A1 (zh)

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