WO2022032497A1 - 用于检测冠状病毒中和抗体的试剂盒、方法 - Google Patents

用于检测冠状病毒中和抗体的试剂盒、方法 Download PDF

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WO2022032497A1
WO2022032497A1 PCT/CN2020/108534 CN2020108534W WO2022032497A1 WO 2022032497 A1 WO2022032497 A1 WO 2022032497A1 CN 2020108534 W CN2020108534 W CN 2020108534W WO 2022032497 A1 WO2022032497 A1 WO 2022032497A1
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protein
coronavirus
converting enzyme
human angiotensin
labeled
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PCT/CN2020/108534
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English (en)
French (fr)
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李可
何建文
于丽娜
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深圳迈瑞生物医疗电子股份有限公司
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    • C—CHEMISTRY; METALLURGY
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • 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 present invention relates to the field of neutralizing antibody determination, in particular to the determination of the neutralizing antibody of coronavirus.
  • Coronaviruses are a class of enveloped, single positive-stranded RNA viruses that exist in humans and other mammals, as well as birds, and cause respiratory, digestive, liver, and nervous system diseases. Six coronaviruses have been reported to cause human diseases, such as colds, Middle East Respiratory Syndrome (MERS) and Severe Acute Respiratory Syndrome (SARS).
  • MERS Middle East Respiratory Syndrome
  • SARS Severe Acute Respiratory Syndrome
  • the new coronavirus (SARS-CoV-2) is a newly discovered coronavirus belonging to the genus ⁇ .
  • SARS-CoV-2 causes novel coronavirus pneumonia (Corona Virus Disease 2019, COVID-19).
  • Common signs of COVID-19 include respiratory symptoms, fever, cough, shortness of breath, and difficulty breathing. In more severe cases, the infection can lead to pneumonia, severe acute respiratory syndrome, kidney failure, and even death.
  • IVIG immunoglobulin for injection
  • knowing the level of SARS-CoV-2 neutralizing antibody in IVIG and the level of SARS-CoV-2 neutralizing antibody in healthy plasma donors from which it is derived can evaluate whether IVIG has a specific anti-SARS-CoV-2 effect, and at the same time Laboratory supporting data may also be provided for clinical use.
  • the expert consensus on the diagnosis and treatment of severe and critical new coronavirus pneumonia recommends: "The use of human convalescent plasma containing new coronavirus antibodies for patients with rapid disease progression, severe and critical illness can be used as a specific treatment. Select.
  • the level of protective antibody titers in plasma should be measured.” That is to say, the detection of neutralizing antibodies is of great significance for the diagnosis and treatment of SARS-CoV-2. At the same time, the determination of neutralizing antibodies can also be used to evaluate the effect of related virus vaccines.
  • a neutralizing antibody detection method based on SARS-CoV-2 pseudovirus has been developed recently.
  • the Spike gene sequence is cloned into a vector, and a replication-defective virus is constructed. Inhibition rate of infection to achieve neutralizing antibody detection.
  • the above pseudovirus-based detection method avoids the use of biosafety level 3 laboratories, it still has certain requirements for safety levels, such as the need to use biosafety level 2 laboratories, and the method involves cell culture, pseudovirus construction and other operations , still has high complexity, professionalism and danger.
  • kits for determining a coronavirus neutralizing antibody comprising:
  • a capture component comprising a solid-phase carrier coated with a protein having the RBD sequence of the S1 subunit of the spike protein of the coronavirus;
  • Human angiotensin-converting enzyme II with a detectable signal label.
  • the detectable signal marker is labeled on human angiotensin-converting enzyme II
  • the human angiotensin-converting enzyme II labeled with the detectable signal marker is used as the detection group in the kit point.
  • the coating on the solid phase carrier is combined with the neutralizing antibodies in the sample; at the same time, a signal marker labeled human angiotensin-converting enzyme II is added. , which competes with neutralizing antibodies in the sample or prevents the binding of human angiotensin-converting enzyme II labeled with a signaling marker to the binding site on the RBD.
  • the content of neutralizing antibody in the sample is determined by the level of the signal value generated by the signal marker, and the content of neutralizing antibody in the sample is inversely proportional to the level of the signal value.
  • the protein-coated solid-phase carrier can be configured as a capture component in a conventional manner (for example, in a suitable buffer), as long as it is suitable for the preservation of the solid-phase carrier and the coating and does not affect the progress of the reaction That's it.
  • the protein-coated solid support may be present, for example, at a concentration of 0.2 mg/mL to 1.0 mg/mL, such as 0.3 mg/mL, 0.4 mg/mL, 0.5 mg/mL, 0.6 mg/mL It is present at concentrations of mg/mL, 0.7 mg/mL, 0.8 mg/mL, 0.9 mg/mL.
  • the solid support is present at a concentration of 0.5 mg/mL to 1.0 mg/mL.
  • the solid support is present at a concentration of 0.5 mg/mL.
  • human angiotensin-converting enzyme II can be present in a conventional manner (eg, in a suitable buffer), as long as it is suitable for the preservation of human angiotensin-converting enzyme II and does not affect the progress of the reaction.
  • human angiotensin-converting enzyme II may for example be present at a concentration of 0.5 ⁇ g/mL to 2 ⁇ g/mL, such as 0.6 ⁇ g/mL, 0.7 ⁇ g/mL, 0.8 ⁇ g/mL, 0.9 ⁇ g/mL mL, 1 ⁇ g/mL, 1.1 ⁇ g/mL, 1.2 ⁇ g/mL, 1.3 ⁇ g/mL, 1.4 ⁇ g/mL, 1.5 ⁇ g/mL, 1.6 ⁇ g/mL, 1.7 ⁇ g/mL, 1.8 ⁇ g/mL, 1.9 ⁇ g/mL concentration exists.
  • human angiotensin-converting enzyme II is present at a concentration of 1 ⁇ g/mL to 2 ⁇ g/mL. In a specific embodiment, human angiotensin-converting enzyme II is present at a concentration of 1 ⁇ g/mL.
  • the capture component and human angiotensin-converting enzyme II can be separately packaged in the kit respectively.
  • a method for determining neutralizing antibodies in a sample from a subject comprising:
  • the capture component, human angiotensin converting enzyme II, and the sample are mixed and incubated for a period of time such that the human angiotensin converting enzyme II and neutralizing antibodies in the sample can compete for binding to the capture component ;
  • the obtained mixture is washed to remove unbound substances
  • the capture component contains a solid-phase carrier coated with a protein, and the protein has the RBD sequence of the spike protein S1 subunit of the coronavirus;
  • the detectable signal marker is labeled on human angiotensin-converting enzyme II.
  • the mixing and incubating for a period of time is first mixing the capture component with the sample and incubating for a period of time, and then adding human angiotensin-converting enzyme II, mixing and incubating for a period of time.
  • the mixing and incubating for a period of time is the simultaneous addition of the capture component, human angiotensin-converting enzyme II and the sample, mixing and incubation for a period of time.
  • incubation for a period of time refers to incubation for at least 5 min; for example, 10 min, 15 min, 20 min. In a specific embodiment, the incubation can be performed for 10 min.
  • the present invention provides a kit for determining a coronavirus neutralizing antibody, comprising:
  • a capture component comprising a solid-phase carrier coated with a protein, and the protein has the RBD sequence of the S1 subunit of the spike protein of the coronavirus;
  • a secondary antibody labeled with a detectable signal label is provided.
  • the detectable signal marker is labeled on the secondary antibody
  • the secondary antibody labeled with the detectable signal marker is used as the detection component in the kit; while human angiotensin-converting enzyme II as a competing component.
  • the added human angiotensin converting enzyme II competes with the neutralizing antibodies in the sample for binding to the coating coated on the solid support.
  • Antibodies that specifically bind to human antibodies are then added.
  • a solid-phase carrier-coating material-neutralizing antibody (primary antibody)-secondary antibody combination is further formed, and a signal is generated by the signal label on the secondary antibody.
  • the content of neutralizing antibody in the sample is determined by the level of the signal value, and the content of neutralizing antibody in the sample is proportional to the level of the signal value.
  • the protein-coated solid-phase carrier can be configured as a capture component in a conventional manner (for example, in a suitable buffer), as long as it is suitable for the preservation of the solid-phase carrier and the coating and does not affect the progress of the reaction That's it.
  • the protein-coated solid support may be present, for example, at a concentration of 0.5 to 1.2 mg/mL, such as 0.6 mg/mL, 0.7 mg/mL, 0.8 mg/mL, 0.9 mg/mL , 1.0 mg/mL, 1.1 mg/mL at concentrations.
  • the protein-coated solid support is present at a concentration of 0.5 mg/mL to 1.0 mg/mL.
  • the protein-coated solid support is present at a concentration of 0.7 mg/mL.
  • human angiotensin-converting enzyme II can be present in a conventional manner (eg, in a suitable buffer), as long as it is suitable for the preservation of human angiotensin-converting enzyme II and does not affect the progress of the reaction.
  • human angiotensin-converting enzyme II may for example be present at a concentration of 0.2 ⁇ g/mL to 1.5 ⁇ g/mL, such as 0.3 ⁇ g/mL, 0.4 ⁇ g/mL, 0.5 ⁇ g/mL, 0.6 ⁇ g It is present at concentrations of /mL, 0.7 ⁇ g/mL, 0.8 ⁇ g/mL, 0.9 ⁇ g/mL, 1 ⁇ g/mL, 1.1 ⁇ g/mL, 1.2 ⁇ g/mL, 1.3 ⁇ g/mL, 1.4 ⁇ g/mL.
  • human angiotensin-converting enzyme II may be present at a concentration of 0.8 ⁇ g/mL to 1.2 ⁇ g/mL. In a specific embodiment, human angiotensin-converting enzyme II is present at a concentration of 1 ⁇ g/mL.
  • the capture component, human angiotensin-converting enzyme II, and the secondary antibody can be separately packaged in the kit respectively.
  • a method for determining neutralizing antibodies in a sample from a subject comprising:
  • the capture component, human angiotensin converting enzyme II, and the sample are mixed and incubated for a period of time such that the human angiotensin converting enzyme II and neutralizing antibodies in the sample can compete for binding to the capture component ;
  • the obtained mixture is washed to remove unbound substances
  • the capture component comprises a solid-phase carrier coated with a protein
  • the protein has the RBD sequence of the S1 subunit of the spike protein of the coronavirus.
  • incubation for a period of time refers to incubation for at least 5 min; for example, 10 min, 15 min, 20 min. In an exemplary embodiment, the incubation is performed for 5 min to 20 min. In a specific embodiment, the incubation may be performed for 5 min.
  • the present invention provides the use of a coating in the preparation of a detection reagent for a coronavirus neutralizing antibody, wherein the coating is an RBD sequence having the spike protein S1 subunit of the coronavirus. protein,
  • the coating material is coated on a solid phase carrier to constitute a capture component.
  • the present invention provides the use of a coating and human angiotensin-converting enzyme II in the preparation of a detection reagent for a coronavirus neutralizing antibody, wherein the coating is a spike with the coronavirus
  • the coating is coated on a solid phase carrier to constitute a capture component; the human angiotensin converting enzyme II can compete with the neutralizing antibody to compete with the capture component.
  • the scheme of the present invention to detect the neutralizing antibody of the coronavirus does not need to use live virus, and also does not need to construct pseudoviruses or culture cells, thereby improving the detection safety and reducing the complexity; on the other hand, the present invention
  • the protocol can obtain detection results that are highly correlated with the virus neutralization test.
  • Figure 1 shows the calibration curve for the detection of SARS-CoV-2 neutralizing antibodies using the kit prepared in Example 1;
  • Figure 2 shows the calibration curve for the detection of SARS-CoV-2 neutralizing antibodies using the kit prepared in the comparative example
  • Fig. 3 shows the consistency analysis result of the detection result of the kit prepared in Example 1 and the virus neutralization test
  • Fig. 4 shows the consistency analysis result of the detection result of the kit prepared by the comparative example and the virus neutralization test
  • Fig. 5 shows the consistency analysis result of the test result and the virus neutralization test under the situation of adopting the sample adding method 2 in Example 3;
  • Fig. 6 shows the consistency analysis result of the detection result of the kit prepared in Example 6 and the virus neutralization test
  • Figure 7 shows the calibration curve for detecting SARS-CoV-2 neutralizing antibodies using the kit prepared in Example 8;
  • Fig. 8 shows the consistency analysis result of the detection result of the kit prepared in Example 8 and the virus neutralization test
  • FIG. 9 shows the consistency analysis results between the detection results of the kit prepared in Example 12 and the virus neutralization test.
  • the detection of neutralizing antibodies in blood is useful in assessing the effectiveness of vaccines.
  • the neutralizing antibody detection of coronavirus adopts virus neutralization test or needs to construct a pseudovirus. Therefore, the detection of neutralizing antibody is a complex and dangerous process that requires a high degree of professionalism.
  • the present invention provides a neutralizing antibody detection kit with high correlation with the detection results of the virus neutralization test, thereby providing a relatively simple, efficient and safe method for the detection of the neutralizing antibody of the coronavirus.
  • the detection of the kit of the present invention has a high correlation, thereby providing an accurate coronavirus Detection of neutralizing antibodies.
  • the present invention provides a kit for measuring coronavirus neutralizing antibodies, comprising:
  • a capture component comprising a solid-phase carrier coated with a protein having the RBD sequence of the S1 subunit of the spike protein of the coronavirus;
  • a detectable signaling marker is labeled on the human angiotensin-converting enzyme II.
  • the detectable signaling marker is labeled on the secondary antibody, in which case it is understood that human angiotensin-converting enzyme II is not labeled with the detectable signaling marker.
  • the terms “having”, “comprising” or “including” or any grammatical variant thereof are used in a non-exclusive manner. Accordingly, these terms may refer to both situations in which no additional features are present in the entity described in this context other than the features introduced by the terms, and situations in which one or more additional features are present.
  • the expressions "A has B,” “A includes B,” and “A includes B” can both refer to situations in which no additional elements other than B are present in A (ie, situations in which B alone and exclusively consists of B). ), and may refer to situations where, in addition to B, there are one or more additional elements in entity A such as element C, elements C and D, or even other elements.
  • kits refers to a collection of components or reagents for detection of the present invention that may or may not be packaged together.
  • the components of the kit may be contained in separate vials (kits of separate parts) or provided in a single vial.
  • the kits of the present invention are useful in practicing the methods described herein.
  • all ingredients are provided in a ready-to-use manner in order to practice the methods herein.
  • the kit contains instructions for practicing the methods herein. Instructions can be provided in paper or electronic form from the user manual.
  • the manual may contain instructions for explaining the results obtained when the above-described methods are carried out using the kit of the invention.
  • a coronavirus refers to a virus belonging to the genus Coronavirus.
  • the coronaviruses of the invention are capable of infecting humans, eg, selected from the group consisting of SARS, MERS, SARS-CoV-2, and combinations thereof.
  • the coronavirus is SARS-CoV-2.
  • solid support is used interchangeably with “solid support”, “solid support” and “solid support”, which means that a coating can be bound and is suitable for, for example, physical means with Any suitable solid surface from which the sample is separated.
  • the solid phase carrier used in the present invention is not particularly limited, and commercial solid phase carriers and any solid phase carrier that can be used for immunological analysis can be used in the present invention.
  • Exemplary solid supports can be magnetic beads (eg, carboxyl magnetic beads), microtiter plates, plastic plates, plastic tubes, latex beads, agarose beads, glass, nitrocellulose membranes, nylon membranes, silica plates, or microarrays. chip, but the present invention is not limited to this.
  • the "capture component” comprises a solid phase carrier to which a coating is bound.
  • the capture component may also include suitable solutions or other components, for example, to facilitate preservation of the solid support and coating.
  • coating material refers to a substance coated on the solid phase carrier of the present invention.
  • Methods of binding coatings to solid surfaces are well known in the art and include, for example, binding by hydrophobic interactions, biotinylation and binding via immobilized streptavidin, covalent Binding, antibody-antigen interactions, etc., or a combination of these interactions.
  • the coating can be selected from the group consisting of the spike protein of the coronavirus, the spike protein S1 subunit of the coronavirus, the RBD of the spike protein S1 subunit of the coronavirus, and combinations thereof, which can of natural origin or produced by recombinant means.
  • a protein having the RBD sequence of the spike protein S1 subunit of the coronavirus refers to a protein comprising the RBD sequence and having the biological function of RBD, and such a protein can be obtained commercially, for example.
  • the RBD sequence of the spike protein S1 subunit of the coronavirus, the spike protein S1 subunit sequence of the coronavirus and the spike protein sequence of the coronavirus are for example recorded in scientific literature, or can be obtained from relevant databases (for example, GenBank/ EMBL/DDBJ).
  • the sequence of the RBD of the spike protein S1 subunit of SARS-CoV-2 can be selected from positions Arg319 to Ser541 in the coding sequence of the gene shown in NCBI accession number YP_009724390.1.
  • Human angiotensin-converting enzyme II is able to interact with the RBD of the S1 subunit of the coronavirus spike protein.
  • "human angiotensin-converting enzyme II” includes human angiotensin-converting enzyme II or an active fragment thereof, which may be of natural origin or produced by recombinant means.
  • the term "secondary antibody” refers to an antibody that can specifically bind to a neutralizing antibody.
  • it can be mouse anti-human antibody, rabbit anti-human antibody, goat anti-human antibody, sheep anti-human antibody, chicken anti-human antibody, but the present invention is not limited thereto.
  • the secondary antibody is a murine anti-human antibody.
  • the term "detectable signal label” refers to a substance having detectable properties, usually optical or/and enzymatic properties; however, the detectable properties may also be radioactive properties.
  • optical property refers to any property that can be detected by an optical instrument.
  • the optically detectable property may be or may comprise at least one property selected from the group consisting of color, reflectivity, refraction, transmission, emission, scattering, fluorescence, phosphorescence, diffraction, and polar chemical.
  • detecting an optically detectable property as used herein includes detecting the presence of a previously undetectable property, detecting the absence of a previously undetectable property, and detecting a quantitative change in a property, i.e. detecting a correlation with at least one optical property The degree of change is related to the change in signal strength.
  • the optically detectable property also involves electrochemiluminescence, which is also referred to as electrochemiluminescence.
  • enzymatic property relates to the property of an indicator that produces a detectable product from a substrate by biocatalysis.
  • the enzymatic property is at least one enzymatic activity selected from the group consisting of phosphatase activity, peroxidase activity, and glycosidase activity.
  • Typical substrates for enzymatic activity are well known in the art; for example, when alkaline phosphatase is used as a label, 3-(2-spiroadamantane)-4-methoxy-4-(3-phosphine can be used Acyl)-phenyl-1,2-dioxetane is used as a luminescent substrate, which is decomposed by alkaline phosphatase, and a phosphate group is removed to generate an unstable intermediate product, which passes through the molecular The internal electron transfer produces methyl meta-oxybenzoate anion, and when the methyl meta-oxybenzoate anion in the excited state returns to the ground state, chemiluminescence is generated.
  • the enzymatic activity produces a product having measurable optical properties as described above, and/or the enzymatic activity produces a product that can be measured electronically.
  • Exemplary detectable signal labels can be alkaline phosphatase, luminol, isoluminol, acridine esters, horseradish peroxidase, beta-galactosidase, ruthenium terpyridine, quantum dots (such as gold quantum dots, CdSe quantum dots, ZnCdSe quantum dots), fluorescent microspheres, but the present invention is not limited thereto.
  • the present invention can be applied to ELISA, chemiluminescence, electrochemiluminescence, POCT, immunochromatography, up-conversion luminescence, down-conversion luminescence, but the present invention Not limited to this.
  • the method of the present invention may comprise steps other than those specifically mentioned. Specifically, the step of providing a sample may be included, or the addition of other compounds to facilitate binding and detection may be included. Additionally, some or all of the steps may be assisted by automated equipment.
  • sample refers to a sample suspected of containing the analyte of the invention (neutralizing antibodies to coronaviruses).
  • the sample includes blood, plasma and/or serum. It will be appreciated that the sample may be further processed in order to carry out the methods of the present invention. Specifically, cells can be removed from a sample by methods and means known in the art. Furthermore, at least one analyte can be extracted and/or purified from the sample by methods and means known in the art.
  • sample may also refer to a preparation containing or suspected of containing at least one analyte, which is diluted, enriched, purified and/or extracted from the sample.
  • Step 1 Preparation of capture components: The SARS-CoV-2 spike protein S1-RBD protein (Beijing Yiqiao Biotechnology, 40159-V02H/40159-V05H1/40159-V08H5) was coated on the surface of magnetic beads (Thermo fisher) magnetic beads.
  • the specific coating method is as follows: first, the S1-RBD protein is pretreated, and the protective components in its buffer matrix are removed by dialysis. Coating was performed at a ratio of 0.5-40 ⁇ g of antigen per milligram of beads. During the reaction, the carboxyl group on the surface of the magnetic beads was coupled with the amino group of the S1-RBD protein under the catalysis of EDC/NHS. Take 20 mg of magnetic beads modified with carboxyl groups on the surface, ultrasonically disperse them in 10 mM MES buffer, add 80 mg of EDC and 120 mg of NHS, and after ultrasonically mix evenly, place on a shaker at 37 °C for 15 min.
  • S1-RBD was added to the treated magnetic beads according to the proportion, mixed well, and placed on a shaker at 37°C for 10-18 hours. After washing and blocking, magnetic beads coated with S1-RBD protein were prepared.
  • Step 2 Preparation of detection components: Human angiotensin-converting enzyme II (ACE2) (Beijing Yiqiao Bio, 10108-H02H) was coupled with alkaline phosphatase (Roche Life Science) to prepare an enzyme-labeled marker .
  • Step 3 Sample detection: In the first step, the sample (5 ⁇ L), magnetic beads coated with the SARS-CoV-2 spike protein S1-RBD protein and human angiotensin-converting enzyme II labeled with alkaline phosphatase ( ACE2) protein was added to the reaction tube and incubated, and the SARS-CoV-2 neutralizing antibody in the sample competed or prevented the human angiotensin-converting enzyme II (ACE2) protein or protein fragment labeled with alkaline phosphatase from SARS-CoV-2. 2 Spike protein S1-RBD protein binding site binding. After the reaction is complete, the magnetic field attracts the beads, washing away unbound material.
  • ACE2 human angiotensin-converting enzyme II
  • the chemiluminescence substrate solution was added to the reaction tube, and the luminescence substrate (3-(2-spiroadamantane)-4-methoxy-4-(3-phosphoryl)-phenyl-1, 2-Dioxetane, AMPPD) is decomposed by alkaline phosphatase, and a phosphate group is removed to generate an unstable intermediate product, which generates methyl m-oxybenzoate anion through intramolecular electron transfer, which is excited. Chemiluminescence occurs when the methyl meta-oxybenzoate anion returns from the excited state to the ground state. The number of photons produced in the reaction was then measured by a photomultiplier tube, and the amount of photons produced was inversely proportional to the content of SARS-CoV-2 neutralizing antibodies in the sample.
  • the calibration master curve is shown in Figure 1.
  • Step 1 Preparation of capture components: The SARS-CoV-2 spike protein S1-RBD protein (Beijing Yiqiao Biotechnology, 40159-V02H/40159-V05H1/40159-V08H5) was coated on the surface of magnetic beads (Thermo fisher) magnetic beads.
  • the specific coating method is as follows: first, the S1-RBD protein is pretreated, and the protective components in its buffer matrix are removed by dialysis. Coating was performed at a ratio of 0.5-40 ⁇ g of antigen per milligram of beads. During the reaction, the carboxyl group on the surface of the magnetic beads was coupled with the amino group of the S1-RBD protein under the catalysis of EDC/NHS. Take 20 mg of magnetic beads modified with carboxyl groups on the surface, ultrasonically disperse them in 10 mM MES buffer, add 80 mg of EDC and 120 mg of NHS, and after ultrasonically mix evenly, place on a shaker at 37 °C for 15 min.
  • S1-RBD was added to the treated magnetic beads according to the proportion, mixed well, and placed on a shaker at 37°C for 10-18 hours. After washing and blocking, magnetic beads coated with S1-RBD protein were prepared.
  • MES 2-morpholinoethanesulfonic acid
  • Step 3 Sample detection: In the first step, add the sample (5 ⁇ L), magnetic beads coated with the SARS-CoV-2 spike protein S1-RBD protein into the reaction tube and incubate, the SARS-CoV-2 antibody in the sample Binds to S1-RBD protein on the surface of magnetic beads. After the reaction is complete, the magnetic field attracts the beads, washing away unbound material.
  • the detection component that is, the mouse antibody specifically bound by the human IgG antibody labeled with the signal marker
  • the conjugate formed in the first step is bound.
  • the magnetic field attracts the magnetic beads. , to wash away unbound material.
  • the chemiluminescence substrate solution was added into the reaction tube, and the luminescence substrate (3-(2-spiroadamantane)-4-methoxy-4-(3-phosphoryl)-phenyl-1, 2-Dioxetane, AMPPD) is decomposed by alkaline phosphatase, and a phosphate group is removed to generate an unstable intermediate product, which generates methyl m-oxybenzoate anion through intramolecular electron transfer, which is excited. Chemiluminescence occurs when the methyl meta-oxybenzoate anion returns from the excited state to the ground state. The number of photons generated in the reaction is then measured by a photomultiplier tube, and the amount of photons generated is proportional to the content of SARS-CoV-2 IgG antibodies in the sample. The calibration curve is shown in Figure 2.
  • Example 2 Investigating the consistency of the neutralizing antibody detection kits of Example 1 and the comparative example and the detection results of the virus neutralization test
  • test kit prepared in Example 1 The test kit prepared in Comparative Example, and the virus neutralization test were respectively used to detect serum samples of 50 patients with COVID-19 in convalescence. The test results of the prepared kit were consistent with the virus neutralization test.
  • the virus neutralization test adopts the fixed virus dilution serum method.
  • the test needs to titrate the virus titer first. During the test, it is diluted to contain 100 TCID50 per unit dose, and then the serum to be tested is double-diluted with an equal amount of 100 TCID50/ml virus solution, and incubated at 37 °C for 2h after mixing. , inoculate a cell culture plate with each dilution, 0.1 ml per well, 5% CO 2 37°C incubator for 4 days, and record the number of CPE wells.
  • the neutralization value was calculated according to Karber's method, that is, the maximum dilution of antiserum when 50% of cells were protected.
  • Embodiment 3 investigates the influence of sample addition mode on detection result
  • sample addition methods are as follows: method 1, after adding the capture component and the detection component, and then incubate (ie, the situation in Example 1).
  • Method 2 Add the capture component for incubation first, and then add the detection component. The rest of the detection steps are the same as in Example 1.
  • Embodiment 4 investigates the influence of incubation time on detection result
  • sample detection In order to study the influence of incubation time on detection results in Example 1 "sample detection", different incubation times were used to test the signal values of different samples and the signal-to-noise ratio was calculated, and the remaining methods were the same as in Example 1.
  • sample 1 is a negative control that does not contain SARS-CoV-2 neutralizing antibodies; the titers log2(1:X) of neutralizing antibodies in samples 2 to 4 are determined by virus neutralization test, among which samples 2 to 4 have The maximum dilutions with 50% inhibition rate were 30, 363, and 757 in sequence. The results are shown in Table 1 below.
  • Example 5 Determination of the concentration of magnetic bead coating and enzyme-labeled human angiotensin-converting enzyme II (ACE2)
  • the capture component was prepared according to the description in Example 1, wherein the amount of S1-RBD used was fixed at 10 ⁇ g/mg, and the effects of the concentration of magnetic bead coating and enzyme-labeled human angiotensin-converting enzyme IIACE2) on the signal value and signal-to-noise ratio were determined. influence.
  • the concentration of immobilized enzyme-labeled human angiotensin-converting enzyme II was 1 ⁇ g/mL, and the signal values of samples 1-4 were tested by the method described in Example 1 using different concentrations of magnetic bead coatings and the results were obtained. The signal-to-noise ratio was calculated. The results are shown in Table 2 below.
  • the concentration of the immobilized magnetic bead coating was 0.5 mg/mL, and different concentrations of enzyme-labeled human angiotensin-converting enzyme II (ACE2) were used to test the signals of samples 1 to 4 according to the method described in Example 1. value and calculated the signal-to-noise ratio. The results are shown in Table 3 below.
  • Step 1 Preparation of capture components: The SARS-CoV-2 spike protein S1 protein (Shenzhen Mindray Biomedical Electronics Co., Ltd.) was coated on magnetic beads (Thermo fisher) surface magnetic beads.
  • the specific coating method is as follows: first, the S1 protein is pretreated, and the protective components in its buffer matrix are removed by dialysis. Coating was performed at a ratio of 0.5-40 ⁇ g of antigen per milligram of beads. During the reaction, the carboxyl group on the surface of the magnetic beads was coupled with the amino group of the S1 protein under the catalysis of EDC/NHS. Take 20 mg of magnetic beads modified with carboxyl groups on the surface, ultrasonically disperse them in 10 mM MES buffer, add 80 mg of EDC and 120 mg of NHS, and after ultrasonically mix evenly, place on a shaker at 37 °C for 15 min.
  • S1 protein was added to the treated magnetic beads according to the proportion, mixed well, and placed on a shaker at 37°C for 10-18 hours. After washing and blocking, magnetic beads coated with S1 protein were prepared.
  • Embodiment 7 investigates the consistency of the test kit of embodiment 6 and the detection result of virus neutralization test
  • test kit and virus neutralization test prepared in Example 6 were used to detect serum samples of 50 patients in the convalescent stage of COVID-19, and the consistency between the test results of the test kit prepared in Example 6 and the virus neutralization test was compared.
  • the method of virus neutralization test is the same as that in Example 2.
  • Step 1 Preparation of capture components: The SARS-CoV-2 spike protein S1-RBD protein (Beijing Yiqiao Biotechnology, 40159-V02H/40159-V05H1/40159-V08H5) was coated on the surface of magnetic beads (Thermo fisher) magnetic beads.
  • the specific coating method is as follows: first, the S1-RBD protein is pretreated, and the protective components in its buffer matrix are removed by dialysis. Coating was performed at a ratio of 0.5-40 ⁇ g of antigen per milligram of beads. During the reaction, the carboxyl group on the surface of the magnetic beads was coupled with the amino group of the S1-RBD protein under the catalysis of EDC/NHS. Take 20 mg of magnetic beads modified with carboxyl groups on the surface, and ultrasonically disperse them in 10 mM MES buffer, add 80 mg of EDC and 120 mg of NHS, and ultrasonically mix evenly, then place on a shaker at 37 °C for 15 min.
  • S1-RBD was added to the treated magnetic beads according to the proportion, mixed well, and placed on a shaker at 37°C for 10-18 hours. After washing and blocking, magnetic beads coated with S1-RBD protein were prepared.
  • MES 2-morpholinoethanesulfonic acid
  • ACE2 Human angiotensin-converting enzyme II
  • Step 4 Sample detection: In the first step, add the sample (5 ⁇ L), magnetic beads coated with SARS-CoV-2 spike protein S1-RBD protein and human angiotensin-converting enzyme II (ACE2) protein to the reaction tube During incubation, the added ACE2 competes with the neutralizing antibody in the sample for binding to S1-RBD coated on magnetic beads. After the reaction is complete, the magnetic field attracts the beads, washing away unbound material.
  • ACE2 human angiotensin-converting enzyme II
  • the detection component that is, the mouse antibody specifically bound by the human IgG antibody labeled with the signal marker
  • the conjugate formed in the first step is bound.
  • the magnetic field attracts the magnetic beads. , to wash away unbound material.
  • the chemiluminescence substrate solution was added into the reaction tube, and the luminescence substrate (3-(2-spiroadamantane)-4-methoxy-4-(3-phosphoryl)-phenyl-1, 2-Dioxetane, AMPPD) is decomposed by alkaline phosphatase, and a phosphate group is removed to generate an unstable intermediate product, which generates methyl m-oxybenzoate anion through intramolecular electron transfer, which is excited Chemiluminescence occurs when the methyl meta-oxybenzoate anion returns from the excited state to the ground state.
  • the number of photons produced in the reaction is then measured by a photomultiplier tube, and the amount of photons produced is proportional to the content of SARS-CoV-2 neutralizing antibodies in the sample.
  • the calibration curve is shown in Figure 7.
  • Example 9 Investigate the consistency of the neutralizing antibody detection kits of Example 8 and the comparative example and the detection results of the virus neutralization test
  • test kit prepared in Example 8 the kit prepared in Comparative Example and the virus neutralization test were respectively used to detect the serum samples of 50 patients in the convalescent stage of COVID-19.
  • the test results of the prepared kit were consistent with the virus neutralization test.
  • the virus neutralization test adopts the fixed virus dilution serum method.
  • the test needs to titrate the virus titer first. During the test, it is diluted to contain 100 TCID50 per unit dose, and then the serum to be tested is double-diluted with an equal amount of 100 TCID50/ml virus solution, and incubated at 37 °C for 2h after mixing. , inoculate a cell culture plate with each dilution, 0.1 ml per well, 5% CO 2 37°C incubator for 4 days, and record the number of CPE wells.
  • the neutralization value was calculated according to Karber's method, that is, the maximum dilution of antiserum when 50% of cells were protected.
  • Embodiment 10 investigates the influence of incubation time on detection result
  • Example 8 The effects of the incubation time in the first step and the incubation time in the second step on the detection results in Example 8 "Sample Detection" were respectively studied.
  • the incubation time of the second step was fixed at 5 min, and the signal values of samples 1-4 were tested with different incubation times of the first step, and the signal-to-noise ratio was calculated.
  • the results are shown in Table 4 below.
  • the capture component was prepared according to the description in Example 8, wherein the amount of S1-RBD used was fixed at 10 ⁇ g/mg, and the effects of magnetic bead coating and human angiotensin-converting enzyme II (ACE2) concentration on signal value and signal-to-noise ratio were determined. influence.
  • ACE2 human angiotensin-converting enzyme II
  • the concentration of immobilized human angiotensin-converting enzyme II was 1 ⁇ g/mL, and the signal values of samples 1-4 were tested according to the method described in Example 8 using different concentrations of magnetic bead-coated materials and calculated. Signal-to-noise ratio. The results are shown in Table 6 below.
  • the concentration of the immobilized magnetic bead coating was 1.0 mg/mL.
  • ACE2 human angiotensin-converting enzyme II
  • Step 1 Preparation of capture components: The SARS-CoV-2 spike protein S1 protein (Shenzhen Mindray Biomedical Electronics Co., Ltd.) was coated on magnetic beads (Thermo fisher) surface magnetic beads.
  • the specific coating method is as follows: first, the S1 protein is pretreated, and the protective components in its buffer matrix are removed by dialysis. Coating was performed at a ratio of 0.5-40 ⁇ g of antigen per milligram of beads. During the reaction, the carboxyl group on the surface of the magnetic beads was coupled with the amino group of the S1 protein under the catalysis of EDC/NHS. Take 20 mg of magnetic beads modified with carboxyl groups on the surface, ultrasonically disperse them in 10 mM MES buffer, add 80 mg of EDC and 120 mg of NHS, and after ultrasonically mix evenly, place on a shaker at 37 °C for 15 min.
  • S1 protein was added to the treated magnetic beads according to the proportion, mixed well, and placed on a shaker at 37°C for 10-18 hours. After washing and blocking, magnetic beads coated with S1 protein were prepared.
  • Example 13 Investigating the consistency of the kit of Example 12 and the detection results of the virus neutralization test
  • the kit and virus neutralization test prepared in Example 12 were used to detect serum samples from 50 patients in the convalescent stage of COVID-19, and the consistency between the test results of the kit prepared in Example 12 and the virus neutralization test was compared.
  • the method of the virus neutralization test is the same as that in Example 9.

Abstract

本发明公开了一种用于测定冠状病毒中和抗体的试剂盒，包括捕获组分，其含有包被有蛋白的固相载体，蛋白具有所述冠状病毒的刺突蛋白S1亚基的RBD序列；人血管紧张素转化酶II；和可检测的信号标记物，其中，可检测的信号标记物标记在人血管紧张素转化酶II上，或者标记在二抗上。还公开了测定中和抗体的方法。

Description

用于检测冠状病毒中和抗体的试剂盒、方法 技术领域
本发明涉及中和抗体测定领域，具体涉及冠状病毒的中和抗体的测定。
背景技术
冠状病毒是一类具有包膜的单正链RNA病毒，存在于人和其他哺乳动物以及鸟类中，并导致呼吸、消化、肝脏和神经系统等类型的疾病。目前已报道的有6种冠状病毒可以引发人类疾病，如可引起感冒以及中东呼吸综合征(MERS)和严重急性呼吸综合征(SARS)等。
新型冠状病毒(SARS-CoV-2)是一种于新发现的冠状病毒，属于β属的冠状病毒，有包膜，颗粒呈圆形或椭圆形，常为多形性，直径60～140nm。SARS-CoV-2引起新型冠状病毒肺炎(Corona Virus Disease 2019,COVID-19)，COVID-19的常见体征有呼吸道症状、发热、咳嗽、气促和呼吸困难等。在较严重的病例中，感染可导致肺炎、严重急性呼吸综合征、肾衰竭，甚至死亡。
COVID-19诊疗快速指南中提出，依据病情重症患者可酌情在早期使用注射用免疫球蛋白(IVIG)。因此，了解IVIG中SARS-CoV-2中和抗体水平，以及其来源的健康献浆员的SARS-CoV-2中和抗体水平，可以评价IVIG是否具有特异的抗SARS-CoV-2作用，同时也可以为临床使用提供实验室支持数据。另外，重型和危重型新型冠状病毒肺炎诊断和治疗专家共识建议：“将含有新型冠状病毒抗体的人恢复期血浆用于病情进展较快、重型和危重型患者，可以作为特异性治疗的一种选择。如使用恢复期血浆，应检测血浆中保护性抗体滴度水平”。也就是说，中和抗体的检测对于SARS-CoV-2的诊疗具有重要意义。同时，中和抗体的测定也可用于相关病毒疫苗的效果评价。
目前，检测SARS-CoV-2中和抗体的方法通常采用活病毒细胞培养的中和试验。然而，由于SARS-CoV-2传染性强、致病性高，该试验必须在生物安全三级实验室内进行。由于利用活病毒细胞培养的中和试验检测，对实验室等级有着极高要求，严重限制了临床大规模的推广使用。
针对这一问题，近期已开发了一种基于SARS-CoV-2假病毒的中和抗体检测方法，在该方法将Spike基因序列克隆到载体，并构建复制缺陷型病毒，随后通过计算样本对 于病毒感染的抑制率来实现中和抗体检测。上述基于假病毒的检测方法虽然避免了生物安全三级实验室的使用，但仍对安全等级有一定要求，例如需要使用生物安全二级实验室，同时该方法涉及细胞培养、假病毒构建等操作，仍具有较高的复杂性、专业性和危险性。
据此，在本领域中存在对更简便、更安全和更高效的中和抗体测定方法的强烈需求。
发明内容
有鉴于此，本发明在第一方面提供了一种用于测定冠状病毒中和抗体的试剂盒，包括：
捕获组分，其含有包被有蛋白的固相载体，并且所述蛋白具有所述冠状病毒的刺突蛋白S1亚基的RBD序列；和
人血管紧张素转化酶II，所述人血管紧张素转化酶II带有可检测的信号标记物。
在本发明的第一方面，由于可检测的信号标记物标记在人血管紧张素转化酶II上，因此标记有可检测的信号标记物的人血管紧张素转化酶II作为试剂盒中的检测组分。
在使用上述试剂盒检测样本中可能存在的中和抗体的情况下，固相载体上的包被物与样本中的中和抗体进行结合；同时加入标记有人血管紧张素转化酶II的信号标记物，其与样本中的中和抗体竞争或阻止标记有信号标记物的人血管紧张素转化酶II与RBD上的结合位点结合。最终通过信号标记物产生的信号值的高低确定样本中中和抗体的含量，样本中的中和抗体含量与信号值高低成反比。
在本发明中，包被有蛋白的固相载体可以常规的方式(例如，于适当的缓冲液中)配置成捕获组分，只要适宜固相载体和包被物的保存且不影响反应的进行即可。在本发明的第一方面中，包被有蛋白的固相载体例如可以0.2mg/mL至1.0mg/mL的浓度存在，例如以0.3mg/mL、0.4mg/mL、0.5mg/mL、0.6mg/mL、0.7mg/mL、0.8mg/mL、0.9mg/mL的浓度存在。在示例性的实施方式中，固相载体以0.5mg/mL至1.0mg/mL的浓度存在。在一个具体的实施方式中，固相载体以0.5mg/mL的浓度存在。
在本发明中，人血管紧张素转化酶II可以常规的方式存在(例如，于适当的缓冲液中)，只要适宜人血管紧张素转化酶II的保存且不影响反应的进行即可。在本发明的第一方面中，人血管紧张素转化酶II例如可以0.5μg/mL至2μg/mL的浓度存在，例如以0.6μg/mL、0.7μg/mL、0.8μg/mL、0.9μg/mL、1μg/mL、1.1μg/mL、1.2μg/mL、1.3μg/mL、 1.4μg/mL、1.5μg/mL、1.6μg/mL、1.7μg/mL、1.8μg/mL、1.9μg/mL的浓度存在。在示例性的实施方式中，人血管紧张素转化酶II以1μg/mL至2μg/mL的浓度存在。在一个具体的实施方式中，人血管紧张素转化酶II以1μg/mL的浓度存在。
在本发明第一方面的试剂盒中，捕获组分、人血管紧张素转化酶II可分别以单独包装的形式存在于试剂盒中。
此外，还提供了一种测定受试者样本中的中和抗体的方法，包括：
将捕获组分、人血管紧张素转化酶II和所述样本混合并孵育一段时间，使得所述人血管紧张素转化酶II与样本中的中和抗体能竞争性地与所述捕获组分结合；
将获得的混合物进行清洗，除去未结合的物质；
对可检测的信号标记物进行检测，以得到信号值；以及
根据所述信号值获得中和抗体的测定结果，
其中，捕获组分含有包被有蛋白的固相载体，并且所述蛋白具有冠状病毒的刺突蛋白S1亚基的RBD序列；
并且其中，所述可检测的信号标记物标记在人血管紧张素转化酶II上。
在一个具体的实施方式中，所述混合并孵育一段时间是先将所述捕获组分与样本混合并孵育一段时间，然后再加入人血管紧张素转化酶II混合并孵育一段时间。
在一个优选的实施方式中，所述混合并孵育一段时间是将所述捕获组分、人血管紧张素转化酶II和样本同时加入，混合并孵育一段时间。通过选择该优选的加入方式，本发明第一方面中测定方法的准确度得以进一步改善。
在本发明第一方面的方法中，孵育一段时间是指孵育至少5min；例如，10min、15min、20min。在一个具体的实施方式中，孵育可进行10min。
在第二方面，本发明提供了一种用于测定冠状病毒中和抗体的试剂盒，包括：
捕获组分，其含有包被有蛋白的固相载体，并且所述蛋白具有所述冠状病毒的刺突蛋白S1亚基的RBD序列；
人血管紧张素转化酶II；和
二抗，所述二抗标记有带有可检测的信号标记物。
在本发明的第二方面，由于可检测的信号标记物标记在二抗上，因此标记有可检测的信号标记物的二抗作为试剂盒中的检测组分；而人血管紧张素转化酶II作为竞争组分。
在使用上述试剂盒检测样本的可能存在的中和抗体的情况下，加入的人血管紧张素 转化酶II与样本中的中和抗体竞争结合包被在固相载体上的包被物。之后加入与人抗体特异性结合的抗体。进一步形成固相载体-包被物-中和抗体(一抗)-二抗的结合物，并通过二抗上的信号标记物产生信号。最终通过信号值的高低确定样本中中和抗体的含量，样本中的中和抗体含量与信号值高低成正比。
在本发明中，包被有蛋白的固相载体可以常规的方式(例如，于适当的缓冲液中)配置成捕获组分，只要适宜固相载体和包被物的保存且不影响反应的进行即可。在本发明的第二方面中，包被有蛋白的固相载体例如可以0.5至1.2mg/mL的浓度存在，例如以0.6mg/mL、0.7mg/mL、0.8mg/mL、0.9mg/mL、1.0mg/mL、1.1mg/mL的浓度存在。在示例性的实施方式中，包被有蛋白的固相载体以0.5mg/mL至1.0mg/mL的浓度存在。在一个具体的实施方式中，包被有蛋白的固相载体以0.7mg/mL的浓度存在。
在本发明中，人血管紧张素转化酶II可以常规的方式存在(例如，于适当的缓冲液中)，只要适宜人血管紧张素转化酶II的保存且不影响反应的进行即可。在本发明的第二方面中，人血管紧张素转化酶II例如可以0.2μg/mL至1.5μg/mL的浓度存在，例如以0.3μg/mL、0.4μg/mL、0.5μg/mL、0.6μg/mL、0.7μg/mL、0.8μg/mL、0.9μg/mL、1μg/mL、1.1μg/mL、1.2μg/mL、1.3μg/mL、1.4μg/mL的浓度存在。在示例性的实施方式人血管紧张素转化酶II可以0.8μg/mL至1.2μg/mL的浓度存在。在一个具体的实施方式中，人血管紧张素转化酶II以1μg/mL的浓度存在。
在本发明第二方面的试剂盒中，捕获组分、人血管紧张素转化酶II、二抗可分别以单独包装的形式存在于试剂盒中。
此外，还提供了一种测定受试者样本中的中和抗体的方法，包括：
将捕获组分、人血管紧张素转化酶II和所述样本混合并孵育一段时间，使得所述人血管紧张素转化酶II与样本中的中和抗体能竞争性地与所述捕获组分结合；
将获得的混合物进行清洗，除去未结合的物质；
在经清洗的混合物中加入带有可检测的信号标记物的二抗，混匀并孵育一段时间，使得所述二抗能与所述捕获组分上结合的中和抗体结合，形成复合物；
对获得的混合物进行清洗，除去未形成复合物的物质；
对可检测的信号标记物进行检测，以得到信号值；以及
根据所述信号值获得中和抗体的测定结果，
其中，捕获组分含有包被有蛋白的固相载体，并且所述蛋白具有冠状病毒的刺突蛋白S1亚基的RBD序列。
在本发明第二方面的方法中，孵育一段时间均是指孵育至少5min；例如，10min、15min、20min。在示例性的实施方式中，孵育进行5min至20min。在具体的实施方式中，孵育可进行5min。
在第三方面，本发明提供了包被物在制备冠状病毒中和抗体的检测试剂中的用途，其中，所述包被物为具有所述冠状病毒的刺突蛋白S1亚基的RBD序列的蛋白，
并且在所述检测试剂中，所述包被物包被在固相载体上以构成捕获组分。
在第四方面，本发明提供了包被物和人血管紧张素转化酶II在制备冠状病毒中和抗体的检测试剂中的用途，其中，所述包被物为具有所述冠状病毒的刺突蛋白S1亚基的RBD序列的蛋白，
并且在所述检测试剂中，所述包被物包被在固相载体上以构成捕获组分；所述人血管紧张素转化酶II能与所述中和抗体竞争性地与所述捕获组分结合。
采用本发明的方案对冠状病毒的中和抗体进行检测，无需使用活病毒，同时也不需要构建假病毒或培养细胞，由此提高了检测安全性并降低了复杂度；另一方面，本发明的方案可以获得与病毒中和试验高相关性的检测结果。
附图说明
图1示出了采用实施例1制备的试剂盒检测SARS-CoV-2中和抗体的定标曲线；
图2示出了采用对比例制备的试剂盒检测SARS-CoV-2中和抗体的定标曲线；
图3示出了实施例1制备的试剂盒的检测结果与病毒中和试验的一致性分析结果；
图4示出了对比例制备的试剂盒的检测结果与病毒中和试验的一致性分析结果；
图5示出了实施例3中采用加样方式2的情况下，测试结果与病毒中和试验的一致性分析结果；
图6示出了实施例6制备的试剂盒的检测结果与病毒中和试验的一致性分析结果；
图7示出了采用实施例8制备的试剂盒检测SARS-CoV-2中和抗体的定标曲线；
图8示出了实施例8制备的试剂盒的检测结果与病毒中和试验的一致性分析结果；
图9示出了实施例12制备的试剂盒的检测结果与病毒中和试验的一致性分析结果。
具体实施方式
下面对本申请实施方式中的技术方式进行清楚、完整地描述，显然，所描述的实施方式仅仅是本申请的一部分实施方式，而不是全部的实施方式。基于本申请中的实施方 式，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施方式，都属于本申请保护的范围。
在整个说明书中，除非另有特别说明，本文使用的术语应理解为如本领域中通常所使用的含义。因此，除非另有定义，本文使用的所有技术和科学术语具有与本发明所属领域技术人员的一般理解相同的含义。若存在矛盾，本说明书优先。
如前文所述，检测血液中的中和抗体例如有利于对疫苗的有效性作出评价。目前，冠状病毒的中和抗体检测采用病毒中和试验或是需要构建假病毒，因此，中和抗体的检测是一个复杂且危险，同时需要很高的专业性的过程。为了解决上述问题，本发明提供了与病毒中和试验检测结果具有高相关度的中和抗体检测试剂盒，从而为冠状病毒的中和抗体的检测提供了相对简便、高效且安全的方法。
另需要指出的是，与广泛认可的中和抗体检测方式，即病毒中和试验的检测结果相比较的，本发明的试剂盒的检测具有高的相关性，从而提供了一种准确的冠状病毒中和抗体的检测方式。
据此，本发明提供了一种用于测定冠状病毒中和抗体的试剂盒，包括：
捕获组分，其含有包被有蛋白的固相载体，所述蛋白具有所述冠状病毒的刺突蛋白S1亚基的RBD序列；
人血管紧张素转化酶II；和
可检测的信号标记物。
在一些实施方式中，可检测的信号标记物标记在所述人血管紧张素转化酶II上。在一些变型的实施方式中，可检测的信号标记物标记在二抗上，在这种情况下，应当理解，人血管紧张素转化酶II上不标记有可检测的信号标记物。
如本发明所使用的，术语“具有”、“包含”或“包括”或其任意语法变体以非排他性的方式使用。因此，这些术语既可以是指其中除了这些术语引入的特征之外在本上下文中描述的实体中没有另外特征存在的情况，而且可以是指其中存在一个或多个另外特征的情况。作为示例，表述“A具有B”、“A包含B”和“A包括B”既可以是指其中除了B之外在A中没有另外要素存在的情况(即其中单独且仅由B组成的情况)，而且可以是指其中除了B之外在实体A中存在一种或多种另外要素诸如要素C、要素C和D或甚至其它要素的情况。
如本发明所使用的，术语“试剂盒”是指可以包装在一起或可以不包装在一起的本发明的用于检测的成分或试剂的集合。试剂盒的成分可以由单独的小瓶(作为单独部分 的试剂盒)包含或提供在单个小瓶中。此外，应当理解，本发明的试剂盒用于实施本文所述的方法。在一个实施方案中，为了实践本文的方法，所有成分都以即用方式提供。此外，在一个实施方案中，试剂盒含有用于实施本文方法的说明书。说明书可以由用户手册以纸张或电子形式提供。例如，所述手册可以包含用于解释当使用本发明的试剂盒实施上述方法时获得的结果的说明书。
在本发明中，冠状病毒是指属于冠状病毒属(Coronavirus)的病毒。在一些实施方式中，本发明的冠状病毒能够感染人，例如选自由SARS、MERS、SARS-CoV-2及其组合所组成的组。在一个具体的实施方式中，冠状病毒为SARS-CoV-2。
在本发明中，术语“固相载体”可与“固相支持物”、“固体支持物”和“固体载体”互换使用，其是指可以结合包被物且适于例如通过物理方式与样本分离的任何合适的固体表面。对用于本发明的固相载体没有特别的限制，商品化的固相载体及任何可用于免疫学分析的固相载体均可用于本发明。示例性的固相载体可以是磁珠(如羧基磁珠)、酶标板、塑料板、塑料管、乳胶珠、琼脂糖珠、玻璃、硝酸纤维素膜、尼龙膜、二氧化硅板或微芯片，但本发明不限于此。
在本发明中，“捕获组分”包含结合有包被物的固相载体。捕获组分例如还可以包括适合溶液或其他组分以有利于固相载体和包被物的保存。
在本发明中，术语“包被物”表示包被在本发明的固相载体上的物质。将包被物(通常为蛋白质或多肽)与固体表面结合的方法是本领域熟知的，并且包括例如，通过疏水相互作用结合、生物素化和经由固定的链霉抗生物素蛋白结合、共价结合、抗体-抗原相互作用等，或这些相互作用的组合。
在一些实施方式中，包被物可选自由冠状病毒的刺突蛋白、冠状病毒的刺突蛋白S1亚基、冠状病毒的刺突蛋白S1亚基的RBD及其组合所组成的组，其可以为天然来源或通过重组产生。
在本发明中，“具有冠状病毒的刺突蛋白S1亚基的RBD序列的蛋白”是指包括该RBD序列且具有RBD生物学功能的蛋白，可以例如通过商购获得这样的蛋白。另外，冠状病毒的刺突蛋白S1亚基的RBD序列、冠状病毒的刺突蛋白S1亚基序列和冠状病毒的刺突蛋白序列例如记载在科技文献，或者可以从相关的数据库(例如，GenBank/EMBL/DDBJ)中获得。例如，SARS-CoV-2的刺突蛋白S1亚基的RBD的序列可以选取自NCBI登录号YP_009724390.1所示基因的编码序列中的第Arg319至Ser541位。
人血管紧张素转化酶II能够与冠状病毒的刺突蛋白S1亚基的RBD相互作用。在本发明中，“人血管紧张素转化酶II”包括人血管紧张素转化酶II或其活性片段，其可以为天然来源或通过重组产生。
在本发明中，术语“二抗(secondary antibody)”表示能与中和抗体特异性结合的抗体。例如可以为鼠抗人抗体、兔抗人抗体、山羊抗人抗体、绵羊抗人抗体、鸡抗人抗体，但本发明不限于此。在一个具体的实施方式中，二抗是鼠抗人抗体。
在本发明中，术语“可检测的信号标记物”表示具有可检测性质的物质，通常为光学或/和酶学性质；然而，可检测性质也可以是发射放射性的性质。
在本发明中，“光学性质”涉及可由光学仪器检测的任何性质。具体地，光学可检测的性质可以是或可以包含选自以下项的至少一种性质：颜色、反射性、折射性、透射性、发射性、散射性、荧光性、磷光性、衍射性和极化性。应当理解，检测如本文中所使用的光学可检测的性质包括检测以前不可检测的性质的存在，检测以前不可检测的性质的不存在，以及检测性质的定量变化，即检测与至少一种光学性质的变化程度相关的信号强度的变化。应当理解，在一些实施方案中，光学可检测的性质也涉及电化学发光，其也称为电致化学发光。
在本发明中，“酶性质”涉及通过生物催化从底物产生可检测产物的指标的性质。通常，酶性质是选自以下项的至少一种酶活性：磷酸酶活性、过氧化物酶活性和糖苷酶活性。酶活性的典型底物是本领域熟知的；例如，当使用碱性磷酸酶作为标记物时，可采用3-(2-螺旋金刚烷)-4-甲氧基-4-(3-磷氧酰)-苯基-1,2-二氧环乙烷作为发光底物，该底物会被碱性磷酸酶所分解，脱去一个磷酸基，生成不稳定的中间产物，该中间产物通过分子内电子转移产生间氧苯甲酸甲酯阴离子，处于激发态的间氧苯甲酸甲酯阴离子从激发态返回基态时，产生化学发光。通常，酶活性产生具有如上文所述的可测定光学性质的产物，和/或所述酶活性产生可由电子仪器测定的产物。
示例性的可检测的信号标记物可以是碱性磷酸酶、鲁米诺、异鲁米诺、吖啶酯、辣根过氧化物酶、β-半乳糖苷酶、三联吡啶钌、量子点(如金量子点、CdSe量子点、ZnCdSe量子点)、荧光微球，但本发明不限于此。
应当理解，根据所采用的固相载体和可检测的信号标记物，本发明可适用于ELISA、化学发光、电化学发光、POCT、免疫层析法、上转换发光、下转换发光，但本发明不限于此。
本发明的方法可以包括除了具体提到的步骤之外的步骤。具体地，可以包括提供样 本的步骤，或者可以包括添加其它化合物以促进结合和检测。此外，一些或所有步骤可以通过自动化设备辅助。
如发明所使用的，术语“样本”涉及怀疑包含本发明分析物(冠状病毒的中和抗体)的样本。在具体的实施方案中，样本包括血液、血浆和/或血清。应当理解，可以进一步处理样品以便实施本发明的方法。具体地，可以通过本领域已知的方法和方式从样品中除去细胞。此外，可以通过本领域已知的方法和方式从样品提取和/或纯化至少一种分析物。因此，术语样品还可以涉及包含或怀疑包含至少一种分析物的制备物，其从样品稀释、富集、纯化和/或提取。
以下通过实施例更详细地说明本发明，但本发明不限于这些实施例。
实施例1 SARS-CoV-2中和抗体检测试剂盒的制备
步骤1：捕获组分的制备：将SARS-CoV-2刺突蛋白S1-RBD蛋白(北京义翘生物，40159-V02H/40159-V05H1/40159-V08H5)包被于磁珠(Thermo fisher)表面磁珠。
具体包被方法为：首先将S1-RBD蛋白进行前处理，通过透析去除其缓冲基质中的保护组分。按照每毫克磁珠加入0.5-40μg的抗原的比例进行包被。在反应过程中磁珠表面的羧基在EDC/NHS催化下与S1-RBD蛋白的氨基进行偶联。取20mg表面修饰有羧基的磁珠，超声分散于10mM MES缓冲液中，加入80mg EDC和120mg NHS，超声混合均匀后，置于37℃摇床15min。之后在处理后的磁珠中，按照比例加入S1-RBD，混匀，并置于37℃摇床反应10-18h。之后清洗封闭后，制备得包被有S1-RBD蛋白的磁珠包被物。将磁珠包被物稀释于50mM MES缓冲液(0.5M NaCl、0.5％BSA、0.05％吐温20，pH＝6.0)，磁珠包被物浓度为0.5mg/mL。
步骤2：检测组分的制备：将人血管紧张素转化酶II(ACE2)(北京义翘生物，10108-H02H)与碱性磷酸酶(Roche Life Science)进行耦联，制备得酶标标记物。将酶标标记物稀释于50mM MES缓冲液(0.5M NaCl、0.5％BSA、0.05％吐温20，pH＝6.0)，酶标标记物浓度为1.0μg/mL。
步骤3：样本检测：第一步，将样本(5μL)、包被有SARS-CoV-2刺突蛋白S1-RBD蛋白的磁珠和标记有碱性磷酸酶的人血管紧张素转化酶II(ACE2)蛋白添加到反应管中孵育，样本中的SARS-CoV-2中和抗体竞争或阻止标记有碱性磷酸酶的人血管紧张素转化酶II(ACE2)蛋白或蛋白片段与SARS-CoV-2刺突蛋白S1-RBD蛋白结合位点结合。反应完成后，磁场吸住磁珠，洗去未结合的物质。
第二步，将化学发光底物液添加到反应管内，发光底物(3-(2-螺旋金刚烷)-4-甲氧基-4-(3-磷氧酰)-苯基-1,2-二氧环乙烷，AMPPD)被碱性磷酸酶所分解，脱去一个磷酸基，生成不稳定的中间产物，该中间产物通过分子内电子转移产生间氧苯甲酸甲酯阴离子，处于激发态的间氧苯甲酸甲酯阴离子从激发态返回基态时，产生化学发光。再通过光电倍增管对反应中所产生的光子数进行测量，所产生光子的量与样本内SARS-CoV-2中和抗体的含量成反比。定标主曲线如图1所示。
对比例SARS-CoV-2 IgG抗体检测试剂盒的制备
步骤1：捕获组分的制备：将SARS-CoV-2刺突蛋白S1-RBD蛋白(北京义翘生物，40159-V02H/40159-V05H1/40159-V08H5)包被于磁珠(Thermo fisher)表面磁珠。
具体包被方法为：首先将S1-RBD蛋白进行前处理，通过透析去除其缓冲基质中的保护组分。按照每毫克磁珠加入0.5-40μg的抗原的比例进行包被。在反应过程中磁珠表面的羧基在EDC/NHS催化下与S1-RBD蛋白的氨基进行偶联。取20mg表面修饰有羧基的磁珠，超声分散于10mM MES缓冲液中，加入80mg EDC和120mg NHS，超声混合均匀后，置于37℃摇床15min。之后在处理后的磁珠中，按照比例加入S1-RBD，混匀，并置于37℃摇床反应10-18h。之后清洗封闭后，制备得包被有S1-RBD蛋白的磁珠包被物。将磁珠包被物稀释于50mM MES缓冲液(0.5M NaCl、0.5％BSA、0.05％吐温20，pH＝6.0)，磁珠包被物浓度为0.5mg/mL。
步骤2：检测组分的制备：将与人IgG抗体特异性结合的鼠抗体(Meridian Life Science)标记信号标记物。使用50mM MES(2-吗啉乙磺酸)pH＝6.0的缓冲液对碱性磷酸酶(Roche Life Science)进行稀释。稀释后信号标记物的浓度为100ng/mL，制备得检测组分。
步骤3：样本检测：第一步，将样本(5μL)、包被有SARS-CoV-2刺突蛋白S1-RBD蛋白的磁珠添加到反应管中孵育，样本中的SARS-CoV-2抗体与磁珠表面的S1-RBD蛋白结合。反应完成后，磁场吸住磁珠，洗去未结合的物质。
第二步，将检测组分，即标记有信号标记物的人IgG抗体特异性结合的鼠抗体加到反应管中孵育，第一步形成的结合物结合，反应完成后，磁场吸住磁珠，洗去未结合的物质。
第三步，将化学发光底物液添加到反应管内，发光底物(3-(2-螺旋金刚烷)-4-甲氧基-4-(3-磷氧酰)-苯基-1,2-二氧环乙烷，AMPPD)被碱性磷酸酶所分解，脱去一个磷酸 基，生成不稳定的中间产物，该中间产物通过分子内电子转移产生间氧苯甲酸甲酯阴离子，处于激发态的间氧苯甲酸甲酯阴离子从激发态返回基态时，产生化学发光。再通过光电倍增管对反应中所产生的光子数进行测量，所产生光子的量与样本内SARS-CoV-2IgG抗体的含量成正比。定标曲线如图2所示。
实施例2考察实施例1和对比例的中和抗体检测试剂盒与病毒中和试验检测结果的一致性
分别采用实施例1所制备的试剂盒、对比例所制备的试剂盒和病毒中和试验对50名COVID-19康复期患者血清样本进行检测，分别比较实施例1所制备的试剂盒、对比例所制备的试剂盒的测试结果与病毒中和试验的一致性。
其中病毒中和试验采用固定病毒稀释血清法。试验需先滴定病毒毒价，试验时将其稀释成每一单位剂量含100个TCID50，再将待检血清倍比稀释加等量100个TCID50/ml的病毒液，混匀后37℃孵育2h，每一稀释度接种细胞培养板，每孔0.1ml、5％CO 2 37℃培养箱培养4天后，记录出现CPE孔数。按Karber法计算中和价，即50％细胞被保护时抗血清的最大稀释度。
结果如图3和图4所示。图3显示了实施例1所制备的SARS-CoV-2中和抗体检测试剂盒与病毒中和试验的相关性，其中r 2＝0.8435；显著高于对比例所制备的SARS-CoV-2 IgG抗体检测试剂盒测试结果与病毒中和试验的相关性r 2＝0.6175(如图4所示)。
实施例3考察加样方式对检测结果的影响
为了研究加样方式对试剂盒检测结果与病毒中和试验的相关性的影响。分别以下加样方式：方式1、加入捕获组分和检测组分后，再进行孵育(即实施例1中的情形)。方式2：先加入捕获组分孵育后，再加入检测组分。其余检测步骤与实施例1相同。
研究这两种方法与病毒中和试验结果的一致性，加样方式1与病毒中和试验的相关性如图3所示，其中r 2＝0.8435，方法学比对较好。加样方式2的测试结果与病毒中和试验的相关性如图5所示，其中r 2＝0.7534。
实施例4考察孵育时间对检测结果的影响
为研究实施例1“样本检测”中孵育时间对检测结果的影响，分别采用不同的孵育 时间测试了不同样本的信号值并计算了信噪比，其余方法与实施例1相同。其中，样本1为不含有SARS-CoV-2中和抗体阴性对照；样本2至样本4中中和抗体的滴度log2(1：X)采用病毒中和试验确定，其中样本2至样本4的50％抑制率的最大稀释度依次为30、363、757。结果如下表1所示。
表1
Figure PCTCN2020108534-appb-000001
由表1可知，当孵育时间低于5min，信号值和信噪比较低。当孵育时间为5-20min，信号值和信噪比提升且无显著差异。
实施例5磁珠包被物及酶标人血管紧张素转化酶II(ACE2)浓度的确定
根据实施例1中的记载制备捕获组分，其中S1-RBD使用量固定为10μg/mg，确定磁珠包被物和酶标人血管紧张素转化酶IIACE2)浓度对信号值和信噪比的影响。
一方面，固定酶标记的人血管紧张素转化酶II(ACE2)的浓度为1μg/mL，采用不同的磁珠包被物浓度按照实施例1记载的方法测试了样本1-4的信号值并计算了信噪比。结果如下表2所示。
表2
Figure PCTCN2020108534-appb-000002
Figure PCTCN2020108534-appb-000003
由表2可知，固相包被物低于0.2mg/mL时，信号值和信噪比较低；固相包被物浓度为0.2-1.0mg/mL时，信号值和信噪比可以接受。提升固相包被物浓度不会显著提升信噪比。
另一方面，固定磁珠包被物的浓度为0.5mg/mL，采用不同的酶标人血管紧张素转化酶II(ACE2)浓度，按照实施例1记载的方法测试了样本1～4的信号值并计算了信噪比。结果如下表3所示。
表3
Figure PCTCN2020108534-appb-000004
由表3可知，酶标人血管紧张素转化酶II(ACE2)低于0.5μg/mL时，信号值和信噪比较低；浓度为0.5-2.0μg/mL时，信号值和信噪比可以接受，提升检测组分浓度不会显著提升信噪比。
实施例6使用S1蛋白(S1亚基)制备SARS-CoV-2中和抗体检测试剂盒
步骤1：捕获组分的制备：将SARS-CoV-2刺突蛋白S1蛋白(深圳迈瑞生物医疗电子股份有限公司)包被于磁珠(Thermo fisher)表面磁珠。
具体包被方法为：首先将S1蛋白进行前处理，通过透析去除其缓冲基质中的保护组分。按照每毫克磁珠加入0.5-40μg的抗原的比例进行包被。在反应过程中磁珠表面的羧基在EDC/NHS催化下与S1蛋白的氨基进行偶联。取20mg表面修饰有羧基的磁珠，超声分散于10mM MES缓冲液中，加入80mg EDC和120mg NHS，超声混合均匀后，置于37℃摇床15min。之后在处理后的磁珠中，按照比例加入S1蛋白，混匀，并置于37℃摇床反应10-18h。之后清洗封闭后，制备得包被有S1蛋白的磁珠包被物。将磁珠包被物稀释于50mM MES缓冲液(0.5M NaCl、0.5％BSA、0.05％吐温20，pH＝6.0)，磁珠包被物浓度为0.2-1.0mg/mL。
检测组分的制备和样本的检测同实施例1。
实施例7考察实施例6的试剂盒与病毒中和试验检测结果的一致性
采用实施例6所制备的试剂盒和病毒中和试验对50名COVID-19康复期患者血清样本进行检测，比较实施例6所制备的试剂盒的测试结果与病毒中和试验的一致性。其中病毒中和试验的方法同实施例2。
结果如图6所示，其中实施例6中使用S1蛋白所制备的中和抗体检测试剂盒与病毒中和试验的相关性r 2＝0.7709。
实施例8 SARS-CoV-2中和抗体检测试剂盒的制备
步骤1：捕获组分的制备：将SARS-CoV-2刺突蛋白S1-RBD蛋白(北京义翘生物，40159-V02H/40159-V05H1/40159-V08H5)包被于磁珠(Thermo fisher)表面磁珠。
具体包被方法为：首先将S1-RBD蛋白进行前处理，通过透析去除其缓冲基质中的保护组分。按照每毫克磁珠加入0.5-40μg的抗原的比例进行包被。在反应过程中磁珠表面的羧基在EDC/NHS催化下与S1-RBD蛋白的氨基进行偶联。取20mg表面修饰有 羧基的磁珠，超声分散于10mM MES缓冲液中，加入80mg EDC和120mg NHS，超声混合均匀后，置于37℃摇床15min。之后在处理后的磁珠中，按照比例加入S1-RBD，混匀，并置于37℃摇床反应10-18h。之后清洗封闭后，制备得包被有S1-RBD蛋白的磁珠包被物。将磁珠包被物稀释于50mM MES缓冲液(0.5M NaCl、0.5％BSA、0.05％吐温20，pH＝6.0)，磁珠包被物浓度为0.7mg/mL。
步骤2：检测组分的制备：将与人IgG抗体特异性结合的鼠抗体(Meridian Life Science)标记信号标记物。使用50mM MES(2-吗啉乙磺酸)pH＝6.0的缓冲液对碱性磷酸酶(Roche Life Science)进行稀释。稀释后信号标记物的浓度为100ng/mL，制备得检测组分。
步骤3：竞争组分的制备：将人血管紧张素转化酶II(ACE2)(北京义翘生物，10108-H08B/10108-H05H)稀释与25mM PBS缓冲液(0.5％BSA、0.05％吐温20，pH＝7.4)，制备得竞争组分，其中人血管紧张素转化酶II(ACE2)的浓度为1.0μg/mL。
步骤4：样本检测：第一步，将样本(5μL)、包被有SARS-CoV-2刺突蛋白S1-RBD蛋白的磁珠和人血管紧张素转化酶II(ACE2)蛋白添加到反应管中孵育，加入的ACE2与样本中的中和抗体竞争结合包被在磁珠上的S1-RBD。反应完成后，磁场吸住磁珠，洗去未结合的物质。
第二步，将检测组分，即标记有信号标记物的人IgG抗体特异性结合的鼠抗体加到反应管中孵育，第一步形成的结合物结合，反应完成后，磁场吸住磁珠，洗去未结合的物质。
第三步，将化学发光底物液添加到反应管内，发光底物(3-(2-螺旋金刚烷)-4-甲氧基-4-(3-磷氧酰)-苯基-1,2-二氧环乙烷，AMPPD)被碱性磷酸酶所分解，脱去一个磷酸基，生成不稳定的中间产物，该中间产物通过分子内电子转移产生间氧苯甲酸甲酯阴离子，处于激发态的间氧苯甲酸甲酯阴离子从激发态返回基态时，产生化学发光。再通过光电倍增管对反应中所产生的光子数进行测量，所产生光子的量与样本内SARS-CoV-2中和抗体的含量成正比。定标曲线如图7所示。
实施例9考察实施例8和对比例的中和抗体检测试剂盒与病毒中和试验检测结果的一致性
分别采用实施例8所制备的试剂盒、对比例所制备的试剂盒和病毒中和试验对50名COVID-19康复期患者血清样本进行检测，分别比较实施例8所制备的试剂盒、对比 例所制备的试剂盒的测试结果与病毒中和试验的一致性。
其中病毒中和试验采用固定病毒稀释血清法。试验需先滴定病毒毒价，试验时将其稀释成每一单位剂量含100个TCID50，再将待检血清倍比稀释加等量100个TCID50/ml的病毒液，混匀后37℃孵育2h，每一稀释度接种细胞培养板，每孔0.1ml、5％CO 2 37℃培养箱培养4天后，记录出现CPE孔数。按Karber法计算中和价，即50％细胞被保护时抗血清的最大稀释度。
结果如图8和图4所示。图8显示了实施例8所制备的SARS-CoV-2中和抗体检测试剂盒与病毒中和试验的相关性r 2＝0.8858；其显著高于对比例所制备的SARS-CoV-2IgG抗体检测试剂盒测试结果与病毒中和试验的相关性r 2＝0.6175(如图4所示)。
实施例10考察孵育时间对检测结果的影响
分别研究了实施例8“样本检测”中第一步中孵育时间、第二步中孵育时间对检测结果的影响。
一方面，将第二步孵育时间固定为5min，采用不同的第一步孵育时间测试了样本1-4的信号值并计算了信噪比。结果如下表4所示。
表4
Figure PCTCN2020108534-appb-000005
由表4可知，当孵育时间低于5min，信号值和信噪比较低。当孵育时间为5-20min，信号值和信噪比可以接受且无显著差异。
另一方面，将第一步孵育时间固定为5min，采用不同的第二步孵育时间测试了样本1-4的信号值并计算了信噪比。结果如下表5所示。
表5
Figure PCTCN2020108534-appb-000006
由表5可知，当孵育时间低于5min，信号值和信噪比较低。当孵育时间为5-20min，信号值和信噪比可以接受且无显著差异。
实施例11磁珠包被物及人血管紧张素转化酶II(ACE2)浓度的确定
根据实施例8中的记载制备捕获组分，其中S1-RBD使用量固定为10μg/mg，确定磁珠包被物和人血管紧张素转化酶II(ACE2)浓度对信号值和信噪比的影响。
一方面，固定人血管紧张素转化酶II(ACE2)的浓度为1μg/mL，采用不同的磁珠包被物浓度按照实施例8中记载的方法测试了样本1-4的信号值并计算了信噪比。结果如下表6所示。
表6
Figure PCTCN2020108534-appb-000007
Figure PCTCN2020108534-appb-000008
由表6可知，固相包被物低于0.5mg/mL时，信号值和信噪比较低；固相包被物浓度为0.5-1.2mg/mL时，信号值和信噪比可以接受。提升固相包被物浓度不会显著提升信噪比。
另一方面，固定磁珠包被物的浓度为1.0mg/mL，采用不同的人血管紧张素转化酶II(ACE2)浓度按照实施例8记载的方法测试了样本1～4的信号值并计算了信噪比。结果如下表7所示。
表7
Figure PCTCN2020108534-appb-000009
由表7可知，人血管紧张素转化酶高于1.5μg/mL时，信号值和信噪比较低；浓度 为0.2-1.5μg/mL时，信号值和信噪比可以接受。
实施例12使用S1蛋白(S1亚基)制备SARS-CoV-2中和抗体检测试剂盒
步骤1：捕获组分的制备：将SARS-CoV-2刺突蛋白S1蛋白(深圳迈瑞生物医疗电子股份有限公司)包被于磁珠(Thermo fisher)表面磁珠。
具体包被方法为：首先将S1蛋白进行前处理，通过透析去除其缓冲基质中的保护组分。按照每毫克磁珠加入0.5-40μg的抗原的比例进行包被。在反应过程中磁珠表面的羧基在EDC/NHS催化下与S1蛋白的氨基进行偶联。取20mg表面修饰有羧基的磁珠，超声分散于10mM MES缓冲液中，加入80mg EDC和120mg NHS，超声混合均匀后，置于37℃摇床15min。之后在处理后的磁珠中，按照比例加入S1蛋白，混匀，并置于37℃摇床反应10-18h。之后清洗封闭后，制备得包被有S1蛋白的磁珠包被物。将磁珠包被物稀释于50mM MES缓冲液(0.5M NaCl、0.5％BSA、0.05％吐温20，pH＝6.0)，磁珠包被物浓度为0.5-1.2mg/mL。
竞争组分、检测组分的制备和样本的检测同实施例8。
实施例13考察实施例12的试剂盒与病毒中和试验检测结果的一致性
采用实施例12所制备的试剂盒和病毒中和试验对50名COVID-19康复期患者血清样本进行检测，比较实施例12所制备的试剂盒的测试结果与病毒中和试验的一致性。其中病毒中和试验的方法同实施例9。
结果如图9所示，其中实施例12中使用S1蛋白所制备的中和抗体检测试剂盒与病毒中和试验的相关性r 2＝0.8308。

Claims (22)

  1. 一种用于测定冠状病毒中和抗体的试剂盒，包括：
    捕获组分，其含有包被有蛋白的固相载体，所述蛋白具有所述冠状病毒的刺突蛋白S1亚基的RBD序列；
    人血管紧张素转化酶II；和
    可检测的信号标记物，
    其中，所述可检测的信号标记物标记在所述人血管紧张素转化酶II上，或者标记在二抗上。
  2. 根据权利要求1所述的试剂盒，其中，所述可检测的信号标记物标记在所述人血管紧张素转化酶II上，标记有可检测的信号标记物的人血管紧张素转化酶II作为检测组分。
  3. 根据权利要求2所述的试剂盒，其中，所述包被有蛋白的固相载体的浓度为0.2mg/mL至1.0mg/mL。
  4. 根据权利要求2所述的试剂盒，其中，标记有可检测的信号标记物的人血管紧张素转化酶II的浓度为0.5μg/mL至2μg/mL，例如为1μg/mL至2μg/mL。
  5. 根据权利要求1所述的试剂盒，其中，所述可检测的信号标记物标记在二抗上，标记有可检测的信号标记物的所述二抗作为检测组分，所述人血管紧张素转化酶II作为竞争组分。
  6. 根据权利要求5所述的试剂盒，其中，所述包被有蛋白的固相载体的浓度为0.5mg/mL至1.2mg/mL。
  7. 根据权利要求5所述的试剂盒，其中，所述人血管紧张素转化酶II的浓度为0.2μg/mL至1.5μg/mL。
  8. 根据权利要求1-7中任一项所述的试剂盒，其中，所述蛋白选自由所述冠状病毒的刺突蛋白、所述冠状病毒的刺突蛋白S1亚基和所述冠状病毒的刺突蛋白S1亚基的RBD组成的组中的一种或多种。
  9. 一种测定受试者样本中的中和抗体的方法，包括：
    将捕获组分、标记有可检测的信号标记物的人血管紧张素转化酶II和所述样本混合并孵育一段时间，使得所述人血管紧张素转化酶II与样本中的中和抗体能竞争性地与所述捕获组分结合；
    将获得的混合物进行清洗，除去未结合的物质；
    测量可检测的信号标记物的信号值；以及
    根据所述信号值获得所述中和抗体的测定结果，
    其中，所述捕获组分含有包被有蛋白的固相载体，所述蛋白具有冠状病毒的刺突蛋白S1亚基的RBD序列。
  10. 根据权利要求9所述的方法，其中，所述混合并孵育一段时间是先将所述捕获组分与样本混合并孵育一段时间，然后再加入标记有可检测的信号标记物的人血管紧张素转化酶II混合并孵育一段时间。
  11. 根据权利要求9所述的方法，其中，所述混合并孵育一段时间是同时将所述捕获组分、标记有可检测的信号标记物的人血管紧张素转化酶II和样本加入，混合并孵育一段时间。
  12. 根据权利要求9-11中任一项所述的方法，其中，所述孵育一段时间是孵育至少5min，例如为5min至10min。
  13. 根据权利要求12所述的方法，其中，所述包被有蛋白的固相载体的浓度为0.2mg/mL至1.0mg/mL。
  14. 根据权利要求9所述的方法，其中，标记有可检测的信号标记物的人血管紧张素转化酶II的浓度为0.5μg/mL至2μg/mL，例如为1μg/mL至2μg/mL。
  15. 一种测定受试者样本中的中和抗体的方法，包括：
    将捕获组分、人血管紧张素转化酶II和所述样本混合并孵育一段时间，使得所述人血管紧张素转化酶II与样本中的中和抗体能竞争性地与所述捕获组分结合；
    将获得的混合物进行清洗，除去未结合的物质；
    在经清洗的混合物中加入带有可检测的信号标记物的二抗，混匀并孵育一段时间，使得所述二抗能与所述捕获组分上结合的中和抗体结合，形成复合物；
    对获得的混合物进行清洗，除去未形成复合物的物质；
    测量可检测的信号标记物的信号值；以及
    根据所述信号值获得中和抗体的测定结果，
    其中，捕获组分含有包被有蛋白的固相载体，并且所述蛋白具有冠状病毒的刺突蛋白S1亚基的RBD序列。
  16. 根据权利要求15所述的方法，其中，所述孵育一段时间均是孵育至少5min，例如为5min至20min。
  17. 根据权利要求15所述的方法，其中，所述包被有蛋白的固相载体的浓度为0.5mg/mL至1.2mg/mL。
  18. 根据权利要求15所述的方法，其中，所述人血管紧张素转化酶II的浓度为 0.2μg/mL至1.5μg/mL。
  19. 根据权利要求9-18中任一项所述的方法，其中，所述蛋白选自由所述冠状病毒的刺突蛋白、所述冠状病毒的刺突蛋白S1亚基和所述冠状病毒的刺突蛋白S1亚基的RBD组成的组中的一种或多种。
  20. 包被物在制备冠状病毒中和抗体的检测试剂中的用途，其中，所述包被物为具有所述冠状病毒的刺突蛋白S1亚基的RBD序列的蛋白，
    并且在所述检测试剂中，所述包被物包被在固相载体上以构成捕获组分。
  21. 包被物和人血管紧张素转化酶II在制备冠状病毒中和抗体的检测试剂中的用途，其中，所述包被物为具有所述冠状病毒的刺突蛋白S1亚基的RBD序列的蛋白，
    并且在所述检测试剂中，所述包被物包被在固相载体上以构成捕获组分；所述人血管紧张素转化酶II能与所述中和抗体竞争性地与所述捕获组分结合。
  22. 根据权利要求20或21所述的用途，其中，所述冠状病毒选自由SARS、MERS和SARS-CoV-2所组成的组，优选的，所述冠状病毒是SARS-CoV-2。
PCT/CN2020/108534 2020-08-11 2020-08-11 用于检测冠状病毒中和抗体的试剂盒、方法 WO2022032497A1 (zh)

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