CN113419061A

CN113419061A - Magnetic particle chemiluminescence kit for detecting SARS-CoV-2 virus neutralizing antibody and application thereof

Info

Publication number: CN113419061A
Application number: CN202110678177.7A
Authority: CN
Inventors: 覃喜建; 汤双双
Original assignee: Nanjing Jinsirui Science and Technology Biology Corp
Current assignee: Nanjing Jinsirui Science and Technology Biology Corp
Priority date: 2020-06-19
Filing date: 2021-06-18
Publication date: 2021-09-21
Anticipated expiration: 2041-06-18
Also published as: WO2021254476A1; CN114509571A; CN113419061B

Abstract

The invention relates to the field of antibody detection, in particular to a magnetic particle chemiluminescence kit for detecting a SARS-CoV-2 virus neutralizing antibody and application thereof, wherein the kit comprises a spike protein part of a novel coronavirus and ACE2 protein or a functional fragment thereof which is specifically combined with the spike protein part of the novel coronavirus; wherein, the spike protein part of the novel coronavirus is connected with a marker, and the ACE2 protein or a functional fragment thereof is connected with a magnetic particle; or the spike protein part of the novel coronavirus is connected with the magnetic particle, and the ACE2 protein or the functional fragment thereof is connected with a marker. The magnetic particle chemiluminescence kit can be used for screening and identifying neutralizing antibodies in biological samples, evaluating the effectiveness of vaccines, and qualitatively or quantitatively detecting the neutralizing antibodies against SARS-CoV-2 virus in samples of subjects.

Description

Magnetic particle chemiluminescence kit for detecting SARS-CoV-2 virus neutralizing antibody and application thereof

Technical Field

The invention relates to the field of antibody detection, in particular to a method for detecting a SARS-CoV-2 virus neutralizing antibody by a technology of inhibiting or blocking magnetic particle chemiluminescence by the SARS-CoV-2 virus neutralizing antibody and a magnetic particle chemiluminescence kit for detecting the SARS-CoV-2 virus neutralizing antibody.

Background

The SARS-CoV-2 virus as pathogen of coronavirus pneumonia (COVID-19), also called 2019Novel coronavirus (2019Novel coronavirus,2019-nCoV), is a RNA coronavirus. 2019 the epidemic situation of coronavirus gradually becomes a global plague, which causes more than 600 million infected patients and more than 30 million patients to die. SARS-CoV-2 virus enters the host cell by binding to the ACE2 receptor on the surface of the host cell via the transmembrane spike glycoprotein (S protein) RBD domain. The SARS-CoV-2 virus neutralizing antibody inhibits SARS-CoV-2 virus from entering host cell by blocking the binding of SARS-CoV-2RBD protein to ACE2 receptor. Research shows that in the treatment process of patients with the novel coronavirus pneumonia, the plasma treatment of patients in the convalescence period achieves better curative effect, and shows the potential of a neutralizing antibody in the treatment aspect of the novel coronavirus pneumonia.

Currently, detection methods for neutralizing antibodies include detection methods based on the traditional neutralization assay (NT) and blocking ELISA detection methods. The NT method is to infect cells after an antibody sample reacts with virus, calculate virus titer by observing cytopathic conditions and adopting a cell culture half infection amount (CCID50) or a half tissue cell infection dose (TCID50) method, and further evaluate the blocking capacity of a neutralizing antibody on virus infection. The neutralizing antibody blocking ELISA method is that the neutralizing antibody in a sample is blocked by using a marked neutralizing antibody to be combined with virus or recombinant antigen coated on an enzyme label plate, chemical enzyme linked color development is carried out, a result is obtained by using an enzyme label analyzer, and then the neutralizing effect of the neutralizing antibody is evaluated. However, in practical application, the NT method has a long experimental period and a large workload, and is difficult to meet the requirements of rapid and high-throughput screening. Neutralizing antibody blocking ELISA methods require the preparation of neutralizing antibodies, which is difficult.

The magnetic particle chemiluminescence analysis method is a novel analysis and detection technology which effectively combines a magnetic separation technology, an immunoassay technology and a chemiluminescence detection technology. The technology uses micro-scale magnetic particles to mark biomolecules such as antibody protein and the like to capture target protein in a sample. The technology uses a chemiluminescent substance as a marker to directly mark biomolecules such as antibody protein and the like, or uses an enzyme capable of catalyzing the chemiluminescent substance as the marker to generate a detection signal. After the reaction, the chemiluminescent substance generates a photon signal which can be detected by a chemiluminescence instrument in the presence of a corresponding oxidant or catalytic enzyme, so that accurate qualitative or quantitative determination of the antibody protein complex to be detected is realized. The most commonly used labels in magnetic particle chemiluminescence immunoassay methods are Alkaline Phosphatase (AP), acridinium ester, horseradish peroxidase (HRP), and the like. The magnetic particle chemiluminescence technology has the advantages of high sensitivity, strong specificity, rapid separation of a magnetic separation system, short detection time, wide linear range, easy realization of automation and the like. In recent years, the method has been widely used in various fields such as clinical diagnosis, biomedicine, food safety inspection, and the like.

There is no report of detecting SARS-CoV-2 virus neutralizing antibody by magnetic particle chemiluminescence analysis method using magnetic particle labeled SARS-CoV-2RBD protein (or ACE2 receptor) and label coupled ACE2 receptor (SARS-CoV-2RBD protein).

Disclosure of Invention

The invention develops a chemiluminescence kit for detecting the SARS-CoV-2 virus neutralizing antibody by utilizing the characteristic that the SARS-CoV-2 virus neutralizing antibody can block the combination of SARS-CoV-2RBD protein and ACE2 receptor. The kit can be universal, is simple and convenient to operate, has low use cost and is easier to popularize and apply.

In one aspect, the present invention provides a chemiluminescent kit for detecting a neutralizing antibody against a novel coronavirus (SARS-CoV-2) or a variant thereof in a sample, characterized in that the kit comprises a spike protein portion of the novel coronavirus or a variant thereof and ACE2 protein or a functional fragment thereof that specifically binds to the spike protein portion of the novel coronavirus or a variant thereof; wherein the spike protein portion of the novel coronavirus or variant thereof is linked to a label, and the ACE2 protein or functional fragment thereof is linked to a magnetic microparticle; or the spike protein part of the novel coronavirus or the variant thereof is connected with magnetic particles, and the ACE2 protein or the functional fragment thereof is connected with a marker; wherein specific binding between the spike protein portion of the novel coronavirus or variant thereof and the ACE2 protein or functional fragment thereof generates a detection signal.

In some embodiments, the label or magnetic particle is attached via a linker or coupling means, preferably via coupling means. In some embodiments, the label or magnetic particle is attached by coupling means. In other embodiments, the label or magnetic particle is attached via a linker. Wherein the linker can be selected from small molecule chemical linkers or peptide linkers, including but not limited to carboxyl, amino, thiol, etc., and the label or magnetic particle can be linked to the spike protein portion of the novel coronavirus or variant thereof or the ACE2 protein or functional fragment thereof through the linker. Wherein the coupling means is linked by forming a covalent bond, which can be formed by covalent reaction of the tag or the magnetic particle with the spike protein portion of the novel coronavirus or variant thereof or the ACE2 protein or functional fragment thereof. In a particular embodiment, the label or magnetic particle is attached by covalent coupling.

In some embodiments, the spike protein portion of the novel coronavirus, or variant thereof, is conjugated to a label and the ACE2 protein, or functional fragment thereof, is conjugated to a magnetic microparticle. In other embodiments, the spike protein portion of the novel coronavirus, or variant thereof, is conjugated to a magnetic microparticle and the ACE2 protein, or functional fragment thereof, is conjugated to a label.

In some embodiments, the label is selected from one or more of alkaline phosphatase, acridinium ester, horseradish peroxidase, ruthenium terpyridyl, isoluminol, or a rare earth element or biotin, preferably alkaline phosphatase, acridinium ester, or horseradish peroxidase. In some embodiments, the label is selected from the group consisting of alkaline phosphatase, acridinium ester, horseradish peroxidase, or ruthenium terpyridyl. In other embodiments, the label is selected from the group consisting of alkaline phosphatase, acridinium ester, and horseradish peroxidase. In a specific embodiment, the marker is alkaline phosphatase. In another embodiment, the label is an acridinium ester. In yet another specific embodiment, the label is horseradish peroxidase.

In some embodiments, the chemiluminescent kit is selected from the group consisting of a horseradish peroxidase magnetic particle chemiluminescent kit, an alkaline phosphatase magnetic particle chemiluminescent kit, an acridinium ester magnetic particle chemiluminescent kit, or a terpyridyl ruthenium magnetic particle electrochemiluminescent kit, preferably an acridinium ester magnetic particle chemiluminescent kit or an alkaline phosphatase magnetic particle chemiluminescent kit. In some embodiments, the chemiluminescent kit is an acridinium ester magnetic particle chemiluminescent kit or an alkaline phosphatase magnetic particle chemiluminescent kit.

In some embodiments, the acridinium ester magnetic particle chemiluminescence kits comprise an acridinium ester-coupled novel coronavirus, or variant spike protein portion thereof, and a magnetic particle-coupled ACE2 protein, or functional fragment thereof. In other embodiments, the acridinium ester magnetic particle chemiluminescence kits comprise an acridinium ester-coupled ACE2 protein or functional fragment thereof and a magnetic particle-coupled portion of a novel coronavirus or variant spike protein thereof.

In some embodiments, the alkaline phosphatase magnetic particle chemiluminescence kit comprises an alkaline phosphatase-conjugated novel coronavirus, or variant spike protein portion thereof, and a magnetic particle-conjugated ACE2 protein, or functional fragment thereof. In other embodiments, the alkaline phosphatase magnetic particle chemiluminescence kits comprise an alkaline phosphatase-conjugated ACE2 protein or functional fragment thereof and a magnetic particle-conjugated novel coronavirus or variant spike protein thereof.

In some embodiments, the horseradish peroxidase magnetic particle chemiluminescence kit comprises a horseradish peroxidase-coupled novel coronavirus, or variant spike protein portion thereof, and a magnetic particle-coupled ACE2 protein, or a functional fragment thereof. In other embodiments, the horseradish peroxidase magnetic particle chemiluminescence kit comprises horseradish peroxidase-conjugated ACE2 protein or a functional fragment thereof and a magnetic particle-conjugated novel coronavirus or variant spike protein portion thereof.

In some embodiments, the ruthenium terpyridyl magnetic particle electrochemiluminescence kit comprises a ruthenium terpyridyl coupled novel coronavirus, or variant spike protein portion thereof, and a magnetic particle coupled ACE2 protein, or functional fragment thereof. In other embodiments, the ruthenium terpyridyl magnetic particle electrochemiluminescence kit comprises ruthenium terpyridyl coupled ACE2 protein or a functional fragment thereof and a magnetic particle coupled novel coronavirus or variant spike protein portion thereof.

In some embodiments, the ruthenium terpyridyl magnetic particle electrochemiluminescence kit comprises a ruthenium terpyridyl coupled novel coronavirus or variant spike protein portion thereof, magnetic particle coupled streptavidin, and biotin-labeled ACE2 protein or a functional fragment thereof. In other embodiments, the ruthenium terpyridyl magnetic particle electrochemiluminescence kit comprises ruthenium terpyridyl coupled ACE2 protein or a functional fragment thereof, magnetic particle coupled streptavidin, and a biotin-labeled novel coronavirus or variant spike protein portion thereof.

In some embodiments, the kit determines the presence of neutralizing antibodies to the novel coronavirus or variant thereof in the sample by detecting a change in a detection signal generated by the test sample that inhibits or blocks the binding of the spike protein portion of the novel coronavirus or variant thereof to the ACE2 protein or functional fragment thereof.

In some embodiments, the novel coronavirus or variant spike protein portion thereof is selected from the group consisting of a novel coronavirus or variant S protein thereof, a novel coronavirus or variant S1 protein thereof, a novel coronavirus or variant RBD protein thereof, or a novel coronavirus or variant RBD protein thereof with a His or Fc tag, preferably a novel coronavirus or variant RBD protein thereof. In some embodiments, the novel coronavirus, or variant spike protein thereof, is a novel coronavirus, or variant S1 protein thereof, or a novel coronavirus, or variant RBD protein thereof. In a specific embodiment, the novel coronavirus, or variant spike protein thereof, is a novel coronavirus, or variant RBD protein thereof.

In some embodiments, the portion of the spike protein of the novel coronavirus, or variant thereof, comprises the amino acid sequence set forth in any one of SEQ ID NOs 1,2, and 4-7, or a sequence at least 70% identical to the amino acid sequence set forth in any one of SEQ ID NOs 1,2, and 4-7. In some embodiments, the spike protein portion of the novel coronavirus, or variant thereof, comprises a sequence at least 70%, at least 75%, at least 80%, at least 83%, at least 85%, at least 87%, at least 89%, at least 91%, at least 93%, at least 95%, at least 97%, or at least 99% identical to an amino acid sequence set forth in any one of SEQ ID NOs 1,2, and 4-7. In other embodiments, the novel coronavirus spike protein moiety comprises a sequence at least 70%, at least 75%, at least 80%, at least 83%, at least 85%, at least 87%, at least 89%, at least 91%, at least 93%, at least 95%, at least 97%, or at least 99% identical to an amino acid sequence set forth in SEQ ID NO. 1 or 2. In some embodiments, the novel coronavirus variant spike protein portion comprises a sequence at least 70%, at least 75%, at least 80%, at least 83%, at least 85%, at least 87%, at least 89%, at least 91%, at least 93%, at least 95%, at least 97%, or at least 99% identical to an amino acid sequence set forth in any one of SEQ ID NOs 4-7. In other embodiments, the spike protein portion of the novel coronavirus, or variant thereof, comprises the amino acid sequence set forth in any one of SEQ ID NOs 1,2, and 4-7. In some embodiments, the novel coronavirus spike protein moiety comprises the amino acid sequence set forth in SEQ ID NO 1 or 2. In other embodiments, the spike protein portion of the novel coronavirus variant comprises the amino acid sequence set forth in any one of SEQ ID NOS 4-7. In a specific embodiment, the amino acid sequence of the spike protein portion of the novel coronavirus or variant thereof is any one of the sequences shown in SEQ ID NOs: 1,2 and 4-7. In another embodiment, the amino acid sequence of the spike protein portion of the novel coronavirus is set forth in SEQ ID NO 1 or 2. In a specific embodiment, the amino acid sequence of the spike protein portion of the novel coronavirus variant is as shown in any one of SEQ ID NOs 4-7.

In some embodiments, the marker-conjugated novel coronavirus, or variant spike protein portion thereof, is at a concentration of 2-200ng/mL, preferably 80 ng/mL. In some embodiments, the concentration of the spike protein moiety of the marker-conjugated novel coronavirus, or variant thereof, is 2ng/mL, 10ng/mL, 20ng/mL, 30ng/mL, 40ng/mL, 50ng/mL, 60ng/mL, 70ng/mL, 80ng/mL, 90ng/mL, 100ng/mL, 110ng/mL, 120ng/mL, 130ng/mL, 140ng/mL, 150ng/mL, 160ng/mL, 170ng/mL, 180ng/mL, 190ng/mL, or 200 ng/mL. In other embodiments, the marker-conjugated novel coronavirus, or variant thereof, spike protein moiety has a concentration of 80 ng/mL. In some embodiments, the marker-conjugated novel coronavirus, or variant spike protein portion thereof, is at a concentration of 100 ng/mL.

In some embodiments, the marker-conjugated novel coronavirus, or variant spike protein moiety thereof, is an acridinium ester-labeled novel coronavirus, or variant RBD protein thereof, or an alkaline phosphatase-labeled novel coronavirus, or variant RBD protein thereof. In other embodiments, the marker-conjugated novel coronavirus, or variant spike protein moiety thereof, is an acridinium ester-labeled novel coronavirus, or variant RBD protein thereof. In some embodiments, the concentration of the acridinium ester-labeled novel coronavirus or variant RBD protein thereof is 2-200ng/mL, preferably 10-100 ng/mL. In some preferred embodiments, the acridinium ester-labeled novel coronavirus or variant thereof RBD protein concentration is 10-100 ng/mL. In other embodiments, the concentration of the acridinium ester-labeled novel coronavirus or variant RBD protein thereof is 2ng/mL, 10ng/mL, 20ng/mL, 30ng/mL, 40ng/mL, 50ng/mL, 60ng/mL, 70ng/mL, 80ng/mL, 90ng/mL, 100ng/mL, 110ng/mL, 120ng/mL, 130ng/mL, 140ng/mL, 150ng/mL, 160ng/mL, 170ng/mL, 180ng/mL, 190ng/mL, or 200 ng/mL. In some embodiments, the marker-conjugated novel coronavirus, or variant spike protein portion thereof, is an alkaline phosphatase-labeled novel coronavirus, or variant RBD protein thereof. In some embodiments, the concentration of the alkaline phosphatase-labeled novel coronavirus, or variant RBD protein thereof, is 2-200ng/mL, preferably 50 ng/mL. In other embodiments, the concentration of the alkaline phosphatase-labeled novel coronavirus, or variant RBD protein thereof, is 2ng/mL, 10ng/mL, 20ng/mL, 30ng/mL, 40ng/mL, 50ng/mL, 60ng/mL, 70ng/mL, 80ng/mL, 90ng/mL, 100ng/mL, 110ng/mL, 120ng/mL, 130ng/mL, 140ng/mL, 150ng/mL, 160ng/mL, 170ng/mL, 180ng/mL, 190ng/mL, or 200 ng/mL. In a preferred embodiment, the concentration of the alkaline phosphatase-labeled novel coronavirus or variant RBD protein thereof is 50 ng/mL.

In some embodiments, the ACE2 protein or functional fragment thereof is derived from a human. In other embodiments, the ACE2 protein or functional fragment thereof is selected from the group consisting of a recombinant ACE2 protein, an extracellular domain of an ACE2 protein, or an Fc-tagged ACE2 protein, an extracellular domain of an ACE2 protein. In some embodiments, the ACE2 protein or functional fragment thereof is a recombinant human ACE2 protein. In other embodiments, the ACE2 protein or functional fragment thereof comprises the amino acid sequence set forth in any one of SEQ ID NOs 3 and 8-12, or a sequence that is at least 70% identical to the amino acid sequence set forth in any one of SEQ ID NOs 3 and 8-12. In some embodiments, the ACE2 protein or functional fragment thereof comprises a sequence that is at least 70%, at least 75%, at least 80%, at least 83%, at least 85%, at least 87%, at least 89%, at least 91%, at least 93%, at least 95%, at least 97%, or at least 99% identical to an amino acid sequence set forth in any of SEQ ID NOs 3 and 8-12. In some embodiments, the ACE2 protein or functional fragment thereof comprises the amino acid sequence set forth in any one of SEQ ID NOs 3 and 8-12. In a specific embodiment, the amino acid sequence of the ACE2 protein or functional fragment thereof is as set forth in any one of SEQ ID NOs 3 and 8-12.

In some embodiments, the concentration of ACE2 protein or a functional fragment thereof in the magnetic particle-coupled ACE2 protein or a functional fragment thereof is 2-15ug/mL, preferably 5-10ug/mL, more preferably 5 ug/mL. In some embodiments, the concentration of ACE2 protein or functional fragment thereof in the magnetic particle-coupled ACE2 protein or functional fragment thereof is 5-10 ug/mL. In some embodiments, the concentration of the ACE2 protein or functional fragment thereof is 2ug/mL, 3ug/mL, 4ug/mL, 5ug/mL, 6ug/mL, 7ug/mL, 8ug/mL, 9ug/mL, 10ug/mL, 11ug/mL, 12ug/mL, 13ug/mL, 14ug/mL, or 15 ug/mL. In a specific embodiment, the concentration of the ACE2 protein or functional fragment thereof is 5 ug/mL.

In other embodiments, the concentration of the magnetic microparticles is from 0.1 to 0.5 mg/mL. In some embodiments, the concentration of the magnetic microparticles is 0.1mg/mL, 0.2mg/mL, 0.3mg/mL, 0.4mg/mL, or 0.5 mg/mL. In a preferred embodiment, the concentration of the magnetic particles is 0.25 mg/mL.

In some embodiments, the kit further comprises a sample diluent and a wash solution.

In other embodiments, the kit further comprises a chemiluminescent substrate. The chemiluminescent substrate is selected from the group consisting of AMPPD, APS5, CSPD, CDP-Star, luminol, and PS-atto. The chemiluminescent substrate of the present invention includes, but is not limited to, direct chemiluminescent acid-base luminescence promoters such as H-containing₂O₂Acid (HNO)₃Or HCl, etc.) and NaOH, indirect chemiluminescent substrates include alkaline phosphatase substrates such as AMPPD (3- (2-spiroadamantane) -4-methoxy-4- (3-phosphonooxy) -phenyl-1, 2-dioxane disodium salt), APS5(9- (4' -chlorophenylthiophosphoroxymethylene) -10-methyl-9, 10-dihydroacridine disodium salt), CSPD (3- (2-spiroadamantane) -4-methoxy-4- (3-phosphonooxy) -phenyl-1, 2-dioxane), CDP-Star (3- (2-spiroadamantane) -4-methoxy-4- (3-phosphonooxy) -phenyl-1, 2-dioxetane) and horseradish peroxidase substrates such as luminol and PS-atto (Lumi-Phos HRP), and the like.

In some embodiments, the kit further comprises a negative control, a sample diluent, and a wash solution, the negative control being the sample diluent.

In other embodiments, the kit further comprises a positive quality control which is a neutralizing antibody against SARS-CoV-2 virus or a variant thereof.

In some embodiments, the sample diluent comprises disodium phosphate, sodium dihydrogen phosphate, sodium chloride, Proclin300, sucrose, bovine serum albumin, or tween 20, at a pH of 7.0-7.5; the lotion comprises Tris, sodium chloride and Tween-20, and the pH value is 7.5-8.0. In some embodiments, the sample diluent comprises 3.0-4.0mg/mL disodium phosphate dodecahydrate, 0.2-0.4mg/mL sodium dihydrogen phosphate dihydrate, 7-9mg/mL sodium chloride, 10-30mg/mL sucrose, 5-20mg/mL bovine serum albumin, Proclin300, and tween 20, at a pH of 7.0-7.5. In a specific embodiment, the sample diluent comprises 3.5mg/mL disodium phosphate dodecahydrate, 0.26mg/mL sodium dihydrogen phosphate dihydrate, 8.5mg/mL sodium chloride, 20mg/mL sucrose, 10mg/mL bovine serum albumin, Proclin300, and Tween 20, and has a pH of 7.0-7.5. In other embodiments, the wash solution comprises 25-35mg/mL Tris, 180 mg/mL sodium chloride, Tween 20, and a pH of 7.5-8.0. In a specific embodiment, the lotion comprises 30.25mg/mL Tris, 219.15mg/mL sodium chloride, Tween 20, pH 7.5-8.0.

In some embodiments, the novel coronavirus variant is selected from SARS-CoV-2B.1.1.7(GISAID accession ID: EPI _ ISL _1234251), B.1.351(GISAID accession ID: EPI _ ISL _1191083), or B.1.617(GISAID accession ID EPI _ ISL _ 1544002).

The invention provides application of the chemiluminescence kit in detecting whether SARS-CoV-2 virus or antibody of variant thereof exists in a sample.

In some embodiments, the sample comprises:

(1) plasma, serum or whole blood infected or suspected of being infected with the novel coronavirus;

(2) plasma, serum or whole blood samples after inoculation with the novel coronavirus vaccine;

(3) plasma, serum or whole blood of animals immunized with the novel coronavirus spike protein; or

(4) A sample of antibodies biologically derived against the novel coronavirus.

The invention also provides the application of the chemiluminescence kit in detecting SARS-CoV-2 virus infection or suspected infection of a subject or a variant thereof.

In a further aspect, the present invention provides the use of a chemiluminescent kit as described above to detect whether a subject has developed effective immunity following vaccination against SARS-CoV-2 or a variant thereof.

In still another aspect, the present invention provides the use of the above-described chemiluminescent kit for the detection of neutralizing antibodies against SARS-CoV-2 or variants thereof obtained by biological methods.

The invention also provides the application of the chemiluminescence kit in detecting whether the antibody of SARS-CoV-2 virus or the variant thereof exists in a subject.

The invention also provides a use method of the chemiluminescence kit, which comprises the following steps:

(1) adding the diluted sample, the positive quality control product or the negative quality control product into a detection tube;

(2) adding a marker-coupled novel coronavirus spike protein part into a detection tube, uniformly mixing, and incubating;

(3) adding ACE2 protein or its functional fragment coupled with magnetic particles, mixing, incubating, magnetically separating, removing supernatant, adding washing solution, and washing repeatedly;

(4) adding a chemiluminescence substrate, mixing uniformly, and detecting the luminous intensity.

In another aspect, the present invention provides a method for using the above chemiluminescence kit, comprising the following steps:

(1) respectively adding the diluted sample, the positive quality control product or the negative quality control product into a detection tube;

(2) adding magnetic particle coupled ACE2 protein or its functional fragment into a detection tube;

(3) adding the marker-conjugated novel coronavirus spike protein part, uniformly mixing, incubating, performing magnetic separation, removing supernatant, adding washing liquid, and repeatedly washing;

The present application also provides a method of detecting the presence or absence of neutralizing antibodies to a novel coronavirus, or variant thereof, in a sample, comprising:

(1) contacting the sample with a novel coronavirus or variant spike protein thereof or an ACE 2-binding fragment thereof;

(2) contacting the mixture of step (1) with an ACE2 protein or fragment thereof that specifically binds to the spike protein or ACE 2-binding fragment thereof;

(3) detecting the level of binding of the spike protein or ACE2 binding fragment thereof to the ACE2 protein or fragment thereof, wherein the spike protein of the novel coronavirus or variant thereof or ACE2 binding fragment thereof is linked to a label and the ACE2 protein or fragment thereof is linked to magnetic microparticles.

The present application further provides a method for detecting the presence or absence of neutralizing antibodies to a novel coronavirus, or variant thereof, in a sample, comprising:

(2) connecting the novel coronavirus or variant spike protein thereof or ACE2 binding fragment thereof with magnetic particles;

(3) adding an ACE2 protein or fragment thereof that specifically binds to the spike protein or ACE 2-binding fragment thereof;

(4) detecting the level of binding of the spike protein or ACE2 binding fragment thereof to the ACE2 protein or fragment thereof, wherein the ACE2 protein or fragment thereof is linked to a label.

The above method further comprises determining the presence or absence of neutralizing antibodies to the novel coronavirus or variant thereof in the sample by inhibiting or blocking the change in the detection signal produced by the binding of the spike protein portion of the novel coronavirus or variant thereof to the ACE2 protein or functional fragment thereof in the sample.

Detailed Description

The magnetic particle chemiluminescence analysis method is a novel analysis and detection technology which effectively combines a magnetic separation technology, an immunoassay technology and a chemiluminescence detection technology. The invention discloses a magnetic particle chemiluminescence detection method for SARS-CoV-2 virus neutralizing antibody, which can quickly and effectively detect SARS-CoV-2 virus neutralizing antibody in human and other animal samples, overcomes the defects of detecting the neutralizing antibody by NT method and blocking ELISA method, is used for screening and identifying the SARS-CoV-2 virus neutralizing antibody, evaluating the effectiveness of vaccines and qualitatively or quantitatively detecting the SARS-CoV-2 virus neutralizing antibody in a sample of a subject.

Magnetic particle chemiluminescence kit and preparation method thereof

The invention provides a magnetic particle chemiluminescence detection kit for detecting a neutralizing antibody of a sample SARS-CoV-2 virus or a variant thereof based on a magnetic particle chemiluminescence analysis method, which comprises the following steps: a. a marker-conjugated SARS-CoV-2 or variant spike protein portion thereof; b. magnetic microparticle-coupled ACE2 protein or a functional fragment thereof; c. a sample diluent; d. positive quality control products; e. negative quality control products; f. a wash solution; and g.

In some embodiments, the magnetic particle chemiluminescent detection kit for SARS-CoV-2 virus or variant neutralizing antibodies comprises: a. a marker-conjugated SARS-CoV-2 or variant spike protein portion thereof; b. magnetic microparticle-coupled ACE2 protein or a functional fragment thereof; c. a sample diluent; d. positive quality control products; e. negative quality control products; f. a wash solution; and g.

In other embodiments, the magnetic particle chemiluminescent detection kit for SARS-CoV-2 virus or variant neutralizing antibodies comprises: a. a marker-conjugated SARS-CoV-2 or variant RBD protein thereof; b. magnetic microparticle-coupled ACE2 protein; c. a sample diluent; d. positive quality control products; e. negative quality control products; f. a wash solution; and g.

In some embodiments, the label is selected from one or more of alkaline phosphatase, acridinium ester, horseradish peroxidase, ruthenium terpyridyl, isoluminol, or a rare earth element, preferably alkaline phosphatase, acridinium ester, or horseradish peroxidase. In some embodiments, the label is selected from the group consisting of alkaline phosphatase, acridinium ester, horseradish peroxidase, or ruthenium terpyridyl. In other embodiments, the label is selected from the group consisting of alkaline phosphatase, acridinium ester, and horseradish peroxidase. In a specific embodiment, the marker is alkaline phosphatase. In another embodiment, the label is an acridinium ester. In yet another specific embodiment, the label is horseradish peroxidase. In other embodiments, the marker-conjugated SARS-CoV-2 or variant RBD protein thereof is selected from the group consisting of alkaline phosphatase-conjugated SARS-CoV-2 or variant RBD protein thereof, acridinium ester-conjugated SARS-CoV-2 or variant RBD protein thereof, and horseradish peroxidase-conjugated SARS-CoV-2 or variant RBD protein thereof.

In some embodiments, the concentration of SARS-CoV-2 or its variant RBD protein conjugated to the marker is 2-200ng/mL, preferably 80 ng/mL. In some embodiments, the concentration of SARS-CoV-2 or its variant RBD protein conjugated to the marker is 2ng/mL, 10ng/mL, 20ng/mL, 30ng/mL, 40ng/mL, 50ng/mL, 60ng/mL, 70ng/mL, 80ng/mL, 90ng/mL, 100ng/mL, 110ng/mL, 120ng/mL, 130ng/mL, 140ng/mL, 150ng/mL, 160ng/mL, 170ng/mL, 180ng/mL, 190ng/mL, or 200 ng/mL. In some embodiments, the marker-conjugated SARS-CoV-2 or variant RBD protein thereof is at a concentration of 50 ng/mL. In another embodiment, in some embodiments, the marker-conjugated SARS-CoV-2 or variant thereof RBD protein has a concentration of 100 ng/mL.

In some embodiments, the marker-conjugated SARS-CoV-2 or variant RBD protein thereof is selected from an acridinium ester-labeled SARS-CoV-2 or variant RBD protein thereof or an alkaline phosphatase-labeled SARS-CoV-2 or variant RBD protein thereof. In other embodiments, the marker-conjugated SARS-CoV-2 or variant RBD protein thereof is an acridinium ester-labeled SARS-CoV-2 or variant RBD protein thereof. In some embodiments, the acridinium ester-labeled SARS-CoV-2 or a variant RBD protein thereof has a concentration of 2-200ng/mL, preferably 10-100 ng/mL. In some preferred embodiments, the acridinium ester-labeled SARS-CoV-2 or a variant RBD protein thereof is present at a concentration of 10-100 ng/mL. In other embodiments, the acridinium ester-labeled SARS-CoV-2 or a variant RBD protein thereof has a concentration of 2ng/mL, 10ng/mL, 20ng/mL, 30ng/mL, 40ng/mL, 50ng/mL, 60ng/mL, 70ng/mL, 80ng/mL, 90ng/mL, 100ng/mL, 110ng/mL, 120ng/mL, 130ng/mL, 140ng/mL, 150ng/mL, 160ng/mL, 170ng/mL, 180ng/mL, 190ng/mL, or 200 ng/mL. In some embodiments, the marker-conjugated SARS-CoV-2 or variant RBD protein thereof is alkaline phosphatase-labeled SARS-CoV-2 or variant RBD protein thereof. In some embodiments, the alkaline phosphatase-labeled SARS-CoV-2 or variant RBD protein thereof has a concentration of 2-200ng/mL, preferably 80 ng/mL. In other embodiments, the alkaline phosphatase-labeled SARS-CoV-2 or variant RBD protein thereof has a concentration of 2ng/mL, 10ng/mL, 20ng/mL, 30ng/mL, 40ng/mL, 50ng/mL, 60ng/mL, 70ng/mL, 80ng/mL, 90ng/mL, 100ng/mL, 110ng/mL, 120ng/mL, 130ng/mL, 140ng/mL, 150ng/mL, 160ng/mL, 170ng/mL, 180ng/mL, 190ng/mL, or 200 ng/mL. In a preferred embodiment, the alkaline phosphatase-labeled SARS-CoV-2 or variant thereof RBD protein has a concentration of 50 ng/mL.

In some embodiments, the ACE2 protein or functional fragment thereof is derived from a human. In other embodiments, the ACE2 protein or functional fragment thereof is selected from the group consisting of a recombinant ACE2 protein or an extracellular domain of an ACE2 protein or an Fc-tagged ACE2 protein or an extracellular domain of an ACE2 protein. In some embodiments, the ACE2 protein or functional fragment thereof is a recombinant human ACE2 protein. In other embodiments, the ACE2 protein or functional fragment thereof comprises the amino acid sequence set forth in any one of SEQ ID NOs 3 and 8-12, or a sequence that is at least 70% identical to the amino acid sequence set forth in any one of SEQ ID NOs 3 and 8-12. In some embodiments, the ACE2 protein or functional fragment thereof comprises a sequence that is at least 70%, at least 75%, at least 80%, at least 83%, at least 85%, at least 87%, at least 89%, at least 91%, at least 93%, at least 95%, at least 97%, or at least 99% identical to an amino acid sequence set forth in any of SEQ ID NOs 3 and 8-12. In some embodiments, the ACE2 protein or functional fragment thereof comprises the amino acid sequence set forth in any one of SEQ ID NOs 3 and 8-12. In a specific embodiment, the amino acid sequence of the ACE2 protein or functional fragment thereof is as set forth in any one of SEQ ID NOs 3 and 8-12.

In some embodiments, the concentration of ACE2 protein or a functional fragment thereof in the magnetic particle-coupled ACE2 protein or a functional fragment thereof is 2-15ug/mL, preferably 5-10ug/mL, more preferably 5 ug/mL. In some embodiments, the concentration of the ACE2 protein or functional fragment thereof is 5-10 ug/mL. In some embodiments, the concentration of the ACE2 protein or functional fragment thereof is 2ug/mL, 3ug/mL, 4ug/mL, 5ug/mL, 6ug/mL, 7ug/mL, 8ug/mL, 9ug/mL, 10ug/mL, 11ug/mL, 12ug/mL, 13ug/mL, 14ug/mL, or 15 ug/mL. In a specific embodiment, the concentration of the ACE2 protein or functional fragment thereof is 5 ug/mL.

In other embodiments, the concentration of the magnetic microparticles is from 0.1 to 0.5 mg/mL. In some embodiments, the concentration of the magnetic microparticles is 0.1mg/mL, 0.2mg/mL, 0.25mg/mL, 0.3mg/mL, 0.4mg/mL, or 0.5 mg/mL.

The invention provides a magnetic particle chemiluminescence qualitative detection kit for SARS-CoV-2 virus neutralizing antibody, which comprises: a. a label-conjugated ACE2 protein or a functional fragment thereof; b. a magnetic microparticle-coupled SARS-CoV-2 or variant spike protein portion thereof; c. a sample diluent; d. positive quality control products; e. negative quality control products; f. a wash solution; and g.

In some embodiments, the magnetic particle chemiluminescent qualitative detection kit for SARS-CoV-2 virus or variant neutralizing antibodies comprises: a. a label-conjugated ACE2 protein or a functional fragment thereof; b. a magnetic microparticle-coupled SARS-CoV-2 spike protein moiety; c. a sample diluent; d. positive quality control products; e. negative quality control products; f. a wash solution; and g.

In other embodiments, the magnetic particle chemiluminescent qualitative detection kit for SARS-CoV-2 virus or variant neutralizing antibodies comprises: a. a label-conjugated ACE2 protein; b. magnetic microparticle-conjugated SARS-CoV-2 or its variant RBD protein; c. a sample diluent; d. positive quality control products; e. negative quality control products; f. a wash solution; and g.

The invention provides a magnetic particle chemiluminescence quantitative detection kit for SARS-CoV-2 virus or variant neutralizing antibody thereof, which comprises: a. a label-conjugated ACE2 protein or a functional fragment thereof; b. a magnetic microparticle-coupled SARS-CoV-2 or variant spike protein portion thereof; c. a sample diluent; d. a calibrator; e. a wash solution; a substrate.

In some embodiments, the magnetic particle chemiluminescence quantitative detection kit for SARS-CoV-2 virus or variant neutralizing antibodies comprises: a. a label-conjugated ACE2 protein or a functional fragment thereof; b. a magnetic microparticle-coupled SARS-CoV-2 or variant spike protein portion thereof; c. a sample diluent; d. a calibrator; e. a wash solution; a substrate.

In other embodiments, the magnetic particle chemiluminescence quantitative detection kit for SARS-CoV-2 virus or variant neutralizing antibody comprises: a. a label-conjugated ACE2 protein; b. magnetic microparticle-coupled SARS-CoV-2RBD or variant proteins thereof; c. a sample diluent; d. a calibrator; e. a wash solution; a substrate.

In some embodiments, the label is selected from one or more of alkaline phosphatase, acridinium ester, horseradish peroxidase, ruthenium terpyridyl, isoluminol, or a rare earth element, preferably alkaline phosphatase, acridinium ester, or horseradish peroxidase. In some embodiments, the label is selected from the group consisting of alkaline phosphatase, acridinium ester, horseradish peroxidase, or ruthenium terpyridyl. In other embodiments, the label is selected from the group consisting of alkaline phosphatase, acridinium ester, and horseradish peroxidase. In a specific embodiment, the marker is alkaline phosphatase. In another embodiment, the label is an acridinium ester. In yet another specific embodiment, the label is horseradish peroxidase. In other embodiments, the label-conjugated ACE2 protein is selected from the group consisting of an alkaline phosphatase-conjugated ACE2 protein, an acridinium ester-conjugated ACE2 protein, and a horseradish peroxidase-conjugated ACE2 protein.

In some embodiments, the spike protein portion of SARS-CoV-2 or a variant thereof comprises the amino acid sequence set forth in any of SEQ ID NOs 1,2, and 4-7, or a sequence that is at least 70% identical to the amino acid sequence set forth in any of SEQ ID NOs 1,2, and 4-7. In some embodiments, the SARS-CoV-2 or mutant spike protein portion thereof comprises a sequence that is at least 70%, at least 75%, at least 80%, at least 83%, at least 85%, at least 87%, at least 89%, at least 91%, at least 93%, at least 95%, at least 97%, or at least 99% identical to an amino acid sequence set forth in any of SEQ ID NOS 1,2, and 4-7. In other embodiments, the SARS-CoV-2 spike protein portion comprises a sequence that is at least 70%, at least 75%, at least 80%, at least 83%, at least 85%, at least 87%, at least 89%, at least 91%, at least 93%, at least 95%, at least 97%, or at least 99% identical to an amino acid sequence set forth in SEQ ID NO. 1 or 2. In some embodiments, the SARS-CoV-2 variant spike protein portion comprises a sequence that is at least 70%, at least 75%, at least 80%, at least 83%, at least 85%, at least 87%, at least 89%, at least 91%, at least 93%, at least 95%, at least 97%, or at least 99% identical to an amino acid sequence set forth in any of SEQ ID NOs 4-7. In some embodiments, the SARS-CoV-2 or variant spike protein portion thereof comprises an amino acid sequence set forth in any of SEQ ID NOs 1,2, and 4-7. In other embodiments, the portion of the SARS-CoV-2 variant spike protein comprises the amino acid sequence set forth in any one of SEQ ID NOS 4-7. In some embodiments, the SARS-CoV-2 spike protein portion comprises the amino acid sequence set forth in SEQ ID NO 1 or 2. In a specific embodiment, the amino acid sequence of the spike protein portion of SARS-CoV-2 or a variant thereof is set forth in SEQ ID NOs 1,2, and 4-7. In another embodiment, the amino acid sequence of the spike protein portion of the novel coronavirus is set forth in SEQ ID NO 1 or 2. In a specific embodiment, the amino acid sequence of the spike protein portion of the novel coronavirus variant is as shown in any one of SEQ ID NO 4-7.

The sample diluent in the chemiluminescence kit comprises disodium hydrogen phosphate, sodium dihydrogen phosphate, sodium chloride, Proclin300, sucrose, bovine serum albumin or Tween 20, and the pH value is 7.0-7.5. In some embodiments, the sample diluent comprises 3.0-4.0mg/mL disodium phosphate dodecahydrate, 0.2-0.4mg/mL sodium dihydrogen phosphate dihydrate, 7-9mg/mL sodium chloride, 10-30mg/mL sucrose, 5-20mg/mL bovine serum albumin, Proclin300, and tween 20, at a pH of 7.0-7.5. In a specific embodiment, the sample diluent comprises 3.5mg/mL disodium phosphate dodecahydrate, 0.26mg/mL sodium dihydrogen phosphate dihydrate, 8.5mg/mL sodium chloride, 20mg/mL sucrose, 10mg/mL bovine serum albumin, Proclin300, and Tween 20, and has a pH of 7.0-7.5.

The negative quality control material in the chemiluminescence kit is a sample diluent. In some embodiments, the negative quality control comprises disodium hydrogen phosphate, sodium dihydrogen phosphate, sodium chloride, Proclin300, sucrose, bovine serum albumin, or tween 20, and the pH is 7.0 to 7.5.

The lotion in the chemiluminescence kit comprises Tris, sodium chloride and Tween-20, and the pH value is 7.5-8.0. In some embodiments, the wash solution comprises 25-35mg/mL of Tris, 180 mg/mL of sodium chloride, Tween 20, and a pH of 7.5-8.0. In a specific embodiment, the lotion comprises 30.25mg/mL Tris, 219.15mg/mL sodium chloride, Tween 20, pH 7.5-8.0.

The positive quality control material in the chemiluminescence kit is a neutralizing antibody of anti-SARS-CoV-2 virus or variant thereof. The neutralizing antibody against SARS-CoV-2 virus or its variant may be any one of those disclosed in the art as a neutralizing antibody against SARS-CoV-2 virus or its variant, i.e., an antibody that binds to the RBD protein of SARS-CoV-2 virus or its variant and blocks the binding of the viral RBD to the human ACE2 receptor, thereby preventing cellular virus invasion. The invention includes but is not limited to the clone 2B2 antibody provided in the examples.

The calibrator in the chemiluminescence kit is anti-SARS-CoV-2 virus or variant neutralizing antibody thereof with different concentrations. The neutralizing antibody against SARS-CoV-2 virus or its variant may be any one of those disclosed in the art as a neutralizing antibody against SARS-CoV-2 virus or its variant, i.e., an antibody that binds to the RBD protein of SARS-CoV-2 virus and blocks the binding of the viral RBD to the human ACE2 receptor, thereby preventing cellular viral entry. The present invention includes, but is not limited to, the a02087 antibody provided in the examples.

The substrate in the above-mentioned chemiluminescence kit of the invention can be an acid-base luminescence initiator of direct chemiluminescence and an indirect chemiluminescence substrate, including but not limited to an acid-base luminescence initiator of direct chemiluminescence such as H-containing₂O₂Acid (HNO)₃Or HCl, etc.) and NaOH, indirect chemiluminescent substrates include alkaline phosphatase substrates such as AMPPD, APS5, CSPD, CDP-Star, and horseradish peroxidase substrates such as luminol and PS-atto, etc.

The preparation method of the chemiluminescence kit comprises the following steps:

(1) preparing a marker-conjugated novel coronavirus, or a variant spike protein portion thereof;

(2) preparing magnetic particle coupled ACE2 protein;

(3) preparing a sample diluent, a negative and positive quality control product or a calibrator and a washing solution;

(4) the reagent is packaged into a kit.

Wherein the fraction of the spike protein of the marker-conjugated novel coronavirus or variant thereof of step (1) comprises SARS-CoV-2 or variant RBD protein thereof conjugated with the marker, and comprises alkaline phosphatase-labeled SARS-CoV-2RBD protein, acridinium ester-labeled SARS-CoV-2RBD protein, alkaline phosphatase-labeled SARS-CoV-2B.1.1.7RBD protein, acridinium ester-labeled SARS-CoV-2B.1.1.7RBD protein, alkaline phosphatase-labeled SARS-CoV-2 B.1.135RBD protein or acridinium ester-labeled SARS-CoV-2B.1.351RBD protein. In some embodiments, the method for preparing alkaline phosphatase-conjugated SARS-CoV-2 or a variant RBD protein thereof comprises: (1) activating SARS-CoV-2 or its variant RBD protein and alkaline phosphatase separately; (2) respectively replacing buffer solution for activated SARS-CoV-2 or its variant RBD protein and alkaline phosphatase by rotating desalting column; (3) uniformly mixing the alkaline phosphatase obtained in the step (2) with a SARS-CoV-2 or variant RBD protein solution, reacting, adding an N-ethylmaleimide solution, and standing; (4) and (4) replacing the buffer solution by the reactant obtained in the step (3) through a rotary desalting column to obtain the SARS-CoV-2 or the variant RBD protein thereof coupled with the AP. In other embodiments, the method for preparing an acridinium ester-conjugated SARS-CoV-2 or a variant RBD protein thereof comprises: (1) activating SARS-CoV-2 or its variant RBD protein, rotating desalting column to replace buffer solution; (2) adding acridine ester solution, mixing uniformly and reacting in a dark place; (3) and (3) adding glycine into the solution after the reaction in the step (2), and rotating the desalting column to replace the buffer solution to obtain SARS-CoV-2 coupled with acridinium ester or variant RBD protein thereof.

Wherein, the preparation of the magnetic particle coupled ACE2 protein in the step (2) comprises the following steps: (1) resuspending the magnetic particles by buffer solution, adding ACE2 protein, mixing uniformly, and reacting at constant temperature; (2) separating the supernatant by magnetic separation, adding confining liquid, and uniformly mixing at constant temperature for reaction; (3) magnetically separating the supernatant, re-suspending the magnetic particles, and repeatedly washing; (4) the magnetic microparticles were resuspended in a single pellet state by adding the sample diluent.

The principle of the magnetic particle chemiluminescence kit for detecting the neutralizing antibody of SARS-CoV-2 virus or the variant thereof provided by the invention is as follows: blocking the magnetic particle coupled SARS-CoV-2 or its variant RBD protein (or ACE2 receptor) and marker coupled ACE2 receptor (or SARS-CoV-2 or its variant RBD protein) interaction by neutralizing antibody of SARS-CoV-2 virus or its variant to generate detection signal. Adding a marker coupled ACE2 receptor (or SARS-CoV-2 or variant RBD protein thereof) and magnetic particles coupled SARS-CoV-2 or variant RBD protein thereof (or ACE2 receptor) in the sample to be detected and the negative quality control product sequentially or simultaneously. Magnetic particle-coupled SARS-CoV-2 or its variant RBD protein (or ACE2 receptor) binds to marker-coupled ACE2 receptor (or SARS-CoV-2RBD protein) when neutralizing antibodies to SARS-CoV-2 virus or its variant are not present in the sample. After washing by magnetic separation, the label bound to the magnetic particle produces a detection signal in the presence of the substrate. When neutralizing antibodies against SARS-CoV-2 virus or variants thereof are present in the sample, the amount of bound magnetic particle-coupled SARS-CoV-2 or variant RBD protein thereof (or ACE2 receptor) and marker-coupled ACE2 receptor (or SARS-CoV-2 or variant RBD protein thereof) is inversely proportional to the concentration of neutralizing antibodies against SARS-CoV-2 virus or variants thereof in the sample. The magnetic particles have reduced labels and the resulting signal is reduced after washing by magnetic separation. And respectively reading a sample value (S) to be measured and a negative sample measured value (N) by a chemiluminescence apparatus, and evaluating the content of the neutralizing antibody in the sample by using the blocking rate (1-S/N). But the blocking rate is more than or equal to 30 percent, which indicates that the neutralizing antibody of SARS-CoV-2 virus or the variant thereof exists in the detected sample. When the blocking rate is < 30%, it is indicated that no neutralizing antibodies against SARS-CoV-2 virus or variants thereof are present in the test sample.

Use method of magnetic particle chemiluminescence kit

(1) respectively adding the diluted sample liquid, the quality control product or the calibrator into a detection tube;

(2) adding a marker-conjugated novel coronavirus or variant spike protein part thereof into a detection tube, uniformly mixing, and incubating;

In another aspect, the present invention provides another method for using the above chemiluminescent kit, comprising the steps of:

(3) adding the spike protein part of the novel coronavirus or the variant thereof coupled with the marker, uniformly mixing, incubating, performing magnetic separation, removing supernatant, adding washing liquid, and repeatedly washing;

The invention also provides a using method of the chemiluminescence kit, which comprises the following steps:

(1) respectively adding the diluted sample solution, the positive quality control product or the negative quality control product into a detection tube;

(2) adding a label-conjugated ACE2 protein to the detection tube;

(3) adding magnetic particle coupled new coronavirus or its variant spike protein part, mixing, incubating, magnetically separating, removing supernatant, adding washing solution, and repeatedly washing;

The invention also provides another using method of the chemiluminescence kit, which comprises the following steps:

(2) adding a spike protein portion of a magnetic microparticle-coupled novel coronavirus, or a variant thereof, to a detection tube;

(3) adding ACE2 protein coupled with marker, mixing, incubating, magnetically separating, removing supernatant, adding washing liquid, and washing repeatedly;

Application of magnetic particle chemiluminescence kit

The invention provides the application of the chemiluminescence detection kit in detecting SARS-CoV-2 virus infection or suspected infection of a subject or a variant thereof. After the body is infected with the novel coronavirus SARS-CoV-2, the immune response of the body is not initiated, so that only the novel coronavirus SARS-CoV-2 antigen can be detected at this time. Thereafter, an immune response is elicited by the body, with the first immunoglobulin appearing being IgM antibodies, followed by IgG antibodies. Therefore, by detecting the existence of the novel coronavirus COVID-19 antigen and the specific IgM antibody and IgG antibody in the organism, the immune response state of the organism to the novel coronavirus SARS-CoV-2 antigen can be diagnosed. For a subject suspected of being infected, the presence of neutralizing antibodies to SARS-CoV-2 virus in serum can be detected by the kit of the invention to determine whether the subject is infected with SARS-CoV-2 virus. Infected subjects can be tested by the kits of the invention for the presence of neutralizing antibodies to SARS-CoV-2 virus in serum to determine whether effective immunity is developed in the body.

The invention provides the application of the chemiluminescence kit in detecting whether effective immunity is generated after a subject is inoculated with a vaccine aiming at SARS-CoV-2 or a variant thereof. The development of SARS-CoV-2 virus vaccine is currently actively underway in clinical practice, and the development of partial vaccines has already entered the clinical stage. The kit of the invention can be used for quickly detecting whether the subject inoculated with the SARS-CoV-2 vaccine generates effective immunity or not and determining whether the serum of the subject inoculated with the SARS-CoV-2 vaccine generates neutralizing antibodies or not. This plays an important role in the development of SARS-CoV-2 vaccine.

In still another aspect, the present invention provides the use of the above-mentioned chemiluminescent kit for detecting SARS-CoV-2 neutralizing antibodies obtained by biological methods. At present, a large number of antibodies against SARS-CoV-2 or variants thereof are under development, and anti-SARS-CoV-2 antibodies obtained by methods common in the art, including animal immunization, phage display library, and the like, need a rapid and efficient method for screening antibodies having neutralizing ability. The immunochromatographic device of the present invention can be used for detecting the neutralizing antibody of SARS-CoV-2 in the serum of immunized animals. Can also be used for detecting the neutralizing antibody in SARS-CoV-2 antibody purified by other different modes. For example, the antibody includes a human anti-SARS-CoV-2 virus neutralizing antibody, a mouse anti-SARS-CoV-2 virus neutralizing antibody, a rat anti-SARS-CoV-2 virus neutralizing antibody, a rabbit anti-SARS-CoV-2 virus neutralizing antibody, a camel anti-SARS-CoV-2 virus neutralizing antibody, an anti-SARS-CoV-2 virus neutralizing antibody Fab fragment and an anti-SARS-CoV-2 virus neutralizing antibody ScFv fragment, a SARS-CoV-2 virus neutralizing antibody screened by a phage antibody library screening technique, a SARS-CoV-2 virus neutralizing antibody sample obtained by a high throughput DNA sequencing technique, a SARS-CoV-2 virus neutralizing antibody prepared by a hybridoma technique, an antibody isolated from serum, and the like.

In yet another aspect, the kits of the invention can be used to detect the presence or absence of SARS-CoV-2 virus neutralizing antibodies in a subject. Wherein the subject includes a healthy subject, a subject who has been infected with the novel coronavirus and has recovered from health, or an infected person who does not exhibit any symptoms. Samples for testing a subject include, but are not limited to, serum, plasma, or whole blood.

The present invention provides a method for detecting the presence or absence of neutralizing antibodies to a novel coronavirus, or variant thereof, in a sample, comprising:

(1) contacting the sample with SARS-CoV-2 virus or a variant spike protein thereof or an ACE 2-binding fragment thereof;

(2) then, the SARS-CoV-2 virus or the variant spike protein or the ACE2 binding fragment thereof is connected to a solid phase carrier;

In some embodiments, the SARS-CoV-2 virus or variant spike protein thereof or ACE2 binding fragment thereof in step (1) is linked to biotin, and the streptavidin is coupled to the solid support in step (2), and the SARS-CoV-2 virus or variant spike protein thereof or ACE2 binding fragment thereof is linked to the solid support by binding of biotin to streptavidin on the solid support. Wherein the solid phase carrier is magnetic particles.

The present invention also provides a method for detecting the presence of neutralizing antibodies to SARS-CoV-2 virus or variants thereof in a sample, comprising:

(1) the amount of SARS-CoV-2 virus or variant spike protein thereof or ACE2 binding fragment thereof linked to the magnetic particles is controlled by the amount of magnetic particles or/and the amount of SARS-CoV-2 virus or variant spike protein thereof or ACE2 binding fragment thereof;

(2) contacting the sample with SARS-CoV-2 virus or variant spike protein thereof or ACE2 binding fragment thereof linked to magnetic microparticles;

In some embodiments, the amount of magnetic particles can be adjusted by controlling the concentration of magnetic particles. In other embodiments, the amount of SARS-CoV-2 virus or variant spike protein or ACE2 binding fragment thereof can be adjusted by controlling the concentration of SARS-CoV-2 virus or variant spike protein or ACE2 binding fragment thereof added or by controlling the density of SARS-CoV-2 virus or variant spike protein or ACE2 binding fragment thereof attached per surface area of the magnetic microparticles. The mole ratio of SARS-CoV-2 virus or its variant spike protein or its ACE2 binding fragment relative to the antibody in the sample is controlled by controlling the amount of magnetic particle or SARS-CoV-2 virus or its variant spike protein or its ACE2 binding fragment, so as to reach the optimal detection range of neutralizing antibody and raise the sensitivity and specificity of the detection method.

The present invention also provides a method for detecting the presence or absence of neutralizing antibodies to a novel coronavirus, or variant thereof, in a sample, comprising:

(3) detecting the level of binding of the spike protein or ACE2 binding fragment thereof to the ACE2 protein or fragment thereof, wherein the spike protein of the novel coronavirus or variant thereof or ACE2 binding fragment thereof is linked to a label and the ACE2 protein or fragment thereof is linked to a solid phase carrier.

In some embodiments, the solid support is a magnetic microparticle.

The method for detecting neutralizing antibodies against the novel coronavirus or the variant thereof as described above, further comprising determining the presence or absence of neutralizing antibodies against the novel coronavirus or the variant thereof in the sample by inhibiting or blocking the change in the detection signal generated by the binding between the spike protein portion of the novel coronavirus or the variant thereof and the ACE2 protein or the functional fragment thereof in the sample.

Interpretation of terms

The invention relates to a novel coronavirus (SARS-CoV-2), which is also called 2019-nCoV, belongs to beta coronavirus, has an envelope, is round or elliptical in particle shape, is usually polymorphic, and has the diameter of 60-140 nm. The gene characteristics of the mutant are obviously different from those of SARSr-Cov and MERSR-CoV. The research shows that the homology of the strain and the bat SARS-like coronavirus (bat-SL-CoVZC45) reaches more than 85 percent. In vitro isolation culture, 2019-nCov can be found in human respiratory epithelial cells within about 96 hours, while in Vero E6 and Huh-7 cell lines, isolation culture takes about 6 days. The "SARS-CoV-2 virus variant" in the present invention refers to a virus having a high sequence homology with the novel coronavirus currently discovered. One coronavirus isolated from manis pentadactyla by the research team now showed 100%, 98.2%, 96.7% and 90.4% amino acid identity with 2019-nCoV in the E, M, N and S genes, respectively. In particular, the receptor binding domain of the S protein of Pangolin coronavirus was virtually identical to that of the S protein of 2019-nCoV, with only one amino acid difference (https:// www.biorxiv.org/content/10.1101/2020.02.17.951335v 1). The gene sequence of the SARS-CoV-2 virus variant of the invention has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the gene sequence of the novel coronavirus. The SARS-CoV-2 mutant can be any mutant, for example B.1.1.7(GISAID accession ID: EPI _ ISL _1234251), B.1.351(GISAID accession ID: EPI _ ISL _1191083), B.1.617(EPI _ ISL _1544002), B.1.617.1(EPI _ ISL _2260468), B.1.617.2(EPI _ ISL _2272895), B.1.617.3(EPI _ ISL _1704535), or any newly-occurring SARS-CoV-2 mutant.

"SARS-CoV-2 virus antigen" refers to the antigen of SARS-CoV-2 whole virus lysate or recombinant SARS-CoV-2 antigen. SARS-CoV-2 virus comprises spike protein (S), envelope protein (E), matrix protein (M) and nucleocapsid protein (N) antigen, wherein the S protein is the largest structural protein of SARS-CoV-2. The S protein can be split into S1 and S2 subunits under the action of host enzyme, wherein the S1 subunit contains a receptor binding region RBD and is a main target antigen. In the present invention, the SARS-CoV-2 virus antigen is selected from the group consisting of spike protein (S), envelope protein (E), matrix protein (M) and/or nucleocapsid protein (N) antigen. Preferably, the SARS-CoV-2 viral antigen is a SARS-CoV-2S1 subunit or SARS-CoV-2S RBD antigen. The SARS-CoV-2S RBD antigen can be produced by a conventional recombinant expression method, and SARS-CoV-2S RBD recombinant protein is obtained by constructing plasmids for expressing SARS-CoV-2S RBD, such as pFastBac1 and pTT5, transfecting expression cells, such as CHO cells and SF9 cells, and expressing and purifying.

The term "neutralizing antibody" as used herein refers to antibodies that prevent a cell from being invaded by an antigen or infectious agent by binding to viral molecules, by inhibiting or even neutralizing some of their biochemical effects. The term "SARS-CoV-2 virus neutralizing antibody" as used herein refers to an antibody that blocks the binding of SARS-CoV-2 virus RBD to the human ACE2 receptor by binding to the SARS-CoV-2 virus RBD protein.

The "ACE 2" or "ACE 2 protein" in the invention may be embodied as recombinant ACE2 protein, may be embodied as soluble ACE2 protein, and may be embodied as Fc-tag ACE2 protein. The "functional fragment of ACE2 protein" in the present invention refers to a fragment of ACE2 protein that can exert binding effect with respect to coronavirus spike protein, particularly SARS-CoV-2 virus RBD protein, and may be a partial region of ACE2 protein that exerts binding function, such as the extracellular domain of ACE2 or the extracellular domain of ACE2 with Fc tag.

The "novel coronavirus or variant spike protein portion thereof" or "novel coronavirus or variant spike protein thereof or ACE 2-binding fragment thereof" in the present invention refers to a spike protein portion including a function of the novel coronavirus or variant thereof, which is capable of specifically binding to ACE2 protein or ACE2 protein extracellular domain. Can be RBD protein of novel coronavirus or its variant, S1 protein and S protein of novel coronavirus or its variant. Preferably SARS-CoV-2 virus or its variant RBD protein, SARS-CoV-2 virus or its variant S1 protein and SARS-CoV-2 virus or its variant S protein, more preferably SARS-CoV-2 virus or its variant RBD protein or SARS-CoV-2 virus or its variant S1 protein.

The term "SARS-CoV-2 virus or variant RBD protein thereof" as used herein can be embodied as recombinant SARS-CoV-2 virus or variant RBD protein thereof (sequence shown in SEQ ID NO:1, 4 or 6), SARS-CoV-2 virus with His tag or variant RBD protein thereof, SARS-CoV-2 virus with Fc tag or variant RBD protein thereof; molecules comprising the SARS-CoV-2 virus or its variant RBD protein, such as the SARS-CoV-2 virus or its variant S1 subunit (sequence shown in SEQ ID NO:2, 5 or 7) and SARS-CoV-2 virus or its variant S protein. Wherein, the SARS-CoV-2 virus variant can be B.1.1.7, B.1.351, B.1.617, B.1.617.1, B.1.617.2, B.1.617.3, or any newly appeared SARS-CoV-2 mutant strain.

The term "magnetic particle" includes magnetic beads that may not be limited to p-toluenesulfonyl-modified (Tosyl magnetic beads), amino-modified magnetic beads, and carboxyl-modified magnetic beads.

The term "label" herein may refer to at least one of horseradish peroxidase (HRP), Alkaline Phosphatase (AP), acridinium ester, isoluminol, or a rare earth element. The label and the luminescent substrate generate chemiluminescence after reaction, for example, luminol generates chemiluminescence after HRP catalytic reaction, and can be detected by a chemiluminescence apparatus.

The "magnetic particle conjugate" or "magnetic particle label" in the present invention may refer to a magnetic particle coupled with a novel coronavirus spike protein moiety or a magnetic particle coupled with ACE2 protein, that is, a magnetic particle covalently coupled with a novel coronavirus spike protein moiety or a magnetic particle covalently coupled with ACE2 protein. Specifically included in the present invention are the RBD protein of magnetic particle-conjugated SARS-Cov-2, the S1 protein of magnetic particle-conjugated SARS-Cov-2 or the ACE2 protein of magnetic particle-conjugated.

"marker conjugate" or "marker linker" in the context of the present invention may refer to a novel coronavirus spike protein moiety or ACE2 protein or functional fragment thereof conjugated to a marker, including but not limited to HRP-conjugated novel coronavirus spike protein moiety, AP-conjugated novel coronavirus spike protein moiety, acridinium ester-conjugated novel coronavirus spike protein moiety, HRP-conjugated ACE2 protein, AP-conjugated ACE2 protein and acridinium ester-conjugated ACE2 protein.

The "conjugate" in the present invention refers to a magnetic particle conjugate in which a coronavirus spike protein moiety or ACE2 protein is labeled to a magnetic particle by a covalent bond, and may refer to a label conjugate in which a coronavirus spike protein moiety or ACE2 protein is labeled to a label by a covalent bond.

The term "linked" in the context of "linked labels or magnetic particles" as used herein means directly coupled to the C-terminus or N-terminus of a protein via a linker or chemical bond, which may be selected from small molecule chemical linkers or peptide linkers. The small molecule chemical linker includes but is not limited to carboxyl, amino and sulfhydryl, etc., the peptide linker can be selected from polypeptide of 1 or more amino acids, and the label or magnetic particle can be connected to the spike protein part of the novel coronavirus or ACE2 protein or functional fragment thereof through the linker. Wherein the coupling mode connection is formed by forming covalent bond connection, and the covalent bond connection can be formed by covalent reaction of a marker or magnetic particles and the novel coronavirus spike protein part or ACE2 protein or functional fragments thereof.

The term "linker" as used herein refers to a compound capable of linking a protein to a label or magnetic particle, and may be a peptide linker, a small molecule chemical linker, or a polymer molecular linker. Wherein, the small molecule chemical linker includes but is not limited to carboxyl, amino and sulfhydryl; the peptide linker may be a polypeptide of up to 20, up to 10, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2, or 1 amino acid.

The term "chemiluminescent substrate" includes, but is not limited to, direct chemiluminescent acid-base luminescence initiators such as H2O 2-containing acids (HNO)₃Or HCl, etc.) and NaOH, indirect chemiluminescence including alkaline phosphatase substrates such as AMPPD, APS5, CSPD, CDP-Star, and horseradish peroxidase substrates such as luminol and PS-atto, etc.

The chemiluminescence analyzer in the invention is composed of an immunoreaction system and a chemiluminescence analysis system. The chemiluminescence analysis system measures the optical quantum signals generated by catalyzing and oxidizing chemiluminescence by using a luminescence signal measuring instrument. The immunoreaction system is to label chemiluminescent substance or enzyme directly on antigen or antibody (immunochemiluminescence analysis). It includes chemiluminescence enzyme immune analysis and electrochemical luminescence analyzer.

The term "subject" as used herein refers to an animal, preferably a mammal, more preferably a human, in need of alleviation, prevention and/or treatment of a disease or disorder, such as a viral infection. The term includes human subjects having or at risk of having infection by a coronavirus, such as SARS-CoV-2. A healthy subject is a healthy animal, preferably a mammal, more preferably a human, not infected with SARS-CoV-2 virus.

Samples of the present invention include, but are not limited to, (1) plasma, serum, or whole blood infected or suspected of being infected with the novel coronavirus; (2) plasma, serum or whole blood after inoculation of the novel coronavirus vaccine; (3) plasma, serum or whole blood of animals immunized with the novel coronavirus spike protein; or (4) a sample of antibodies against the novel coronavirus obtained by a biological method. The sample can be derived from plasma, serum, whole blood, pleural effusion, cerebrospinal fluid or tissue specimen of human or animal infected or suspected to be infected with SARS-CoV-2 virus, such as mouse, rat, rabbit, or can be derived from serum, plasma, whole blood or antibody solution against SARS-CoV-2 coronavirus of animal prepared by biological method such as animal immunization.

"percent (%) amino acid sequence identity" with respect to a peptide or polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to amino acid residues in the particular peptide or polypeptide sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment to determine percent amino acid sequence identity can be performed in a variety of ways within the skill in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or megalign (dnastar) software. One skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms required to obtain maximum alignment over the full length of the sequences being compared.

Advantageous technical effects

No SARS-CoV-2 neutralizing antibody magnetic particle chemiluminescence detection kit product is sold in the market at present. The method for developing the magnetic particle chemiluminescence kit for detecting the neutralizing antibody of the SARS-CoV-2 virus is novel and is developed and utilized by utilizing the characteristic that the neutralizing antibody can block the combination of the spike protein part of the novel coronavirus and the ACE2 protein or a functional fragment thereof. The magnetic particle chemiluminescence detection of SARS-CoV-2 neutralizing antibody has not been exploited in the value of SARS-CoV-2 virus-assisted diagnosis, antibody drug screening and vaccine evaluation. The SARS-CoV-2 neutralizing antibody blocking magnetic particle chemiluminescence detection kit provided by the invention detects SARS-CoV-2 virus neutralizing antibody in a sample by the inhibition rate of chemiluminescence signals, and the method has the characteristics of large detection flux, high automation degree, high accuracy, strong specificity, high detection speed, simple and convenient operation and the like.

Drawings

FIG. 1 is a schematic diagram of a magnetic particle chemiluminescence kit for a virus neutralizing antibody of SARS-CoV-2;

FIG. 2 is a graph showing the effect of blocking 4 strains of SARS-CoV-2 antibody by magnetic particle chemiluminescence detection kit for SARS-CoV-2 virus neutralizing antibody alkaline phosphatase.

FIG. 3 is a graph showing the results of testing 30 negative samples and 20 positive samples with the magnetic particle chemiluminescence assay kit for SARS-CoV-2 virus-neutralizing antibody alkaline phosphatase.

FIG. 4 is a calibration graph of the magnetic particle chemiluminescence detection kit for SARS-CoV-2 virus and mutants B.1.1.7 and B.1.351 neutralizing antibodies.

FIG. 5 shows a magnetic particle chemiluminescence detection kit for SARS-CoV-2 virus neutralizing antibody alkaline phosphatase and a commercial kit cPass^TMSARS-CoV-2 neutralizing antibody detection kit (FDA EUA) tests correlation chart of 40 samples.

Detailed Description

Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The present invention is further illustrated by the following examples, which should not be construed as limiting the invention. Any modification, equivalent replacement or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

EXAMPLE 1 preparation of magnetic microparticle chemiluminescence kit for SARS-CoV-2 virus-neutralizing antibody, alkaline phosphatase, and Performance evaluation

1.1 kit Components

The kit consists of the following 7 reagents:

AP-coupled SARS-CoV-2RBD protein;

b. magnetic microparticle coupling ACE 2;

c. a sample diluent;

d. positive quality control products;

e. negative quality control products;

f. a wash solution;

g. a chemiluminescent substrate;

1.2 preparation of AP-coupled SARS-CoV-2RBD protein

1) To a 2mLEP tube, 1mL of a 1mg/mL solution of recombinant SARS-CoV-2RBD protein (King Share, Cat No. T80302) was added.

2) 60ul of 1.376mg/mL Traut's Reagent (Thermo, Cat No.26101) solution was added to the above EP tube, mixed well and allowed to stand at room temperature for 20 min.

3) To another 2mL EP tube was added 600ul of an alkaline phosphatase solution at a concentration of 10 mg/mL.

4) 11ul of a 17.5mg/mL (N-maleimidomethyl) cyclohexane-1-carboxylic acid sulfosuccinimide sodium salt (Thermo, CatNo.22322) solution was added to the above-mentioned EP tube containing the alkaline phosphatase solution, mixed well and allowed to stand at room temperature for 20 min.

5) Adding 6-24uL of l M glycine solution into the activated recombinant SARS-CoV-2RBD protein solution, mixing, and standing at room temperature for 10 min.

6) Adding lM glycine solution 4.4-8.8uL into the activated alkaline phosphatase solution, mixing, and standing at room temperature for 10 min.

7) With Zeba^TMSpin desalting column (Thermo, cat No. 8989891) protein displacement buffer from step 5) (0.1M triethanolamine; 5mM Mg²⁺；1mMZn²⁺(ii) a pH7.2-7.4), and collecting the activated recombinant SARS-CoV-2RBD protein solution.

8) With Zeba^TMSpin desalting column (Thermo, cat No.89882) alkaline phosphatase replacement buffer (0.1M triethanolamine; 5mM Mg²⁺；1mMZn²⁺(ii) a pH7.2-7.4), and collecting the alkaline phosphatase solution after activation.

9) Adding 4-6mg of the Alkaline Phosphatase (AP) solution into 1mg of recombinant SARS-CoV-2RBD protein solution, mixing, standing at 2-8 deg.C for reaction for 18-24 hr;

10) 10-15ul of 12.5mg/mL N-ethylmaleimide solution was added to the AP-conjugated SARS-CoV-2RBD protein reaction and allowed to stand at room temperature for 30 min.

11) With Zeba^TMPurification by rotating desalting column (Thermo, Cat No. 8989891) by displacement of buffer (0.1M triethanolamine; 5mM MG)²⁺；1mM Zn²⁺(ii) a 0.05% ProClin300 (v/v); pH7.2-7.6) to obtain AP coupled SARS-CoV-2RBD concentrated solution.

12) Diluting the AP-coupled SARS-CoV-2RBD concentrated solution obtained in the above step with the diluent prepared in step 1.4 to 2-200ng/mL to obtain AP-coupled SARS-CoV-2RBD protein.

1.3 preparation of magnetic microparticle coupling ACE2

1) 500ul of magnetic particles (JSR, MS300/Tosyl) at a concentration of 100mg/mL were placed in a 10mL centrifuge tube, and the supernatant was removed by magnetic separation.

2) Resuspend the magnetic particles in 5mL boric acid buffer (0.1mol/LpH 9.2.2), add 1mg ACE2 protein (Kinserin, Cat No. Z03484) into the centrifuge tube, mix uniformly at 37 deg.C and react for 16-24 h.

3) The supernatant was removed by magnetic separation, 50ul of blocking solution (10% (w/v) BSA) was added to the centrifuge tube, and the mixture was mixed at a constant temperature of 37 ℃ and reacted for more than 6 hours.

4) The supernatant was removed magnetically and washed with 5mL of 1 × TBST (10mM Tris; 150mM NaCl; 0.05% Tween-20 (v/v); pH 7.4-7.5), mixing for 1-5min, and magnetically separating out supernatant. The washing step was repeated 2-3 times.

5) And (3) adding 10mL of the diluent prepared in the step 1.4, uniformly mixing by shaking for 1-5min, re-suspending the magnetic particles to 5mg/mL (the content of ACE2 is 100ug/mL), and dispersing the agglomerated magnetic beads in the suspension into a single particle state by using an ultrasonic oscillator to obtain a magnetic separation reagent concentrated solution.

6) And diluting the liquid into magnetic particles with the concentration of 0.1-0.5mg/mL and the concentration of 2-10ug/mL of ACE2 by using a magnetic separation reagent diluent, and coupling the ACE2 with the magnetic particles.

1.4 preparation of sample dilutions

Adding 800mL of purified water, 3.5g of disodium hydrogen phosphate dodecahydrate and 0.26g of sodium dihydrogen phosphate dihydrate into a container, uniformly mixing by stirring, adding 8.5g of sodium chloride and 0.3mL of Proclin300, stirring until the sodium phosphate dodecahydrate and the sodium dihydrogen phosphate dihydrate are completely dissolved, wherein the pH value is 7.0-7.5, adding 20g of sucrose, stirring until the sodium phosphate dodecahydrate and the sodium dihydrogen phosphate dihydrate are completely dissolved, adding 0.5mL of Tween 20 and 10g of bovine serum albumin into the container, stirring until the sodium phosphate dodecahydrate and the sodium dihydrogen phosphate dihydrate are completely dissolved, fixing the volume to 1L by using purified water, filtering by using a 0.22 mu m filter, and keeping the obtained sample diluent at 2-8 ℃ for later use.

1.5 preparation of Positive quality control

The sample diluent prepared in the above procedure was used to prepare a positive quality control of 200ng/mL of neutralizing antibody against SARS-CoV-2 virus (Kingsry, clone 2B 2). The positive quality control material is Kinry Cpass^TMThe kit (Cat No. L00847) was confirmed to be a neutralizing antibody.

1.6 preparation of negative quality control Material

The negative control was the sample dilution prepared in example 1.4.

1.7 preparation of lotion

Adding 800mL of purified water, 30.25g of Tris and 219.15g of NaCl into a 1L container, and fully stirring until the mixture is completely dissolved; and adding 12.5mL of Tween-20, fully stirring until the mixture is completely mixed, adjusting the pH value to 7.5-8.0, fixing the volume to 1L, filtering by using a 0.2 mu m filter, storing at room temperature, adding purified water to 1L of the cleaning solution per 40mL before use, and mixing uniformly.

2 handling of the kit

1) Adding 50uL positive quality control substance, 50uL negative quality control substance or 50uL serum or plasma sample detection tube after 1:10 dilution.

2) Add 50uL of AP-conjugated SARS-CoV-2RBD protein to the tube, mix well, incubate at 37 ℃ for 30 min.

3) Adding 50uL of magnetic particle coupled ACE2 protein into a detection tube, mixing uniformly, incubating for 15min at 37 ℃, performing magnetic separation, and removing supernatant.

4) Adding 300uL of cleaning solution into the detection tube, mixing uniformly, and carrying out magnetic separation to remove the supernatant. The washing step was repeated twice.

5) Adding 150uL of chemiluminescent substrate (CatNo. APSUB-1, Beijing Avided biotechnology Co., Ltd.) into the detection tube, mixing, and detecting the luminescence intensity with chemiluminescence apparatus.

6) Results calculation [ see formula (1) -formula (3) ]:

n2: detection value of negative control 2.

P2: detection value of positive control 2:

s: sample detection value

7) The test is satisfied under the conditions: the inhibition rate of the standard positive control is more than or equal to 30 percent, the experimental result is true, and if the test is not true, repeated detection is carried out.

8) If the inhibition rate of the detected sample is more than or equal to 30 percent, the sample can be judged to be positive. If the inhibition rate of the detected sample is less than 30%, the sample can be judged to be negative.

Evaluation of blocking Effect of 3SARS-CoV-2 antibody

1) 4 strains of SARS-CoV-2 monoclonal antibody (SARS-CoV-2mAb-1/-2/-3/-4, antibody prepared by Kinserin) were analyzed using the SARS-CoV-2 virus neutralizing antibody alkaline phosphatase magnetic particle chemiluminescence kit.

2) SARS-CoV-2mAb-1 was diluted with sample dilutions to 7 concentration gradient samples "50000 pM", "16667 pM", "5556 pM", "1852 pM", "617 pM", "206 pM" and "23 pM".

3) Different concentration gradient samples of SARS-CoV-2mAb-2/-3/-4 antibody were prepared according to the procedure of 3.2).

4) Adding 50uL of positive quality control substance, 50uL of negative quality control substance or 50uL of sample prepared in the step 3.1-3.2 into a detection tube.

5) Add 50uL of AP-conjugated SARS-CoV-2RBD protein to the tube, mix well, incubate at 37 ℃ for 30 min.

6) Adding 50uL of magnetic particle coupled ACE2 into detection tube, mixing, incubating at 37 deg.C for 15min, performing magnetic separation, and removing supernatant.

7) Adding 300uL of cleaning solution into the detection tube, mixing uniformly, and carrying out magnetic separation to remove the supernatant. The washing step was repeated twice.

8) Adding 150uL of chemiluminescent substrate (CatNo. APSUB-1, Beijing Avided biotechnology Co., Ltd.) into the detection tube, mixing, and detecting the luminescence intensity with chemiluminescence apparatus.

9) The inhibition rate of each sample prepared in steps 3.2-3.3 was calculated according to the following formula. [ see formula (1) -formula (3) ]:

n2: detection value of negative control 2.

P2: detection value of positive control 2:

s: sample detection value

Using GraphPadprism 6, plotted as the abscissa of the log antibody concentration and the ordinate of the inhibition rate, see FIG. 2, the results show that the binding blocking effect of SARS-CoV-2mAb-1 on the AP-conjugated marker SARS-CoV-2RBD protein and magnetic microparticle-conjugated ACE2 is not significant. The SARS-CoV-2mAb-2, SARS-CoV-2mAb-3 and SARS-CoV-2mAb-4 antibodies can obviously block the binding effect of AP coupled marker SARS-CoV-2RBD protein and magnetic particle coupled ACE 2. Wherein SARS-CoV-2mAb-4 can block the binding effect of AP coupled marker SARS-CoV-2RBD protein and magnetic particle coupled ACE2 under low concentration.

4 Performance evaluation of the kit

4.1) evaluation of reproducibility

The kit in the embodiment is used for repeatedly detecting samples with the concentration of 200 plus or minus 40ng/mL and 500 plus or minus 100ng/mL for 10 times respectively, the mean value M and the standard deviation SD of 10 times of measurement results are calculated, the coefficient of variation CV is obtained according to the formula CV as SD/M multiplied by 100 percent, and the Coefficient of Variation (CV) of the method is not more than 10 percent.

4.2) evaluation of inter-batch Difference

The kits of examples were prepared in three lots, each of which was measured for samples having concentrations ranging from 200 ± 40ng/mL and 500 ± 100mg/mL, the measurement was repeated 10 times for each lot, the mean (μ) and Standard Deviation (SD) of the 30 measurement results were calculated, and the Coefficient of Variation (CV) was calculated according to the formula CV ═ SD/mx 100%, and the Coefficient of Variation (CV) was not more than 15%.

4.3) evaluation of specificity

30 samples containing no neutralizing antibody were tested by SARS-CoV-2 virus neutralizing antibody alkaline phosphatase magnetic particle chemiluminescence kit and all were negative (inhibition < 30% was negative), as shown in FIG. 3.

4.4) sensitivity assessment

Detection of 20 positive samples containing varying concentrations of anti-SARS-CoV-2 neutralizing antibody (from Kinry Cpass) by the SARS-CoV-2 virus neutralizing antibody alkaline phosphatase magnetic particle chemiluminescence kit^TMA kit (Cat No. l00847) that tests positive) prepared from 5B7D7 (kasei, Cat No. a02056), 6D11F2 (kasei, Cat No. a02055), R2B2 (kasei, Cat No. a02051), 11D5D3 (prepared by kasei) in sample dilutions to give solutions of 39, 78, 156, 313 and 625 ng/ml. The detection results of the magnetic particle chemiluminescence kit are positive (the inhibition rate is more than or equal to 30 percent, and the result is shown in figure 3.

EXAMPLE 2 preparation of anti-SARS-CoV-2 Virus and mutant B.1.1.7 and B.1.351 neutralizing antibody alkaline phosphatase magnetic particle chemiluminescence quantitation kit and Performance evaluation

1.1 kit Components

The kit consists of the following 8 reagents:

AP-coupled SARS-CoV-2RBD recombinant protein;

AP-coupled SARS-CoV-2 mutant (B.1.1.7) RBD recombinant protein

AP-coupled SARS-CoV-2 mutant (B.1.351) RBD recombinant protein

d. Magnetic particle coupling recombinant human ACE 2;

e. a sample diluent;

f. a calibrator;

g. a wash solution;

h. a chemiluminescent substrate;

1.2 preparation of AP-coupled SARS-CoV-2RBD and variants thereof

AP-conjugated SARS-CoV-2RBD and variants thereof were prepared as described in 1.2 of example 1, using the following protocol:

1) to a 2mL LEP tube was added 1mL of a 1mg/mL solution of recombinant SARS-CoV-2RBD protein (Kingsry, Cat No. T80302), SARS-CoV-2 mutant (B.1.1.7) RBD recombinant protein (Kingsry, Cat No. Z03533), or SARS-CoV-2 mutant (B.1.351) RBD recombinant protein (Kingsry, Cat No. Z03537).

5) Adding 6-24uL of l M glycine solution into activated recombinant SARS-CoV-2 and mutant RBD protein solution, mixing, and standing at room temperature for 10 min.

7) With Zeba^TMSpin desalting column (Thermo, cat No. 8989891) protein displacement buffer from step 5) (0.1M triethanolamine; 5mM Mg²⁺；1mM Zn²⁺(ii) a pH7.2-7.4), and collecting activated recombinant SARS-CoV-2RBD, SARS-CoV-2 mutant strain (B.1.1.7) RBD recombinant protein or SARS-CoV-2 mutant strain (B.1.351) RBD recombinant protein solution.

8) With Zeba^TMSpin desalting column (Thermo, cat No.89882) alkaline phosphatase replacement buffer (0.1M triethanolamine; 5mM Mg²⁺；1mM Zn²⁺(ii) a pH7.2-7.4), and collecting the alkaline phosphatase solution after activation.

9) Adding 4-6mg of the above Alkaline Phosphatase (AP) solution into 1mg of recombinant SARS-CoV-2RBD, SARS-CoV-2B.1.1.7RBD or SARS-CoV-2B.1.351RBD recombinant protein solution, mixing, standing at 2-8 deg.C for 18-24 hr;

10) adding 10-15ul of 12.5mg/mL N-ethylmaleimide solution into AP-conjugated SARS-CoV-2RBD, SARS-CoV-2B.1.1.7RBD or SARS-CoV-2B.1.351RBD recombinant protein reactant, and standing at room temperature for 30 min.

11) With Zeba^TMPurification by spin desalting column (Thermo, Cat No. 8989891) by displacement of buffer (0.1M triethanolamine; 5mM Mg²⁺；1mM Zn²⁺(ii) a 0.05% ProClin300 (v/v); pH7.2-7.6) to obtain AP coupled SARS-CoV-2RBD, SARS-CoV-2B.1.1.7RBD or SARS-CoV-2B.1.351RBD recombinant protein concentrated solution.

12) And (3) diluting the concentrated solution of the AP-conjugated SARS-CoV-2RBD or the variant thereof obtained in the step (1) with the diluent prepared in the step (6) to 50ng/mL to obtain the RBD protein of the AP-conjugated SARS-CoV-2 or the variant thereof in the kit.

1.3 preparation of magnetic particle coupled human ACE2

1) 500ul of magnetic microparticles (JSR, MS160/Carboxyl) at a concentration of 100mg/mL were placed in a 10mL centrifuge tube and the supernatant was removed by magnetic separation.

2) Resuspend the magnetic particles in 5mL MES buffer (0.1mol/LpH 5.0.0), mix well for 1-5min, and magnetically separate the supernatant. The washing step was repeated 1 time.

3) Adding 3.2 mM MES buffer solution (0.1mol/LpH 5.0.0) into the centrifuge tube, resuspending the magnetic particles, and mixing for 1-5 min.

4) To the tube, 1.15ml of a 10mg/ml NHS (Thermo, cat 2450) solution and 0.5ml of a 10mg/ml EDC (Thermo, cat 22980) solution were added, and the mixture was mixed at room temperature for 30 minutes.

5) After the reaction is finished, the supernatant is removed by magnetic separation, 4.35 mM MES buffer solution (0.1mol/L pH 5.0) is added to resuspend the magnetic particles, and the mixture is mixed evenly for 1 to 5 min.

6) 1mg of ACE2 ectodomain-Fc protein (Kisry, Cat No. Z03484) was added to the tube and mixed well at room temperature for 3-20 h.

7) 250ul of confining liquid (JSR, CE210) is added into the centrifuge tube, and the mixture is mixed uniformly at room temperature and reacted for 18-24 h.

8) The supernatant was magnetically separated and washed with 5mL of wash solution (25mM Tris; 150mM NaCl; 0.1% BSA; 0.05% Tween-20 (v/v); 0.1% ProClin 300; pH 7.4-7.5), mixing for 1-5min, and magnetically separating out supernatant. The washing step was repeated 2-3 times.

9) And (3) adding 10mL of the diluent prepared in the step 1.4, uniformly mixing by shaking for 1-5min, re-suspending the magnetic particles to 5mg/mL (the content of ACE2 is 100ug/mL), and dispersing the agglomerated magnetic beads in the suspension into a single particle state by using an ultrasonic oscillator to obtain a magnetic separation reagent concentrated solution.

10) The liquid is diluted into magnetic particles with the concentration of 0.25mg/mL and the concentration of 5ug/mL of ACE2 to be coupled with ACE2 by using a magnetic separation reagent diluent.

1.4 preparation of sample dilutions

The preparation method and components are the same as the preparation steps and components of 1.4 of example 1.

1.5 preparation of calibrator

anti-SARS-CoV-2 virus neutralizing antibody standard (kasumi, cat No. a02087) was formulated as a series of concentration points using sample dilutions prepared as described above: 6000U/ml, 3000U/ml, 1000U/ml, 400U/ml, 150U/ml and 0U/ml.

1.6 preparation of lotion

The washing solution was prepared by the same method and composition as in 1.7 of example 1.

2 handling of the kit

Prepare 3 groups of 25uLSARS-CoV-2 virus neutralizing antibody calibrator into the detection tube, then add 25uL sample dilution. 50uL of the AP-conjugated SARS-CoV-2RBD, SARS-CoV-2 mutant (B.1.1.7) RBD and SARS-CoV-2 mutant (B.1.351) RBD recombinant protein prepared above were added to 3 sets of assay tubes, respectively. Then respectively adding 50uL of magnetic particles to couple ACE2 protein into a detection tube, uniformly mixing at 37 ℃ for reaction for 30min, carrying out magnetic separation, and removing supernatant. Add 300uL of washing solution to the detection tube, mix well, and magnetically separate the supernatant. The washing step was repeated twice. Adding 150uL of chemiluminescent substrate (Cat No. APSUB-1, Beijing Avided biotechnology, Inc.) into the detection tube, mixing, and detecting the luminescence intensity with chemiluminescence apparatus.

Four parameters were used to fit the calibration curves using calibrator concentrations and signals, and 3 sets of RBDs were used to fit 3 calibration curves, see fig. 4. The condition that the curve is established is as follows: and if the correlation coefficient r of the calibration curve is more than or equal to 0.99, the result of the curve is true, and if the result is false, repeated detection is carried out.

The sample to be tested can be tested by the same procedure as the SARS-CoV-2 virus neutralizing antibody calibrator.

3 Performance evaluation of the kit

3.1) evaluation of detection Limit

Taking 2 batches of the kit in the embodiment, continuously detecting 5 blank samples and 5 low-value samples for 3 days, repeating each sample for 4 times, and calculating blank detection limit LOB according to 60 results of 2 batches of blank samples; calculating the lowest detection limit LOD through 60 results of 2 batches of low-value samples; from 60 results of 2 batches of low value samples, the limit of quantitation LOQ at CV of 20% was calculated.

RBD type	LOB	LOD	LOQ
				Wild type	146.32U/ml	259.32U/ml	326U/ml
B.1.1.7 mutant	266.24U/ml	452.06U/ml	523U/ml
				B.1.351 mutant	446.12U/ml	537.17U/ml

3.2) Linear evaluation

Diluting with high value sample (approaching linear range upper limit (6000U/ml)) at a certain ratio to 5 concentration points, repeating for 3 times each concentration, calculating concentration mean value, performing straight line fitting on the result mean value and dilution ratio according to least square method, and calculating linear correlation coefficient r, wherein the linear r should be not less than 0.99 within the specified linear range.

3.3) recovery evaluation

The detection is carried out by using samples with three concentrations, namely low concentration, medium concentration and high concentration, each sample is repeated for 3 times, the concentration mean value is calculated, and the recovery rate of each concentration is in a range of (80 percent and 120 percent).

3.4) evaluation of repeatability

The detection is carried out by using samples with the concentrations of 750 +/-150U/mL and 1500 +/-300U/mL, each sample is repeated for 10 times, the average value M and the standard deviation SD of 10 measurement results are calculated, the coefficient of variation CV is obtained according to the formula CV which is SD/M multiplied by 100 percent, and the Coefficient of Variation (CV) of the method is not more than 10 percent.

3.5) inter-batch Difference assessment

The kits of examples were prepared in three lots, each of which was measured for samples having concentrations in the range of 750 ± 150U/mL and 1500 ± 300U/mL, and the measurement was repeated 10 times for each lot, and the mean value (μ) and Standard Deviation (SD) of the measurement results were calculated 30 times, and the Coefficient of Variation (CV) was calculated according to the formula CV ═ SD/mx 100%, and the Coefficient of Variation (CV) was not more than 15%.

3.6) methodological comparison evaluation

Kit and cPass for detecting neutralizing antibody against wild type SARS-CoV-2RBD using this example^TMSARS-CoV-2 neutralizing antibody detection kit (FDA EUA) (Kingsry, product number L00847) detects 40 samples (obtained by diluting commercial SARS-CoV-2 neutralizing antibody standard (Kingsry, Cat No.3SH344) with matrix human serum (Scantibodies, Cat No.3SH344) to different concentrations ranging from 0U/ml to 7200U/ml), and linear fitting is performed on the test concentrations of the examples and the cPass test concentration by using a least square method to calculate a correlation coefficient R²See fig. 5. R²The correlation between the example kit and the cPass kit is good as more than or equal to 0.95.

EXAMPLE 3 preparation of magnetic microparticle chemiluminescence kit for SARS-CoV-2 virus neutralizing antibody acridinium ester

1.1 kit Components

The kit consists of the following 7 reagents:

a. acridinium ester coupling SARS-CoV-2RBD protein;

b. magnetic microparticle label ACE 2;

c. a sample diluent;

d. positive quality control products;

e. negative quality control products;

f. a wash solution;

g. substrates (pre-excitation liquid a and excitation liquid B);

1.2 preparation of acridinium ester-coupled SARS-CoV-2RBD protein

1) 1mL of a 1mg/mL solution of recombinant SARS-CoV-2RBD protein (Kinseriy, CatNo. T80302) was applied to a Zeba^TMThe column was then transferred to 20mM phosphate buffer (pH7.4) and placed in a 2mL EP tube.

2) Adding 80uL of 5mM acridine ester solution (Shenzhen MeyKate, CatNo. Intlus-B05) into an EP tube, shaking and uniformly mixing for 30-60s, and standing at 2-8 ℃ in the dark for reaction for 18-24 h.

3) To the reaction product of the acridinium ester-conjugated marker SARS-CoV-2RBD protein, 10ul of 50mM glycine solution was added, and the mixture was left standing at room temperature in the dark for 30 min.

4) With Zeba^TMPurifying by rotary desalting column displacement buffer (20mM PB; 150mM NaCl; pH7.4) to obtain concentrated solution of SARS-CoV-2RBD protein coupled with acridinium ester.

5) Diluting the acridinium ester coupling marker SARS-CoV-2RBD protein concentrated solution prepared in the step 4) to 10-100ng/mL by using the sample diluent prepared in the step 1.4 to obtain the reagent.

1.3 preparation of magnetic microparticle coupling ACE2

2) The magnetic particles were resuspended in 5mL of boric acid buffer (0.1mol/LpH 9.2.2), 1mg of ACE2 ectodomain-Fc protein (Kinseri, CatNo. Z03484) was added to the centrifuge tube, and the mixture was mixed at 37 ℃ for 16-24 hours.

3) The supernatant was removed by magnetic separation, 50ul of blocking solution (10% (w/v) BSA) was added to the centrifuge tube and mixed at 37 ℃ for reaction for more than 6 h.

5) Adding 10mL of magnetic separation reagent diluent, shaking and uniformly mixing for 1-5min, re-suspending the magnetic particles to 5mg/mL (the content of ACE2 is 100ug/mL), and dispersing the agglomerated magnetic beads in the suspension into a single particle state by using an ultrasonic oscillator to obtain the magnetic separation reagent concentrated solution.

6) Diluting the sample diluent prepared in the step 1.4 to dilute the concentrated solution of the magnetic particle coupled ACE2 obtained in the step 5) into the magnetic particle coupled ACE2 with the magnetic particle concentration of 0.1-0.5mg/mL and the ACE2 concentration of 2-10 ug/mL.

1.4 preparation of sample dilutions

1.5 preparation of Positive quality control

An anti-SARS-CoV-2 virus neutralizing antibody (Kinry, clone: 2B2, CatNo. A02051) was formulated as a positive quality control at 200ng/mL using the sample dilutions.

1.6 preparation of negative quality control Material

The negative control was the sample dilution prepared in example 1.4.

1.7 preparation of lotion

2 handling of the kit

1) Adding 50uL positive quality control substance, 50uL negative quality control substance or 50uL serum or plasma sample diluted by 1:10 into a detection tube.

2) Add 50uL of acridinium ester conjugated SARS-CoV-2RBD protein into the detection tube, mix well, incubate 30min at 37 ℃.

3) Adding 50uL of magnetic particle coupled ACE2 into detection tube, mixing, incubating at 37 deg.C for 15min, performing magnetic separation, and removing supernatant.

5) 50uL of pre-excitation solution A (0.1M HCl; 1.32% (w/v) H₂O₂) And 50uL of excitation liquid B (0.35M NaOH; 0.025% (w/v) CTAC (cetyltrimethylammonium chloride)) to a detection tube, uniformly mixing, and detecting the luminous intensity by a chemiluminescence instrument.

6) Results calculation [ see formula (1) -formula (3) ]:

n2: detection value of negative control 2.

P2: detection value of positive control 2:

s: sample detection value

3 Performance evaluation of the kit

3.1 evaluation of reproducibility

The kit of this example was used to repeatedly detect samples having neutralizing antibody concentrations of 200 ± 40ng/mL and 500 ± 100ng/mL 10 times each, the mean μ M and standard deviation SD of the 10 measurements were calculated, and the coefficient of variation CV was obtained according to the formula CV ═ SD/mx 100%, and the Coefficient of Variation (CV) of the method was not more than 10%.

3.2 evaluation of inter-batch Difference

The kits of examples were prepared in three lots, each of which was a sample in which the concentration of neutralizing antibody was in the range of 200 ± 40ng/mL and 500 ± 100mg/mL, and the measurement was repeated 10 times per lot, and the mean value (μ) and Standard Deviation (SD) of the results of 30 measurements were calculated, and the Coefficient of Variation (CV) was calculated according to the formula CV ═ SD/mx 100%, and the Coefficient of Variation (CV) of the method was not more than 15%.

3.3 evaluation of specificity

The result of 20 samples containing no SARS-CoV-2 neutralizing antibody was negative by using the SARS-CoV-2 virus neutralizing antibody blocking acridinium ester magnetic particle chemiluminescence detection kit.

3.4 sensitivity assessment

Detection of 20 positive samples containing different concentrations of SARS-CoV-2 neutralizing antibody (obtained from Kisry Cpass) by SARS-CoV-2 virus neutralizing antibody blocking acridinium ester magnetic particle chemiluminescence detection kit^TMThe kit (Cat No. L00847) confirms that the antibody is a neutralizing antibody), and the result is positive.

Sequence information:

SARS-CoV-2 spike protein RBD protein sequence (SEQ ID NO:1)

RVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNF

SARS-CoV-2 spike protein S1 subunit sequence (SEQ ID NO:2)

SQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSPRRAR

Human ACE2 protein sequence (SEQ ID NO:3)

QSTIEEQAKTFLDKFNHEAEDLFYQSSLASWNYNTNITEENVQNMNNAGDKWSAFLKEQSTLAQMYPLQEIQNLTVKLQLQALQQNGSSVLSEDKSKRLNTILNTMSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWESWRSEVGKQLRPLYEEYVVLKNEMARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFEEIKPLYEHLHAYVRAKLMNAYPSYISPIGCLPAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFVSVGLPNMTQGFWENSMLTDPGNVQKAVCHPTAWDLGKGDFRILMCTKVTMDDFLTAHHEMGHIQYDMAYAAQPFLLRNGANEGFHEAVGEIMSLSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEKWRWMVFKGEIPKDQWMKKWWEMKREIVGVVEPVPHDETYCDPASLFHVSNDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLHKCDISNSTEAGQKLFNMLRLGKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVGWSTDWSPYADQSIKVRISLKSALGDKAYEWNDNEMYLFRSSVAYAMRQYFLKVKNQMILFGEEDVRVANLKPRISFNFFVTAPKNVSDIIPRTEVEKAIRMSRSRINDAFRLNDNSLEFLGIQPTLGPPNQPPVSIWLIVFGVVMGVIVVGIVILIFTGIRDRKKKNKARSGENPYASIDISKGENNPGFQNTDDVQTSF

SARS-CoV-2 mutant (B.1.1.7) spike protein RBD protein sequence (SEQ ID NO:4)

RVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTYGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNF

SARS-CoV-2 mutant (B.1.1.7) spike protein S1 subunit sequence (SEQ ID NO:5)

SQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAISGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTYGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIDDTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQGVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSHRRAR

SARS-CoV-2 mutant (B.1.351) spike protein RBD protein sequence (SEQ ID NO:6)

RVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVKGFNCYFPLQSYGFQPTYGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNF

SARS-CoV-2 mutant (B.1.351) spike protein S1 subunit sequence (SEQ ID NO:7)

SQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFANPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRGLPQGFSALEPLVDLPIGINITRFQTLHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVKGFNCYFPLQSYGFQPTYGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQGVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSPRRAR

Human ACE21 isomer (SEQ ID NO:8)

MSSSSWLLLSLVAVTAAQSTIEEQAKTFLDKFNHEAEDLFYQSSLASWNYNTNITEENVQNMNNAGDKWSAFLKEQSTLAQMYPLQEIQNLTVKLQLQALQQNGSSVLSEDKSKRLNTILNTMSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWESWRSEVGKQLRPLYEEYVVLKNEMARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFEEIKPLYEHLHAYVRAKLMNAYPSYISPIGCLPAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFVSVGLPNMTQGFWENSMLTDPGNVQKAVCHPTAWDLGKGDFRILMCTKVTMDDFLTAHHEMGHIQYDMAYAAQPFLLRNGANEGFHEAVGEIMSLSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEKWRWMVFKGEIPKDQWMKKWWEMKREIVGVVEPVPHDETYCDPASLFHVSNDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLHKCDISNSTEAGQKLFNMLRLGKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVGWSTDWSPYADQSIKVRISLKSALGDKAYEWNDNEMYLFRSSVAYAMRQYFLKVKNQMILFGEEDVRVANLKPRISFNFFVTAPKNVSDIIPRTEVEKAIRMSRSRINDAFRLNDNSLEFLGIQPTLGPPNQPPVSIWLIVFGVVMGVIVVGIVILIFTGIRDRKKKNKARSGENPYASIDISKGENNPGFQNTDDVQTSF

Human ACE21 type isomer extracellular domain (SEQ ID NO:9)

QSTIEEQAKTFLDKFNHEAEDLFYQSSLASWNYNTNITEENVQNMNNAGDKWSAFLKEQSTLAQMYPLQEIQNLTVKLQLQALQQNGSSVLSEDKSKRLNTILNTMSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWESWRSEVGKQLRPLYEEYVVLKNEMARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFEEIKPLYEHLHAYVRAKLMNAYPSYISPIGCLPAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFVSVGLPNMTQGFWENSMLTDPGNVQKAVCHPTAWDLGKGDFRILMCTKVTMDDFLTAHHEMGHIQYDMAYAAQPFLLRNGANEGFHEAVGEIMSLSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEKWRWMVFKGEIPKDQWMKKWWEMKREIVGVVEPVPHDETYCDPASLFHVSNDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLHKCDISNSTEAGQKLFNMLRLGKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVGWSTDWSPYADQSIKVRISLKSALGDKAYEWNDNEMYLFRSSVAYAMRQYFLKVKNQMILFGEEDVRVANLKPRISFNFFVTAPKNVSDIIPRTEVEKAIRMSRSRINDAFRLNDNSLEFLGIQPTLGPPNQPPVS

Human ACE22 isomer (SEQ ID NO:10)

MSSSSWLLLSLVAVTAAQSTIEEQAKTFLDKFNHEAEDLFYQSSLASWNYNTNITEENVQNMNNAGDKWSAFLKEQSTLAQMYPLQEIQNLTVKLQLQALQQNGSSVLSEDKSKRLNTILNTMSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWESWRSEVGKQLRPLYEEYVVLKNEMARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFEEIKPLYEHLHAYVRAKLMNAYPSYISPIGCLPAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFVSVGLPNMTQGFWENSMLTDPGNVQKAVCHPTAWDLGKGDFRILMCTKVTMDDFLTAHHEMGHIQYDMAYAAQPFLLRNGANEGFHEAVGEIMSLSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEKWRWMVFKGEIPKDQWMKKWWEMKREIVGVVEPVPHDETYCDPASLFHVSNDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLHKCDISNSTEAGQKLL

Human ACE2(SEQ ID NO:11)

MSSSSWLLLSLVAVTAAQSTIEEQAKTFLDKFNHEAEDLFYQSSLASWNYNTNITEENVQNMNNAGDKWSAFLKEQSTLAQMYPLQEIQNLTVKLQLQALQQNGSSVLSEDKSKRLNTILNTMSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWESWRSEVGKQLRPLYEEYVVLKNEMARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFEEIKPLYEHLHAYVRAKLMNAYPSYISPIGCLPAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFVSVGLPNMTQGFWENSMLTDPGNVQKAVCHPTAWDLGKGDFRILMCTKVTMDDFLTAHHEMGHIQYDMAYAAQPFLLRNGANEGFHEAVGEIMSLSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEKWRWMVFKGEIPKDQWMKKWWEMKREIVGVVEPVPHDETYCDPASLFHVSNDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLHKCDISNSTEAGQKLFNMLRLGKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVGWSTDWSPYADQSIKVRISLKSALGDRAYEWNDNEMYLFRSSVAYAMRQYFLKVKNQMILFGEEDVRVANLKPRISFNFFVTAPKNVSDIIPRTEVEKAIRMSRSRINDAFRLNDNSLEFLGIQPTLGPPNQPPVSIWLIVFGVVMGVIVVGIVILIFTGIRDRKKKNKARSGENPYASIDISKGENNPGFQNTDDVQTSF

Human ACE2 extracellular domain (SEQ ID NO:12)

QSTIEEQAKTFLDKFNHEAEDLFYQSSLASWNYNTNITEENVQNMNNAGDKWSAFLKEQSTLAQMYPLQEIQNLTVKLQLQALQQNGSSVLSEDKSKRLNTILNTMSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWESWRSEVGKQLRPLYEEYVVLKNEMARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFEEIKPLYEHLHAYVRAKLMNAYPSYISPIGCLPAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFVSVGLPNMTQGFWENSMLTDPGNVQKAVCHPTAWDLGKGDFRILMCTKVTMDDFLTAHHEMGHIQYDMAYAAQPFLLRNGANEGFHEAVGEIMSLSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEKWRWMVFKGEIPKDQWMKKWWEMKREIVGVVEPVPHDETYCDPASLFHVSNDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLHKCDISNSTEAGQKLFNMLRLGKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVGWSTDWSPYADQSIKVRISLKSALGDRAYEWNDNEMYLFRSSVAYAMRQYFLKVKNQMILFGEEDVRVANLKPRISFNFFVTAPKNVSDIIPRTEVEKAIRMSRSRINDAFRLNDNSLEFLGIQPTLGPPNQPPVS

Claims

1. A chemiluminescent kit for detecting a neutralizing antibody to a novel coronavirus or variant thereof in a sample, wherein the kit comprises a spike protein portion of a novel coronavirus or variant thereof and an ACE2 protein or functional fragment thereof that specifically binds to the spike protein portion of the novel coronavirus or variant thereof;

wherein the spike protein portion of the novel coronavirus or variant thereof is linked to a label, and the ACE2 protein or functional fragment thereof is linked to a magnetic microparticle; or the spike protein part of the novel coronavirus or the variant thereof is connected with magnetic particles, and the ACE2 protein or the functional fragment thereof is connected with a marker;

wherein specific binding between the spike protein portion of the novel coronavirus or variant thereof and the ACE2 protein or functional fragment thereof generates a detection signal.

2. The chemiluminescent kit of claim 1 wherein the labels or magnetic particles are attached by a linker or coupling means, preferably by coupling means.

3. The chemiluminescent kit according to claim 1 or 2, wherein the spike protein moiety of the novel coronavirus or variant thereof is conjugated to a label and the ACE2 protein or functional fragment thereof is conjugated to magnetic microparticles.

4. The chemiluminescent kit of any one of claims 1 to 3, the label is selected from one or more of alkaline phosphatase, acridinium ester, horseradish peroxidase, ruthenium terpyridyl, biotin, isoluminol or a rare earth element, preferably alkaline phosphatase, acridinium ester or horseradish peroxidase.

5. The chemiluminescent kit of claim 1 comprising:

the spike protein part of the novel acridinium ester-conjugated coronavirus or a variant thereof and the ACE2 protein or a functional fragment thereof coupled with magnetic particles, or the spike protein part of the novel acridinium ester-conjugated ACE2 protein or a functional fragment thereof and the magnetic particles.

6. The chemiluminescent kit of claim 1 comprising:

an alkaline phosphatase-conjugated novel coronavirus or a variant spike protein thereof having a moiety and a magnetic particle-conjugated ACE2 protein or a functional fragment thereof, or an alkaline phosphatase-conjugated ACE2 protein or a functional fragment thereof and a magnetic particle-conjugated novel coronavirus or a variant spike protein thereof.

7. A chemiluminescent kit according to any one of claims 1 to 6 wherein the presence or absence of neutralizing antibodies to the novel coronavirus or variant thereof in the sample is determined by detecting a change in the detection signal generated by the test sample inhibiting or blocking the binding between the spike protein portion of the novel coronavirus or variant thereof and the ACE2 protein or functional fragment thereof.

8. The chemiluminescent kit of any one of claims 1 to 7, wherein the novel coronavirus or variant spike protein moiety thereof is selected from the group consisting of a novel coronavirus or variant S protein thereof, a novel coronavirus or variant S1 protein thereof, a novel coronavirus or variant RBD protein thereof, or a His-or Fc-tagged novel coronavirus or variant RBD protein thereof, preferably a novel coronavirus or variant RBD protein thereof.

9. The chemiluminescent kit of any one of claims 1 to 8 wherein the spike protein portion of the novel coronavirus or variant thereof comprises the amino acid sequence set forth in any one of SEQ ID NOs 1,2 and 4-7, or a sequence at least 70% identical to the amino acid sequence set forth in any one of SEQ ID NOs 1,2 and 4-7.

10. The chemiluminescent kit of any one of claims 1 to 9 wherein the concentration of the marker-conjugated novel coronavirus or variant spike protein moiety thereof is from 2 to 200 ng/mL.

11. The chemiluminescent kit of claim 10 wherein the marker conjugated novel coronavirus or variant spike protein moiety thereof is an acridinium ester-labeled or alkaline phosphatase-labeled novel coronavirus or variant RBD protein thereof.

12. The chemiluminescent kit according to claim 11, wherein the concentration of the acridinium ester or alkaline phosphatase labeled novel coronavirus or variant RBD protein thereof is 2-200ng/mL, preferably 10-100 ng/mL.

13. The chemiluminescent kit of any one of claims 1 to 12, wherein the ACE2 protein or functional fragment thereof is derived from human.

14. The chemiluminescent kit of any one of claims 1 to 13, wherein the ACE2 protein or functional fragment thereof is selected from the group consisting of a recombinant ACE2 protein or an extracellular domain of an ACE2 protein or an Fc-tagged ACE2 protein or an extracellular domain of an ACE2 protein.

15. The chemiluminescent kit of claim 13 or 14, wherein the ACE2 protein or functional fragment thereof comprises the amino acid sequence set forth in any one of SEQ ID NOs 3 and 8-12 or a sequence at least 70% identical to the amino acid sequence set forth in any one of SEQ ID NOs 3 and 8-12.

16. The chemiluminescent kit of any one of claims 1 to 15, wherein the concentration of ACE2 protein or functional fragment thereof in the magnetic particle-coupled ACE2 protein or functional fragment thereof is 2-15ug/mL, preferably 5-10 ug/mL.

17. The chemiluminescent kit of claim 16 wherein the concentration of the magnetic particles is 0.1-0.5 mg/mL.

18. The chemiluminescent kit of any one of claims 1 to 17 further comprising a chemiluminescent substrate selected from AMPPD, APS5, CSPD, CDP-Star, luminol or PS-atto.

19. The chemiluminescent kit of any one of claims 1 to 18 further comprising a negative control, a sample diluent and a wash solution, the negative control being a sample diluent.

20. The chemiluminescent kit of any one of claims 1-19 further comprising a positive quality control which is a neutralizing antibody against SARS-CoV-2 virus or a variant thereof.

21. The chemiluminescent kit of any one of claims 1-20, the novel coronavirus variant selected from the group consisting of SARS-CoV-2b.1.1.7, SARS-CoV-2b.1.351, or SARS-CoV-2 b.1.617.

22. Use of a chemiluminescent kit of any one of claims 1 to 21 for detecting the presence of antibodies to a novel coronavirus or variant thereof in a sample.

23. Use of the chemiluminescent kit of any one of claims 1 to 21 or the chemiluminescent kit of claim 22 wherein said sample comprises:

(1) plasma, serum or whole blood infected or suspected of being infected with the novel coronavirus or variant thereof;

(2) plasma, serum or whole blood samples after vaccination with a novel coronavirus or variant thereof;

(3) plasma, serum or whole blood following immunization of an animal with the novel coronavirus or variant spike protein thereof; or

(4) Biological methods obtain samples of antibodies against the novel coronavirus or variant thereof.

24. The method of using the chemiluminescent kit of any one of claims 1 to 21 comprising the steps of:

25. The method of using the chemiluminescent kit of any one of claims 1 to 21 comprising the steps of:

26. A method for detecting the presence or absence of neutralizing antibodies to a novel coronavirus, or variant thereof, in a sample, comprising:

(3) detecting the level of binding between the spike protein or ACE2 binding fragment thereof and the ACE2 protein or fragment thereof,

wherein the novel coronavirus or variant spike protein thereof or ACE2 binding fragment thereof is linked to a label and the ACE2 protein or fragment thereof is linked to a magnetic microparticle.

27. A method for detecting the presence or absence of neutralizing antibodies to a novel coronavirus, or variant thereof, in a sample, comprising:

(4) detecting the level of binding between the spike protein or ACE2 binding fragment thereof and the ACE2 protein or fragment thereof,

wherein the ACE2 protein or fragment thereof is linked to a label.

28. The method of claim 26 or 27, further comprising determining the presence or absence of neutralizing antibodies to the novel coronavirus or variant thereof in the sample by inhibiting or blocking the change in the detection signal produced by the binding of the spike protein of the novel coronavirus or variant thereof or the ACE 2-binding fragment thereof and the ACE2 protein or functional fragment thereof in the sample.