CN112964872A - SARS-CoV-2 detection reagent kit - Google Patents
SARS-CoV-2 detection reagent kit Download PDFInfo
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56983—Viruses
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6854—Immunoglobulins
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
- G01N2333/08—RNA viruses
- G01N2333/165—Coronaviridae, e.g. avian infectious bronchitis virus
Abstract
The invention relates to a SARS-CoV-2 detection kit. S the kit comprises: a) a solid phase conjugate; the solid phase conjugate consists of a solid phase carrier, and an antigen and an antibody coated on the solid phase carrier; the antibody is a monoclonal antibody for resisting a new coronavirus nucleocapsid N protein; the antigen comprises: n protein of recombinant SARS-CoV-2; b) an antigen labeled with a chemiluminescent label, and an antibody and/or an anti-antibody labeled with a chemiluminescent label; the antibody is a monoclonal antibody of the N protein of the new coronavirus nucleocapsid, but the epitope bound by the antibody in a) is different; the antigen comprises: n protein of recombinant SARS-CoV-2; the anti-antibody is at least one of an anti-human IgG secondary antibody, an anti-human IgA secondary antibody and an anti-human IgM secondary antibody; the anti-antibody is a monoclonal antibody or a polyclonal antibody; wherein, in a) and b), the N protein of the recombinant SARS-CoV-2 does not have an epitope recognized by the monoclonal antibody against the N protein of the neocoronavirus nucleocapsid.
Description
Technical Field
The invention relates to the field of biological medicine, in particular to a SARS-CoV-2 detection kit.
Background
The novel coronavirus (SARS-CoV-2) belongs to the genus of beta, and the particle is circular or elliptical, has a diameter of 60 nm-140 nm, and has higher infectivity than SARS. The novel coronavirus has strong interpersonal transmission capability, is generally susceptible to the crowd and is the most important infection source for patients, so that the early discovery, early isolation and early treatment of asymptomatic infectors, suspected and confirmed cases are still necessary. How to accurately detect the virus, quickly identify a new coronary infected patient, and reduce cross infection; and how to effectively monitor disease progression and antibody production will be of central importance. Based on epidemiological investigation, the incubation period of the disease COVID-19 caused by the novel coronavirus is 1-14 days, most of the disease is 3-7 days, symptoms mainly comprise fever, dry cough and hypodynamia, severe patients mostly have dyspnea and/or hypoxemia after one week of morbidity, and severe patients can rapidly develop acute respiratory distress syndrome, septic shock, metabolic acidosis which is difficult to correct, blood coagulation dysfunction, multiple organ failure and the like.
The novel coronavirus comprises 4 major structural proteins such as Spike protein (Spike protein), Envelope protein (Envelope protein), Membrane protein (Membrane protein), Nucleocapsid protein (Nucleocapsid protein) and the like. Wherein the spike protein forms the outer layer of coronavirus, forms obvious spikes on the surface of the virus by forming trimer, and plays a key role in inducing neutralizing antibody and T cell response protective immunity, the spike protein comprises large type 1 transmembrane proteins of two subunits S1 and S2, S1 mainly comprises a Receptor Binding Domain (RBD) which is responsible for recognizing the protein of a cell surface Receptor ACE2 (angiotensin converting enzyme 2) and mediating virus invasion into cells; s2 contains essential elements required for membrane fusion. The envelope protein is a structural protein. Once the virus enters the interior of the cell, it can lock in proteins, thereby helping to open and close the genome of the infected cell; membrane proteins are another structural protein that forms the outer shell of the virus. The N protein located in the virus can protect the virus RNA and keep the virus RNA stable in the virus, and a plurality of N proteins are connected together in a long spiral shape to wrap the RNA and are relatively conserved in sequence. The structural proteins play a key role in the recognition adsorption, membrane fusion, replication assembly and release cycles of viruses, can induce antibody and T cell reaction protective immunity to different degrees, and are also main design targets for serum antigen antibody detection.
The current method for determining COVID-19 is to detect the nucleic acid positivity of a novel coronavirus or virus gene sequencing by using real-time fluorescent RT-PCR, but the problem of missed detection of a nucleic acid detection reagent is exposed again, the positive detection rate is only 30-50 percent, and the detection is mainly caused by the limitation of a sampling mode. Nucleic acid detection is generally performed by collecting a nasopharyngeal swab of a suspected patient, extracting RNA fragments in the nasopharyngeal swab, and performing fluorescence RT-PCR, wherein a large amount of human cells and bacteria are mixed in the sampling process, so that the abundance of RNA to be detected is low, a detection system cannot detect the RNA, and finally false negative is generated. Meanwhile, the sampling mode can cause the sampling personnel to be exposed to the environment possibly containing pathogens, and the potential infection risk exists. The sample preparation process needs many steps, has long operation time and has high requirements on the technical level of detection personnel.
The chemiluminescence technology is mainly based on the principle that the concentration of an object to be detected in a detection system and the chemiluminescence intensity of the system form a quantitative linear relationship under a certain condition, and the chemiluminescence intensity is detected by using a matched instrument, so that the content of the object to be detected is determined.
According to the immunological principle of antigen-antibody combination, the virus can also be detected by using a chemiluminescence technology. The current assays associated with the new coronavirus pneumonias focus on IgG and IgM with fewer assays directed to the antigen. This is because the virus titer in the blood of a patient is low at the early stage of infection, and a highly sensitive detection method is required, and in addition, the virus in the blood may be recognized by the relevant antibody, and it is difficult to release the epitope without processing the sample, thereby causing false negative test results.
According to the early research result on SARS, after a patient is infected with virus, IgM antibodies can be generated in about 3-7 days, IgG antibodies can also rise after 1 week, and when IgG appears, the titer can be continuously increased and exists for a long time; IgM continued to decrease until it disappeared. Therefore, accurate observation of the dynamic changes of both contributes to the diagnosis of new coronavirus infection in the case of false negative nucleic acid detection. Patients who are clinically suspected of new coronary pneumonia and negative for nucleic acid detection, as well as patients in convalescent phase and negative for nucleic acid detection, can be diagnosed by antibody detection.
However, the sensitivity of antibody detection is still unsatisfactory in the prior art.
Disclosure of Invention
The first aspect of the present invention relates to a SARS-CoV-2 detection kit comprising:
a) a solid phase conjugate;
the solid phase conjugate consists of a solid phase carrier, and an antigen and an antibody coated on the solid phase carrier;
the antibody is a monoclonal antibody for resisting a new coronavirus nucleocapsid N protein;
the antigen comprises: n protein of recombinant SARS-CoV-2, and optional S1 subunit, S2 subunit, RBD, M protein and E protein of SARS-CoV-2Spike protein;
b) an antigen labeled with a chemiluminescent label, and an antibody and/or an anti-antibody labeled with a chemiluminescent label;
the antibody is a monoclonal antibody of the N protein of the new coronavirus nucleocapsid, but the epitope bound by the antibody in a) is different;
the antigen comprises: n protein of recombinant SARS-CoV-2, and optional S1 subunit, S2 subunit, RBD, M protein and E protein of SARS-CoV-2Spike protein;
the anti-antibody is at least one of an anti-human IgG secondary antibody, an anti-human IgA secondary antibody and an anti-human IgM secondary antibody;
the anti-antibody is a monoclonal antibody or a polyclonal antibody;
wherein, in a) and b), the N protein of the recombinant SARS-CoV-2 does not have an epitope recognized by the monoclonal antibody against the N protein of the neocoronavirus nucleocapsid.
Alternatively, the kit as described above, wherein the N protein of recombinant SARS-CoV-2 is modified by any one of chemical modification, amino acid substitution and truncation mutation to remove the epitope recognized by the monoclonal antibody against the N protein of neocoronavirus nucleocapsid in a) and b).
Optionally, in the kit as described above, the chemiluminescent label is an acridinium chemiluminescent substance.
Optionally, a kit as described above, further comprising an antigen releasing component;
the antigen releasing component comprises: 10 mM-50 mM citrate, 0.5 mM-2 mM organic acid, 0.1 v/v% -2 v/v% surfactant, and pH is 3-4.5.
Optionally, the kit as described above, wherein the surfactant comprises a Tween, preferably at least one of Tween-20, Tween-80 and Tween-100.
Optionally, the kit as described above, wherein the antigen releasing component further comprises a preservative, preferably at a concentration of 0.1 w/v% to 0.5 w/v%.
Optionally, in the kit as described above, the solid-phase carrier is a test tube, an EP tube, a multi-well plate, a microplate well, a microbead or a disc.
Optionally, in the kit as described above, the material of the solid phase carrier is one or more of polystyrene, plastic, cellulose, polyacrylamide, polyethylene polypropylene, cross-linked dextran, glass, silicone rubber, and agarose gel.
Alternatively, the kit as described above, in a), the antibodies are multiple and respectively directed to different epitopes of the N protein.
Alternatively, the kit as described above, wherein in b) the species origin of the antibody and/or anti-antibody is the same as the antibody in step a).
A second aspect of the invention relates to a method of non-diagnostic interest for the detection of SARS-CoV-2 based on a kit as described above, comprising:
i) treating the sample to be detected to release SARS-CoV-2 antigen and antibody therein, and incubating the sample with the solid phase conjugate;
ii) washing away unbound SARS-CoV-2 antigen and antibody;
iii) adding the antigen labeled with the chemiluminescent label, and an antibody and/or an anti-antibody, and detecting the signal intensity of the chemiluminescent label.
Compared with the prior art, the invention has the beneficial effects that:
the invention integrates SARS-CoV-2 antigen detection and antibody detection into a single detection kit, only one sample is needed, the virus antigen and total antibody content in the patient can be detected quickly, the operation time and cost are saved, the operation is convenient and quick, and the cross contamination is reduced; the kit is expected to be used for screening and managing clinical patients and close contacts, and is applied to screening of high-risk area populations, epidemiological investigation and other application scenes. The invention can be well used for detecting serum samples due to high detection sensitivity.
The total antibody detection not only contains the advantages of early diagnosis of the novel coronavirus through IgM antibody detection, but also has the characteristic of detecting the prior infection (including patients with decreased IgM in the convalescent period) of the IgG antibody, can better prevent omission and shorten the diagnosis window period, and has higher specificity and sensitivity.
Detailed Description
Reference will now be made in detail to embodiments of the invention, one or more examples of which are described below. Each example is provided by way of explanation, not limitation, of the invention. In fact, it will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the scope or spirit of the invention. For instance, features illustrated or described as part of one embodiment, can be used on another embodiment to yield a still further embodiment.
Unless defined otherwise, all 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 terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.
As used in the specification and examples of the invention, the singular forms "a", "an" and "the" include both the singular and the plural, unless the context clearly dictates otherwise. For example, "antibody" refers to one or more than one antibody(s).
As used herein, the terms "comprising," "including," and "comprising" are synonymous, inclusive or open-ended, and do not exclude additional, unrecited members, elements, or method steps.
The recitation of numerical ranges by endpoints includes all numbers and fractions subsumed within that range and the recited endpoints.
Aiming at the difficulties and possible defects of the prior art, the kit is developed according to the principles of an immune sandwich method and an indirect method of a direct chemiluminescence technology. The kit is matched with a corresponding chemiluminescence determinator, and can realize automation (automatic sampling, automatic detection and automatic reporting), high flux (300 persons/hour) and rapid detection (30min reporting) of the novel coronavirus antigen and total antibody.
The invention relates to a SARS-CoV-2 detection kit, comprising:
a) a solid phase conjugate;
the solid phase conjugate consists of a solid phase carrier, and an antigen and an antibody coated on the solid phase carrier;
the antibody is a monoclonal antibody for resisting a new coronavirus nucleocapsid N protein;
the antigen comprises: n protein of recombinant SARS-CoV-2;
b) an antigen labeled with a chemiluminescent label, and an antibody and/or an anti-antibody labeled with a chemiluminescent label;
the antibody is a monoclonal antibody of the N protein of the new coronavirus nucleocapsid, but the epitope bound by the antibody in a) is different;
the antigen comprises: n protein of recombinant SARS-CoV-2;
the anti-antibody is at least one of an anti-human IgG secondary antibody, an anti-human IgA secondary antibody and an anti-human IgM secondary antibody;
the anti-antibody is a monoclonal antibody or a polyclonal antibody;
wherein, in a) and b), the N protein of the recombinant SARS-CoV-2 does not have an epitope recognized by the monoclonal antibody against the N protein of the neocoronavirus nucleocapsid.
The invention integrates SARS-CoV-2 antigen detection and antibody detection into a single detection kit, only one sample is needed, the virus antigen and total antibody content in the patient can be detected quickly, the operation time and cost are saved, the operation is convenient and quick, and the cross contamination is reduced; the kit is expected to be used for screening and managing clinical patients and close contacts, and is applied to screening of high-risk area populations, epidemiological investigation and other application scenes. The invention can be well used for detecting serum samples due to high detection sensitivity.
The total antibody detection not only contains the advantages of early diagnosis of the novel coronavirus through IgM antibody detection, but also has the characteristic of detecting the prior infection (including patients with decreased IgM in the convalescent period) of the IgG antibody, can better prevent omission and shorten the diagnosis window period, and has higher specificity and sensitivity.
In some embodiments, the antigen in a) further comprises at least one of the S1 subunit, S2 subunit, RBD, M protein, and E protein of SARS-CoV-2Spike protein.
In some embodiments, the antigen in b) further comprises at least one of S1 subunit, S2 subunit, RBD, M protein, and E protein of SARS-CoV-2Spike protein.
In some embodiments, the N protein of recombinant SARS-CoV-2 is modified by any of chemical modification, amino acid substitution, and truncation mutation to remove an epitope recognized by the monoclonal antibody against the N protein of a) and b) of a neocoronavirus nucleocapsid.
In some embodiments, the methods of chemical modification include, but are not limited to, at least one of phosphorylation, acetylation, ubiquitination, nitrosylation, lipidation, methylation, and glycosylation modification of one or more amino acids at an epitope recognized by an antibody.
In some embodiments, chemical modifications may be performed preferentially to the more immunogenic amino acids.
In some embodiments, amino acid substitution selections replace more immunogenic amino acids with less immunogenic amino acids (e.g., glycine, serine, etc.); in some embodiments, amino acid substitutions should avoid conservative substitutions, which are generally considered to be substitutions in the aliphatic amino acids Ala, Val, Leu and he for each other, the exchange of hydroxyl residues Ser and Thr, the exchange of acidic residues Asp and Glu, the exchange of amide residues Asn and gin, the exchange of basic residues Lys and Arg and the exchange of aromatic residues Phe, Tyr.
In some embodiments, the number of amino acids deleted by the truncation mutation may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more, preferably the more immunogenic amino acids are deleted.
In the present invention, the "more immunogenic amino acid" is known to those skilled in the art, and includes, for example, aromatic amino acids (particularly tyrosine) having a more complex structure.
In some embodiments, the chemiluminescent label is an acridinium-based chemiluminescent substance.
Further, acridine chemiluminescent substances include acridinium esters and acridinium sulfonamides.
Further, the acridine chemiluminescent substance includes acridine ester AE-NHS, acridine ester DMAE-NHS, acridine ester Me-DMAE-NHS, acridine ester NSP-DMAE-NHS, acridine salt NSP-SA-NHS, acridine hydrazide NSP-SA-ADH, etc.
In some embodiments, the kit further comprises an antigen releasing component;
the antigen releasing component comprises: 10 mM-50 mM citrate, 0.5 mM-2 mM organic acid, 0.1 v/v% -2 v/v% surfactant, and pH is 3-4.5.
The pH can also be selected from 3.5, 4, etc.
The antigen release component of the reagent is proved by research, under the condition of ensuring the activity of the antigen and the antibody, the combined antigen and the antibody are dissociated to obtain the optimal condition, wherein a buffer system and acetic acid can ensure that the integral pH is 3-4.5 after the antigen release component is added into a serum sample, which is the condition most beneficial to the dissociation of the antigen and the antibody and can also ensure the activity of the antigen; the detection rate and accuracy are improved.
In some embodiments, the organic acid is at least one of acetic acid, oxalic acid, malic acid, salicylic acid.
In some embodiments, the surfactant comprises Tween. In some embodiments, the Tween comprises at least one of Tween-20, Tween-80, Tween-100.
In some embodiments, the antigen releasing component further comprises a preservative, preferably at a concentration of 0.1 w/v% to 0.5 w/v%.
In some embodiments, the solid support is a test tube, an EP tube, a multi-well plate, a microplate well, a microbead or a wafer.
In some embodiments, the multi-well plate is an elisa plate, which may contain 16, 32, 48, 64, 96 or more wells.
In the present invention, the term "microsphere" may be a sphere, a nearly sphere, a cube, a polyhedron or an irregular shape. The diameter of the microspheres is preferably 10nm to 1mm, for example 100nm, 500nm, 1 μm, 10 μm, 100 μm, 500 μm; preferably 400nm to 10 μm.
The microspheres have specific binding properties for the substance of interest (target or analyte) to be assayed on their surface.
The microspheres are preferably magnetic beads, and the magnetic material is contained in the composition. Magnetic propertyThe substance can be metal (metal simple substance or alloy), nonmetal, or composite formed by metal and nonmetal. Metals such as iron, alnico, and the like; non-metals, e.g. ferrite non-metals (preferably Fe)2O3Or Fe3O4Magnetic nanoparticles); a composite of metal and non-metal such as neodymium iron boron rubber magnetic composite.
The surface of the microsphere is modified with one or more active functional groups, wherein the active functional groups comprise-OH, -COOH and-NH2-CHO, and-SO3H. In some embodiments, the coated antigen and antibody are conjugated or bound to the microsphere by physisorption or direct chemical conjugation (e.g., bridging by a bridge).
In some embodiments, the material of the solid phase carrier is one or more of polystyrene, plastic, cellulose, polyacrylamide, polyethylene polypropylene, cross-linked dextran, glass, silicone rubber and agarose gel.
In a preferred embodiment, in a), the antibodies are multiple, each directed against a different epitope of the N protein. The desired antibody should cover a substantial portion of the active epitopes of the N protein in order to increase the sensitivity of the detection of the N protein antigen of SARS-CoV-2.
Obviously, the antibodies described in b) can also be a plurality of antibodies, each of which is paired with a different antibody in a).
In the present invention, the source of the antibody in a) and the antibody in b) can be independently selected from animals including humans and all animal husbandry (such as domestic animals and pets) and wild and avian birds, including, but not limited to, cattle, horses, dairy cattle, pigs, sheep, goats, rats, mice, dogs, cats, rabbits, camels, donkeys, deer, mink, chickens, ducks, geese, turkeys, fighting chickens, etc.
The term "antibody" includes naturally occurring antibodies as well as non-naturally occurring antibodies, including, for example, chimeric (chimeric), bifunctional (bifunctional), humanized (humanized) antibodies and human antibodies, as well as related synthetic isomeric forms (isoantibodies). In addition, antibodies also include those capable of bindingFunctional fragments of the desired antigen, e.g. Fab, F (ab')2Fd, Fv, scFv, diabodies and antibody minimal recognition units, as well as single chain derivatives of these antibodies and fragments, such as scFv-Fc and the like. The type of antibody can be selected from IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, IgD.
In some embodiments, in b), the species origin of the antibody and/or anti-antibody is the same as the antibody in step a).
The antibody from the same species can effectively reduce cross reaction.
According to a further aspect of the invention, it also relates to a method for detecting SARS-CoV-2 based on a kit as described above, said method comprising:
i) treating the sample to be detected to release SARS-CoV-2 antigen and antibody therein, and incubating the sample with the solid phase conjugate;
ii) washing away unbound SARS-CoV-2 antigen and antibody;
iii) adding the antigen labeled with the chemiluminescent label, and an antibody and/or an anti-antibody, and detecting the signal intensity of the chemiluminescent label.
The term "detecting" and similar terms are used in this application to generally refer to a process or the discovery or determination of the presence or absence, as well as the degree, quantity or level, or probability of occurrence of something. For example, the term "detecting" when used in reference to a target nucleic acid sequence can refer to finding or determining the presence, absence, level, or amount, and the probability or likelihood of the presence or absence, of the sequence. It is to be understood that the expressions "detecting the presence or absence", "detecting the presence or absence" and related expressions include both qualitative and quantitative detections. For example, quantitative detection includes determining the level, amount, or quantity of SARS-CoV-2 associated antigen or antibody in a sample.
The term "sample" refers to any composition that contains or is suspected of containing SARS-CoV-2 associated antigen or antibody from an individual. The term includes purified or isolated components of cells, tissues or blood. In a specific embodiment, the analysis is performed on a whole blood sample. As used herein, a "whole blood sample" includes blood drawn from the body from which components such as plasma or platelets have not been removed. Typically, the sample is unmodified except for the presence of an anticoagulant. Samples may also refer to other types of biological samples, such as plasma, serum, blood components (buffy coat), and dried blood spots (dried blood spots). The blood sample is preferably peripheral blood, the sample may also include components and constituents of an in vitro culture of cells obtained from an individual, including cell lines.
The method may be used for diagnostic or non-diagnostic purposes.
Embodiments of the present invention will be described in detail with reference to examples.
Example 1
The kit mainly comprises a calibrator and a detection reagent, wherein the detection reagent consists of 3 parts:
1.1R1 magnetic bead component:
the magnetic bead coated protein consists of 7 paramagnetic magnetic beads and a magnetic bead diluent, wherein the magnetic bead coated protein is combined as follows:
anti-new coronavirus N protein mouse monoclonal antibody;
② SARS-CoV-2N protein;
③ SARS-CoV-2Spike protein S1 subunit;
(iv) SARS-CoV-2Spike protein S2 subunit;
SARS-CoV-2Spike protein S1 subunit Receptor Binding Domain (RBD) protein;
sixthly, SARS-CoV-2M protein;
seventhly, SARS-CoV-2E protein;
wherein the antiviral N protein mouse monoclonal antibody can be obtained by mouse ascites extraction or hybridoma cell in vitro culture, and can be a plurality of monoclonal antibodies aiming at different N protein different immune sites; the S1, S2, RBD, M and E proteins coated on the magnetic beads can be naturally extracted or can be expressed by recombination.
First and second do not specifically bind.
The concentration of the magnetic beads was 0.1mg/mL, and the magnetic bead diluent was a commonly used diluent containing 50mM MES, 3% BSA, preservative, etc., and had a pH of 6.0.
1.2R2 acridine component: consists of an N protein mouse monoclonal antibody, an anti-antibody and antigen component and an acridine diluent, wherein the N protein mouse monoclonal antibody is respectively marked with acridine ester.
The acridinium ester combination comprises:
the mouse monoclonal antibody resisting the new coronavirus N protein and the monoclonal antibody in the R1 magnetic bead component may constitute paired antibody for detecting N protein antigen.
② SARS-CoV-2N protein;
③ SARS-CoV-2Spike protein S1 subunit;
(iv) SARS-CoV-2Spike protein S2 subunit;
SARS-CoV-2Spike protein S1 subunit Receptor Binding Domain (RBD) protein;
sixthly, SARS-CoV-2M protein;
seventhly, SARS-CoV-2E protein;
first and second do not specifically bind.
The concentration of the acridine component is 20ug/mL, and the acridine diluent is a diluent containing 30mM PBS, 3% BSA, Mouse IgG, preservative and other components, and the pH is 7.5.
1.3R3 antigen releasing component: is prepared from 30mM citrate, 1mM acetic acid, 1% Tween-80, and 0.3% NaN3Composition, pH 3.5.
Example 2
The kit mainly comprises a calibrator and a detection reagent, wherein the detection reagent consists of 3 parts:
1.1R1 magnetic bead component:
the magnetic bead coated protein consists of 7 paramagnetic magnetic beads and a magnetic bead diluent, wherein the magnetic bead coated protein is combined as follows:
anti-new coronavirus N protein mouse monoclonal antibody;
② SARS-CoV-2N protein;
③ SARS-CoV-2Spike protein S1 subunit;
(iv) SARS-CoV-2Spike protein S2 subunit;
SARS-CoV-2Spike protein S1 subunit Receptor Binding Domain (RBD) protein;
sixthly, SARS-CoV-2M protein;
seventhly, SARS-CoV-2E protein;
wherein the antiviral N protein mouse monoclonal antibody can be obtained by mouse ascites extraction or hybridoma cell in vitro culture, and can be a plurality of monoclonal antibodies aiming at different N protein different immune sites; the S1, S2, RBD, M and E proteins coated on the magnetic beads can be naturally extracted or can be expressed by recombination.
First and second do not specifically bind.
The concentration of the magnetic beads was 0.05mg/ml, and the magnetic bead diluent was a commonly used diluent containing 20mM MES, 5% BSA, preservative and the like, and had a pH of 5.5.
1.2R2 acridine component: consists of an N protein mouse monoclonal antibody marked with acridine sulfamide, an antigen component and an acridine diluent.
The combination comprises:
the mouse monoclonal antibody resisting the new coronavirus N protein and the monoclonal antibody in the R1 magnetic bead component may constitute paired antibody for detecting N protein antigen.
② SARS-CoV-2N protein;
③ SARS-CoV-2Spike protein S1 subunit;
(iv) SARS-CoV-2Spike protein S2 subunit;
SARS-CoV-2Spike protein S1 subunit Receptor Binding Domain (RBD) protein;
sixthly, SARS-CoV-2M protein;
seventhly, SARS-CoV-2E protein;
first and second do not specifically bind.
The concentration of the acridine component is 100ug/mL, and the acridine diluent is a diluent containing 10mM PBS, 5% BSA, Mouse IgG, preservative and other components, and the pH is 8.5.
1.3R3 antigen releasing component: consists of 10mM citrate, 0.5mM acetic acid, 0.1% Tween-20, 0.5% ProClin-300, pH 3.
Example 3
The kit mainly comprises a calibrator and a detection reagent, wherein the detection reagent consists of 3 parts:
1.1R1 magnetic bead component:
the magnetic bead coated protein consists of 5 paramagnetic magnetic beads and a magnetic bead diluent, wherein the magnetic bead coated protein comprises the following components in percentage by weight:
the mouse monoclonal antibody resisting the new coronavirus N protein and the monoclonal antibody in the R1 magnetic bead component may constitute paired antibody for detecting N protein antigen.
② SARS-CoV-2N protein;
③ SARS-CoV-2Spike protein S1 subunit;
(iv) SARS-CoV-2Spike protein S2 subunit;
SARS-CoV-2Spike protein S1 subunit Receptor Binding Domain (RBD) protein;
first and second do not specifically bind.
Wherein the antiviral N protein mouse monoclonal antibody can be obtained by mouse ascites extraction or hybridoma cell in vitro culture, and can be a plurality of monoclonal antibodies aiming at different N protein different immune sites; the S1, S2, RBD, M and E proteins coated on the magnetic beads can be naturally extracted or can be expressed by recombination.
The concentration of the magnetic beads was 0.5mg/ml, and the magnetic bead diluent was a commonly used diluent containing 100mM MES, 0.5% BSA, preservative and the like, and had a pH of 6.5.
1.2R2 acridine component: consists of an N protein mouse monoclonal antibody and an antigen which are respectively marked with acridine ester and an acridine diluent.
The combination comprises:
the mouse monoclonal antibody resisting the new coronavirus N protein and the monoclonal antibody in the R1 magnetic bead component may constitute paired antibody for detecting N protein antigen.
② SARS-CoV-2N protein;
③ SARS-CoV-2Spike protein S1 subunit;
(iv) SARS-CoV-2Spike protein S2 subunit;
SARS-CoV-2Spike protein S1 subunit Receptor Binding Domain (RBD) protein;
first and second do not specifically bind.
The concentration of the acridine component is 200ug/mL, and the acridine diluent is a diluent containing 50mM PBS, 0.1% BSA, Mouse IgG, preservative and other components, and the pH is 5.5.
1.3R3 antigen releasing component: is prepared from 10-50 mM of citrate, 2mM of malic acid, 2% of surfactant Tween-100 and 0.1% of NaN3Composition, pH 4.5.
Example 4
This example is a method of using a kit comprising:
the reagent can be matched with an iFlash-3000 series chemiluminescence determinator produced by the company for use, wherein the steps of cleaning solution, pre-excitation solution, excitation solution and corresponding cleaning and luminescence reading are default settings of the determinator, and other steps are finished by manual setting programs and processing by the determinator.
First step sample processing: setting the program, sucking 20uL of sample into the reaction cup, adding 100uL of R3 component, and incubating at 37 ℃ for 5min to dissociate and fully release the possibly combined antigen and antibody.
And step two, adding magnetic beads: adding 50uL of the R1 reagent component into the reaction cup, and incubating for 10min at 37 ℃, wherein the virus N protein antigen in the sample is combined with the anti-N protein murine monoclonal antibody coated on the magnetic beads; the total antibodies of the new coronavirus in the sample are respectively combined with N protein, S1, and/or S2, and/or RBD, and/or M, and/or E protein antigens which are possibly coated on the magnetic beads, and then a washing procedure is carried out to wash away the unbound and adsorbed samples;
and step three, adding acridine: 100uL of the above-mentioned R2 fraction was added to the reaction cuvette and incubated at 37 ℃ for 10min to allow the antigen-antibody to react sufficiently to form an immune complex. A washing procedure is then performed to wash away unbound components. Immune complexes that may form (including but not limited to) are as follows:
antigen: mouse monoclonal antibody-virus N protein-mouse monoclonal antibody
Total antibody (double antigen sandwich):
acridinium ester labeled murine anti-human secondary antibody:
n protein-anti-N protein antibody-N protein; s1 protein-anti-S1 antibody-S1 protein; s2 protein-anti-S2 antibody-S2 protein; RBD protein-anti-RBD antibody-RBD protein; m protein-anti-M antibody-M protein; protein E-anti-E antibody-protein E;
acridinium ester labeled goat anti-human polyclonal antibody:
n protein-anti-N protein antibody-goat anti-human polyclonal antibody; s1 protein-anti-S1 antibody-goat anti-human polyclonal antibody; s2 protein-anti-S2 antibody-goat anti-human polyclonal antibody; RBD protein-anti-RBD antibody-goat anti-human polyclonal antibody; m protein-anti-M antibody-goat anti-human polyclonal antibody; protein E-anti-E antibody-goat anti-human polyclonal antibody.
The fourth step is to determine the concentration of the substance to be detected: exciting a chemiluminescent marker combined on the magnetic beads through a series of reactions according to a preset luminescence program of the instrument, and reading a luminescence value by a chemiluminescent determinator;
the fifth step is to generate a report: according to the calibration curve and the reference interval, the content of the novel coronavirus antigen and the total antibody in the patient blood sample is reported so as to assist clinical judgment of the disease condition.
Comparative example 1
The same as example 1, except that the R1 magnetic bead component only contains the anti-neocoronavirus N protein murine monoclonal antibody;
the R2 acridinium component contains only murine mab against the new coronavirus N protein.
Comparative example 2
The same as example 1, except that the R1 magnetic bead component contains only SARS-CoV-2Spike protein S1 subunit Receptor Binding Domain (RBD) protein;
the R2 acridinium component contains only acridinium-labeled RBD protein.
The test of example 1, comparative example 1 and comparative example 2 was carried out by the method of example 4, the test sample was serum, and the test results are shown in tables 1 and 2:
TABLE 1
TABLE 2
As can be seen from tables 1 and 2, the detection method provided by the invention has detection sensitivity significantly better than that of comparative examples 1 and 2, and can be effectively used for detecting neocoronal serum samples.
The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
Claims (10)
- A SARS-CoV-2 detection kit, comprising:a) a solid phase conjugate;the solid phase conjugate consists of a solid phase carrier, and an antigen and an antibody coated on the solid phase carrier;the antibody is a monoclonal antibody for resisting a new coronavirus nucleocapsid N protein;the antigen comprises: n protein of recombinant SARS-CoV-2, and optional S1 subunit, S2 subunit, RBD, M protein and E protein of SARS-CoV-2Spike protein;b) an antigen labeled with a chemiluminescent label, and an antibody and/or an anti-antibody labeled with a chemiluminescent label;the antibody is a monoclonal antibody of the N protein of the new coronavirus nucleocapsid, but the epitope bound by the antibody in a) is different;the antigen comprises: n protein of recombinant SARS-CoV-2, and optional S1 subunit, S2 subunit, RBD, M protein and E protein of SARS-CoV-2Spike protein;the anti-antibody is at least one of an anti-human IgG secondary antibody, an anti-human IgA secondary antibody and an anti-human IgM secondary antibody;the anti-antibody is a monoclonal antibody or a polyclonal antibody;wherein, in a) and b), the N protein of the recombinant SARS-CoV-2 does not have an epitope recognized by the monoclonal antibody against the N protein of the neocoronavirus nucleocapsid.
- 2. The kit of claim 1, wherein the N protein of recombinant SARS-CoV-2 is modified by any one of chemical modification, amino acid substitution and truncation mutation to remove the epitope recognized by the monoclonal antibody against the N protein of neocoronavirus nucleocapsid in a) and b).
- 3. The kit of claim 1, wherein the chemiluminescent label is an acridinium chemiluminescent substance.
- 4. The kit of claim 1, further comprising an antigen releasing component;the antigen releasing component comprises: 10 mM-50 mM citrate, 0.5 mM-2 mM organic acid, 0.1 v/v% -2 v/v% of surfactant, and the pH value is 3-4.5;optionally, the surfactant comprises Tween, preferably at least one of Tween-20, Tween-80 and Tween-100.
- 5. The kit according to claim 4, wherein the antigen releasing component further comprises a preservative, preferably at a concentration of 0.1 w/v% to 0.5 w/v%.
- 6. The kit of claim 1, wherein the solid support is a test tube, an EP tube, a multi-well plate, a microplate well, a microbead or a disc.
- 7. The kit of claim 6, wherein the solid phase carrier is made of one or more of polystyrene, plastic, cellulose, polyacrylamide, polyethylene polypropylene, sephadex, glass, silicone rubber and agarose gel.
- 8. The kit according to any one of claims 1 to 7, wherein in a), the antibodies are multiple and respectively directed to different epitopes of the N protein.
- 9. The kit according to any one of claims 1 to 7, wherein in b) the species of the antibody and/or anti-antibody is derived from the same species as the antibody in step a).
- 10. A method for detecting SARS-CoV-2 for non-diagnostic purposes based on the kit of any one of claims 1 to 9, comprising:i) treating the sample to be detected to release SARS-CoV-2 antigen and antibody therein, and incubating the sample with the solid phase conjugate;ii) washing away unbound SARS-CoV-2 antigen and antibody;iii) adding the antigen labeled with the chemiluminescent label, and an antibody and/or an anti-antibody, and detecting the signal intensity of the chemiluminescent label.
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