CN112964874B - SARS-CoV-2 detecting reagent kit based on indirect method - Google Patents

SARS-CoV-2 detecting reagent kit based on indirect method Download PDF

Info

Publication number
CN112964874B
CN112964874B CN202110197051.8A CN202110197051A CN112964874B CN 112964874 B CN112964874 B CN 112964874B CN 202110197051 A CN202110197051 A CN 202110197051A CN 112964874 B CN112964874 B CN 112964874B
Authority
CN
China
Prior art keywords
antibody
protein
kit
antigen
sars
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
CN202110197051.8A
Other languages
Chinese (zh)
Other versions
CN112964874A (en
Inventor
钱纯亘
汪大明
程方明
龚育清
吴力强
胡鹍辉
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Shenzhen Yhlo Biotech Co Ltd
Original Assignee
Shenzhen Yhlo Biotech Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Shenzhen Yhlo Biotech Co Ltd filed Critical Shenzhen Yhlo Biotech Co Ltd
Priority to CN202110197051.8A priority Critical patent/CN112964874B/en
Publication of CN112964874A publication Critical patent/CN112964874A/en
Application granted granted Critical
Publication of CN112964874B publication Critical patent/CN112964874B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6854—Immunoglobulins
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • 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

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Chemical & Material Sciences (AREA)
  • Hematology (AREA)
  • Urology & Nephrology (AREA)
  • Food Science & Technology (AREA)
  • Biochemistry (AREA)
  • Cell Biology (AREA)
  • Biotechnology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Microbiology (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Virology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Peptides Or Proteins (AREA)

Abstract

The invention relates to the field of biological medicine, in particular to a SARS-CoV-2 detection kit based on an indirect method. 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: s1 subunit, S2 subunit, RBD, N protein and E protein of SARS-CoV-2Spike protein; b) An anti-antibody and an 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 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.

Description

SARS-CoV-2 detecting reagent kit based on indirect method
Technical Field
The invention relates to the field of biological medicine, in particular to a SARS-CoV-2 detection kit based on an indirect method.
Background
The new type coronavirus (SARS-CoV-2) belongs to the genus beta coronavirus, the particle is circular or elliptical, the diameter is 60 nm-140 nm, and compared with SARS, it has higher infection capacity. The novel coronavirus has strong interpersonal transmission capability, is generally susceptible to crowds, and is the most important infection source for patients, so that early discovery, early isolation and early treatment of asymptomatic infectors, suspected and confirmed cases are still the main lines of prevention and control of next epidemic situations. How to accurately detect the virus, quickly identify a new coronary infected patient, and reduce cross infection; how to effectively monitor the disease process and the antibody generation condition is the key point for fighting against the epidemic situation. Based on epidemiological investigation, the incubation period of the disease COVID-19 caused by the novel coronavirus is 1-14 days and is mostly 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 quickly 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 spike on the surface of the virus by forming trimer, plays a key role in inducing neutralizing antibody and T cell reaction protective immunity, the spike protein comprises large type 1 transmembrane protein 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.
According to the rules in the diagnosis and treatment scheme for novel coronavirus pneumonia (trial sixth edition), the conventional method for diagnosing COVID-19 is to detect nucleic acid positivity of the novel coronavirus or virus gene sequencing by using real-time fluorescence RT-PCR (reverse transcription-polymerase chain reaction), but the problem of missed detection of a nucleic acid detection reagent is exposed again when the epidemic situation develops so far, and the positive detection rate is only 30-50%, which 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 novel coronavirus pneumonia focus on IgG and IgM with fewer assays directed against 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 antibody can be generated in about 3-7 days, igG antibody can also rise after 1 week, and when IgG appears, the titer can be continuously increased, and the SARS 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. In the diagnosis and treatment of new coronavirus pneumonia (trial eighth edition), it has been pointed out that: 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: s1 subunit, S2 subunit, RBD, M protein and E protein of SARS-CoV-2Spike protein;
b) An anti-antibody and an antibody labeled with a chemiluminescent label;
the antibody is a monoclonal antibody of a new coronavirus nucleocapsid N protein, but is different from an antigen epitope bound by the antibody in a);
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.
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, 0.1v/v% -2 v/v% surfactant, and pH = 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 0.1w/v% to 0.5w/v%.
Optionally, in the kit as described above, the solid support 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 of the anti-antibody is derived from the same species as the antibody in step a).
A second aspect of the invention relates to a method for the non-diagnostic purpose of detecting 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 anti-antibody and the antibody which are marked with the chemiluminescent markers, and detecting the signal intensity of the chemiluminescent markers.
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. Indeed, 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 (30 min reporting) of the novel coronavirus antigen and total antibody.
The invention relates to a SARS-CoV-2 detection reagent box, 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: s1 subunit, S2 subunit, RBD, M protein and E protein of SARS-CoV-2Spike protein;
b) An anti-antibody and an 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 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.
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 previous infection of the IgG antibody (including a patient in recovery IgM decline), can better prevent omission and shorten the diagnosis window period, and has higher specificity and sensitivity.
In some embodiments, the chemiluminescent label is an acridinium-based chemiluminescent substance.
Further, acridine chemiluminescent substances include acridinium esters and acridine 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, 0.1v/v% -2 v/v% surfactant, and pH = 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 value 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 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 0.1w/v% to 0.5w/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 components thereof contain a magnetic substance. Magnetic fieldThe material may be metal (metal simple substance or alloy), nonmetal, or composite of 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.
In the present invention, the source of the antibody in a) and the anti-antibody in b) can be independently selected from the group consisting of human and all domestic animals (such as domestic animals and pets) and wild animals and 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). Furthermore, antibodies also include functional fragments capable of binding to the desired antigen, such as Fab, F (ab')2Fd, fv, scFv, diabody and antibody minimal recognition unit,and 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, and IgD.
"Secondary antibody" refers to an antibody directed against the Fc region of an antibody.
In some embodiments, in b), the species source of the 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 anti-antibody and the antibody which are marked with the chemiluminescent markers, and detecting the signal intensity of the chemiluminescent markers.
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 purposes 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:
1.1 R1 magnetic bead component:
the magnetic bead coated protein consists of 6 paramagnetic magnetic beads and a magnetic bead diluent, wherein the magnetic bead coated protein is combined as follows:
(1) mouse monoclonal antibody of anti-new coronavirus N protein;
(2) SARS-CoV-2Spike protein S1 subunit;
(3) SARS-CoV-2Spike protein S2 subunit;
(4) SARS-CoV-2Spike protein S1 subunit Receptor Binding Domain (RBD) protein;
(5) SARS-CoV-2M protein
(6) 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 recombinantly expressed.
The concentration of each magnetic bead was 0.1mg/mL, and the magnetic bead dilution was a usual dilution containing 50mM MES, 3% BSA, preservative and other components, and had a pH of 6.0.
1.2 R2 acridine component: consists of an N protein mouse monoclonal antibody and a mouse anti-human IgG/IgA/IgM second antibody which are respectively marked with acridine ester, and an acridine diluent.
The acridinium ester combination comprises:
(1) the anti-new coronavirus N protein mouse monoclonal antibody and the monoclonal antibody in the R1 magnetic bead component can form a paired antibody to detect the N protein antigen.
(2) A mouse anti-human IgG secondary antibody, a mouse anti-human IgA secondary antibody, and a mouse anti-human IgM secondary antibody.
The acridine component concentration was 20ug/mL, and the acridine diluent was a diluent containing 30mM PBS, 3% BSA, mouse IgG, preservative, etc., and had a pH of 7.5.
1.3 R3 antigen releasing component: 1% by weight of Tween-80, 0.3% by 30mM citrate, 1mM acetic acid3Composition, pH 3.5.
Example 2
The kit mainly comprises a calibrator and a detection reagent, wherein the detection reagent consists of 3 parts:
1.1 R1 magnetic bead component:
the magnetic bead coated protein consists of 6 paramagnetic magnetic beads and a magnetic bead diluent, wherein the magnetic bead coated protein is composed of the following components in percentage by weight:
(1) mouse monoclonal antibody of anti-new coronavirus N protein;
(2) SARS-CoV-2Spike protein S1 subunit;
(3) SARS-CoV-2Spike protein S2 subunit;
(4) SARS-CoV-2Spike protein S1 subunit Receptor Binding Domain (RBD) protein;
(5) SARS-CoV-2M protein
(6) 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 immune sites of different N proteins; the S1, S2, RBD, M and E proteins coated on the magnetic beads can be naturally extracted or recombinantly expressed.
The concentration of the magnetic beads was 0.05mg/ml, and the magnetic bead dilution was a commonly used dilution containing 20mM MES, 5% BSA, preservative and other components, and had a pH of 5.5.
1.2 R2 acridine component: consists of an N protein mouse monoclonal antibody and an sheep anti-human polyclonal antibody which are respectively marked with acridine sulfamide and an acridine diluent.
The combination comprises:
(1) the anti-new coronavirus N protein mouse monoclonal antibody and the monoclonal antibody in the R1 magnetic bead component can form a paired antibody to detect the N protein antigen.
(2) Goat anti-human IgG polyclonal antibody, mouse anti-human IgA polyclonal antibody, and mouse anti-human IgM polyclonal antibody.
The acridine component concentration was 100ug/mL, and the acridine diluent was a diluent containing 10mM PBS, 5% BSA, mouse IgG, preservatives, etc., and had a pH of 8.5.
1.3 R3 antigen releasing component: composition ProClin-300% by 10mM citrate, 0.5mM acetic acid, 0.1% Tween-20, 0.5%, pH 3.
Example 3
The kit mainly comprises a calibrator and a detection reagent, wherein the detection reagent consists of 3 parts:
1.1 R1 magnetic bead component:
the magnetic bead coated protein consists of 6 paramagnetic magnetic beads and a magnetic bead diluent, wherein the magnetic bead coated protein is combined as follows:
(1) mouse monoclonal antibody of anti-new coronavirus N protein;
(2) SARS-CoV-2Spike protein S1 subunit;
(3) SARS-CoV-2Spike protein S2 subunit;
(4) SARS-CoV-2Spike protein S1 subunit Receptor Binding Domain (RBD) protein;
(5) SARS-CoV-2M protein
(6) 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 coating the magnetic beads can be naturally extracted or recombinantly expressed.
The concentration of the magnetic beads was 0.5mg/ml, and the magnetic bead dilution was a usual dilution containing 100mM MES, 0.5% BSA, preservative and the like, and had a pH of 6.5.
1.2 R2 acridine component: consists of an N protein mouse monoclonal antibody and a mouse anti-human IgG/IgA/IgM second antibody which are respectively marked with acridine ester, and an acridine diluent.
The combination comprises:
(1) the anti-new coronavirus N protein mouse monoclonal antibody and the monoclonal antibody in the R1 magnetic bead component can form a paired antibody to detect the N protein antigen.
(2) A mouse anti-human IgG secondary antibody, a mouse anti-human IgA secondary antibody and a mouse anti-human IgM secondary antibody.
The acridine component concentration was 200ug/mL, and the acridine dilution was a dilution containing 50mM PBS, 0.1% BSA, mouse IgG, preservatives, etc., and pH 5.5.
1.3 R3 antigen releasing component: 10-50 mM citrate, 2mM malic acid, 2% surfactant Tween-100, 0.1% NaN3Composition, pH 4.5.
Example 4
This embodiment 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.
The first step of sample processing: setting the program, sucking 20ul of sample into the reaction cup, adding 100uL of R3 component, and incubating for 5min at 37 ℃ to dissociate and fully release the possibly combined antigen and antibody.
Second step magnetic bead addition: adding 50uL of the R1 reagent component into a reaction cup, incubating for 10min at 37 ℃, and combining the virus N protein antigen in the sample with the anti-N protein mouse monoclonal antibody coated on the magnetic beads; binding the total antibodies of the new coronavirus to S1, and/or S2, and/or RBD, and/or M, and/or E protein antigens possibly coated on magnetic beads respectively, and then performing a washing procedure to wash away unbound and adsorbed samples;
and step three, adding acridine: 100uL of the above R2 fraction was added to the reaction cuvette and incubated at 37 ℃ for 10min to allow the antigen-antibody reaction to proceed sufficiently, thereby forming 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:
acridinium ester labeled murine anti-human secondary antibody:
s1 protein-anti-S1 antibody-murine anti-human IgG/IgM/IgA secondary antibody; s2 protein-anti-S2 antibody-murine anti-human IgG/IgM/IgA secondary antibody; RBD protein-anti-RBD antibody-murine anti-human IgG/IgM/IgA secondary antibody; m protein-anti-M antibody-murine anti-human IgG/IgM/IgA secondary antibody; e protein-anti-E antibody-murine anti-human IgG/IgM/IgA secondary antibody
Acridinium ester labeled 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; e protein-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 contained only murine mAb against the N protein of neocoronavirus;
the R2 acridine 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 acridine component contains only a mouse anti-human IgG secondary antibody, a mouse anti-human IgA secondary antibody and a mouse anti-human IgM secondary antibody.
The example 1, the comparative example 1 and the comparative example 2 were tested by the method of example 4, the test sample was serum, and the test results are shown in tables 1 and 2:
TABLE 1
Figure BDA0002947315220000131
Figure BDA0002947315220000141
Figure BDA0002947315220000151
Figure BDA0002947315220000161
Figure BDA0002947315220000171
Figure BDA0002947315220000181
Figure BDA0002947315220000191
TABLE 2
Figure BDA0002947315220000192
Figure BDA0002947315220000201
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.
All possible combinations of the technical features of the above embodiments may not be described for the sake of brevity, but should be considered as within the scope of the present disclosure 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 (11)

  1. 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: s1 subunit, S2 subunit, RBD, M protein and E protein of SARS-CoV-2Spike protein;
    b) An anti-antibody and an antibody labeled with a chemiluminescent label;
    the antibody is a monoclonal antibody of the N protein of the neocoronavirus nucleocapsid, but the epitope of the antigen combined with the antibody in a) is different;
    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;
    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, 0.1v/v% -2 v/v% surfactant, pH = 3-4.5; the surfactant comprises Tween;
    the organic acid comprises at least one of acetic acid, oxalic acid, malic acid and salicylic acid.
  2. 2. The kit of claim 1, wherein the chemiluminescent label is an acridinium chemiluminescent substance.
  3. 3. The kit of claim 1, wherein the surfactant comprises at least one of Tween-20, tween-80, and Tween-100.
  4. 4. The kit of claim 1, wherein the antigen-releasing component further comprises a preservative.
  5. 5. The kit of claim 4, wherein the preservative is at a concentration of 0.1w/v% to 0.5w/v%.
  6. 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 wafer.
  7. 7. The kit of claim 6, wherein the solid phase carrier is made of one or more of plastic, cellulose, sephadex, glass, silicone rubber and agarose gel.
  8. 8. The kit of claim 7, wherein the plastic comprises at least one of polystyrene, polyacrylamide, polyethylene, and polypropylene.
  9. 9. The kit of claim 1~8 wherein in a) said antibody is in a plurality, each directed to a different epitope of the N protein.
  10. 10. The kit of claim 1~8 wherein in b) the species source of the anti-antibody is the same as the antibody of step a).
  11. 11. A non-diagnostic method for detecting SARS-CoV-2, which is based on the kit according to any one of claims 1 to 10, 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 anti-antibody and the antibody which are marked with the chemiluminescent markers, and detecting the signal intensity of the chemiluminescent markers.
CN202110197051.8A 2021-02-22 2021-02-22 SARS-CoV-2 detecting reagent kit based on indirect method Active CN112964874B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202110197051.8A CN112964874B (en) 2021-02-22 2021-02-22 SARS-CoV-2 detecting reagent kit based on indirect method

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202110197051.8A CN112964874B (en) 2021-02-22 2021-02-22 SARS-CoV-2 detecting reagent kit based on indirect method

Publications (2)

Publication Number Publication Date
CN112964874A CN112964874A (en) 2021-06-15
CN112964874B true CN112964874B (en) 2022-11-01

Family

ID=76285481

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202110197051.8A Active CN112964874B (en) 2021-02-22 2021-02-22 SARS-CoV-2 detecting reagent kit based on indirect method

Country Status (1)

Country Link
CN (1) CN112964874B (en)

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
IT202100020042A1 (en) * 2021-07-27 2023-01-27 Univ Degli Studi Magna Graecia Di Catanzaro Rapid test device for the detection of SARS-CoV-2 viruses and related antibody production
CN114736274A (en) * 2022-01-25 2022-07-12 伊莱瑞特(武汉)生物技术有限公司 New coronavirus S protein total antibody ELISA detection kit and preparation method thereof

Family Cites Families (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104792987B (en) * 2015-04-29 2017-03-08 中国食品药品检定研究院 For diagnosing method and the kit of hepatitis e virus infection
CN212459720U (en) * 2020-02-17 2021-02-02 浙江大学医学院附属第一医院 Alkaline phosphatase protein chip kit for detecting novel coronavirus antibody
CN111024954A (en) * 2020-03-09 2020-04-17 深圳市易瑞生物技术股份有限公司 Colloidal gold immunochromatography device for combined detection of COVID-19 antigen and antibody and use method thereof
CN111366735B (en) * 2020-03-20 2021-07-13 广州市康润生物科技有限公司 Novel early stage coronavirus screening method
CN112305218A (en) * 2020-08-18 2021-02-02 上海纳米技术及应用国家工程研究中心有限公司 Novel coronavirus antibody colloidal gold immune lateral chromatography detection method and application thereof
CN111999496A (en) * 2020-08-20 2020-11-27 必欧瀚生物技术（合肥）有限公司 SARS-CoV-2 antigen-antibody combined detection kit and its preparation method

Also Published As

Publication number Publication date
CN112964874A (en) 2021-06-15

Similar Documents

Publication Publication Date Title
CN112964873B (en) SARS-CoV-2 detecting reagent kit based on sandwich method
JP5710699B2 (en) Test method and test agent for chronic liver disease by autotaxin measurement
US20060115896A1 (en) Diagnostic test for west nile virus
CN112964874B (en) SARS-CoV-2 detecting reagent kit based on indirect method
CN112679605B (en) Antibodies or antigen binding fragments thereof against novel coronavirus nucleocapsid proteins and uses thereof
CN112964872B (en) SARS-CoV-2 detection reagent kit
CN113447658B (en) Kit for detecting anti-peroxiredoxin-1-IgG antibody
ES2845656T3 (en) Kits and procedures for detecting endothelial cell antibodies in allograft rejection
EP3564673B1 (en) L-fabp immunoassay method and assay reagent used in said method
CN111273017A (en) Fluorescence immunochromatography kit for rapidly detecting novel coronavirus
CN113791212B (en) Novel coronavirus neutralizing antibody magnetic bead fluorescence detection kit and detection method thereof
CN113447649B (en) Kit for detecting anti-adhesion plaque protein-IgG antibody
EP4365195A1 (en) Antibody against nucleocapsid protein of sars-cov-2
US20220034885A1 (en) Compositions and methods for determining coronavirus neutralization titers
EP1423543A2 (en) Detection of bovine viral diarrhea virus in tissue samples
US9658227B2 (en) Recombinant GRA antigens and the use of same for early diagnosis of toxoplasmosis
US8628916B2 (en) Method for isolating hepatitis A virus or spring viremia of carp virus
JPWO2009122592A1 (en) Virus antigen and virus antibody detection kit provided with reagent that can be stored at room temperature, and virus antigen and virus antibody detection method
US7459269B2 (en) Detection of bovine viral diarrhea virus in hair samples
Li et al. Advance of modified ELISA and their application
CN113325173B (en) Novel coronavirus detection kit
CN114527275A (en) Diagnostic kit for hepatic fibrosis
CN114910649A (en) Application of reagent for detecting anti-alpha-enolase-IgG antibody in preparation of kit for detecting vascular endothelial injury
WO2017204349A1 (en) Methods and assays for estimating acid-fast bacteria viability, and kits therefor
CN114924081A (en) Application of neuroblast differentiation related protein-IgG in preparation of vascular endothelial injury kit

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant