Receptor reagent for detecting novel coronavirus and application thereof

Abstract

The application relates to a receptor reagent for detecting a novel coronavirus and application thereof. The receptor comprises receptor microspheres capable of reacting with active oxygen to generate a detectable chemiluminescence signal; the inside of the receptor microspheres is filled with a chemiluminescence agent, and the surface of the receptor microspheres is connected with a novel coronavirus antibody 1. A kit comprising the receptor reagent detects the novel coronavirus by using a double-antibody sandwich method, can effectively shorten the window period, has a fast detection result, can report the result within 30 minutes, has a large detection flux, can reach 200-500 tests / hour, is suitable for large-scale sample detection, and can reduce pollution of high-risk viruses.

Description

[0001] This application is a divisional application of Chinese patent application No. 202110220410.7, filed on February 26, 2021, entitled "A receptor reagent for detecting novel coronavirus and its application therein". Technical Field

[0002] This invention belongs to the field of immunoassay technology, specifically relating to a receptor reagent for detecting the novel coronavirus and its application. Background Technology

[0003] Coronaviruses are a group of enveloped, positive-sense, single-stranded RNA viruses belonging to the order Nidovirales, family Coronaviridae, and subfamily Coronavirinae. There are 26 known species, classified into four genera (α, β, γ, and δ) based on different antigenic cross-reactivity and genetic composition. Only the α- and β- genera contain strains pathogenic to humans. Coronaviruses have long been important animal pathogens, causing respiratory and intestinal diseases in mammals and birds. Six known coronaviruses can cause human disease, including HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, SARS-CoV, and MERS-CoV. The first four are localized epidemics, mainly causing mild, self-limiting illness, while the latter two can cause severe illness. SARS-CoV and MERS-CoV, discovered in 2002 and 2012 respectively, belong to the β-coronavirus family and are listed on the WHO's high-threat list due to their high threat to humans. The high morbidity caused by coronaviruses poses a continuing threat to human health. The novel coronavirus (2019-nCoV) has become the seventh discrete coronavirus species capable of causing human disease, characterized as a β-coronavirus.

[0004] Because 2019-nCoV is distributed in lower respiratory tract secretions, a deep cough is required from the subject to obtain a satisfactory sample when collecting a throat swab specimen, posing a significant risk of exposure to healthcare workers during the collection process. Furthermore, after obtaining the specimen, healthcare workers need to immediately send it for nucleic acid isolation and testing. Nucleic acid testing uses polymerase chain reaction (PCR) technology, which is far more complex than immunological detection methods, making it difficult to perform in general laboratories.

[0005] The detection window for 2019-nCoV antibodies is relatively long (from viral infection to antibody production in the human body), which can easily lead to false negatives and delay diagnosis. Therefore, clinical laboratories urgently need an in vitro diagnostic kit for serological detection of 2019-nCoV. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention provides a receptor reagent for detecting the novel coronavirus and its application. When using a kit containing the receptor reagent to detect the novel coronavirus, the window period can be effectively shortened, and the kit is simple to operate, precise, specific, fast, and has a high throughput.

[0007] Therefore, the first aspect of the present invention provides a receptor reagent for detecting the novel coronavirus, comprising receptor microspheres capable of reacting with reactive oxygen species to generate a detectable chemiluminescent signal; the receptor microspheres are filled with a chemiluminescent agent, and the surface of the receptor microspheres is attached with a novel coronavirus antibody 1.

[0008] In some embodiments of the present invention, the surface of the receptor microspheres is not coated with polysaccharides.

[0009] In other embodiments of the present invention, the surface of the receptor microspheres is coated with polysaccharides, the novel coronavirus antibody 1 is linked to the polysaccharides, and the total sugar content per milligram of the receptor microspheres is not less than 25 micrograms; preferably, the total sugar content per milligram of the receptor microspheres is not less than 30 micrograms; more preferably, the total sugar content per milligram of the receptor microspheres is not less than 35.1 micrograms; and even more preferably, the total sugar content per milligram of the receptor microspheres is not less than 45.8 micrograms.

[0010] In some embodiments of the present invention, the total sugar content is detected by the anthrone method;

[0011] Preferably, the sugar is selected from carbohydrates containing three or more unmodified or modified monosaccharide units, and more preferably from dextran, starch, glycogen, inulin, fructan, mannan, agarose, galactan, carboxyglucan and aminoglucan; more preferably from dextran, starch, glycogen and polyribose.

[0012] In other embodiments of the invention, the zeta potential of the receptor microspheres in the receptor reagent is between -5 mV and -45 mV; preferably between -5 mV and -43.4 mV; more preferably between -25.2 mV and -30.6 mV.

[0013] A second aspect of the present invention provides a kit for detecting the novel coronavirus, comprising:

[0014] A receptor reagent, said receptor reagent as described in the first aspect of the present invention;

[0015] A capture reagent, wherein the capture reagent comprises one of a specific paired member linked to SARS-CoV-2 antibody 2;

[0016] The novel coronavirus antibody 2 and the novel coronavirus antibody 1 linked on the receptor microspheres in the receptor reagent can simultaneously bind specifically to the novel coronavirus to be tested.

[0017] In some embodiments of the present invention, the antigenic epitopes targeted by the novel coronavirus antibody 1 and the novel coronavirus antibody 2 are N antigens, S antigens or N+S fusion antigens; preferably N antigens.

[0018] In other embodiments of the present invention, the N antigen, S antigen, and N+S fusion antigen are full-length fragments or partial fragments of the corresponding antigens.

[0019] In some embodiments of the present invention, the S antigen includes S1 protein, S1-RBD protein and S2 protein.

[0020] In other embodiments of the present invention, the specific pairing member is selected from a pair of substances consisting of an antibody, antibody fragment, ligand, oligonucleotide, oligonucleotide-binding protein, lectin, hapten, antigen, immunoglobulin-binding protein, avidin, or biotin; preferably, the specific pairing member is biotin-avidin.

[0021] In some embodiments of the present invention, the biotin is selected from biotin with different activating groups, preferably from biotin with NHS activating groups that can react with amino groups, and more preferably from NHS-LC-LC-biotin.

[0022] A third aspect of the present invention provides a method for detecting the novel coronavirus in a test sample using a kit as described in the second aspect of the present invention, comprising: firstly preparing a complex comprising receptor microspheres-novel coronavirus antibody 1-novel coronavirus-novel coronavirus antibody 2-donor microspheres; then treating the complex with energy or an active compound to excite the donor microspheres to generate reactive oxygen species, and the receptor microspheres reacting with the received reactive oxygen species to generate a detectable chemiluminescent signal; and finally analyzing the chemiluminescent signal to determine whether the novel coronavirus is present in the test sample and the content of the novel coronavirus.

[0023] In some embodiments of the present invention, when the value of the chemiluminescence signal is greater than or equal to the chemiluminescence signal value of the qualitative reference sample, the sample to be tested is a positive sample; when the value of the chemiluminescence signal is less than the chemiluminescence signal value of the qualitative reference sample, the sample to be tested is a negative sample.

[0024] In some embodiments of the present invention, the sample to be tested is selected from human serum, nasopharyngeal swabs, or pharyngeal swabs.

[0025] The beneficial effects of this invention are as follows: For the detection of the novel coronavirus, nucleic acid testing is prone to false negatives due to sampling methods and other factors; similarly, the long window period for detecting 2019-nCoV antibodies also easily leads to false negatives and delays in diagnosis. The kit containing the aforementioned receptor reagent utilizes a double-antibody sandwich method to detect the novel coronavirus, effectively shortening the window period and providing rapid results (reported within 30 minutes); it also boasts a high throughput of 200-500 tests / hour, suitable for large-scale sample testing. The kit can be used with the LiCA500 system for novel coronavirus detection, requiring no cleaning, using disposable tip heads, eliminating the need for wastewater treatment, and reducing contamination by high-risk viruses. Detailed Implementation

[0026] To facilitate understanding of the present invention, it will be described in detail below. However, before describing the present invention in detail, it should be understood that the present invention is not limited to the specific embodiments described. It should also be understood that the terminology used herein is for describing specific embodiments only and is not intended to be restrictive.

[0027] Where numerical ranges are provided, it should be understood that every intermediate value between the upper and lower limits of the range and any other specified or intermediate value within the specified range is covered by this invention. The upper and lower limits of these smaller ranges may be independently included in the smaller range and are also covered by this invention, subject to any explicitly excluded limits within the specified range. Where a specified range includes one or two limits, the range excluding any or both of those included limits is also included by this invention.

[0028] Unless otherwise defined, all terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. While any methods and materials similar to or equivalent to those described herein may also be used in the practice or testing of this invention, preferred methods and materials are now described.

[0029] II. Specific Implementation Plan

[0030] The present invention will now be described in detail.

[0031] This invention includes a kit containing the aforementioned receptor reagent, which uses a double-antibody sandwich structure followed by homogeneous chemiluminescence for the detection of the novel coronavirus. Photo-induced chemiluminescence (PET) is a homogeneous chemiluminescence process, a continuous chemical and luminescent reaction induced by photoexcitation. Donor microspheres and receptor microspheres can approach each other via antigen-antibody binding, creating conditions for energy transfer and generating a light signal under laser irradiation. Conversely, if antigen-antibody binding does not occur, there is a certain distance between the donor and receptor microspheres, and the receptor microspheres do not meet the conditions for receiving energy-induced luminescence, thus no light signal is generated. Therefore, without separation and washing, the light signal can be directly detected, and the light signal intensity is positively correlated with antigen-antibody binding. PET analysis is a homogeneous luminescent immunoassay, a trace substance quantitative analysis technique based on the principles of PET and antigen-antibody binding. It involves no separation or washing process, is simple and rapid, and is characterized by a unique "double-sphere" and "double-label" approach. The solid-phase microspheres have good suspension properties, which facilitates uniform diffusion and the collision and binding of antigen or antibody molecules on the microsphere surface with the antibody or antigen to be detected.

[0032] In the kit of this invention, firstly, a pair of specific antibodies are selected to coat receptor microspheres (FG-Ag) and labeled biotin (Bio-Ag), respectively, serving as receptor reagent (R1) and capture reagent (R2); donor microspheres are coated with avidin (e.g., neutral avidin) as a universal solution (donor reagent). Next, R1 and R2, along with the sample to be tested and quality control, are added to the microwells. After starting the first-stage incubation, a double-antibody sandwich complex forms on the surface of the receptor microspheres. Without washing, the universal solution is added directly, and the second-stage incubation is started, causing biotin to bind to avidin, bringing the two microspheres closer together. At this point, a laser beam excitation induces a photo-induced chemiluminescence reaction to generate a light signal.

[0033] Therefore, the receptor reagent for detecting the novel coronavirus according to the first aspect of the present invention comprises receptor microspheres capable of reacting with reactive oxygen species to generate a detectable chemiluminescent signal; the interior of the receptor microspheres is filled with a chemiluminescent agent, and the surface of the receptor microspheres is connected with a novel coronavirus antibody 1.

[0034] In some embodiments of the present invention, the surface of the receptor microspheres is not coated with polysaccharides.

[0035] In other embodiments of the present invention, the surface of the receptor microspheres is coated with polysaccharides, the novel coronavirus antibody 1 is linked to the polysaccharides, and the total sugar content per milligram of the receptor microspheres is not less than 25 micrograms; preferably, the total sugar content per milligram of the receptor microspheres is not less than 30 micrograms; more preferably, the total sugar content per milligram of the receptor microspheres is not less than 35.1 micrograms; and even more preferably, the total sugar content per milligram of the receptor microspheres is not less than 45.8 micrograms.

[0036] In some specific embodiments of the present invention, the total sugar content per milligram of the receptor microspheres can be 25 micrograms, 30 micrograms, 35 micrograms, 35.1 micrograms, 40 micrograms, 45.8 micrograms, 50 micrograms, 80 micrograms, 100 micrograms, 150 micrograms, or 200 micrograms.

[0037] In some embodiments of the present invention, the total sugar content is detected by the anthrone method;

[0038] Preferably, the sugar is selected from carbohydrates containing three or more unmodified or modified monosaccharide units, and more preferably from dextran, starch, glycogen, inulin, fructan, mannan, agarose, galactan, carboxyglucan and aminoglucan; more preferably from dextran, starch, glycogen and polyribose.

[0039] In other embodiments of the present invention, the zeta potential of the receptor microspheres in the receptor reagent is between -5 mV and -45 mV; preferably between -5 mV and -43.4 mV; more preferably between -25.2 mV and -30.6 mV. In some specific embodiments of the present invention, the zeta potential of the receptor microspheres in the receptor reagent can be -5 mV, -10 mV, -15 mV, -20 mV, -25.2 mV, -30 mV, -30.6 mV, -40 mV, -43.4 mV, or -45 mV. The inventors of this application have discovered that precisely controlling the zeta potential of the receptor microspheres in the receptor reagent within a suitable range enables the kit to have the advantages of strong anti-interference ability and excellent testing performance.

[0040] The Zeta potential value described in this invention refers to the potential value of the acceptor microspheres in a dispersion system with a pH of 6-9. The Zeta potential of a microsphere refers to the potential at the shear plane; that is, the potential difference between the continuous phase and the fluid-stabilized layer attached to the microsphere. Because the dispersed particles carry a charge on their surface, they attract surrounding anti-charge ions. These anti-charge ions are distributed in a diffuse state at the interface between the two phases, forming a diffuse electric double layer. According to the Stern double layer theory, the double layer can be divided into two parts: the Stern layer and the diffuse layer. The Stern layer is defined as a planar layer composed of a layer of ionic (IHP or OHP) charge centers adsorbed on the electrode surface. The potential of this planar layer relative to a point in the fluid far from the interface is called the Stern potential. The interface between the stationary layer (including the stern layer and the portion of the diffusion layer within the slipping plane) and the dispersion medium within the diffusion layer, where relative movement occurs, is called the slipping plane. The potential at this point relative to a point in the fluid far from the interface is called the zeta potential or electrodynamic potential (ζ-potential). In other words, the zeta potential is the potential difference between the continuous phase and the fluid stationary layer attached to the dispersed particles. It can be directly measured through electrodynamic phenomena. Currently, the main methods for measuring the zeta potential include electrophoresis, electroosmosis, flow potential analysis, and ultrasound, with electrophoresis being the most widely used.

[0041] The term "receptor microsphere" as used in this invention refers to a microsphere containing a compound capable of reacting with reactive oxygen species to generate a detectable signal. The donor microsphere is activated by energy or an active compound, releasing high-energy reactive oxygen species. These high-energy reactive oxygen species are captured by nearby receptor particles, thereby transferring energy to activate the receptor microsphere. The receptor microsphere may have different functional groups, such as aldehyde, carboxyl, and amino groups. Aldehyde-based receptor microspheres and carboxyl-based receptor microspheres that can couple with free amino groups of proteins are preferred, and aldehyde-based receptor microspheres are more preferred. The receptor microsphere itself may be sugar-free or sugar-coated. Receptor microspheres coated with polysaccharides are preferred, and receptor microspheres coated with at least two consecutive polysaccharide coatings are more preferred.

[0042] The term "reactive oxygen species" as used in this invention refers to a general term for substances composed of oxygen in the body or natural environment that contain oxygen and are reactive in nature. It is mainly an excited-state oxygen molecule, including the one-electron reduction product of oxygen, superoxide anion (O2·-), the two-electron reduction product, hydrogen peroxide (H2O2), the three-electron reduction product, hydroxyl radical (·OH), as well as nitric oxide and singlet oxygen (1O2), etc.

[0043] In some specific embodiments of the present invention, the active oxygen is singlet oxygen.

[0044] A second aspect of the present invention relates to a kit for detecting the novel coronavirus, comprising:

[0045] A receptor reagent, said receptor reagent as described in the first aspect of the present invention;

[0046] A capture reagent, wherein the capture reagent comprises one of a specific paired member linked to SARS-CoV-2 antibody 2;

[0047] The novel coronavirus antibody 2 and the novel coronavirus antibody 1 linked on the receptor microspheres in the receptor reagent can simultaneously bind specifically to the novel coronavirus to be tested.

[0048] In some embodiments of the present invention, the antigenic epitopes targeted by the novel coronavirus antibody 1 and the novel coronavirus antibody 2 are N antigens, S antigens or N+S fusion antigens; preferably N antigens.

[0049] In some embodiments of the present invention, the N antigen, S antigen, and N+S fusion antigen are full-length fragments or partial fragments of the corresponding antigens.

[0050] In some embodiments of the present invention, the S antigen includes S1 protein, S1-RBD protein and S2 protein.

[0051] In this invention, an antibody against the N antigen (e.g., a monoclonal antibody) refers to an antibody (e.g., a monoclonal antibody) that can specifically bind to the N antigen.

[0052] The term "N antigen" used in this invention refers to the nucleocapsid protein of the novel coronavirus, which is the most abundant protein in coronaviruses. During virion assembly, the N protein binds to viral RNA, leading to the formation of a helical nucleocapsid. The nucleocapsid protein is a highly immunogenic phosphoprotein involved in viral genome replication and the regulation of cellular signaling pathways. Due to its sequence conservation (94% homology with SARS) and strong immunogenicity, the N protein is often used as a diagnostic tool for coronaviruses. The term "S antigen" used in this invention refers to the spike protein of the novel coronavirus, which is the most important surface membrane protein of coronaviruses and contains two subunits, S1 and S2. S1 mainly contains the receptor-binding domain (RBD), responsible for recognizing cellular receptors. S2 contains the basic elements required for membrane fusion. The S protein has approximately 75% homology with SARS (68% homology for S1 and 94% homology for S2). Because the N antigen has strong immunogenicity and is produced in large quantities, its antibody detection has high sensitivity; however, due to its relatively conserved sequence, its specificity is relatively poor. The S antigen (especially the S1 protein), due to its low sequence homology, has better specificity.

[0053] In some embodiments of the present invention, the novel coronavirus antibody 1 and novel coronavirus antibody 2 are each independently selected from at least one of monoclonal antibodies and polyclonal antibodies; preferably, monoclonal antibodies. In the present invention, theoretically, monoclonal antibodies and polyclonal antibodies can be co-coated with receptor particles, or receptor particles coated with monoclonal antibodies and polyclonal antibodies can be mixed as receptor reagents; simultaneously, monoclonal antibodies and polyclonal antibodies can be simultaneously labeled with biotin as capture reagents.

[0054] In some embodiments of the present invention, the kit further includes a donor reagent comprising donor microspheres capable of generating reactive oxygen species in an excited state; preferably, the donor reagent binds to another member of a specific pairing member.

[0055] The term "donor microsphere" as used in this invention refers to a microsphere containing a sensitizer that, upon activation by energy or an active compound, can generate an active intermediate, such as reactive oxygen species, that reacts with the acceptor microsphere. Donor microspheres can be photoactivated (e.g., dyes and aromatic compounds) or chemically activated (e.g., enzymes, metal salts, etc.). In some specific embodiments of this invention, the donor microsphere is a polymeric microsphere filled with a photosensitizer. The photosensitizer can be a photosensitizer known in the art, preferably a relatively photostable compound that does not react effectively with singlet oxygen. Non-limiting examples include, for example, compounds such as methylene blue, rose red, porphyrin, phthalocyanine, and chlorophyll disclosed in US Patent 5709994 (which is incorporated herein by reference in its entirety), and derivatives of these compounds having 1-50 substituents, which are used to make these compounds more lipophilic or more hydrophilic, and / or as linking groups to specifically binding pairing members. Other examples of photosensitizers known to those skilled in the art may also be used in this invention, such as those described in U.S. Patent US6406913, which is incorporated herein by reference.

[0056] The term "specific pairing members" as used in this invention refers to a pair of substances capable of specifically binding to each other. The term "specific binding" as used in this invention refers to a mutually discriminative and selective binding reaction between two substances; from a stereostructural perspective, it refers to the conformational correspondence between the corresponding reactants.

[0057] In some specific embodiments of the present invention, the specific pairing member is selected from a pair of substances composed of antibodies, antibody fragments, ligands, oligonucleotides, oligonucleotide-binding proteins, lectins, haptens, antigens, immunoglobulin-binding proteins, avidin, avidin, or biotin; preferably, the specific pairing member is biotin-avidin. In the present invention, the avidin can be streptavidin or neutral avidin.

[0058] In some embodiments of the present invention, the biotin is selected from biotin with different activating groups, preferably from biotin with NHS activating groups that can react with amino groups, and more preferably from NHS-LC-LC-biotin.

[0059] The term "biotin" as used in this invention is widely found in animal and plant tissues. Its molecule has two ring structures: an imidazoline ring and a thiophene ring. The imidazoline ring is the primary site for binding to streptavidin. Activated biotin can be coupled to almost all known biomolecules, including proteins, nucleic acids, polysaccharides, and lipids, mediated by protein cross-linking agents.

[0060] In addition to the reagents described above, the kit of the present invention may optionally include a negative control: hormone-free human serum, a positive control: hormone-free human serum containing 2019-nCoV-Ag, and a reference sample (quality control): hormone-free human serum containing 2019-nCoV-Ag.

[0061] A third aspect of the present invention relates to a method for detecting novel coronavirus in a test sample using a kit as described in the second aspect of the present invention, comprising: firstly preparing a complex comprising receptor microspheres-novel coronavirus antibody 1-novel coronavirus-novel coronavirus antibody 2-donor microspheres; then treating the complex with energy or an active compound to excite the donor microspheres to generate reactive oxygen species, and the receptor microspheres reacting with the received reactive oxygen species to generate a detectable chemiluminescent signal; and finally analyzing the chemiluminescent signal to determine whether the test sample contains novel coronavirus and the content of novel coronavirus.

[0062] In some embodiments of the present invention, when the value of the chemiluminescence signal is greater than or equal to the chemiluminescence signal value of the qualitative reference sample, the sample to be tested is a positive sample; when the value of the chemiluminescence signal is less than the chemiluminescence signal value of the qualitative reference sample, the sample to be tested is a negative sample. In the present invention, the value of the chemiluminescence signal generated when detecting the sample to be tested can be represented as S, and the chemiluminescence signal value of the qualitative reference sample can be represented as CO. Therefore, when S / CO ≥ 1, it is determined to be reactive (i.e., the sample to be tested is a positive sample), and when S / CO < 1, it is determined to be non-reactive (i.e., the sample to be tested is a negative sample).

[0063] In this invention, the term "qualitative reference sample" refers to a critically positive sample, whose emission signal value is used to determine whether a test sample is positive. When the emission signal value of the test sample is not lower than that of the qualitative reference sample, the test sample is a positive sample. Conversely, when the emission signal value of the test sample is lower than that of the qualitative reference sample, the test sample is a negative sample.

[0064] In this invention, the kit used to prepare the complex comprising receptor microspheres-novel coronavirus antibody 1-novel coronavirus-novel coronavirus antibody 2-donor microspheres is the kit described in the second aspect of this invention.

[0065] In some embodiments of the present invention, the method specifically includes the following steps:

[0066] S1, the test sample, receptor reagent and capture reagent are mixed to obtain the first mixture;

[0067] S2, the donor reagent is mixed with the first mixture to obtain the second mixture;

[0068] S3, treat the second mixture with energy or an active compound to excite the donor microspheres to produce reactive oxygen species, and the acceptor reacts with the reactive oxygen species to generate a detectable chemiluminescent signal;

[0069] S4, Analyze the chemiluminescence signal to determine whether the novel coronavirus exists in the sample to be tested and the content of the novel coronavirus.

[0070] The receptor reagent described above is the receptor reagent as described in the first aspect of the present invention, or the receptor reagent in the kit as described in the second aspect of the present invention; the capture reagent and donor reagent described above are the capture reagent and donor reagent in the kit as described in the second aspect of the present invention.

[0071] In the method of this invention, the reagents can be incubated as needed after mixing. Specifically, the incubation temperature can be 35-40℃, and the incubation time can be 10-20 min; preferably, the incubation temperature can be selected from 36℃, 37℃, 38℃, 39℃, or 40℃; the incubation time can be selected from 10 min, 12 min, 15 min, 18 min, or 20 min; more preferably, the incubation temperature is 37℃, the incubation time in step S1 is 15 min, and the incubation time in step S2 is 10 min.

[0072] In this invention, the sample volume, receptor reagent, and capture reagent are each independently added in an amount of 15-35 μL; preferably, the sample volume, receptor reagent, and capture reagent are each independently added in amounts of 15 μL, 18 μL, 20 μL, 22 μL, 25 μL, 28 μL, 30 μL, or 35 μL. More preferably, the sample volume, receptor reagent, and capture reagent are all added in an amount of 25 μL.

[0073] In some embodiments of the present invention, the sample to be tested is selected from human serum, nasopharyngeal swabs, or pharyngeal swabs.

[0074] Example

[0075] To make the present invention easier to understand, the present invention will be further described in detail below with reference to embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of application of the present invention. Unless otherwise specified, the raw materials or components used in the present invention can be obtained commercially or by conventional methods.

[0076] The raw materials used in the following examples are as follows:

[0077] Raw material group 1 (for detecting S protein):

[0078] Ab1: Genscript: Antibody to the S1 subunit of the SARS-CoV-2 spike protein;

[0079] Ab2: Beijing Yiqiao Shenzhou: 2019-nCoV spike antibody, rabbit monoclonal antibody;

[0080] Ag: Beijing Yiqiao Shenzhou: SARS-CoV-2 (2019-nCoV) spike protein (S1 subunit, His tag).

[0081] Raw material group 2 (for detecting N protein):

[0082] Ab1: Beijing Yiqiao Shenzhou: SARS-CoV-2 (2019-nCoV) nucleocapsid protein / N antibody;

[0083] Ab2: Beijing Yiqiao Shenzhou: 2019-nCoV nucleocapsid protein / N antibody, rabbit monoclonal antibody;

[0084] Ag: Genscript: 2019-nCoV nucleocapsid protein

[0085] Example 1: Preparation of receptor microspheres

[0086] 1.1 Synthesis of polystyrene latex microspheres

[0087] Prepare a 100mL three-necked flask, add 40mmol styrene, 3mmol methacrylic acid, and 10mL water, stir for 10min, and then purge with N2 for 30min.

[0088] 1) Weigh 0.11g of ammonium persulfate and 0.2g of sodium chloride, dissolve them in 40mL of water to prepare an aqueous solution. Add this aqueous solution to the reaction system in step 1), and continue to purge with N2 for 30min;

[0089] 2) Heat the reaction system to 70℃ and react for 15 hours;

[0090] 3) Cool the emulsion after the reaction is complete to room temperature and filter it with a suitable filter cloth. Wash the resulting emulsion by centrifugation and sedimentation with deionized water multiple times until the conductivity of the supernatant after centrifugation is close to that of deionized water. Then dilute it with water and store it in emulsion form.

[0091] 1.2. Filling process of chemiluminescent agent

[0092] 1) Prepare a 25 mL round-bottom flask, add 0.1 g of dimethylthiophene derivative and 0.1 g of europium(III) complex (MTTA-EU) 3+ Add 10 mL of 95% ethanol, stir magnetically, and heat in a water bath to 70°C to obtain the complex solution;

[0093] 2) Prepare a 100mL three-necked flask, add 10mL of 95% ethanol, 10mL of water and 10mL of polystyrene latex microspheres obtained in step 1.1 with a concentration of 10%, stir magnetically, and heat in a water bath to 70℃;

[0094] 3) Slowly add the complex solution from step 1) to the three-necked flask from step 2), react at 70°C for 2 hours, then stop stirring and allow to cool naturally.

[0095] 4) Centrifuge the above emulsion for 1 hour at 30,000 g. After centrifugation, discard the supernatant to obtain polystyrene microspheres filled with chemiluminescent agent, i.e., receptor microspheres. Adjust the volume with 20 mM HEPES buffer to a final concentration of 20 mg / mL.

[0096] 1.3 Surface coating of receptor microspheres with dextran

[0097] 1) Take 50 mg of aminoglucan solid into a 20 mL round bottom flask, add 5 mL of 50 mM / pH=10 carbonate buffer, and stir to dissolve at 30 °C in the dark;

[0098] 2) Take 100 mg of the prepared polystyrene microspheres filled with the luminescent composition and add them to the aminodextran solution and stir for 2 h;

[0099] 3) Dissolve 10 mg of sodium borohydride in 0.5 mL of 50 mM / pH=10 carbonate buffer solution and add it dropwise to the above reaction solution. React overnight at 30°C in the dark.

[0100] 4) Centrifuge the reaction mixture at 30,000 g, discard the supernatant, and add 50 mM / pH=10 carbonate buffer for ultrasonic dispersion. Repeat centrifugation and washing three times, then bring the volume up to 20 mg / mL with 50 mM / pH=10 carbonate buffer.

[0101] 5) Take 100 mg of aldehyde dextran solid into a 20 mL round-bottom flask, add 5 mL of 50 mM / pH=10 carbonate buffer, and stir to dissolve at 30 °C in the dark.

[0102] 6) Add the above microspheres to the aldehyde dextran solution and stir for 2 hours;

[0103] 7) Dissolve 15 mg of sodium borohydride in 0.5 mL of 50 mM / pH=10 carbonate buffer solution and add it dropwise to the above reaction solution. React overnight at 30°C in the dark.

[0104] 8) After centrifuging the reaction mixture at 30,000 g, discard the supernatant and add 50 mM / pH=10 carbonate buffer for ultrasonic dispersion. Repeat centrifugation and washing three times, then bring the volume up to 20 mg / mL with 50 mM / pH=10 carbonate buffer.

[0105] 9) The average particle size of the microspheres at this time, as measured by a nanoparticle size analyzer, is 241.6 nm, with a coefficient of variation (CV) of 14%.

[0106] Example 2: Preparation of the reagent kit

[0107] 2.1 Preparation of receptor reagents

[0108] 1) Antibody treatment: Dialyze the SARS-CoV-2 spike S1 subunit antibody (or SARS-CoV-2 (2019-nCoV) nucleocapsid protein / N antibody) and replace it with coating buffer, and measure the protein concentration;

[0109] 2) Receptor microsphere treatment: The receptor microspheres prepared in Example 1 were replaced with coating buffer through processes such as centrifugation and sonication;

[0110] 3) Conjugation: The treated receptor microspheres and the treated 2019-nCoV antibody 1 are mixed and reacted. After reduction, blocking and other processes, receptor microspheres-2019-nCoV antibody 1 are obtained. The mixture is then brought to a final volume using a preservation solution and stored to obtain the receptor reagent.

[0111] 2.2 Preparation of the capture reagent

[0112] 1) Antibody treatment: Dialyze the 2019-nCoV spike antibody (or 2019-nCoV nucleocapsid protein / N antibody), replace it with labeling buffer, and measure the protein concentration;

[0113] 2) Labeling reaction: The treated 2019-nCoV antibody 2 was mixed with activated biotin and reacted for labeling;

[0114] 3) Dialysis: The labeled biotin-2019-nCoV antibody 2 was dialyzed to remove unlabeled free biotin;

[0115] 4) Preservation: The protein concentration of the biotin-2019-nCoV antibody 2 after dialysis was measured, and glycerol was added and then preserved to obtain the capture reagent.

[0116] Example 3: Determination of sugar content using the anthrone method

[0117] 3.1 Microsphere Sample Pretreatment:

[0118] The receptor reagent containing 1 mg of receptor microspheres from Example 2 was centrifuged at 20000G for 40 min. After discarding the supernatant, it was ultrasonically dispersed with purified water. The centrifugation and dispersion were repeated three times. Each sample was then diluted with purified water to 1 mg / mL as the test sample.

[0119] 3.2 Preparation of glucose standard solution:

[0120] The 1 mg / mL glucose stock solution was prepared into standard solution curves with concentrations of 0 mg / mL, 0.025 mg / mL, 0.05 mg / mL, 0.075 mg / mL, 0.10 mg / mL, and 0.15 mg / mL using purified water.

[0121] 3.3 Preparation of anthrone solution: Prepare a solution of 2 mg / mL using 80% sulfuric acid solution.

[0122] 3.4 Add 0.1 mL of glucose standard solution of various concentrations and the sample to be tested to centrifuge tubes respectively, and add 1 mL of anthrone test solution to each tube.

[0123] 3.5 Incubate at 85℃ for 30 minutes.

[0124] 3.6 Centrifuge the sample reaction tube at 15000G for 40 min. Use a pipette tip to aspirate the clear liquid from the bottom of the tube for absorbance measurement, avoiding aspirating the suspended matter at the top.

[0125] 3.7 Return to room temperature and measure absorbance at 620 nm.

[0126] 3.8 Using the concentration of the standard as X and the absorbance as Y, a linear regression was performed to obtain the absorbance values ​​of the standard curve shown in Table 1. Based on this, the sugar content of each milligram of receptor microspheres was determined to be 50 μg.

[0127] Table 1

[0128] Serial Number Concentration mg / mL Absorbance A Absorbance B average absorbance 1 0.15 0.415 0.411 0.4130 2 0.1 0.293 0.302 0.2975 3 0.075 0.214 0.227 0.2205 4 0.05 0.146 0.153 0.1495 5 0.025 0.101 0.098 0.0995 6 0 0.032 0.031 0.0315

[0129] Example 4: Method of using the reagent kit of the present invention

[0130] 1) The receptor reagent and capture reagent prepared in Example 2 were diluted with buffer to 50 μg / mL and 0.8 μg / mL, respectively, to prepare reagent R1 and reagent R2;

[0131] 2) Add 25 μL of the sample to be tested, 25 μL of reagent R1 and 25 μL of reagent R1 solution to the microplate respectively, and incubate at 37℃ for 15 min;

[0132] 3) Add 175 μL of universal solution for photo-induced chemiluminescence analysis system (donor reagent), incubate at 37°C for 10 min, and then use... HT takes the reading.

[0133] Example 5: Effect of different sugar contents of receptor microspheres on the level of on-machine detection signal.

[0134] Following the method described in Example 1, a series of kits containing receptor microspheres with different sugar contents were prepared (as shown in the table below). Then, the method shown in Example 4 was used to detect the performance of each kit on the same batch of samples produced by Boyang Biotechnology (Shanghai) Co., Ltd. The signal levels were compared on the HT photochemiluminescence instrument, and the results are shown in Table 2.

[0135] Table 2

[0136]

[0137] Test results showed that when the sugar content in each milligram of the receptor particle was ≥25 μg, the signal level detected by the instrument was relatively high.

[0138] Example 6: Test on the influence of the ZETA potential of receptor microspheres in the receptor reagent on the level of on-machine detection signal

[0139] Using a NICOMP 380Z3000 instrument, after calibration with standards, the zeta potential of the receptor reagents was measured to prepare a series of receptor reagents with different zeta potentials. Then, the method shown in Example 4 was used to detect the different reagents for the same batch of samples produced by Boyang Biotechnology (Shanghai) Co., Ltd. The signal levels were compared on the HT photochemiluminescence instrument, and the results are shown in Table 3.

[0140] Table 3

[0141]

[0142] The results showed that the average level of the detection signal of the receptor microspheres was relatively high when the ZETA potential in the receptor reagent was between -5mV and -45mV, and even higher when the ZETA potential was between -25mV and -31mV.

[0143] Example 7: Sample detection performance test of the kit

[0144] The recombinant antigen of the novel coronavirus was serially diluted with negative serum and tested using the above-mentioned kit. The test results are shown in Table 4.

[0145] Table 4: Detection of Recombinant Antigens

[0146]

[0147] The results showed that the recombinant antigen concentration of 0.8 ng / mL was still detectable as reactive, indicating that the kit has high detection sensitivity.

[0148] The above kit was used to test 94 normal negative serum samples, and the results are shown in Table 5.

[0149] Table 5: Results of Normal Negative Serum Detection

[0150]

[0151]

[0152] Table 5 shows that no false positive results were found in the 94 normal negative random serum samples, with a specificity of 100% (95% CI: 95.1%-100%), indicating that the kit has good specificity.

[0153] The above-mentioned kit was used to test the low and high value quality controls to test the accuracy of the kit. The test results are shown in Table 6.

[0154] Table 6: Precision Test Results

[0155]

[0156]

[0157] As shown in Table 6, the CVs for low and high value quality control were 1.10% and 1.56%, respectively, indicating that the kit described in this invention has good precision.

[0158] It should be noted that the embodiments described above are only for explaining the present invention and do not constitute any limitation on the present invention. The present invention has been described with reference to typical embodiments, but it should be understood that the words used therein are descriptive and explanatory terms, not limiting terms. Modifications can be made to the present invention within the scope of the claims, and revisions can be made to the present invention without departing from the scope and spirit of the present invention. Although the present invention described herein relates to specific methods, materials, and embodiments, it does not mean that the present invention is limited to the specific examples disclosed herein; on the contrary, the present invention can be extended to all other methods and applications with the same function.

Claims

1. A receptor reagent for detecting the novel coronavirus, comprising receptor microspheres capable of reacting with reactive oxygen species to generate a detectable chemiluminescent signal; the receptor microspheres are filled with a chemiluminescent agent, and the surface of the receptor microspheres is attached with a novel coronavirus antibody 1; The surface of the receptor microspheres is coated with dextran, the novel coronavirus antibody 1 is linked to the dextran, and the total sugar content per milligram of the receptor microspheres is not less than 25 micrograms; The zeta potential of the receptor microspheres in the receptor reagent is between -25.2 mV and -43.4 mV.

2. The receptor reagent according to claim 1, characterized in that, The total sugar content of each milligram of the receptor microspheres is not less than 30 micrograms.

3. The receptor reagent according to claim 2, characterized in that, The total sugar content of each milligram of the receptor microspheres is not less than 35.1 micrograms.

4. The receptor reagent according to claim 2, characterized in that, The total sugar content of each milligram of the receptor microspheres is not less than 45.8 micrograms.

5. The receptor reagent according to claim 2, characterized in that, The total sugar content was determined by the anthrone method.

6. The receptor reagent according to claim 1, characterized in that, The zeta potential of the receptor microspheres in the receptor reagent is between -25.2 mV and -30.6 mV.

7. A kit for detecting the novel coronavirus, comprising: The receptor reagent, as described in any one of claims 1-6; A capture reagent, wherein the capture reagent comprises one of a specific paired member linked to SARS-CoV-2 antibody 2; A donor reagent, comprising donor microspheres capable of generating reactive oxygen species in an excited state, wherein the donor reagent binds to another member of a specific pairing group. The novel coronavirus antibody 2 and the novel coronavirus antibody 1 linked on the receptor microspheres in the receptor reagent can simultaneously bind specifically to the novel coronavirus to be tested.

8. The reagent kit according to claim 7, characterized in that, The antigenic epitopes targeted by the novel coronavirus antibody 1 and novel coronavirus antibody 2 are N antigen, S antigen or N+S fusion antigen.

9. The reagent kit according to claim 7, characterized in that, The antigenic epitopes targeted by the novel coronavirus antibody 1 and novel coronavirus antibody 2 are N antigens.

10. The reagent kit according to claim 8, characterized in that, The N antigen, S antigen, and N+S fusion antigen are full-length fragments or partial fragments of the corresponding antigens.

11. The reagent kit according to claim 8, characterized in that, The S antigen includes S1 protein, S1-RBD protein and S2 protein.

12. The kit according to claim 7, characterized in that, The specific pairing member is biotin-avidin.

13. The reagent kit according to claim 12, characterized in that, The biotin is selected from biotin with different activating groups.

14. The kit according to claim 12, characterized in that, The biotin is selected from biotin with an NHS activating group that can react with amino groups.

15. The kit according to claim 12, characterized in that, The biotin is NHS-LC-LC-Biotin.

16. The use of the kit according to any one of claims 7-15 in the preparation of a product for detecting the novel coronavirus in a sample to be tested.

17. The application according to claim 16, characterized in that, The test sample is selected from human serum, nasopharyngeal swabs, or pharyngeal swabs.

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