A method for early screening of monkeypox virus infection

Abstract

The present invention provides the use of a reagent capable of specifically binding to at least one state of monkeypox virus in EEV and IEV in a subject sample in the preparation of a kit for early screening of monkeypox virus infection. Further, the subject refers to an individual without obvious epidermal symptoms. The reagent provided by the present invention that can bind to at least one state of monkeypox virus has higher accuracy and sensitivity when applied to the preparation of a kit for early screening of monkeypox virus infection.

Description

Technical Field

[0001] The present invention relates to the field of in vitro diagnostic reagents. Specifically, the present invention relates to a monkeypox virus detection reagent, a kit containing the reagent, and applications thereof. Background Art

[0002] Monkeypox is a zoonotic viral infectious disease caused by the monkeypox virus (MPV). It is highly contagious and can be secondarily transmitted among humans. The main manifestations are fever, rash, and lymphadenopathy, and it may lead to a series of complications. Severe cases may even result in death. On July 23, 2022, the World Health Organization issued the highest-level alert for global public health emergencies regarding the monkeypox infection, believing that the monkeypox epidemic spreading in 75 countries and regions has constituted a "public health emergency of international concern". This also means that the monkeypox epidemic poses a great challenge to human health and social stability.

[0003] The incubation period of monkeypox (the interval from infection to the appearance of symptoms) is long, ranging from 5 to 21 days, mostly 6 to 13 days. The infection can be divided into two stages: the onset stage and the skin rash stage. The clinical symptoms during the virus latency period are atypical, often manifested as: fever, headache, lymphadenopathy, back pain, muscle pain, and listlessness, etc. And the rash will appear 1 to 3 days after the onset. This makes the monkeypox virus have a strong potential for infectious hazards. Therefore, it is particularly important to detect the virus in the early stage of its invasion into the body.

[0004] Monkeypox virus is an enveloped double-stranded DNA virus belonging to the Orthopoxvirus genus of the Poxviridae family. Common viruses in the same genus as monkeypox virus also include: Variola virus (VAR), Cowpox virus (CPV), and Vaccinia virus (VAC). Poxviruses exist in two forms: mature virus particles (MV) and extracellular enveloped virus particles (EV). Among them, EV is based on MV and is wrapped by a lipid membrane derived from the endoplasmic reticulum membrane. This membrane can decompose after contacting the cell, exposing the outer membrane and then directly fusing with the cell membrane, and then proceeding with downstream infection. MV can directly fuse with the cell membrane or be endocytosed into the cell through macropinocytosis. In the endosome, the outer membrane fuses with the endosome membrane to release the virus core wrapped by the inner membrane. The virus invades the cell through attachment, entry into the cell, uncoating, genome replication, protein synthesis and processing, assembly, and release of progeny viruses, and finally spreads in the body through the blood. Monkeypox virus can be roughly divided into four different states according to these two forms: intracellular mature virus (IMV), intracellular enveloped virus (IEV), cell-associated enveloped virus (CEV), and extracellular enveloped virus (EEV). Among them, IMV is the earliest virus form to appear. Antibodies are produced by the body under the stimulation of antigens. During the latent period of the virus, the antibody titer is low and difficult to detect. Therefore, detecting antigens can ensure the sensitivity of early detection. Monkeypox antigens are generally present in a large amount in the fluid of skin lesions after pox formation. However, once pox is formed, it means losing the opportunity to control potential transmission routes. Therefore, in order to achieve early detection, it is necessary to start with serum with stable antigen content and detect the virus in it with a highly sensitive detection method.

[0005] Currently, the existing technical solutions mainly have the following technical problems: (1) PCR detection is a commonly used technical means for monkeypox detection, but this method takes a long time and is not conducive to quickly obtaining detection results, bringing a window period for controlling the spread of the virus; (2) There is a lack of highly sensitive and highly specific detection reagents and technical methods that can diagnose during the latent period (i.e., early stage) of monkeypox-infected individuals. Summary of the Invention

[0006] The present invention mainly provides the use of a reagent in the preparation of a kit for early screening of monkeypox virus infection.

[0007] According to one aspect of the present invention, the use of a reagent in the preparation of a kit for early screening of monkeypox virus infection, the reagent comprising one or more reagents that specifically bind to at least one state of monkeypox virus in EEV and IEV in a subject sample; the subject refers to an individual without obvious epidermal symptoms.

[0008] Furthermore, the reagent further comprises a reagent that can specifically bind to monkeypox virus in the IMV state in a subject sample.

[0009] Further, the reagent includes antibodies and / or antigen-binding fragments that can specifically recognize target proteins B6R and / or A35R.

[0010] Furthermore, the target proteins further include: A29L and / or M1R; preferably, the target proteins are: A35R and A29L.

[0011] Further, the sequence of B6R includes at least: SEQ ID NO:1, and the sequence of A35R includes at least: SEQ ID NO:2.

[0012] Furthermore, the sequence of A29L includes at least: SEQ ID NO:3, and the sequence of M1R includes at least: SEQ ID NO:4.

[0013] Further, the antibodies and / or antigen-binding fragments include: monoclonal antibodies, polyclonal antibodies, single-chain antibodies, recombinant antibodies, chimeric antibodies, antibody fragments, ligands, peptidomimetics, aptamers, or cofactors, etc.

[0014] Further, the antibodies and / or antigen-binding fragments are labeled with signal generators, and the signal generators are selected from: horseradish peroxidase, alkaline phosphatase, fluorescein isothiocyanate, rhodamine isothiocyanate, isoluminol, acridinium ester, ruthenium tris(bipyridine), isotopes, biotin, colloidal gold, etc.

[0015] Further, the antibodies and / or antigen-binding fragments are coated on a solid phase, and the solid phase is selected from: latex microspheres, magnetic beads, cellulose membranes, glass, aluminum silicate, polystyrene, etc. Preferably, the solid phase is a cellulose membrane.

[0016] Further, the subject samples include: serum, plasma, and whole blood.

[0017] Furthermore, the epidermal symptoms refer to visible skin lesion symptoms such as rashes, macules, papules, vesicles, pustules, etc.

[0018] The present invention provides the use of a reagent that can specifically bind to at least one state of monkeypox virus in EEV and IEV in a subject sample in the preparation of a kit for early screening of monkeypox virus infection. Further, the subject refers to an individual without obvious epidermal symptoms. Furthermore, the epidermal symptoms refer to visible skin lesion symptoms such as rashes, macules, papules, vesicles, pustules, etc. The reagent provided by the present invention that can bind to at least one state of monkeypox virus has higher accuracy and sensitivity when applied in the preparation of a kit for early screening of monkeypox virus infection. Detailed implementation manners

[0019] It should be noted that, without conflict, the embodiments in this application and the features in the embodiments can be combined with each other. The present invention will be described in detail below with reference to the embodiments.

[0020] Term Explanation:

[0021] IMV: Intracellular mature virus, which is the earliest formed virus particle, present in the cytoplasm 6 hours after infection and spreads between hosts.

[0022] IEV: Intracellular enveloped virus, a part of the intracellular mature virus forms by obtaining a double membrane from early endosomes or the trans-Golgi network through a complex encapsulation mechanism.

[0023] CEV: Cell-associated enveloped virus, the intracellular enveloped virus is transported from the encapsulation site to the cell surface along microtubules, and the virus particles retained on the cell surface are mainly used for intercellular transmission.

[0024] EEV: Extracellular enveloped virus, the intracellular enveloped virus is transported from the encapsulation site to the cell surface along microtubules and released from the cytoplasm by the fusion of its outermost membrane with the plasma membrane.

[0025] Antibody fragment: Refers to the fragment formed by protease hydrolysis of an antibody.

[0026] Aptamer: A short oligonucleotide sequence that can bind to a target molecule with extremely high affinity and specificity.

[0027] Peptidomimetic: Peptide mimetic, designed according to the conformation, topochemistry and electrical characteristics of a bioactive peptide and its corresponding receptor, a non-peptide analog compound with similar biological activity.

[0028] Ligand: Any molecule that binds to an anchor protein.

[0029] Cofactor: Refers to a non-protein compound that binds to an enzyme (enzyme) and is necessary in a catalytic reaction.

[0030] As used herein, the term "screening" includes determining the susceptibility of a subject to a disease or disorder or whether the subject has a specific disease or disorder.

[0031] As used herein, the term "subject" refers to any individual in need of treatment, including any subject who does not show clinical symptoms and participates in a clinical study, or any subject who participates in an epidemiological test or serves as a control.

[0032] Monkeypox virus is a highly lethal infectious virus that poses a great risk to human society and individual health. However, due to its long incubation period and the lack of obvious symptoms in the early stage of infection, current detection methods can only diagnose after the onset of the disease, and the sensitivity is relatively low, which is not conducive to early diagnosis.

[0033] In a typical embodiment of the present application, in view of the above deficiencies, a detection reagent for monkeypox virus that can specifically bind to at least one state of EEV and IEV in a subject sample is provided.

[0034] In some embodiments, the above detection reagent can be used to screen early monkeypox virus infected individuals or monkeypox virus carriers, where the early monkeypox virus infected individuals or monkeypox virus carriers refer to individuals who have not shown obvious epidermal symptoms. The epidermal symptoms refer to visible skin lesion symptoms such as rashes, macules, papules, herpes, pustules, etc.

[0035] The above detection reagent includes an antibody and / or antigen-binding fragment that specifically binds to at least one state of the virus of EEV and IEV.

[0036] In some embodiments of the present application, the detection reagent for detecting at least one state of the virus of EEV and IEV in the subject sample includes an antibody and / or antigen-binding fragment that specifically binds to a target protein, where the target protein includes B6R and / or A35R.

[0037] Preferably, the detection reagent for detecting the virus in two states of EEV and IEV in the subject sample includes an antibody and / or antigen-binding fragment that specifically binds to a target protein, and the target protein is A35R.

[0038] In some embodiments of the present application, the above antibody and / or antigen-binding fragment can specifically bind to the B6R protein, and the sequence of the B6R protein includes at least SEQ ID NO:1, and / or can specifically bind to the A35R protein, and the sequence of the A35R protein includes at least SEQ ID NO:2.

[0039] Preferably, the above antibody and / or antigen-binding fragment specifically binds to the A35R protein, and the sequence of the A35R protein includes at least SEQ ID NO:2.

[0040] In some embodiments of the present application, the detection reagent for detecting at least one state of the virus of EEV and IEV in the subject sample further includes a detection reagent for detecting the virus in the IMV state.

[0041] In some embodiments of the present application, the detection reagent for detecting the virus in the IMV state includes an antibody and / or antigen-binding fragment that specifically binds to a target protein, where the target protein includes A29L and / or M1R.

[0042] Preferably, the detection reagent for detecting the virus in the IMV state in the subject sample includes an antibody and / or antigen-binding fragment that specifically binds to a target protein, and the target protein is A29L.

[0043] In some embodiments of the present application, the above-mentioned antibody and / or antigen-binding fragment specifically binds to the A29L protein, the sequence of which at least includes SEQ ID NO:3, and / or can specifically bind to the M1R protein, the sequence of which at least includes SEQ ID NO:4.

[0044] Preferably, the above-mentioned antibody and / or antigen-binding fragment specifically binds to the A29L protein, the sequence of which at least includes SEQ ID NO:3.

[0045] According to the above solution, the present invention selects various states of monkeypox virus and its target proteins as targets, which can greatly reduce the non-specific recognition of virus detection and improve the specificity and sensitivity of the detection results.

[0046] In the present application, a reagent that specifically binds to at least one of the monkeypox virus in the states of EEV, IEV, EEV and IMV, IEV and IMV in the subject sample can be used to prepare a kit for early screening of monkeypox virus infection. Among them, EEV refers to extracellular enveloped virus, and intracellular enveloped virus is transported along microtubules from the wrapping site to the cell surface and released from the cytoplasm through the fusion of its outermost membrane with the plasma membrane; IEV refers to intracellular enveloped virus, and a part of the intracellular mature virus obtains a double membrane through a complex wrapping mechanism from early endosomes or the trans-Golgi network; IMV refers to intracellular mature virus, which is the earliest formed virus particle, exists in the cytoplasm 6 hours after infection, and spreads between hosts.

[0047] In the present application, the detection of a specific state of monkeypox virus in a subject sample is achieved by binding to the virus target protein in this state. Among them, the target proteins include: B6R and / or A35R, and also include A29L and / or M1R. B6R (31.6KD) is a host range protein located on the membrane of the extracellular enveloped virus particles (EEV) of monkeypox virus and participates in the negative regulation of complement activation. A35R (181AA) is located on the envelopes of IEV and EEV and plays a role in antibody-resistant transmission. A29L (110AA) is a surface envelope protein encoded by the A29 gene in the A region of the intracellular mature virus (IMV) of monkeypox virus, mediating the recognition of the virus to host cells. M1R (250AA) is a surface transmembrane protein of IMV and is also an additional component of the virus entry device.

[0048] In some embodiments of the present application, the analytical method for detecting the target protein in a sample is a double-antibody sandwich assay. A pair of antibodies against the same target protein needs to be selected. In a heterogeneous immunoassay system, one of the antibodies is connected to a solid-phase carrier and can capture the target protein in the sample to be tested; the other antibody is attached with a signal generator and can generate a corresponding detection signal. When the target protein is captured by the capture antibody and forms a sandwich form with the antibody attached with the signal generator, the signal generator can generate a signal that can be detected, indicating the presence of the target protein at this time, as well as the amount present, etc. The antibodies used for detection are not limited to monoclonal antibodies or polyclonal antibodies, but also include one or more of single-chain antibodies, recombinant antibodies, chimeric antibodies, etc. The antibodies can be obtained by conventional technical means, and these techniques can be any one of various different techniques well-known to those skilled in the art. For example, to prepare polyclonal antibodies, an immunogen containing the target protein, such as cells with the target protein polypeptide or isolated target protein polypeptide, needs to be injected into a suitable animal, such as a mouse, rabbit, or sheep. Preferably, one or more injections are performed to enhance the immunity, and blood is taken from the immunized animal regularly. Then, polyclonal antibodies specific to the target protein are purified from these antisera by affinity chromatography using a polypeptide conjugated to a suitable solid-phase support.

[0049] The double-antibody sandwich includes two antibodies. One of the antibodies is connected to a solid-phase carrier and serves as a capture antibody, and the other antibody is connected with a signal generator and serves as a labeled antibody. Any non-competitive combination of antibodies can be used with the method disclosed herein, including monoclonal antibodies, polyclonal antibodies, and combinations thereof.

[0050] In some embodiments of the present application, the detection reagent for detecting at least one state of the virus in the subject sample of EEV and IEV includes at least a pair of antibody pairs. The two antibodies recognize two non-overlapping different epitopes, and these two antibodies can simultaneously recognize a certain state of the monkeypox virus.

[0051] The antibody pair can be obtained by conventional technical means. To facilitate the subsequent screening of the antibody pair, the prepared antibodies should be monoclonal antibodies, and monoclonal antibodies are usually prepared using the hybridoma technique. Also, to prevent the inability to find a pairable antibody, multiple animals can be immunized when immunizing animals to obtain more monoclonal antibodies. Usually, the double antibody sandwich ELISA method is used to screen the antibody pair. After obtaining the monoclonal antibodies, two of them are taken out, one as the capture antibody and the other as the labeled antibody (labeled with HRP enzyme). The capture antibody is coated on the antigen plate, the antigen is first added and the unbound antigen is washed away after incubation, then the labeled antibody is added and the unbound labeled antibody is removed after incubation, and finally the chromogenic solution is added. If color develops, it indicates that the two antibodies are paired antibodies; if no color develops, it indicates that the two antibodies cannot be paired. In some embodiments of the present application, at least one of the at least two antibodies is labeled with a signal generating substance, and the signal generating substance can be horseradish peroxidase (HRP), alkaline phosphatase (AP), fluorescein isothiocyanate (FITC), rhodamine isothiocyanate (TRITC), isoluminol (ABEI), acridinium ester (AE), ruthenium tris(bipyridyl), isotope, biotin, colloidal gold, etc.

[0052] In some embodiments of the present application, the paired antibodies are referred to as antibody pairs. In a kit for detecting the state of monkeypox virus, there are at least two antibody pairs, and these antibody pairs can respectively bind to specific proteins of monkeypox virus in different states, so as to form a double antibody sandwich state, and through the signal generating substance, indicate the presence of the target protein, thereby judging the presence of monkeypox virus.

[0053] In some embodiments of the present application, in the above reagent for detecting the state of monkeypox virus, the antibody is labeled on colloidal gold. According to the principle of colloidal gold labeling, only when the pH is close to and slightly higher than the isoelectric point of the protein, the adsorption force of colloidal gold to the protein is the strongest; too high or too low pH is not conducive to the combination of the two. Therefore, a precise pH measuring instrument or test strip is selected for labeling, and different methods are used to repeat the calibration, and gradient tests are carried out to find the optimal labeling pH. The preferred diameter range of the particle size of the colloidal gold is 40nm - 60nm. When the particle size of the colloidal gold is within this range, the specificity and sensitivity of the detection can be ensured.

[0054] In some embodiments of the present application, at least one of the at least two antibodies is coated on a solid phase, and the solid phase can be: latex microspheres, magnetic beads, cellulose membranes, glass, aluminum silicate, polystyrene, etc. Preferably, the solid phase is a cellulose membrane.

[0055] In some embodiments of the present application, the analytical method for detecting a target protein in a sample is an immunocompetition assay, and an antibody capable of specifically binding to the target protein and a labeled antigen conjugated with a signal generator need to be prepared. The antibody is coated on a solid phase and incubated together with the labeled antigen and the antigen in the test sample. The two antigens compete for binding to the capture antibody coated on the solid phase. The more the antigen to be detected in the sample, the stronger the competition, the less the amount of the labeled antigen binding to the capture antibody, and the weaker the signal emitted.

[0056] The labeled antigen that specifically binds to the capture antibody can be one or more and has one or more labels.

[0057] The above antigen conjugate for detecting the state of the monkeypox virus needs to be conjugated with colloidal gold particles of a specific particle size, wherein the particle size range of the colloidal gold is 40 nm - 60 nm. The particle size of the colloidal gold within this range can ensure sensitivity and avoid a certain degree of non-specific adsorption.

[0058] In some embodiments of the present application, the analytical method for detecting a target protein in a sample is a latex turbidimetry assay. The specific antibody corresponding to the target protein needs to be coated on latex particles with a size of 10 nm - 1 μm to increase the immune complex of the antigen and antibody, enlarge the volume of the antigen-antibody conjugate, and thus increase the signal to form turbidity. By detecting the change in light intensity or the angle of light deflection, etc., the content of the antigen in the test sample can be calculated.

[0059] The specific antibody includes one or more antibodies. The antibody is coated on a solid phase, and the solid phase can be a latex microsphere, etc. The latex microsphere can be a microsphere with different particle sizes.

[0060] In another typical embodiment of the present application, there is provided the use of a detection reagent for detecting at least one of the states of EEV and IEV in a subject sample in the preparation of a kit for early screening of monkeypox virus infection. The kit includes one or more antibodies that specifically recognize the target protein, and one of the antibodies is coated on a solid phase as a capture antibody; another antibody or a competitive binding antigen is conjugated with a signal generator, and the signal generator can emit a signal by forming an immune complex, and thus the presence or amount of the target protein can be detected.

[0061] In some embodiments of the present application, in the reagent for detecting the state of monkeypox virus, the judgment rule for the detection result is as follows: if at least one pair of antibodies binds to the target protein in the sample to form a double-antibody sandwich and indicates its presence, the detection result is judged to be positive, that is, the presence of monkeypox virus; if no pair of antibodies binds to the target protein in the sample to form a double-antibody sandwich and the presence of the signal indicator cannot be observed, the detection result is judged to be negative, that is, the absence of monkeypox virus. In some embodiments of the present application, when the reagent is used to detect other viruses that may potentially have cross-reactions (such as: rubella virus, cytomegalovirus, herpes simplex virus, etc.), if at least one pair of antibodies binds to a certain protein in the sample to form a double-antibody sandwich and indicates its presence, the detection result is judged to be positive; if no pair of antibodies binds to a certain protein in the sample to form a double-antibody sandwich and the presence of the signal indicator cannot be observed, the detection result is judged to be negative.

[0062] In the above-mentioned kit, in addition to including the above-mentioned conjugate reagent, it also includes other reagents for convenient detection, including but not limited to any one or more of calibration products, quality control products, reagent buffers, and sample extraction solutions, etc. The calibration product is used to calibrate the built-in main curve of the reagent into a new working curve. The quality control product is used to test whether the reagent can be used normally. The reagent buffer can be added according to needs or configured separately during actual detection. The sample extraction solution is used in the pretreatment step of the collected sample, mainly to lyse the virus and expose or release the target protein, making it more convenient for the detection reagent to bind to it.

[0063] In the above-mentioned kit, preferably, the calibration product includes a first calibration product (i.e., the low-point calibration product) and a second calibration product (i.e., the high-point calibration product) with different concentrations. More preferably, the concentration of the low-point calibration product is preferably 0.05 pg / ml - 10 pg / ml, and the concentration of the high-point calibration product is preferably 100 pg / ml - 1000 pg / ml; preferably, the quality control product includes a negative quality control product and a positive quality control product. Further preferably, the concentration of the negative quality control product is 0 pg / ml, and the concentration of the positive quality control product is 500 pg / ml.

[0064] In the above-mentioned kit, a conventional buffer can be used as the reagent buffer. In the present application, the reagent buffer includes but not limited to carbonate buffer, phosphate buffer, borate buffer, or Tris-HCl buffer, etc.

[0065] In the above-mentioned kit, a sample extraction solution is further included, and the components of the sample extraction solution include: Tris, Triton X-100, absolute ethanol, NaCl, BSA, NaN3, Procline 300. The sample extraction solution can improve the sensitivity and specificity of monkeypox antigen detection. The sample of the exudate stored by it is compatible with conventional commercial nucleic acid extraction kits for nucleic acid extraction and can also be compatible with antigen-antibody detection. During the storage of the sample by the sample extraction solution, the activity of the enveloped virus can be inactivated, reducing the infection risk of laboratory personnel during the operation process.

[0066] In the above-mentioned kit, the kit of the present invention is used as a rapid test kit, including an immersion rod type immunochromatographic strip for diagnosis or detection. The immunochromatographic strip may include: (a) a sample pad for absorbing the sample; (b) a conjugate pad capable of binding to the target antigen of the subject sample; (c) a reaction membrane on which a reaction line and a control line including antibodies against the antigen are formed; (d) an absorbent pad for absorbing the residual sample; and (e) a support or backing material. The antibodies used have high specificity and high affinity for each biomarker and have almost no cross-reactivity with other antigens. Such antibodies can be monoclonal antibodies, polyclonal antibodies or recombinant antibodies. In addition, the rapid test kit may include antibodies specific for the control material. Other rapid test kits may include other materials required for diagnosis, such as reagents capable of detecting the bound antibodies, for example, nitrocellulose membranes immobilized with specific antibodies and secondary antibodies, membranes bound with antibodies, absorbent pads and sample pads, etc.

[0067] The detection step of the kit includes dropping the sample solution pre-treated with the sample extraction solution into the sample well. Due to the capillary action of the microporous filter membrane, the sample solution slowly chromatographs to the other end, preferentially binds to the colloidal gold signal generating substance, and is captured by the capture antibody coated on the solid phase. The unbound substances (including free labeled antibodies) are separated from the binding region, and finally the determination result is judged by the color strip of the colloidal gold.

[0068] The sample types for the above detection include: whole blood, serum, plasma.

[0069] According to another aspect of the present application, there is also provided an application of a reagent for detecting monkeypox virus in the preparation of a kit for early screening of monkeypox virus infection. Further, the above application is for screening early monkeypox virus infected individuals or monkeypox virus carriers, and the early virus infected individuals refer to individuals without obvious epidermal symptoms. The epidermal symptoms refer to visible skin lesion symptoms such as rashes, macules, papules, herpes, pustules, etc.

[0070] The beneficial effects of the present application will be further described below in conjunction with specific embodiments.

[0071] It should be noted that the patient samples in the following examples are all from clinically diagnosed samples.

[0072] Example 1: Preparation of Specific Monoclonal Antibodies

[0073] To prepare monoclonal antibodies with high specificity and sensitivity, A29L, B6R, A35R, and M1R with specific sequences were used as immunogens to stimulate the immune system of mice to produce specific antibodies, ensuring the high affinity of the antibodies for the corresponding antigen epitopes.

[0074] 1. Specific Antigen Sequences:

[0075] (1) B6R: SEQ ID NO:1: gyhsldpnav cetdkwkyen pckkmctvsd yvselydkplyevnstmtls cngetkyfrc eekngntswn dtvtcpnaec qplqlehgsc qpvkekysfg eymtincdvgyevigvsyis ctanswnvip

[0076] (2) A35R: SEQ ID NO:2: mvisllsmit msaflivrqn qcmsaneaai tdsavavaaassthrkvass ttqydhkesc nglyyqgscy ilhsdyksfe

[0077] (3) A29L: SEQ ID NO:3: reaivkayg ddneetlkqr ltnlekkitn ittkfeqiekcckhndevlf rlenha

[0078] (4) M1R: SEQ ID NO:4: qtkcdieign fyirqnhgcn itvknmcsad adaqldavlsaatetysglt peqkayvpam ftaalniqts vntvvrdfen yvkqtcnssa vvdnklkiqn viidecygapgsptnl

[0079] 2. Immunize Animals with Antigens

[0080] Balb / c mice were selected for injection. The antigen-antibody complex stimulated the cloning of corresponding B lymphocytes, activated and proliferated them, and differentiated them into sensitized B lymphocytes.

[0081] 3. Preparation of Myeloma Cells

[0082] 48 h before fusion, myeloma cells were cultured with R / MINI-1640 medium containing 20% fetal bovine serum for proliferation. 24 h before fusion, the medium was changed once with the same medium. The myeloma cells were taken, centrifuged and washed, and then suspended in serum-free medium.

[0083] 4. Preparation of immune mouse spleen cells

[0084] Mice on the third day after the last immunization were bled from the orbital cavity, and the serum was separated and stored frozen for later use. The spleen was taken under aseptic conditions to prepare a spleen cell suspension.

[0085] 5. Preparation of feeder cells

[0086] The mice were sacrificed, surface disinfected and fixed, and then 10 mL of medium was injected into the abdominal cavity. Subsequently, the injected medium was aspirated, and the supernatant was discarded. The precipitated cells were suspended in HAT medium and placed in a CO2 incubator for later use.

[0087] 6. Cell fusion

[0088] Myeloma cells and mouse spleen cells were mixed at a ratio of 1:5 - 1:10, added with 50 mL of R / MINI 1640 solution, centrifuged at 1000 r / min, and the supernatant was discarded.

[0089] The centrifuge tube was placed in a 37 °C water bath. 1 mL of 50% PEG was aspirated and dropped into the centrifuge tube for static placement, then R / MINI 1640 solution was added, followed by 10 mL of R / MINI-1640 solution, and then R / MINI-1640 solution was added to 50 mL to terminate the action of PEG. Centrifuge at 800 r / min for 10 min, and discard all the supernatant. Add HAT-R / MINI-1640 selection medium, and then inoculate the suspended cells into a 96-well culture plate.

[0090] 7. Selective culture

[0091] In HAT medium, only the fused hybridoma cells can survive and proliferate in HAT medium because they have obtained hypoxanthine-guanine phosphoribosyl transferase from spleen cells and have the characteristic of infinite proliferation of myeloma cells.

[0092] 8. Screening and cloning of hybridoma positive clones

[0093] Enzyme-linked immunosorbent assay (ELISA) was used to screen for hybridoma positive clones:

[0094] The antigen was used as a continuous 1:2 gradient dilution at a concentration of 1 μg / mL with the coating buffer CBS at the optimal coating concentration, 100 μl / well was added to the ELISA plate, sealed with a film, and coated at 4 °C for more than 12 h. At the same time for blocking, the negative / positive sera were serially diluted, 100 μl was added to the ELISA plate wells, then the secondary antibody and substrate were added sequentially for color development, and the OD value was read. The wells with the highest positive values were selected and reserved for subcloning.

[0095] Cloning technology: The cell line to be recloned was aspirated from the culture well using the limiting dilution method and cell counting was performed to count the number of cells in 1 mL. The positive wells with single clone growth were selected for cloning again, generally until a 100% positive well rate was achieved to ensure that the antibody was produced by a single clone.

[0096] 9. Purification of antibody by affinity chromatography

[0097] An appropriate amount of ascites was centrifuged to obtain the supernatant, diluted 4-fold with PBS, incubated overnight at 4 °C, and centrifuged to obtain the supernatant. The purified antigen was immobilized on the silica filler in the affinity chromatography column, and the sequence of the antigen was selected from: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4. The sample was loaded at 1 / 10 of the column volume, eluted with 0.01 mol / L PBS solution at pH 7.4 with a flow rate of 1 ml / min, and then the antibody was eluted with citrate buffer. The eluted components were collected, which were the purified antibody.

[0098] The protein content in each tube was measured using a spectrophotometer, and the purified antibody was aliquoted and stored at 2 - 8 °C.

[0099] Example 2: Preparation of antibody pairs

[0100] Even if two antibodies target different antigenic determinants on the same antigen molecule, it does not necessarily mean that these two antibody molecules can bind to the antigen molecule simultaneously. When one antibody binds to the antigen, it may cause a conformational change in other binding sites (antigenic determinants), resulting in other antibodies being unable to bind to the antigen normally. In addition, due to the steric hindrance effect, antibodies with relatively close binding sites may also be unable to bind to the antigen simultaneously. Therefore, to obtain paired antibodies that can bind to the antigen molecule simultaneously for use in detection methods, the antibodies need to be screened.

[0101] 1. Preparation of HRP-labeled antibody:

[0102] (1) Weigh 5 mg of HRP enzyme and dissolve it in 0.5 mL of distilled water;

[0103] (2) Add 0.5 mL of freshly prepared sodium periodate solution (0.1 mol / L), and let it stand at 4 °C for 30 min;

[0104] (3) Add 0.5 mL of ethylene glycol solution (0.16 mol / L), mix well in the dark at room temperature, and let it stand for 30 min;

[0105] (4) Add 1 mL of an aqueous solution containing 5 mg of antibody, mix well, transfer it into a dialysis bag, dialyze against pH 9.5 carbonate buffer, and place it at 4 °C overnight;

[0106] (5) Add 0.2 mL of freshly prepared sodium borohydride solution (0.5 mg / mL), mix well, and let it stand at 4 °C for 2 h;

[0107] (6) While stirring, slowly add an equal volume of saturated ammonium sulfate solution, and let it stand at 4 °C for 1 h;

[0108] (7) Centrifuge at 3000 r / min for 30 min, discard the supernatant, and wash the precipitate twice with semi-saturated ammonium sulfate;

[0109] (8) Dissolve the precipitate in a small amount of phosphate buffer (0.15 mol / L, pH 7.4), dialyze overnight to remove ammonium ions, then centrifuge at 10000 r / min for 30 min to remove the precipitate. The supernatant is the enzyme conjugate.

[0110] 2. Screening of paired antibodies:

[0111] The double antibody sandwich ELISA method is used to screen antibody pairs:

[0112] Take two monoclonal antibodies with high titers. One is used as the capture antibody, and the other is used as the labeled antibody (HRP-labeled antibody). Coat the capture antibody on the solid phase carrier, first add the antigen, incubate, and then wash away the unbound antigen to form an antibody-antigen complex on the solid phase carrier and separate it from other substances in the liquid. Then add the labeled antibody, incubate, and wash away the unbound labeled antibody. Finally, add the chromogenic solution for color development. If color development occurs, it indicates that the capture antibody and the labeled antibody are a pair of paired antibodies. Qualitative or quantitative analysis can be further carried out according to the depth of the color. If no color development occurs, it indicates that the capture antibody and the labeled antibody are not paired antibodies. If the two selected monoclonal antibodies cannot be paired, two other monoclonal antibodies need to be reselected and the experiment repeated until paired antibodies are found.

[0113] (1) Dilution: Dilute the monoclonal antibody to 5 μg / mL with carbonate buffer (0.01 mol / L, pH 9.6);

[0114] (2) Incubation: After coating the enzyme-linked immunosorbent assay (ELISA) plate with the capture antibody, incubate at 4 °C overnight;

[0115] (3)Washing: Wash the plate 3 times with PBS / T20 for 3 min each time;

[0116] (4)Blocking: Block with PBS / T20 containing 3% calf serum, incubate at 37 °C for 1 h, and wash the plate;

[0117] (5)Adding antigen: Add serially diluted antigen solution, incubate at 37 °C for 45 min, and wash the plate;

[0118] (6)Adding enzyme-labeled antibody: Add HRP-monoclonal antibody, incubate at 37 °C for 30 min, and wash the plate;

[0119] (7)Color development: Add TMB chromogenic solution, gently shake and mix well, and react at 37 °C in the dark for 15 min;

[0120] (8)Termination: Add 50 μL of termination solution to terminate the reaction;

[0121] (9)Measurement: Read the OD450 value of each well on an enzyme-linked immunosorbent assay reader.

[0122] Example 3: Verification of antibody specificity

[0123] After the specific antibody is prepared, it is necessary to further experimentally verify whether it will react with other antigens other than the target antigen, that is, the degree of cross-reactivity, using ELISA (enzyme-linked immunosorbent assay) for verification. Since the antibodies screened out through the above examples are antibody pairs, it is also necessary to test the two antibodies of each antibody pair with four antigens respectively.

[0124] 1. Coating antigen:

[0125] Dilute A29L, B6R, A35R, and M1R proteins to 20 μg / ml in PBS respectively. Transfer 50 μl of the antigen dilution to the micro-wells of the bottom plate to coat the micro-wells with antigen. Cover the microtiter plate with an adhesive plastic product and incubate at room temperature for 2 h. Discard the coating solution, add 200 μl of PBS to the wells, wash the titer plate 3 times, and remove the remaining droplets in the micro-wells after the last wash.

[0126] 2. Blocking:

[0127] Add 200 μl of blocking buffer to each well. The blocking buffer is 5% serum / PBS. Block the remaining protein binding sites in the coated wells and incubate at room temperature for at least 2 h. After completion, wash the titer plate 2 times with PBS.

[0128] 3. Incubation:

[0129] Add 100 μl of the primary antibody dilution solution to each well and incubate at room temperature for 2 h. Wash the titration plate 4 times with PBS, and remove the residual droplets in the microwells after the last wash. Add 100 μl of the labeled secondary antibody to each well, incubate at room temperature for 1 - 2 h, and wash the titration plate 4 times with PBS.

[0130] 4. Detection:

[0131] Add 100 μl of the substrate to each microwell, incubate for 30 min, take it out and read the value in the microplate reader.

[0132] The test results are shown in Table 1. In Table 1, "+" indicates that the detection result is that the corresponding antigen can be detected, and "-" indicates that the corresponding antigen is not detected.

[0133] Table 1: Specificity detection of different antibodies

[0134]

[0135] The experimental results show that the antibody pair prepared according to Example 2 has strong specificity, can sensitively detect the corresponding antigen, and does not produce cross-reaction.

[0136] Example 4: Preparation of colloidal gold kit

[0137] Use the antibody pair prepared in Example 2 to prepare a colloidal gold kit, and use the kit prepared in this example for subsequent example verification. The specific preparation method is as follows:

[0138] 1. Preparation of colloidal gold: Prepare colloidal gold by the sodium citrate reduction method

[0139] Take 990 g of ultrapure water into a flask, add 10 mL of 1% chloroauric acid solution. After heating to boiling, add 10 mL of 1% sodium citrate dihydrate solution, stop heating 15 min after the solution changes color, and cool at room temperature.

[0140] 2. Preparation of immunocolloidal gold: ① Test line (T line): Mouse anti-human IgG antibody with a concentration of 1 mg / mL. ② Control line (C line): Goat anti-mouse IgG antibody with a concentration of 1 mg / mL.

[0141] 3. Preparation of immunocolloidal gold pad:

[0142] ① Pretreatment of gold-labeled pad: Cut the gold-labeled pad into gold-labeled pad strips, and use a gold-spraying and membrane-scratching instrument to pretreat each gold-labeled pad strip and then dry for 16 h. ② Spraying of immunocolloidal gold: Use a gold-spraying and membrane-scratching instrument to spray gold on each pretreated gold-labeled pad strip. After spraying, dry for 16 h and store for later use.

[0143] 4. Preparation of immunonitrocellulose membrane:

[0144] Cut the NC membrane and paste it at the corresponding position on the PVC bottom plate. The membrane scribing coating diluent is 0.01M PB buffer. Prepare the T line with 0.01M PB buffer, and prepare the antibody ② in the corresponding antibody pair prepared in Example 2 to a concentration of 1.0 mg / mL; prepare the C line with 0.01M PB buffer, and prepare the goat anti-mouse antibody to a concentration of 1.0 mg / mL. Dry the immunized NC membrane after scribing for 24 h and store it for later use.

[0145] 5. Sample pad pretreatment:

[0146] Prepare the sample pad pretreatment solution. Soak the sample pad in the sample pad pretreatment solution, take out the sample pad with forceps, drain the water, dry it, cut it after drying and collect it in a drying oven for later use. The components of the sample pad pretreatment solution are: 0.5% BSA, 10 mM Tris, 0.1% PVP10, 0.1% PEG20000, 0.5% Tween-20, 0.15% Proclin300.

[0147] Since in the technical solution of this application, there will be a solution in which multiple antibodies are mixed and coated on the T line, it is necessary to explain each solution here. Table 2 lists the solutions that will be encountered in the subsequent examples and the concentrations of each coated antibody:

[0148] Table 2: Solutions for coating antibodies on the T line in each example

[0149]

[0150] Example 5: Detection of early-stage patients by different solutions

[0151] Use the colloidal gold test kit prepared in Example 4 for testing. Verify the early diagnosis rate of early-stage patients through two different solutions of ① IMV, EEV and ② IMV, EEV, IEV. The definition of early-stage patients is: patients who have not shown visible symptoms such as rashes but are infected with the virus. The total number of case samples included in the experimental verification is: 60 cases, including 30 early-stage patient samples and 30 negative samples. All these patients have been verified as monkeypox-infected by PCR experiments.

[0152] During detection, the sample is first added to the sample extraction solution to fully expose the proteins in the sample. The components of the sample extraction solution include: Tris, Triton X-100, absolute ethanol, NaCl, BSA, NaN3, and Procline 300. After 3 - 5 minutes of the extraction process, 30 μl of the mixed liquid is taken and added to the sample pad. Through chromatography, the viral proteins (antigens) in the sample enter the gold conjugate pad. Antibodies against different proteins react and bind with the antibodies labeled with colloidal gold on the gold conjugate pad, forming antigen-antibody complexes with gold labels. The complexes continue to chromatograph to the T line on the NC membrane and bind to the capture antibody coated on the surface of the NC membrane, forming a double-antibody sandwich complex. This complex continuously accumulates and develops color. At this time, the excess labeled antibody continues to chromatograph to the C line and is captured by the goat anti-mouse IgG antibody, forming an immune complex and enriching to show the quality control line. If the corresponding protein is not present in the sample, the T line does not appear, only the C line appears, and the test result is negative, denoted as "-"; if the corresponding protein is present in the sample, the T line appears and the C line also appears, and the test result is positive, denoted as "+"; if the C line does not appear, it indicates that this test is invalid.

[0153] The calculation methods for sensitivity and specificity are as follows: Sensitivity = True positive cases / (True positive + False negative) * 100%, Specificity = True negative cases / (True negative + False positive) * 100%.

[0154] Table 3: Early diagnosis rates of different antibody combination schemes for detecting the status of IMV and EEV

[0155]

[0156] When detecting viruses in the IMV and EEV status, the combination scheme of B6R + A29L has the highest specificity for diagnosing early-stage monkeypox virus infections, which is 90%; the combination scheme of B6R + A29L + M1R has the highest sensitivity, which is 90%, but the lowest specificity.

[0157] Table 4: Early diagnosis rates of different antibody combination schemes for detecting the status of IMV, EEV, and IEV

[0158]

[0159] When detecting viruses in the IMV, EEV, and IEV status, the combination scheme of A35R + A29L has the highest specificity for diagnosing early-stage monkeypox virus infections, which is 96.33%; the combination scheme of A35R + A29L + B6R + M1R has the highest sensitivity, which is 100%, but the lowest specificity.

[0160] Considering the 9 detection schemes verified in this example, the combination scheme of A35R + A29L has high sensitivity and high specificity for diagnosing early-stage monkeypox virus infections and is the most preferred scheme.

[0161] Example 6: Comparison of Detection Rates with Other Schemes

[0162] The technical solution aims to detect multiple targets simultaneously to improve the detection rate. In this example, the preferred solution in the previous example is compared with the single-target solution and the double-target solution that only detects one virus state, and the verification method is to use the colloidal gold kit prepared in Example 4 for the experiment. The total number of case samples included in the experimental verification is 60, including 30 early patient samples and 30 negative samples. The calculation methods of sensitivity and specificity are: sensitivity = true positive cases / (true positive + false negative) * 100%, specificity = true negative cases / (true negative + false positive) * 100%.

[0163] Table 5: Comparison of Detection Rates with Other Schemes

[0164]

[0165] Compared with the single-target solution (B6R, A29L, A35R, M1R) and the double-target solution that only detects one virus state (A29L + M1R), the preferred solution (A29L + A35R) has high sensitivity and high specificity.

[0166] Example 7: Specificity of Different Combination Schemes

[0167] Relevant virus samples containing potential cross-reactions are selected for specificity research. Among them, the sample sizes included in the experiment are: 45 healthy controls (negative samples), 38 rubella virus samples, 47 cytomegalovirus samples, 40 herpes simplex virus type I samples, and 44 herpes simplex virus type II samples. The verification method is to use the colloidal gold prepared in Example 4 for the experiment. The preferred solutions in Example 5 and Example 6 are used as the evaluation criteria.

[0168] Table 6: Detection of Other Viruses by Different Antibody Combinations

[0169]

[0170] In the experiment on the specificity of the detection scheme, for the preferred solution: A35R + A29L, the positive rate remains 0% in the tests of other viruses that may have potential cross-reactions; while the results of other schemes show that the more targets can be detected and the more virus state types can be detected, the higher the possibility of cross-reaction and the lower the specificity.

[0171] Compared with the related technology, the present application has the following beneficial effects:

[0172] This application uses the technical means of immunochromatography to shorten the detection time, quickly obtain the detection results, and eliminate the window period for suppressing the spread of the virus;

[0173] The technical solution of this application can perform highly sensitive and highly specific detection during the incubation period (i.e., the early stage) of monkeypox-infected individuals.

[0174] The above are only the preferred embodiments of the present invention and are not used to limit the present invention. For those skilled in the art, the present invention can have various changes and modifications. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. Use of a reagent in the preparation of a kit for early screening of monkeypox virus infection, the reagent comprising one or more reagents that specifically bind to monkeypox virus in at least one state selected from EEV and IEV in a subject sample and a reagent that specifically binds to monkeypox virus in the IMV state in a subject sample; the reagent comprises an antibody and / or antigen-binding fragment that can specifically recognize a target protein selected from A35R and A29L, or an antibody and / or antigen-binding fragment of a target protein selected from B6R and A29L; the subject refers to an individual without obvious epidermal symptoms; The sequence of B6R is: SEQ ID NO:1, the sequence of A35R is: SEQ ID NO:2, and the sequence of A29L is: SEQ ID NO:

3.

2. The use according to claim 1, wherein, The antibody and / or antigen-binding fragment includes: monoclonal antibody, polyclonal antibody, single-chain antibody, recombinant antibody, chimeric antibody, ligand, peptidomimetic, aptamer or cofactor, etc.

3. The use according to claim 2, wherein, The antibody and / or antigen-binding fragment is labeled with a signal generator, and the signal generator is selected from: horseradish peroxidase, alkaline phosphatase, fluorescein isothiocyanate, rhodamine isothiocyanate, isoluminol, acridinium ester, ruthenium tris(bipyridine), isotope, biotin or colloidal gold, etc.

4. The use according to claim 3, wherein, The antibody and / or antigen-binding fragment is coated on a solid phase, and the solid phase is selected from: latex microspheres, magnetic beads, cellulose membrane, glass, aluminosilicate or polystyrene, etc.

5. The use according to claim 4, wherein, The solid phase is a cellulose membrane.

6. The use according to claim 1, wherein, The subject sample includes: serum, plasma and whole blood.

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