WO2024041493A1 - 一种猴痘病毒检测方法和试剂盒 - Google Patents

一种猴痘病毒检测方法和试剂盒 Download PDF

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WO2024041493A1
WO2024041493A1 PCT/CN2023/114106 CN2023114106W WO2024041493A1 WO 2024041493 A1 WO2024041493 A1 WO 2024041493A1 CN 2023114106 W CN2023114106 W CN 2023114106W WO 2024041493 A1 WO2024041493 A1 WO 2024041493A1
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antibody
monkeypox virus
protein
fragment
virus
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PCT/CN2023/114106
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English (en)
French (fr)
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李瑞净
张翼
秦汤
周俊
文飘
刘春艳
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广东菲鹏生物有限公司
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Priority claimed from CN202211239858.4A external-priority patent/CN117659170A/zh
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Publication of WO2024041493A1 publication Critical patent/WO2024041493A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances

Definitions

  • the present disclosure relates to the field of protein detection. Specifically, the present invention relates to detection methods and kits for monkeypox virus.
  • Monkeypox is a zoonotic disease caused by monkeypox virus (MPXV), which was first discovered in the virgin forests of western and central Africa. Monkeypox virus is similar to vaccinia virus and smallpox virus. The clinical manifestations are fever 10-12 days after exposure and skin rash 2-3 days after the fever. The rash changes from maculopapular rash to small blisters and pustules, with scabs appearing in about 10 days. The virus can be transmitted from animals to humans through direct close contact. The main routes of infection include blood, body fluids, skin or mucous membrane wounds. Secondary transmission to humans is mainly through airborne droplets, which can infect the mucous membranes of the eyes, nose and throat.
  • MPXV monkeypox virus
  • polymerase chain reaction PCR is the main detection method for detecting monkeypox virus at home and abroad, but it requires equipment and is prone to contamination. There is a need to develop simple and popular immunological detection methods for monkeypox virus.
  • Monkeypox virus A29L protein is the surface envelope protein of monkeypox virus (NCBI sequence number NP_536566.1). It is 110 amino acids in length and is similar to vaccinia virus 162 protein (CPXV-162) and vaccinia virus A27L protein (VACA27L). Same origin.
  • the inventor After extensive theoretical research and experimental exploration, the inventor fully considered the structural characteristics of the A29L protein of monkeypox virus, analyzed and studied various A29L antigen intervals and detection antibodies to be tested, and obtained a combination of A29L antigen regions that can be used for A29L detection.
  • the disclosure may include one or more of the following:
  • a monkeypox virus antibody which includes a first antibody that binds to 19-25aa of the monkeypox virus A29L protein.
  • a monkeypox virus antibody combination which includes the first antibody described in item 1 and which binds to monkeypox virus Secondary antibody to 81-110aa of A29L protein,
  • detectable label is selected from metal particles, fluorescent labels, chromophore labels, electron-dense labels, chemiluminescent labels, radioactive labels, or enzyme labels.
  • the detectable label can be rhodamine, fluorescein, fluorescent microspheres, colloidal gold, acridinium ester, luciferase, horseradish peroxidase, alkaline phosphate Enzyme, beta-galactosidase, glucoamylase, lysozyme, sugar oxidase, glucose oxidase, galactose oxidase or glucose-6-phosphate dehydrogenase label.
  • the binding partner is selected from biotin/avidin, biotin/streptavidin; for example, the labeled antibody is labeled by the binding partner and a detectable label .
  • the solid phase is, for example, magnetic particles, latex particles, microtiter plates, nitrocellulose membranes or microfluidic chips;
  • the binding partner is, for example, biotin/ Avidin, biotin/streptavidin.
  • a kit comprising a first antibody; optionally, a second antibody; wherein the first antibody and the second antibody are as defined in any one of items 1-10.
  • monkeypox virus antibody as described in item 1 or the antibody combination as described in any one of items 2-10 in the preparation of a kit for detecting monkeypox virus.
  • An antigen comprising the 19-25aa fragment of the A29L protein.
  • Fragment 1 Amino acid fragment 19-25aa of monkeypox virus A29L protein
  • Fragment 2 Monkeypox virus A29L protein amino acid fragment 81-110aa;
  • kits of the present disclosure include a primary antibody that binds 19-25 aa of the monkeypox virus A29L protein.
  • the first antibody binds or specifically binds to 17-49aa of the monkeypox virus A29L protein; in some embodiments, the first antibody binds or specifically binds to 2-37aa of the monkeypox virus A29L protein; in some In embodiments, the first antibody binds or specifically binds to 19-31aa of the monkeypox virus A29L protein; in some embodiments, the first antibody binds or specifically binds to 19-25aa of the monkeypox virus A29L protein.
  • the first antibody does not bind or does not specifically bind to 37-81 aa of the monkeypox virus A29L protein. In some embodiments, the first antibody does not bind or does not specifically bind to 61-93 aa of the monkeypox virus A29L protein. In some embodiments, the first antibody does not bind or does not specifically bind to 81-110 aa of the monkeypox virus A29L protein. In some embodiments, the first antibody does not bind or does not specifically bind to 25-37 aa of the monkeypox virus A29L protein. In some embodiments, the first antibody does not bind or does not specifically bind to 31-43 aa of the monkeypox virus A29L protein. In some embodiments, the first antibody does not bind or does not specifically bind to 37-49aa of the monkeypox virus A29L protein.
  • the second antibody binds or specifically binds to 81-110aa of the monkeypox virus A29L protein. In some embodiments, the second antibody binds or specifically binds to 94-110aa of the monkeypox virus A29L protein. In some embodiments, the second antibody does not bind or specifically binds monkeypox virus A29L protein 17-49aa. In some embodiments, the second antibody does not bind or specifically binds monkeypox virus A29L protein 19-61aa. In some embodiments, the second antibody does not bind or specifically binds monkeypox virus A29L protein 37-81aa. In some embodiments, the second antibody does not bind or specifically binds monkeypox virus A29L protein 61-93aa.
  • Binding refers to the ability of the antibody or antigen-binding fragment to bind or capture the analyte or target molecule.
  • binding of an affinity-reactive antibody to a target molecule is employed, for example, an analyte-binding antibody has an affinity of KD ⁇ 10 -7 M for its target molecule (KD represents the equilibrium dissociation constant, i.e., the affinity constant).
  • KD represents the equilibrium dissociation constant, i.e., the affinity constant.
  • the antibody has an affinity for its target molecule of 10 "8 M or even 10 "9 M.
  • the antibody has an affinity for its target molecule of at least 10 -10 M.
  • an ELISA method is used to react the binding of an antibody to a target molecule, such as the reactivity measured in the embodiments of the present disclosure.
  • the antibody's reactivity to its target molecule such as an OD reading value of greater than or equal to 0.5, or greater than or equal to 1.0, or greater than or equal to 1.5 or greater than or equal to 2.0, or greater than or equal to 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0.
  • the term specific is used to indicate that an antibody or antigen-binding fragment binds significantly to a target molecule but not to other molecules.
  • the reactivity of the antibody to bind similar molecules is the binding target.
  • the reactivity of the molecule is at most 50%, at most 40%, at most 30%, at most 20%, at most 10%, at most 5% or less.
  • the binding affinity of the antibody to a similar molecule is unmeasurable or has a KD greater than 10 -7 M compared to the target molecule.
  • the antibody binds similar molecules with a reactivity of less than 0.5, less than 0.4, less than 0.3, less than 0.2, or less than 0.1.
  • kits of the present disclosure further comprise an antibody that binds 81-110 aa of the A29L protein.
  • the present disclosure can utilize antibodies that bind different amino acid fragments to recognize multiple positions on the antigen, reducing the risk of missed detection and increasing the detection rate.
  • the kit includes a test reagent card (test strip).
  • the kit includes an Elisa plate.
  • the kit includes magnetic beads.
  • the present disclosure can be performed by, or include reagents for, performing fluorescent immunochromatography. In some embodiments, the present disclosure may be performed by, or include reagents for, colloidal gold immunochromatography. The present disclosure may be performed by time-resolved fluorescence immunochromatography or include reagents for performing time-resolved fluorescence immunochromatography. The present disclosure may be performed by quantum dot chromatography or include reagents for performing quantum dot chromatography. In some embodiments, the present disclosure can be performed by magnetic bead immunochromatography or include reagents for performing magnetic bead immunochromatography.
  • the antibodies of the present disclosure may be monoclonal antibodies or polyclonal antibodies. In some embodiments, the antibodies of the present disclosure can be full-length antibodies or antibody fragments. In some embodiments, the antibodies of the present disclosure can be prepared using methods known in the art. In some embodiments, the antibodies of the present disclosure can be prepared by immunizing an animal with an antigen comprising an amino acid fragment described herein. To increase immunogenicity, carrier proteins (including but not limited to BSA, ovalbumin, KLH, etc.) can be coupled to immunoreactive substances (eg, epitope peptides). Carrier proteins can include proteins or polypeptides that can function as immunogen carriers.
  • Polypeptides of these types include albumins, serum proteins, globulins, crystallins, lipoproteins and/or fragments thereof.
  • immunoreactive substances eg, but not limited to, monkeypox virus A29L protein fragments
  • monkeypox virus A29L protein fragments can be used to generate antibodies with affinity for the monkeypox virus A29L protein.
  • the antibody binds to the amino acid fragment corresponding to the monkeypox virus A29L protein. Binding may mean that the antibody is able to bind to the amino acid fragment, but the amino acid fragment is not necessarily the smallest binding fragment.
  • the effects of the disclosed antibodies can be tested using any suitable in vitro assay, cell-based assay, in vivo assay, animal model, etc.
  • the assay can include, for example, ELISA, FACS binding assay, Biacore, competitive binding assay, and the like.
  • the reactivity of an antibody of the present disclosure in binding to an antigen (antigenic peptide) is characterized in an ELISA, for example, a reaction value ⁇ 0.5 at 405 nm read by a peroxidase-labeled ELISA method is determined to be good. Reactive and can be used in immunoassays.
  • the kit includes a first antibody; in some embodiments, the kit includes a first antibody, a second antibody; in some embodiments, the kit includes a first antibody, a second antibody, and Third antibody; In some embodiments, other antibodies can also be used as coating antibodies or labeled antibodies.
  • the coated antibody binds to a solid phase.
  • the coating antibody may be bound to the solid phase directly or indirectly.
  • the coating antibodies can be used to coat solid supports.
  • the solid support is not particularly limited and may be, for example, magnetic particles, latex particles, microtiter plates, nitrocellulose membranes, or microfluidic chips.
  • the coated antibody binds to a binding partner, such as biotin/avidin, biotin/streptavidin; in some embodiments, the coated antibody is The binding partner is attached to the solid phase.
  • the labeled antibody is labeled with a detectable label.
  • the labeled antibody and detectable label may be directly or indirectly bound.
  • the detectable label is, for example, a metal particle, a fluorescent label, a chromophore label, an electron dense label, a chemiluminescent label, a radioactive label, or an enzyme label; in some embodiments, the detectable label can be, for example, Rhodamine, luciferin, fluorescent microspheres, colloidal gold, acridinium ester, luciferase, horseradish peroxidase, alkaline phosphatase, ⁇ -galactosidase, glucoamylase, lysozyme, sugar oxidation enzyme, glucose oxidase, galactose oxidase or glucose-6-phosphate dehydrogenase label.
  • the labeled antibody is linked to a binding partner, such as biotin/avidin, biotin/streptavidin. In some embodiments, the labeled antibody is labeled with a binding partner and a detectable label.
  • kits of the present disclosure include reagents suitable for performing immunoassays.
  • kits of the present disclosure can be used to perform immunoassays such as ELISA, indirect immunofluorescence assay IFA, radioimmunoassay RIA, and other non-enzyme-linked antibody binding assays or methods.
  • the present disclosure provides an antibody combination for detecting monkeypox virus A29L protein, including a first antibody and a second antibody as defined above; in some embodiments, a third antibody as defined above is also included.
  • the present disclosure provides a method for preparing an antibody for detecting monkeypox virus A29L protein, comprising immunizing an animal with an immunoreactive polypeptide comprising an amino acid fragment described herein as an antigen or hapten, respectively, thereby preparing Antibodies such as monoclonal antibodies or polyclonal antibodies that detect monkeypox virus A29L protein.
  • Monoclonal or polyclonal antibodies can be produced by methods known in the art.
  • the disclosure provides the use of an immunoreactive polypeptide comprising an amino acid fragment described herein in the preparation of a reagent or kit for detecting monkeypox virus A29L protein.
  • mice Take 8 to 12 weeks old BALB/c mice of the same strain as myeloma cells, mix A29L antigen containing 100 ⁇ g/mouse of protein with an equal amount of Freund's complete adjuvant, and inject into the abdominal cavity of the mice. Every 2 weeks, 100 ⁇ g/mouse of A29L antigen was thoroughly mixed with an equal amount of Freund's incomplete adjuvant, and injected multiple times into the abdominal cavity of mice to enhance immunization. After testing the mouse sera (indirect ELISA method), those with a titer above 1:2000 can be used for fusion. The mice were intraperitoneally boosted again 3 days before fusion, with a dose of 50 ⁇ g/mouse.
  • BALB/c mouse peritoneal macrophages were used as feeder cells.
  • the BALB/c mice were sacrificed by neck pull, and their whole bodies were immersed in 75% alcohol.
  • the abdominal skin was cut open with scissors under aseptic operation to expose the peritoneum, and 5 mL of RPMI 1640 basic culture medium was injected intraperitoneally with a syringe.
  • Rinse repeatedly, recover the rinse fluid, centrifuge at 1000 rpm for 5 minutes, leave the precipitate, resuspend in RPMI 1640 screening culture medium (RPMI 1640 complete culture medium containing HAT), adjust the cell concentration to 1 ⁇ 10 5 cells/mL, and add to 96 wells Plate, 150 ⁇ L/well, incubate overnight at 37°C, 5% CO2 .
  • mice Three days after the final immunization of the mice, remove the spleen under sterile conditions, place it in a dish, rinse it once with RPMI 1640 basic culture medium, grind it on a nylon mesh in a small beaker and filter it to make a cell suspension. Centrifuge, discard the supernatant, and resuspend in RPMI 1640 basic culture medium. Repeat three times and count.
  • Immune mouse splenocytes and mouse myeloma cells were treated with PEG to form a mixture of various cellular components, including unfused myeloma cells and immune splenocytes, synkaryosomes of myeloma cells and immune splenocytes. symkaryosomes, as well as nucleosomes in myeloma cells and immune splenocytes. Only the latter can form hybridoma cells. To this end, unfused cells and homofused symkaryosomes must be removed from this diverse cell mixture and true hybrid cells must be selected. Therefore, on days 1, 3, 5, and 7 after cell fusion, the culture medium was replaced with the aforementioned HAT culture medium.
  • Hybridoma cells were cloned using the limiting dilution method. After culture, a single cell can proliferate into a homologous cell clone; through reactivity screening, a cell line with good reactivity and stable secretion of anti-A29L monoclonal antibody can be obtained.
  • Microtiter plates (Nunc, Maxisorb) were coated with 100 ⁇ L/well of coating buffer containing 2.5 ⁇ g/mL monkeypox virus A29L protein (as antigen) for 1 hour at room temperature while stirring.
  • Post-coating treatment was performed by incubating in PBS buffer and 1% monoclonal antibody for 30 minutes. Subsequently, washing is performed with wash buffer. Incubate 100 ⁇ L/well antibody sample for 1 hour at room temperature with stirring. Then wash 2 more times with washing solution. Then incubate with 100 ⁇ L/well of detection antibody peroxidase-conjugated goat anti-mouse IgG diluted 1:40000 in PBS buffer for 1 hour at room temperature while stirring.
  • ELISA By ELISA, the cross-reactivity of the antibodies secreted by the cell lines with monkeypox virus A29L protein, vaccinia virus 162 protein and vaccinia virus A27L protein was identified.
  • the ELISA operation is as follows:
  • Coating Coat the microtiter plate with 100ul/well of MKPVA29L, VCA27L, and CPXV162 at 0.5 ⁇ g/ml and 0.05 ⁇ g/ml respectively, and incubate at 37°C for 2 hours.
  • Blocking Take 120ul of BSA blocking solution and incubate at 37°C for 2 hours.
  • the antibodies secreted by the cell lines were diluted with PBS to 0.5ug/ml, 0.05ug/ml, 50ul/well, and incubated at 37°C for 30 minutes.
  • Coat T line Coat T1 line and T2 line respectively with 56# antibody and 23# antibody diluted to 2.0 mg/mL with coating diluent; place in 37°C incubator for 60 minutes.
  • Process the sample pad Spray the colloidal gold-labeled 14# antibody solution on the sample pad.
  • the sample pad is 0.6cm wide ⁇ 10cm long, and 280 ⁇ L is spread.
  • the readings of the colloidal gold card include C1, C2, C3, C4, C5, C6, C7, C8, C9, and B, a total of 10 gradients, and the colors of the T lines corresponding to these 10 gradients from left to right are in order. becomes lighter, C1 means the T line is the darkest, C9 means the T line is the lightest, B means no color, and B+ means almost no color; "C3+” in Table 2 means a little darker than C3 but not C2. Others with "+” are analogous.
  • step 1 colloidal gold is used to label the 52# antibody
  • step 4 the 64# antibody is used to coat the T2 line.
  • the T1 line was coated with 2#, 9# and 56# antibodies respectively in parallel.
  • the test results of antibody pairing verification 1 and 2 are shown in Table 2 (52# antibody can also be replaced by 32# antibody with the same epitope. These three antibodies share the same epitope, which will be experimentally proven later; similarly, 64# antibody can also be used with 23 #Antibody substitution, these two antibodies share the same epitope, which will be experimentally proven later).
  • the two antibodies can be used interchangeably as labeled antibodies or coated antibodies, and the final detection results are also equivalent.
  • the results show that the combination of an antibody that binds to the 17-49aa segment of the monkeypox virus A29L protein and an antibody that binds to the monkeypox virus A29L protein but does not bind to the 17-49aa segment of the monkeypox virus A29L protein can effectively detect the monkeypox virus A29L protein. And it can be distinguished from the highly homologous vaccinia virus A27L protein and vaccinia virus 162 protein.
  • the experimental method is the same as the ELISA method in the previous embodiment, using different segments of A29L antigen peptides to coat the micropores respectively, using PBS as the diluent, diluting the monoclonal antibody concentration to 0.5-2 ⁇ g/mL, and using goat anti-mouse IgG-HRP as the diluent.
  • Secondary antibodies determine the specific A29L fragment that the monoclonal antibody binds to based on the response of each monoclonal antibody to different antigens.
  • the synthesized verification peptides are shown in Table 3 below, among which A29LA1-A29LA4 were used to refine and identify the specific epitopes of antibody 64# and antibody 23#.
  • A29LB-A29LF was used to refine and identify the specific epitope of the antibody that binds to the monkeypox virus A29L protein but does not bind to the 17-49aa segment of the monkeypox virus A29L protein.
  • the verification results are shown in Tables 4 and 5.
  • the specific epitopes of antibodies 64# and 23# are 19-25aa, while the specific epitopes of antibodies such as 52# are 81-110aa.
  • the antibody that binds to monkeypox virus A29L protein 19-25aa has a high degree of specificity.

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Abstract

一种猴痘病毒检测试剂盒，可用于检测猴痘病毒；采用猴痘病毒检测试剂盒，可以有效提高检测的特异性和/或检出率。

Description

一种猴痘病毒检测方法和试剂盒
相关申请的交叉引用
本公开要求于2022年8月23日提交至中国专利局的申请号为202211013297.6，发明名称为“一种猴痘病毒检测方法和试剂盒”的中国专利申请的优先权，其全部内容通过引用并入本公开。
本公开要求于2022年10月11日提交至中国专利局的申请号为202211239858.4，发明名称为“一种猴痘病毒检测方法和试剂盒”的中国专利申请的优先权，其全部内容通过引用并入本公开。
技术领域
本公开涉及蛋白检测领域。具体而言，本发明涉及猴痘病毒的检测方法和试剂盒。
背景技术
猴痘(Monkeypox)是一种由猴痘病毒(MPXV)引起的人畜共患病，最早发现于非洲西部和中部地区原始森林。猴痘病毒与痘苗病毒、天花病毒相似，临床表现为暴露10-12天后发热，发热2-3天后皮肤出疹。皮疹的变化，从斑丘疹，一直到小水疱、脓疱，大约10天左右出现结痂。病毒可以通过直接密切接触由动物传染给人，传染途径主要包括血液、体液、皮肤或黏膜伤口。人类二次传播主要通过空气飞沫传播，飞沫可感染眼睛、鼻子和喉咙的黏膜。
目前，聚合酶链反应PCR是国内外检测猴痘病毒的主要检测方法，但需要依赖设备，且较易产生污染。需要开发简便易普及的猴痘病毒免疫学检测方法。
发明内容
猴痘病毒A29L蛋白(MKPVA29L)是猴痘病毒的表面包膜蛋白(NCBI序列号NP_536566.1)，长度为110个氨基酸，与牛痘病毒162蛋白(CPXV-162)和痘苗病毒A27L蛋白(VACA27L)同源。
发明人经过大量理论研究和实验摸索，充分考虑猴痘病毒的A29L蛋白结构特性，对各种待测A29L抗原区间和检测抗体进行分析研究，获得了能够用于A29L检测的A29L抗原区域组合。
在一些实施方案中，本公开可以包括下述一项或多项：
1.一种猴痘病毒抗体，其包括结合猴痘病毒A29L蛋白的19-25aa的第一抗体。
2.一种猴痘病毒抗体组合，其包括项目1所述的第一抗体，并且包括结合猴痘病毒 A29L蛋白的81-110aa的第二抗体，
3.如项目2所述的猴痘病毒抗体组合，第一抗体为标记抗体时第二抗体为包被抗体；或第二抗体为标记抗体时第一抗体为包被抗体。
4.如项目2-3中任一项所述的抗体组合，其中所述标记抗体用可检测标记物和/或结合配偶体标记。
5.如项目4所述的抗体组合，所述可检测标记物选自金属粒子，荧光标记，发色团标记，电子致密标记，化学发光标记，放射性标记，或酶标记。
6.如项目5所述的抗体组合，所述可检测标记物可以是罗丹明，荧光素，荧光微球，胶体金，吖啶酯，荧光素酶，辣根过氧化物酶，碱性磷酸酶，β-半乳糖苷酶，葡糖淀粉酶，溶菌酶，糖氧化酶，葡萄糖氧化酶，半乳糖氧化酶或葡萄糖-6-磷酸脱氢酶标记。
7.如项目4所述的抗体组合，所述结合配偶体选自生物素/抗生物素蛋白，生物素/链霉抗生物素蛋白；例如标记抗体通过结合配偶体与可检测标记物进行标记。
8.如项目2-3中任一项所述的抗体组合，其中所述包被抗体连接至固相和/或结合配偶体。
9.如项目2-3任一项所述的抗体组合，所述固相例如是磁性颗粒，胶乳粒子、微量滴定板、硝酸纤维素膜或微流控芯片；结合配偶体例如是生物素/抗生物素蛋白，生物素/链霉抗生物素蛋白。
10.如项目8所述的抗体组合，所述包被抗体通过结合配偶体与固相进行连接。
11.一种试剂盒，包括第一抗体；任选地，还包括第二抗体；其中，第一抗体、第二抗体根据项目1-10中任一项所定义。
12.如项目1所述的猴痘病毒抗体或项目2-10中任一项所述抗体组合在制备检测猴痘病毒的试剂盒中的用途。
13.如项目12所述的用途，其中所述试剂盒用于对猴痘病毒进行检测。
14.一种抗原，所述抗原包括A29L蛋白的19-25aa片段。
15.一种制备项目1所述抗体或项目2所述的抗体组合的方法，所述方法包括：
1)使用含有下述片段1和/或片段2的肽段作为抗原或半抗原免疫动物：
片段1：猴痘病毒A29L蛋白氨基酸片段19-25aa；
片段2：猴痘病毒A29L蛋白氨基酸片段81-110aa；
和
2)从所述动物获得分别结合所述片段1或片段2的抗体。
16.一种编码项目1所述的抗体的核酸。
17.一种含有项目16所述的核酸的载体。
18.一种细胞，其含有项目16所述的核酸或项目17所述的载体。
具体实施方式
在一些实施方案中，本公开的试剂盒包括结合猴痘病毒A29L蛋白的19-25aa的第一抗体。
在一些实施方案中，第一抗体结合或特异性结合猴痘病毒A29L蛋白的17-49aa；在一些实施方案中，第一抗体结合或特异性结合猴痘病毒A29L蛋白的2-37aa；在一些实施方案中，第一抗体结合或特异性结合猴痘病毒A29L蛋白的19-31aa；在一些实施方案中，第一抗体结合或特异性结合猴痘病毒A29L蛋白的19-25aa。
在一些实施方案中，第一抗体不结合或不特异性结合猴痘病毒A29L蛋白的37～81aa。在一些实施方案中，第一抗体不结合或不特异性结合猴痘病毒A29L蛋白的61～93aa。在一些实施方案中，第一抗体不结合或不特异性结合猴痘病毒A29L蛋白的81～110aa。在一些实施方案中，第一抗体不结合或不特异性结合猴痘病毒A29L蛋白的25～37aa。在一些实施方案中，第一抗体不结合或不特异性结合猴痘病毒A29L蛋白的31～43aa。在一些实施方案中，第一抗体不结合或不特异性结合猴痘病毒A29L蛋白的37～49aa。
在一些实施方案中，第二抗体结合或特异性结合猴痘病毒A29L蛋白的81-110aa。在一些实施方案中，第二抗体结合或特异性结合猴痘病毒A29L蛋白的94-110aa。在一些实施方案中，第二抗体不结合或不特异性结合猴痘病毒A29L蛋白17～49aa。在一些实施方案中，第二抗体不结合或不特异性结合猴痘病毒A29L蛋白19～61aa。在一些实施方案中，第二抗体不结合或不特异性结合猴痘病毒A29L蛋白37～81aa。在一些实施方案中，第二抗体不结合或不特异性结合猴痘病毒A29L蛋白61～93aa。
结合是指抗体或抗原结合片段能够结合或捕获分析物或靶分子。在一个实施方案中，采用亲和力反应抗体与靶分子的结合，例如分析物结合抗体对于其靶分子具有KD≤10-7M的亲和力(KD表示平衡解离常数即亲和力常数)。在其他实施方案中，抗体对于其靶分子具有10-8M或甚至10-9M的亲和力。在一个实施方案中，抗体对其靶分子的亲和力为至少10-10M。在一些实施方案中，采用ELISA方法反应抗体与靶分子的结合，如本公开实施例测定的反应性，抗体对于其靶分子反应性例如OD读值大于等于0.5，或大于等于1.0，或大于等于1.5或大于等于2.0，或大于等于0.5、0.6、0.7、0.8、0.9、1.0、1.1、1.2、 1.3、1.4、1.5、1.6、1.7、1.8、1.9、2.0。如本领域技术人员所了解的，术语特异性用于指示抗体或抗原结合片段显著结合靶分子，但对其他分子不显著结合。不显著结合通常是指抗体与类似分子的结合能力显著低于靶分子，在一些方面，例如本公开实施例所述，采用ELISA对反应性进行测试时，抗体结合类似分子的反应性为结合靶分子的反应性的至多50％，至多40％，至多30％、至多20％，至多10％，至多5％或更低。在一个实施方案中，与靶分子相比，抗体与类似分子的结合亲和力是无法测量的，或者为高于10-7M的KD。在一个实施方案中，抗体结合类似分子的反应性低于0.5、低于0.4、低于0.3、低于0.2或低于0.1。
在一些实施方案中，本公开的试剂盒进一步包括结合A29L蛋白的81-110aa的抗体。
在一些实施方案中，本公开能够利用结合不同氨基酸片段的抗体识别抗原上的多个位置，降低漏检的风险，提高检出率。
在一些实施方案中，试剂盒包括检测试剂卡(试纸条)。
在一些实施方案中，试剂盒包括Elisa板。
在一些实施方案中，试剂盒包括磁珠。
在一些实施方案中，本公开可以通过荧光免疫层析进行或包括进行荧光免疫层析的试剂。在一些实施方案中，本公开可以通过胶体金免疫层析进行或包括进行胶体金免疫层析的试剂。本公开可以通过时间分辨荧光免疫层析进行或包括进行时间分辨荧光免疫层析的试剂。本公开可以通过量子点层析进行或包括进行量子点层析的试剂。在一些实施方案中，本公开可以通过磁珠免疫层析进行或包括进行磁珠免疫层析的试剂。
本文中的术语“抗体”在最广义上使用。在一些实施方案中，本公开的抗体可以是单克隆抗体或多克隆抗体。在一些实施方案中，本公开的抗体可以是全长抗体或抗体片段。在一些实施方案中，可以采用本领域已知的方法制备本公开的抗体。在一些实施方案中，可以以包含本文描述的氨基酸片段的抗原免疫动物制备本公开的抗体。为了增加免疫原性，可以将载体蛋白(包括但不限于BSA、卵清蛋白、KLH等)与免疫反应性物质(例如表位肽)偶联。载体蛋白可以包括蛋白质或多肽，其可以起免疫原载体的作用。这些类型的多肽包括白蛋白、血清蛋白、球蛋白、晶状体蛋白、脂蛋白和/或其片段。在一些实施方案中，免疫反应性物质(例如猴痘病毒A29L蛋白片段，但不限于此)可以用于产生具有对猴痘病毒A29L蛋白亲和力的抗体。
在一些实施方案中，抗体结合猴痘病毒A29L蛋白对应的氨基酸片段，结合可以指抗体能够结合所述氨基酸片段，但该氨基酸片段不一定是最小结合片段。
在一些实施方案中，可以使用任何适当的体外测定、基于细胞的测定、体内测定、动物模型等检测本公开抗体的效果如结合活性和/或交叉反应性。在一些实施方案中，所述测定可以包括例如ELISA，FACS结合测定，Biacore，竞争性结合测定等。在一些实施方案中，例如在ELISA中表征本公开的抗体与抗原(抗原肽)结合的反应性，例如通过过氧化物酶标记的ELISA法读取405nm处的反应值≥0.5确定为有较好反应性，可用于免疫测定。
在一些实施方案中，试剂盒中包括第一抗体；在一些实施方案中，试剂盒中包括第一抗体、第二抗体；在一些实施方案中，试剂盒中包括第一抗体、第二抗体和第三抗体；在一些实施方案中，还可以使用其他抗体作为包被抗体或标记抗体。
在一些实施方案中，所述包被抗体结合至固相。在一些实施方案中，所述包被抗体结合至固相的方式可以是直接或间接。在一些实施方案中，所述包被抗体可以用于包被固相支持物。在一些实施方案中，固相支持物没有特别限制，其可以是例如磁性颗粒，胶乳粒子、微量滴定板、硝酸纤维素膜或微流控芯片。在一些实施方案中，所述包被抗体结合至结合配偶体，结合配偶体例如是生物素/抗生物素蛋白，生物素/链霉抗生物素蛋白；在一些实施方案中，包被抗体通过结合配偶体与固相进行连接。
在一些实施方案中，所述标记抗体用可检测标记物标记。在一些实施方案中，所述标记抗体与可检测标记物可以是直接或间接结合。在一些实施方案中，可检测标记物例如金属粒子，荧光标记，发色团标记，电子致密标记，化学发光标记，放射性标记，或酶标记；在一些实施方案中，可检测标记物例如可以是罗丹明，荧光素，荧光微球，胶体金，吖啶酯，荧光素酶，辣根过氧化物酶，碱性磷酸酶，β-半乳糖苷酶，葡糖淀粉酶，溶菌酶，糖氧化酶，葡萄糖氧化酶，半乳糖氧化酶或葡萄糖-6-磷酸脱氢酶标记。在一些实施方案中，所述标记抗体与结合配偶体连接，结合配偶体例如是生物素/抗生物素蛋白，生物素/链霉抗生物素蛋白。在一些实施方案中，标记抗体通过结合配偶体与可检测标记物进行标记。
在一些实施方案中，本公开的试剂盒包括适合进行免疫测定的试剂。在一些实施方案中，本公开的试剂盒可以用于进行免疫测定，例如ELISA，间接免疫荧光测定IFA，放射免疫测定RIA以及其它非酶联抗体结合试验或方法。
在一些实施方案中，本公开提供用于检测猴痘病毒A29L蛋白的抗体组合，包括前述定义的第一抗体和第二抗体；在一些实施方案中，还包括前述定义的第三抗体。
在一些实施方案中，本公开提供制备检测猴痘病毒A29L蛋白的抗体的方法，包括将包含本文描述的氨基酸片段的免疫反应性多肽作为抗原或半抗原分别免疫动物，从而制备 检测猴痘病毒A29L蛋白的抗体如单克隆抗体或多克隆抗体。单克隆抗体或多克隆抗体可以通过本领域已知的方法进行。
在一些实施方案中，本公开提供包含本文描述的氨基酸片段的免疫反应性多肽在制备检测猴痘病毒A29L蛋白的试剂或试剂盒中的用途。
下面主要结合具体实施例对本公开作进一步详细的说明。提供以下实施例以说明本公开的实施方案，并非意在限制本公开。本公开可以任选包括未通过实施例示出的实施方案。
一、猴痘病毒A29L蛋白单克隆抗体的制备
1.免疫动物：取8～12周龄与骨髓瘤细胞同种系的BALB/c小鼠，以含蛋白质100μg/只的A29L抗原与等量福氏完全佐剂充分混匀，注入小鼠腹腔内，每隔2周100μg/只的A29L抗原与等量福氏不完全佐剂充分混匀，多次注入小鼠腹腔内加强免疫。经检测小鼠血清(间接ELISA法)，滴度在1:2000以上者可用于融合，融合前3天经小鼠腹腔内再次加强免疫，剂量为50μg/只。
2.饲养细胞的制备
以BALB/c鼠腹腔巨噬细胞作饲养细胞。在融合前1天，BALB/c鼠拉颈处死，75％酒精全身浸泡，超净台内，无菌操作下用剪刀剪开腹部皮肤，暴露腹膜，用注射器腹腔注入RPMI 1640基础培养液5mL，反复冲洗，回收冲洗液，1000rpm，离心5分钟，留沉淀，用RPMI 1640筛选培养液(含HAT的RPMI 1640完全培养液中)重悬，调整细胞浓度1×105个/mL，加入96孔板，150μL/孔，37℃，5％CO2培养过夜。
3.免疫脾细胞的制备
小鼠末次免疫后三天，在无菌条件下取出脾脏，置于平皿中，RPMI 1640基础培养液冲洗一次，放于小烧杯的尼龙网上磨碎过滤，制成细胞悬液。离心，弃上清，RPMI 1640基础培养液重悬，如此重复三次，计数。
4.细胞融合
(1)取40mL HAT培养液，15mL DMEM无血清培养液和1mL 50％PEG(M12000)分别置于37℃水浴中预温；
(2)分别取小鼠骨髓瘤细胞Sp2/0(菲鹏生物股份有限公司保存)(2-5×107)、上述免疫脾细胞(108)悬液加入50mL离心管中混匀，并加DMEM无血清培养液至40mL。离心10分钟，倒尽上清液，混匀；
(3)将离心管置于37℃预温的水中，取0.7mL预温的50％PEG溶液，静置90秒钟。立即滴加37℃15mL预温的无血清培养液；
(4)补加DMEM无血清培养液至40mL，离心10分钟，倒尽上清液。加40mL含15％～20％胎牛血清的HAT培养液。用吸管混匀，滴加到已含有饲养细胞的4块96孔细胞培养板的小孔中，每孔2滴，置37℃、7％CO2的培养箱中培养。
5.杂交瘤细胞的选择培养
免疫小鼠脾细胞与小鼠骨髓瘤细胞，经PEG处理后，形成多种细胞成分的混合体，其中包括未融合的骨髓瘤细胞和免疫脾细胞，骨髓瘤细胞的共核体和免疫脾细胞的共核体，以及骨髓瘤细胞和免疫脾细胞的导核体。仅后者才能形成杂交瘤细胞。为此，在这多种细胞混合体中必须除去未融合的细胞和同种融合的共核体，并选择出真正的杂交细胞。因此，在细胞融合后第1，3，5，7天用前述的HAT培养液换液培养。
6.抗体的检测及杂交瘤细胞克隆化
吸取每个培养孔的上清液，用间接ELISA法检测出培养液中含A29L蛋白的抗体的培养孔。采用有限稀释法使杂交瘤细胞克隆化。经过培养后单个细胞可增殖为同源性细胞克隆；经通过反应性筛选得到反应性较好且稳定分泌抗A29L单克隆抗体的细胞株。
反应性筛选的方法：在室温下，在搅拌时微量滴定板(Nunc，Maxisorb)用100μL/孔含2.5μg/mL猴痘病毒A29L蛋白(作为抗原)的包被缓冲液包被1小时。包被后处理在PBS缓冲液和1％单克隆抗体中进行孵育30分钟。随后，用洗涤缓冲液进行洗涤。在室温下，在搅拌时将100μL/孔抗体样品孵育1小时。然后用洗涤溶液再洗涤2次。然后在室温下，在搅拌时再与100μL/孔按1:40000用PBS缓冲液稀释的检测抗体过氧化物酶缀合的羊抗鼠IgG孵育1小时。用洗涤缓冲液再次洗涤后，用常规方法测定过氧化物酶活性(例如用ELISA读板器读取405nm处的反应值)，保留OD405≥0.5(有较好反应性)的抗体，得到包括56#、23#、14#、2#、9#和64#在内的24个细胞株。
二、抗体交叉反应性的鉴定
通过ELISA，鉴定细胞株分泌抗体与猴痘病毒A29L蛋白、牛痘病毒162蛋白和痘苗病毒A27L蛋白的交叉反应。ELISA操作如下：
包被：分别用0.5μg/ml和0.05μg/ml的MKPVA29L、VCA27L、CPXV162以100ul/孔包被微量滴定板，37℃孵育2h。
封闭:取120ul的BSA封闭液，在37℃孵育2h。
一抗/样品：细胞株分泌抗体均用PBS稀释至0.5ug/ml、0.05ug/ml，50ul/孔，37℃孵育30min。
阴性对照：加入PBS孵育
二抗：100ul/孔，羊抗鼠IgG-HRP 5K稀释in HIV-ED，37℃孵育30min。
显色：各加50ulA、B液，10min后加50ul终止液，读数。
结果如下表所示：
表1

56#、23#、14#、2#、9#、64#和52#六个抗体都会结合MKPVA29L，但只有23#和64#结合MKPVA29L的17-49aa区段；56#、14#、2#、9#和52#都会结合VACA27L和CPXV162，23#和64#不结合VACA27L和CPXV162，说明56#、14#、2#、9#和52#结合的是MKPVA29L、VACA27L和CPXV162的共同表位，23#和64#结合的是MKPVA29L的特有表位。
三、胶体金平台检测验证
1.抗体配对验证
1、胶体金标记：取10mL 4/万(40nm)的胶体金，加入60μL 0.2M K2CO3(pH≈6.8～7.2之间)，搅拌5min，加入100μg的14#抗体(所加抗体体积＝100μg/抗体浓度)，搅拌10min，再加入100μL 10％BSA封闭终止，标记搅拌10min；离心10000rpm，5min，去上清，沉淀用金子复溶液，复溶，最后用金子复溶液定容到1mL(即1/10胶体金溶液体积)，制得胶体金标记的14#抗体溶液。
2、配制金子工作液：用金子稀释液将步骤1制得的胶体金标记的14#抗体溶液最终稀释到10OD配制成金子工作液，铺于玻璃纤维上。
3、制备干燥好的金子：将铺好的金子放入冻干机冻干(2h)或者放37℃干燥房干燥过夜。
4、包被T线：将用包被稀释液稀释至2.0mg/mL的56#抗体和23#抗体分别包被T1线和T2线；放37℃恒温箱60min。
5、处理样品垫：将胶体金标记的14#抗体溶液喷涂在样品垫上。样品垫0.6cm宽×10cm长，铺280μL。
6、制备金标条：用切条机将金标条按需要的宽度切条，组装后加样进行检测。
分别测试MKPVA29L、VACA27L和CPXV162样品。需要说明的是，胶体金色卡的读数有C1、C2、C3、C4、C5、C6、C7、C8、C9、B一共10个梯度，且这10个梯度从左至右对应的T线颜色依次变浅，C1表示T线颜色为最深，C9表示T线颜色最浅，B表示不显色，B+为几乎不显色；表2中“C3+”是指比C3颜色更深一点但未达到C2，其他带“+”类推。
2.抗体配对验证
操作流程同上，只是步骤1中用胶体金标记52#抗体，步骤4中用64#抗体包被T2线， 平行地分别用2#、9#和56#抗体包被T1线。抗体配对验证1和2的测试结果见表2(52#抗体也可用表位相同的32#抗体替代，这三株抗体共享相同表位，后文有实验证明；同理64#抗体也可用23#抗体替代，这两株抗体共享相同表位，后文有实验证明)，对于双抗体夹心法检测，两个抗体可以互换地作为标记抗体或包被抗体，最终检测结果也相当。
表2
结果表明用结合猴痘病毒A29L蛋白17-49aa区段的抗体和结合猴痘病毒A29L蛋白但不结合猴痘病毒A29L蛋白17-49aa区段的抗体组合，能有效检测出猴痘病毒A29L蛋白，并且能与高度同源的痘苗病毒A27L蛋白以及牛痘病毒162蛋白区分开。
四、抗体结合片段的细化鉴定
实验方法同前述实施例中的ELISA法，采用不同区段的A29L抗原肽分别包被微孔，以PBS为稀释液，稀释单抗浓度到0.5-2μg/mL，以羊抗鼠IgG-HRP为二抗，依据各单抗对不同抗原的反应情况来确定单抗的具体所结合的A29L片段。所合成的验证肽段如下表3所示，其中A29LA1-A29LA4用于细化鉴定64#抗体和23#抗体的具体表位。A29LB-A29LF用于细化鉴定结合猴痘病毒A29L蛋白但不结合猴痘病毒A29L蛋白17-49aa区段的抗体的具体表位，验证结果如表4和表5所示。64#抗体和23#抗体的具体表位是19-25aa，而52#等抗体的具体表位是81-110aa。
表3
表4

表5
综合实施例中数据可以证明，结合猴痘病毒A29L蛋白19-25aa的抗体具有高度的特异性。
以上所述的具体实施例，对本公开的目的、技术方案和有益效果进行了进一步详细说明，应理解的是，以上所述仅为本公开的具体实施例而已，并不用于限制本公开，凡在本公开的精神和原则之内，所做的任何修改、等同替换、改进等，均应包含在本公开的保护范围之内。

Claims (18)

  1. 一种猴痘病毒抗体，其特征在于，其包括结合猴痘病毒A29L蛋白的19-25aa的第一抗体。
  2. 一种猴痘病毒抗体组合，其特征在于，其包括权利要求1所述的第一抗体，并且包括结合猴痘病毒A29L蛋白的81-110aa的第二抗体。
  3. 根据权利要求2所述的猴痘病毒抗体组合，其特征在于，第一抗体为标记抗体时第二抗体为包被抗体；或第二抗体为标记抗体时第一抗体为包被抗体。
  4. 如权利要求2-3中任一项所述的抗体组合，其特征在于，其中所述标记抗体用可检测标记物和/或结合配偶体标记。
  5. 如权利要求4所述的抗体组合，其特征在于，所述可检测标记物选自金属粒子，荧光标记，发色团标记，电子致密标记，化学发光标记，放射性标记，或酶标记。
  6. 如权利要求5所述的抗体组合，其特征在于，所述可检测标记物可以是罗丹明，荧光素，荧光微球，胶体金，吖啶酯，荧光素酶，辣根过氧化物酶，碱性磷酸酶，β-半乳糖苷酶，葡糖淀粉酶，溶菌酶，糖氧化酶，葡萄糖氧化酶，半乳糖氧化酶或葡萄糖-6-磷酸脱氢酶标记。
  7. 如权利要求4所述的抗体组合，其特征在于，所述结合配偶体选自生物素/抗生物素蛋白，生物素/链霉抗生物素蛋白。
  8. 如权利要求2-3中任一项所述的抗体组合，其特征在于，其中所述包被抗体连接至固相和/或结合配偶体。
  9. 如权利要求2-3中任一项所述的抗体组合，其特征在于，所述固相例如是磁性颗粒，胶乳粒子、微量滴定板、硝酸纤维素膜或微流控芯片；所述结合配偶体例如是生物素/抗生物素蛋白，生物素/链霉抗生物素蛋白。
  10. 如权利要求8所述的抗体组合，其特征在于，所述包被抗体通过结合配偶体与固相进行连接。
  11. 一种试剂盒，其特征在于，包括第一抗体；任选地，还包括第二抗体；其中，第一抗体、第二抗体根据权利要求1-10中任一项所定义。
  12. 如权利要求1所述的猴痘病毒抗体或权利要求2-10中任一项所述抗体组合在制备检测猴痘病毒的试剂盒中的用途。
  13. 如权利要求12所述的用途，其特征在于，其中所述试剂盒用于对猴痘病毒进行 检测。
  14. 一种抗原，其特征在于，所述抗原包括A29L蛋白的19-25aa片段。
  15. 一种制备权利要求1所述的抗体或权利要求2所述的抗体组合的方法，其特征在于，所述方法包括：
    1)使用含有下述片段1和/或片段2的肽段作为抗原或半抗原免疫动物：
    片段1：猴痘病毒A29L蛋白氨基酸片段19-25aa；
    片段2：猴痘病毒A29L蛋白氨基酸片段81-110aa；
    和
    2)从所述动物获得分别结合所述片段1或片段2的抗体。
  16. 一种编码权利要求1所述的抗体的核酸。
  17. 一种含有权利要求16所述的核酸的载体。
  18. 一种细胞，其特征在于，其含有权利要求16所述的核酸或权利要求17所述的载体。
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