CN116239680B - Monkey poxvirus A29 mouse monoclonal coated antibody and detection antibody - Google Patents
Monkey poxvirus A29 mouse monoclonal coated antibody and detection antibody Download PDFInfo
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Classifications
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- 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
- C07K16/081—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from DNA viruses
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56983—Viruses
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—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
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
- C07K2317/565—Complementarity determining region [CDR]
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
- G01N2333/01—DNA viruses
- G01N2333/065—Poxviridae, e.g. avipoxvirus
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2469/00—Immunoassays for the detection of microorganisms
- G01N2469/10—Detection of antigens from microorganism in sample from host
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention discloses a monkey pox virus A29 mouse monoclonal coating antibody, which comprises the following components: a heavy chain comprising or consisting essentially of SEQ ID NO:1, and consists of or consists essentially of the heavy chain variable region CDR-H1 shown in SEQ ID NO:2, and consists of or consists essentially of the heavy chain variable region CDR-H2 shown in SEQ ID NO:3, and a heavy chain variable region CDR-H3; a light chain comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO:4, and consists of or consists essentially of the light chain variable region CDR-L1 shown in SEQ ID NO:5, and consists of or consists essentially of the light chain variable region CDR-L2 shown in SEQ ID NO:6 and a light chain variable region CDR-L3. Also discloses a monkey pox virus A29 mouse monoclonal detection antibody and a corresponding kit. The coated antibody and the detection antibody developed by the invention can qualitatively or quantitatively detect the level of the monkey pox virus A29 protein in a sample.
Description
Technical Field
The invention relates to a monkey pox virus A29 mouse monoclonal coating antibody and a detection antibody, belonging to the technical field of virus detection.
Background
The monkey pox virus A29 mouse monoclonal detection antibody pair mainly comprises an anti-monkey pox virus A29 protein mouse monoclonal coating antibody, a monkey pox virus A29 protein mouse monoclonal detection antibody and a recombinant expressed monkey pox A29 protein standard (40891-V08E). The monoclonal antibody is obtained by immunizing animals with recombinant monkey pox A29 protein, screening by using a Beacon photoconductive platform, and purifying by using a protein A purification and antigen affinity purification technology. The enzyme-labeled antibody was labeled with horseradish peroxidase (HRP) according to a conventional method. The recombinant monkey pox A29 protein is obtained by HEK293 transient expression.
Monkey pox virus (MPXV) is an enveloped double-stranded DNA virus that is zoonotic and belongs to the genus orthopoxvirus of the family poxviridae. Orthopoxviruses also include smallpox virus, vaccinia virus, and vaccinia virus. The monkey poxvirus has two major branches, the congo basin branch and the western non branch. There are two forms of poxviruses: mature Virions (MVs), and a membrane-encased morphology (EV) outside the MVs. MV comprises a viral core component containing double-stranded DNA, protein side bodies and an outer membrane containing 20 viral proteins. MV is very stable and is thought to mediate transmission between hosts, whereas EV has a fragile outer membrane and mediates transmission between the same host cells. Vaccinia virus (VACV) a27 protein can interact with Heparan Sulfate (Heparan Sulfate) on the cell surface, mediating viral binding to cells and catalyzing cell fusion. The A29 protein is highly homologous with the vaccinia A27 protein, is a monkey pox virus intracellular Mature Virus (MV) surface envelope protein, and an epitope of a specific monoclonal antibody prepared by using the monkey pox virus is positioned on the A29 protein, so the A29 protein is a potential target for monkey pox antigen and antibody detection.
However, the current society is still under study on the disease treatment mechanism of the monkey pox virus, and the development of serological and immunological detection reagents based on the antibody of the key target spot of the monkey pox virus is also quite rare, so that the rapid detection of the monkey pox virus and the timely discovery of the infection cases of the monkey pox are faced with serious challenges.
Disclosure of Invention
The invention aims to provide a monkey pox virus A29 mouse monoclonal coated antibody and a detection antibody, which can qualitatively or quantitatively detect the level of a monkey pox virus A29 protein in a sample.
The invention adopts the technical scheme that:
a monkey poxvirus a29 mouse monoclonal coated antibody, characterized in that the antibody comprises:
a heavy chain comprising or consisting essentially of SEQ ID NO:1, and consists of or consists essentially of the heavy chain variable region CDR-H1 shown in SEQ ID NO:2, and consists of or consists essentially of the heavy chain variable region CDR-H2 shown in SEQ ID NO:3, and a heavy chain variable region CDR-H3;
a light chain comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO:4, and consists of or consists essentially of the light chain variable region CDR-L1 shown in SEQ ID NO:5, and consists of or consists essentially of the light chain variable region CDR-L2 shown in SEQ ID NO:6 and a light chain variable region CDR-L3.
Preferably, the amino acid sequence of the heavy chain is as shown in SEQ ID NO:7, the amino acid sequence of the light chain is shown as SEQ ID NO: shown at 8.
The application of the monkey pox virus A29 mouse monoclonal coated antibody in preparing a reagent for detecting monkey pox is provided.
The invention also discloses a kit for detecting the monkey pox, which comprises the monkey pox virus A29 mouse monoclonal coating antibody.
The invention discloses a monkey pox virus A29 mouse monoclonal detection antibody which comprises:
a heavy chain comprising or consisting essentially of SEQ ID NO:9, and consists of or consists essentially of the heavy chain variable region CDR-H1 shown in SEQ ID NO:10, and consists of or consists essentially of the heavy chain variable region CDR-H2 shown in SEQ ID NO:11, and a heavy chain variable region CDR-H3, as shown in seq id no;
a light chain comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO:12, and a light chain variable region CDR-L1 represented by or consisting essentially of SEQ ID NO:13, and consists of or consists essentially of the light chain variable region CDR-L2 shown in SEQ ID NO:14, and a light chain variable region CDR-L3.
Preferably, the amino acid sequence of the heavy chain is as shown in SEQ ID NO:15, the amino acid sequence of the light chain is shown as SEQ ID NO: shown at 16.
The application of the monkey pox virus A29 mouse monoclonal detection antibody in preparing a reagent for detecting monkey pox is provided.
The invention also discloses a kit for detecting the monkey pox, which comprises the monkey pox virus A29 mouse monoclonal detection antibody.
Preferably, the monkey poxvirus A29 mouse monoclonal coating antibody is also included.
According to the invention, by combining with a Beacon photoconductive platform, the monkey pox A29 immunized mouse monoclonal antibody is screened by single B cells, and the developed coated antibody and detection antibody can qualitatively or quantitatively detect the level of the monkey pox virus A29 protein in a sample. The development of the antibody pair can effectively relieve the shortage of monkey pox virus immunological detection reagents, and has wide market application prospect and great economic and social benefits.
Drawings
FIG. 1A standard curve of the A29 protein of monkey poxvirus.
FIG. 2 shows a schematic diagram of a mouse monoclonal coated antibody WB, wherein the monkey pox virus A29 mouse monoclonal coated antibody 1:1000 was diluted, lane A10 ng of the monkey pox virus A29 recombinant protein (Cat# 40891-V08E), goat anti-mouse secondary antibody/HRP 1:10000 was diluted.
FIG. 3 shows a schematic diagram of a mouse monoclonal antibody WB, wherein the monkey pox virus A29 mouse monoclonal antibody was diluted 1:1000, lane A10 ng of the monkey pox virus A29 recombinant protein (Cat# 40891-V08E), lane B10 ng of the vaccinia A27L recombinant protein, goat anti-mouse secondary antibody/HRP 1:10000.
Detailed Description
The present invention is further illustrated by the following examples, which are not intended to limit the scope of the invention in any way.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs, and the terms used herein in this description of the invention are for the purpose of describing particular embodiments only and are not intended to be limiting of the invention.
The materials or instruments used in the following examples, if not specifically described, were available from conventional commercial sources.
EXAMPLE 1 mouse immunization
Immunizing Balb/c mice, wherein each mouse has a single immunization dose of 50 mug, and the immunogen and an equal volume of complete Freund's adjuvant are prepared into an emulsifier by first immunization, and the emulsifier is injected subcutaneously in abdomen at multiple points; the same dose of immunogen was taken at 2 weeks intervals and an equal volume of incomplete Freund's adjuvant was formulated as an emulsifier for subcutaneous multipoint injection in the abdomen. Blood was taken one week after three immunizations and serum binding titers were determined by an indirect ELISA method. The standard of qualified titer is that the OD450-blank is more than 1.0 when the serum of the mice is diluted by 1:16000 times. The ELISA titer-failed mice continue to immunize until the titers are qualified, and a maximum of five immunizations are recommended; mice were selected for booster immunization, and spleen of the mice was taken 3 days after booster immunization for Beacon sorting.
EXAMPLE 2Beacon single B cell screening
Taking immune tissues such as spleen, bone marrow, lymph node and the like of the immunized mice, grinding the immune tissues to single cell suspension,
red blood cells are lysed, magnetic beads of mouse CD138 antibody are added for incubation, and CD138 plasma cells are obtained through positive selection. The separated plasma cells were programmed into a chip and the cells were introduced into a NanoPen cell (NanoPen) by the optical electrical localization (OEP) technique. The microspheres coated with the antigen and the fluorescent secondary antibody are led into a chip channel, and positive cells secreting specific antibodies are screened through multiple rounds of photographing. Analyzing the data to determine the position of the positive cells at the Nanopen, and exporting the cells from the Nanopen to a 96-well plate containing a cell lysate by an OEP mode.
EXAMPLE 3 Single B cell variable region amplification and vector construction
After single cells derived from Beacon are lysed, oligo-d (T), dNTP and other reverse transcription systems are added into the lysate, and cDNA is prepared by a two-step reverse transcription method. The cDNA is used as a template, and the variable region genes of the heavy and light chains of the antibody are gradually amplified by adopting a multi-round progressive amplification mode and taking the PCR product of the previous round as the template for the next round of gene amplification. Adding the heavy and light chain variable region fragments into membrane binding solution, fully and uniformly mixing, adding into a purification column, and eluting by using a nucleic-Free Water to obtain purified heavy and light chain variable region gene fragments. The gene variable region fragment was inserted into an expression vector with constant region sequence, and the ligation product transformed competent cells, cultured overnight at 37 ℃. And (3) sequencing the obtained monoclonal amplified antibody to obtain the heavy and light chain sequence of the antibody. The correct vector was sequenced to extract the plasmid and deliver the pilot expression.
EXAMPLE 4 monoclonal antibody expression and purification
HEK293 cells are transfected by expression plasmids, cell culture is carried out by using a shake flask or a bioreactor, cell culture supernatant is collected, purification is carried out by using a conventional protein A affinity chromatography column, the purity and the specificity of the obtained antibody are identified by SDS-PAGE electrophoresis and an indirect ELISA method, and then split charging is carried out, and the obtained antibody is preserved at a low temperature of minus 20 ℃ for standby.
EXAMPLE 5 Western immunoblotting
The protein sample to be detected is added with 2×loading buffer in equal volume, and the concentration of the protein sample is more than 1mg/mL and 20 mu L/hole. Constant pressure was set at 100V until the bottom of the offset plate where bromophenol blue runs. The membrane was placed in methanol for 2 minutes and transferred at a constant pressure of 110V for 1 hour. 5% skim milk was blocked overnight at 4 ℃. The primary antibody is diluted with 5% skim milk according to the ratio of 1:1000, and the diluted primary antibody is added into a corresponding antibody incubation box and incubated for 2 hours at room temperature. The membrane was eluted 3 times, 5 minutes each time using a decolorizing shaker. Diluted secondary antibody (HRP-labeled goat anti-mouse secondary antibody) was added and reacted at room temperature for 2 hours. The membrane was eluted 3 times, 5 minutes each time using a decolorizing shaker. Color development was performed using a chemiluminescent imaging system. Wherein FIG. 2 shows the result of detecting the affinity of the coated antibody when the primary antibody is a monkey pox virus A29 mouse monoclonal coated antibody, and FIG. 3 shows the result of detecting the affinity of the detected antibody when the primary antibody is a monkey pox virus A29 mouse monoclonal detected antibody.
Example 6 monkey pox A29 protein ELISA detection
And taking out the coated ELISA plate and the freeze-dried standard substance, wherein the ELISA plate is coated by adopting a monkey pox virus A29 mouse monoclonal coating antibody in advance, and adding 1mL of sample diluent to dissolve the standard substance. Left at room temperature for 20 minutes. The standard was diluted 2-fold from 1500pg/mL, 7 concentration gradients were added, and 100. Mu.L of each dilution was added to a 96-well ELISA plate. Taking a sample to be detected, adding 100 mu L/hole into a reaction hole, and incubating for 2 hours at room temperature; the plate was washed 3 times with wash solution, 200. Mu.L/well, and the ELISA plate was dried by pipetting. HRP-labeled monkey pox virus A29 mouse monoclonal detection antibody was diluted to 1. Mu.g/mL with sample dilution, added to the reaction wells, incubated at 100. Mu.L/well for 1 hour at room temperature, washed 3 times with wash solution, 200. Mu.L/well, and the ELISA plates were blotted dry. 200. Mu.L of freshly prepared substrate chromogenic solution was added, the reaction was carried out at room temperature for 20 minutes, and then stopped by adding 50. Mu.L of stop solution, and the absorbance was read at a wavelength of 450nm by an ELISA reader. And (3) drawing a standard curve (figure 1) by adopting linear regression with the concentration of the standard substance as an abscissa and the OD value as an ordinate, and calculating the content of the monkey pox virus A29 protein in the sample according to the measured OD value of the sample. If the sample is diluted, the A29 concentration in the sample can be obtained by multiplying the calculated result by the dilution times. The linear range of detection is 23.44-1500pg/mL, with a minimum detection concentration of 2.65pg/mL. The lowest detection concentration was calculated as zero-well OD450 absorbance plus three standard deviations of the corresponding concentration of a 29.
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.
Claims (6)
1. A monkey poxvirus a29 mouse monoclonal coated antibody, characterized in that the antibody comprises:
a heavy chain comprising a sequence consisting of SEQ ID NO:1, and a heavy chain variable region CDR-H1 consisting of SEQ ID NO:2, and a heavy chain variable region CDR-H2 consisting of SEQ ID NO:3, and a heavy chain variable region CDR-H3;
a light chain comprising a sequence consisting of SEQ ID NO:4, and a light chain variable region CDR-L1 consisting of SEQ ID NO:5, and a light chain variable region CDR-L2 consisting of SEQ ID NO:6 and a light chain variable region CDR-L3.
2. The monkey poxvirus a29 mouse monoclonal coated antibody according to claim 1, characterized in that:
the amino acid sequence of the heavy chain is shown in SEQ ID NO:7, the amino acid sequence of the light chain is shown as SEQ ID NO: shown at 8.
3. Use of a monkey pox virus a29 mouse monoclonal coated antibody according to claim 1 or 2 for the preparation of a reagent for detecting monkey pox.
4. A kit for detecting monkey pox, comprising the monkey pox virus a29 mouse monoclonal coated antibody of claim 1 or 2.
5. The kit of claim 4, further comprising a monkey pox virus a29 mouse monoclonal detection antibody, the monkey pox virus a29 mouse monoclonal detection antibody comprising:
a heavy chain comprising a sequence consisting of SEQ ID NO:9, and a heavy chain variable region CDR-H1 consisting of SEQ ID NO:10, and a heavy chain variable region CDR-H2 consisting of SEQ ID NO:11, and a heavy chain variable region CDR-H3;
a light chain comprising a sequence consisting of SEQ ID NO:12, and a light chain variable region CDR-L1 consisting of SEQ ID NO:13, and a light chain variable region CDR-L2 consisting of SEQ ID NO:14, and a light chain variable region CDR-L3.
6. The kit according to claim 5, wherein the amino acid sequence of the heavy chain of the monkey poxvirus a29 mouse monoclonal antibody is as set forth in SEQ ID NO:15, the amino acid sequence of the light chain is shown as SEQ ID NO: shown at 16.
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CN202311518779.1A CN117700533A (en) | 2023-02-09 | 2023-02-09 | Monkey poxvirus A29 mouse monoclonal coated antibody and detection antibody |
CN202310090089.4A CN116239680B (en) | 2023-02-09 | 2023-02-09 | Monkey poxvirus A29 mouse monoclonal coated antibody and detection antibody |
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A highly specific monoclonal antibody against monkeypox virus detects the heparin binding domain of A27;Laura J Hughes等;《Virology .》;全文 * |
Kinetic and Structural Aspects of Glycosaminoglycan-Monkeypox Virus Protein A29 Interactions Using Surface Plasmon Resonance;Shi Deling等;《Molecules .》;第27卷(第18期);全文 * |
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