CN116444658A - New crown antibody and application thereof - Google Patents

New crown antibody and application thereof Download PDF

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CN116444658A
CN116444658A CN202211089322.9A CN202211089322A CN116444658A CN 116444658 A CN116444658 A CN 116444658A CN 202211089322 A CN202211089322 A CN 202211089322A CN 116444658 A CN116444658 A CN 116444658A
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antibody
epitope
seq
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cov
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刘春艳
张翼
易嘉乐
罗沛
钟振宇
李瑞净
刘旭霞
秦汤
吴仁贞
阳馨滢
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Fapon Biotech Inc
Guangdong Fapon Biotech Co Ltd
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Guangdong Fapon Biotech Co Ltd
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    • C07—ORGANIC CHEMISTRY
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    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
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    • G01N33/56983—Viruses
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    • 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
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    • 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
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    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
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    • G01N2333/08—RNA viruses
    • G01N2333/165—Coronaviridae, e.g. avian infectious bronchitis virus
    • 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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Abstract

The present disclosure relates to a novel crown antibody and uses thereof. The antibody provided by the disclosure has high sensitivity, good specificity and no cross reaction, and has important effect on diagnosis of novel coronaviruses.

Description

New crown antibody and application thereof
Cross Reference to Related Applications
The present disclosure claims priority from chinese patent application No. CN202111063235.1, entitled "a novel crown antibody or antigen binding fragment thereof and use thereof," filed on 10/09/2021, the entire contents of which are incorporated herein by reference.
Technical Field
The present disclosure is in the field of protein detection. More particularly, it relates to a novel crown antibody or antigen-binding fragment thereof and uses thereof.
Background
Coronavirus is a enveloped single-stranded positive-strand RNA virus. Coronaviruses are divided into four genera based on their genetic evolution, serology and host-specific differences: alpha coronavirus genus, beta coronavirus genus, gamma coronavirus genus and delta coronavirus genus. Viruses of the genus betacoronavirus can be divided into four groups A, B, C, D. The alpha and beta coronaviruses primarily infect mammals including humans, domestic animals, pets, and the gamma and delta coronaviruses primarily infect birds and mammals. The novel coronavirus belongs to the genus beta-coronavirus, and from the position of the clade, 2019-nCoV (i.e., 2019 novel coronavirus, also known as "SARS-CoV-2") is adjacent to the class of SARS and SARS-like viruses, whose evolutionarily common outer group is an HKU9-1 coronavirus parasitic to fruit bats. 2019-nCoV is coated, and the particles are round or elliptic, often polymorphic, and have diameters of 50-200nm.
Currently, there is a global need for the sensitivity, specificity, and presence or absence of cross-reactions of reagents for screening, detecting, and diagnosing novel coronaviruses.
Disclosure of Invention
SARS-CoV-2 nucleocapsid protein is 419 amino acids in length and its sequence is well known, e.g., from GenBank: UGZ64655.1.
The present disclosure provides an antibody that binds to amino acid fragments 44-180 of SARS-CoV-2 nucleocapsid protein.
Further, the antibody does not bind to the amino acid fragments at positions 44-84, 65-103, 96-136, and 130-180 of SARS-CoV-2 nucleocapsid protein.
Still further, the antibody binds to an epitope that is identical to an epitope comprising SEQ ID NO:1 and the heavy chain variable region of SEQ ID NO:2 or an epitope to which an antibody comprising the light chain variable region of SEQ ID NO:5 and SEQ ID NO:6, the epitope to which the antibody of the light chain variable region binds is identical.
The present disclosure provides an antibody combination comprising antibody 1 and antibody 2, the antibody 1 binding to an epitope that is complementary to an epitope comprising SEQ ID NO:1 and the heavy chain variable region of SEQ ID NO:2, and said antibody 2 binds to an epitope that is identical to an epitope comprising the light chain variable region of SEQ ID NO:5 and SEQ ID NO:6, the epitope to which the antibody of the light chain variable region binds is identical.
The present disclosure also provides an antibody combination comprising antibody 3 and antibody 4, wherein the antibody 3 is an antibody that binds to amino acid fragments 44-180 of SARS-CoV-2 nucleocapsid protein but does not bind to amino acid fragments 44-84, 65-103, 96-136, and 130-180 of SARS-CoV-2 nucleocapsid protein, and the antibody 4 is an antibody that binds to amino acid fragments 65-103 of SARS-CoV-2 nucleocapsid protein.
Further, antibody 3 binds to an epitope that is identical to an epitope comprising SEQ ID NO:1 and the heavy chain variable region of SEQ ID NO:2 or an epitope to which an antibody comprising the light chain variable region of SEQ ID NO:5 and SEQ ID NO:6, the epitope to which the antibody of the light chain variable region binds is identical.
Further, antibody 4 binds to an epitope that is identical to an epitope comprising SEQ ID NO:9 and SEQ ID NO:10, and the epitope to which the antibody of the light chain variable region binds is the same.
The disclosure also provides a kit comprising the antibody, or the combination of antibodies.
The disclosure also provides for the use of the antibodies, or the antibody combinations, in the preparation of a kit.
The present disclosure also provides a method for preparing the antibody, which comprises preparing the antibody by hybridoma technology or phage display technology by using the 44 th-180 th amino acid fragment of SARS-CoV-2 nucleocapsid protein as antigen.
The present disclosure also provides a method of preparing the antibody, the method comprising: 1) Immunizing an animal with an antigen or hapten comprising an amino acid fragment of SARS-CoV-2 nucleocapsid protein at positions 44-180; and 2) obtaining antibodies binding to amino acid fragments 44-180 of SARS-CoV-2 nucleocapsid protein from ascites of said animal.
In some embodiments, the method of making the antibody further comprises screening for antibodies that do not bind to amino acid fragments above by SARS-CoV-2 nucleocapsid protein amino acid fragments 44-84, amino acid fragments 65-103, amino acid fragments 96-136, and amino acid fragments 130-180.
The disclosure also provides the use of the antibodies, or the combination of antibodies, or the kit, for detecting SARS-CoV-2.
The present disclosure also provides a method of detecting SARS-Cov-2, comprising:
a) Contacting the antibody, or the combination of antibodies, or the kit, with a sample under conditions sufficient for a binding reaction to occur; and
b) Detecting immune complexes generated by the binding reaction.
The present disclosure also provides a method of diagnosing a subject as being infected with SARS-Cov-2 or a disease associated with SARS-Cov-2 infection, comprising:
a) Contacting the antibody, or the combination of antibodies, or the kit with a sample from the subject under conditions sufficient for a binding reaction to occur; and
b) Detecting immune complexes generated by the binding reaction.
In some embodiments, the disease associated with SARS-Cov-2 infection includes respiratory symptoms, fever, cough, breathlessness, dyspnea, pneumonia, severe acute respiratory syndrome, renal failure.
Detailed Description
SARS-CoV-2 belongs to the family of coronaviruses and is a non-segmented, single-stranded positive-strand RNA virus. It encodes two large overlapping open reading frames (ORF 1a and ORF1 b), 4 structural proteins (S, E, M and N proteins), and 9 helper proteins. Wherein, the N protein is the core component of the virion, which binds to viral genomic RNA, and packages the RNA into Ribonucleoprotein (RNP) complexes. In addition to assembly, N proteins play an important role in viral mRNA transcription and replication and are involved in immunomodulation. It has been reported that N protein can bind to double-stranded RNA and has RNAi inhibitory activity, and can combat host RNAi-mediated antiviral response, and at the same time, N protein can induce humoral and cellular immune responses after infection, making it a key target for early rapid diagnosis and vaccine development.
SARS-CoV-2N protein is 419 amino acids in length and 43-50kDa in size. The N protein has three relatively conserved domains, one of which can intertwine with viral genomic RNA to form a viral nucleocapsid, which plays an important role in the synthesis of viral RNA, and is involved in viral genome replication and in regulating cell signaling pathways. The N protein is a phosphorylated protein, and the phosphorylation can regulate the conformation of the N protein, enhance the conformation with viral proteins and enhance the affinity with viral RNA. During nucleocapsid packaging, the N protein can interact with the M protein, facilitating nucleocapsid packaging into virions. The organism can produce high-level antibodies against N protein in early infection, and a method for rapidly detecting 2019-nCoV serum antibodies can be established by utilizing the N protein. Therefore, the N protein is often used as a diagnostic and detection tool for coronaviruses and is a core raw material of an immunological rapid diagnostic reagent.
In conclusion, 2019-nCoV N protein detection plays an important role in diagnosis of novel coronaviruses, and development of monoclonal antibodies for 2019-nCoV N protein detection has an important significance.
Some embodiments of the present disclosure provide an antibody, wherein the antibody binds to amino acid fragments 44-180 of SARS-CoV-2 nucleocapsid protein. In some embodiments, the antibody may also not bind to amino acid fragments 44-84, 65-103, 96-136, and 130-180 of SARS-CoV-2 nucleocapsid protein.
As used herein, the term "antibody" is used in its broadest sense and may include full length antibodies and antigen binding fragments thereof, and may include monospecific, bispecific or multispecific antibodies, as well as chimeric antibodies so long as they exhibit the desired biological activity. The term "antigen binding fragment" is a substance comprising a portion or all of the CDRs (complementarity determining regions) of an antibody that lacks at least some of the amino acids present in the full-length chain but is still capable of specifically binding to an antigen. Such fragments are biologically active in that they bind to an antigen and can compete with other antigen binding molecules (including intact antibodies) for binding to a given epitope. Such fragments are selected from Fab (antigen binding fragment) (consisting of an intact light chain and Fd), fv (consisting of VH and VL), scFv (single chain antibody, with a linker peptide between VH and VL) or single domain antibody (consisting of VH only). Such fragments may be produced by recombinant nucleic acid techniques, or may be produced by enzymatic or chemical cleavage of antigen binding molecules, including intact antibodies.
In some embodiments, the antibody binds to an epitope that binds to a polypeptide comprising SEQ ID NO:1 and the heavy chain variable region of SEQ ID NO:2 or an epitope to which an antibody comprising the light chain variable region of SEQ ID NO:5 and SEQ ID NO:6, the epitope to which the antibody of the light chain variable region binds is identical. In some embodiments, the antibody binds to an epitope that binds to a polypeptide comprising SEQ ID NO:3 and SEQ ID NO:4 or an epitope to which an antibody comprising the light chain of SEQ ID NO:7 and SEQ ID NO:8, and the epitope bound by the antibody of the light chain is the same.
In the present disclosure, whether an antibody recognizes the same epitope as other antibodies can be confirmed by competition of both for the epitope. Competition between antibodies can be assessed by competition binding assays by: ELISA, fluorescent energy transfer assay, or fluorescent microassay techniques, etc.
In some embodiments, an antibody that binds to an amino acid fragment corresponding to SARS-CoV-2 nucleocapsid protein refers to an antibody that is capable of binding to the amino acid fragment, but the amino acid fragment is not necessarily the smallest binding fragment.
In some embodiments, the disclosure provides an antibody combination comprising antibody 1 and antibody 2, the antibody 1 binding to an epitope that is complementary to an epitope comprising SEQ ID NO:1 and the heavy chain variable region of SEQ ID NO:2, and said antibody 2 binds to an epitope that is identical to an epitope comprising the light chain variable region of SEQ ID NO:5 and SEQ ID NO:6, the epitope to which the antibody of the light chain variable region binds is identical.
In some embodiments, the disclosure provides an antibody combination comprising antibody 1 and antibody 2, the antibody 1 binding to an epitope that is complementary to an epitope comprising SEQ ID NO:3 and SEQ ID NO:4, and the antibody 2 binds to an epitope that is identical to an epitope comprising the light chain of SEQ ID NO:7 and SEQ ID NO:8, and the epitope bound by the antibody of the light chain is the same.
In some embodiments, the disclosure also provides an antibody combination comprising antibody 3 and antibody 4.
In some embodiments, the antibody 3 binds to amino acid fragments 44-180 of SARS-CoV-2 nucleocapsid protein. In some embodiments, antibody 3 may also not bind to amino acid fragments 44-84, 65-103, 96-136, and 130-180 of SARS-CoV-2 nucleocapsid protein. In some embodiments, the antibody 3 binds to an epitope that is identical to an epitope comprising SEQ ID NO:1 and the heavy chain variable region of SEQ ID NO:2 or an epitope to which an antibody comprising the light chain variable region of SEQ ID NO:5 and SEQ ID NO:6, the epitope bound by the antibody of the light chain variable region is the same; the antibody 4 binds to amino acid fragments 65-103 of SARS-CoV-2 nucleocapsid protein.
In some embodiments, the antibody 4 binds to an epitope that comprises SEQ ID NO:9 and SEQ ID NO:10, and the epitope to which the antibody of the light chain variable region binds is the same. The antibody 4 binds to an epitope that is identical to an epitope comprising SEQ ID NO:11 and SEQ ID NO:12, and the epitope bound by the antibody of the light chain is the same.
In some embodiments, the disclosure further provides a kit, wherein the kit comprises the antibodies, the combination of antibodies.
In some embodiments, the effects of antibodies of the present disclosure, such as binding activity and/or cross-reactivity, can be detected using any suitable in vitro assay, cell-based assay, in vivo assay, animal model, and the like. In some embodiments, the assay may include, for example, ELISA (enzyme linked immunosorbent assay), FACS (flow cytometry fluorescence sorting technique) binding assay, biacore, competitive binding assay, and the like. In some embodiments, the reactivity of the antibodies of the present disclosure with antigen (antigenic peptide) binding is characterized, for example, in ELISA, e.g., a reaction value ∈ 0.5 at 405nm as determined by peroxidase-labeled ELISA is better reactive and useful in immunoassays.
In some embodiments, wherein the antibody is optionally labeled with a detectable label. In some embodiments, the detectable label is, for example, colloidal gold, radiolabel, luminescent material, colored material, enzyme, for example, fluorescent label, chromophore label, electron dense label, for example, radioisotope, fluorophore, rhodamine and its derivatives, luciferase, luciferin, horseradish peroxidase, alkaline phosphatase, β -galactosidase, glucoamylase, lysozyme, carbohydrate oxidase, glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase, biotin/avidin, spin label.
In some embodiments, the disclosure also provides for the use of the antibodies, or the combination of antibodies, in the preparation of a kit.
In some embodiments, the disclosure also provides methods of making the antibodies.
The antibody is prepared by using SARS-CoV-2 nucleocapsid protein 44-180 amino acid fragment as antigen by hybridoma technique or phage display technique.
Some embodiments of the present disclosure provide a method of making the antibody, which is a monoclonal antibody, comprising making the monoclonal antibody with the amino acid fragments at positions 44-180 of the SARS-CoV-2 nucleocapsid protein as an antigen.
Some embodiments of the present disclosure also provide a method of preparing the antibody, the method comprising: 1) Immunizing an animal with an antigen or hapten comprising an amino acid fragment of SARS-CoV-2 nucleocapsid protein at positions 44-180; and 2) obtaining antibodies binding to amino acid fragments 44-180 of SARS-CoV-2 nucleocapsid protein from ascites of said animal.
Some embodiments of the present disclosure also provide the use of the antibody, or the combination of antibodies, or the kit for detecting SARS-CoV-2.
Some embodiments of the present disclosure also provide a method of detecting SARS-Cov-2, comprising:
a) Contacting the antibody, or the combination of antibodies, or the kit, with a sample under conditions sufficient for a binding reaction to occur; and
b) Detecting immune complexes generated by the binding reaction.
Some embodiments of the present disclosure also provide a method of diagnosing whether a subject is infected with SARS-Cov-2 or has a disease associated with SARS-Cov-2 infection, comprising:
a) Contacting the antibody, or the combination of antibodies, or the kit with a sample from the subject under conditions sufficient for a binding reaction to occur; and
b) Detecting immune complexes generated by the binding reaction.
In some embodiments, the disease associated with SARS-Cov-2 infection comprises at least one of respiratory symptoms, fever, cough, breathlessness, dyspnea, pneumonia, severe acute respiratory syndrome, renal failure.
The antibody provided by the disclosure has high sensitivity, good specificity and no cross reaction, and has important effect on diagnosis of novel coronaviruses.
Examples
Embodiments of the present disclosure will be described in detail below with reference to examples.
EXAMPLE 1 preparation of novel crown N protein monoclonal antibodies
1. Immunization of animals
Taking 8-12 week old BALB/c mice with the same line as myeloma cells, fully and uniformly mixing 2019-nCoV N protein recombinant antigen containing 100 mug/mouse with equal amount of Freund's complete adjuvant, injecting into the abdominal cavity of the mice, fully and uniformly mixing 2019-nCoV N protein recombinant antigen containing 100 mug/mouse every 2 weeks with equal amount of Freund's Incomplete Adjuvant (FIA), and injecting into the abdominal cavity of the mice for multiple times to strengthen immunity. The serum of the mice is detected (indirect ELISA method), the titer is more than 1:2000, the mice can be used for fusion, the mice are boosted in abdominal cavity for 3 days before fusion, and the dosage is 50 mug/mouse.
2. Preparation of feeder cells
BALB/c murine peritoneal macrophages were used as feeder cells. 1 day before fusion, BALB/c mice were sacrificed by pulling the neck, 75% alcohol was soaked whole body, in a super clean bench, the abdominal skin was cut off with scissors under aseptic operation, the peritoneum was exposed, 5mL of RPMI 1640 basal medium was injected with a syringe, washing was repeated, the washing liquid was recovered, 1000rpm, centrifugation was carried out for 5 minutes, precipitation was left, the culture broth (in the complete culture broth of RPMI 1640 containing HAT) was screened with RPMI 1640, and the cell concentration was adjusted to 1X 10 5 Individual cells/mL, add 96-well plate, 150. Mu.L/well, 37 ℃,5% CO 2 Culturing overnight.
3. Preparation of immune spleen cells
Three days after the last immunization of the mice, the spleens were taken out under aseptic conditions, placed in a plate, rinsed once with RPMI 1640 basal culture solution, placed on a nylon mesh of a small beaker, and ground and filtered to prepare a cell suspension. Centrifugation, discarding supernatant, re-suspending the RPMI 1640 basal medium, repeating the above steps three times, and counting.
4. Cell fusion
(1) Taking 40mL of HAT culture solution, 15mL of DMEM serum-free culture solution and 1mL of 50% PEG (M12000), and respectively placing in a water bath at 37 ℃ for pre-heating;
(2) The mouse myeloma cells Sp2/0 (stored by the Phpeng organism Co., ltd.) were taken separately (2.about.5X10) 7 ) The above-mentioned immune spleen cells (10) 8 ) The suspension was added to a 50mL centrifuge tube and mixed well, and DMEM serum-free medium was added to 40mL. Centrifuging for 10 min, pouring out supernatant, and mixing;
(3) The centrifuge tube was placed in 37℃pre-warmed water, 0.7mL of pre-warmed 50% PEG solution was taken and allowed to stand for 90 seconds. Immediately dropwise adding 15mL of preheated serum-free culture solution at 37 ℃;
(4) The DMEM serum-free medium was added to 40mL, centrifuged for 10 minutes, and the supernatant was discarded. 40mL of HAT culture solution containing 15% -20% (v) fetal bovine serum is added. Mixing with a pipette, dripping into small holes of 4 96-well cell culture plates containing feeder cells, 2 drops per well, placing at 37deg.C and 7% CO 2 Is cultured in an incubator of (a).
5. Selective culture of hybridoma cells
Immunizing a mouse spleen cell and a mouse myeloma cell, and forming a mixture of a plurality of cell components after PEG treatment, wherein the mixture comprises unfused myeloma cells and immune spleen cells; the synuclei of myeloma cells and the synuclei of immune spleen cells, and the heterokaryons of myeloma cells and immune spleen cells. Only the latter is able to form hybridoma cells. For this purpose, unfused cells and homofusion co-nuclei must be removed from the mixture of cells and true hybrid cells must be selected. Therefore, the HAT medium was used for the culture at 1, 3, 5 and 7 days after cell fusion.
6. Detection of specific antibodies and hybridoma cell cloning
The supernatant from each well was aspirated and wells containing antibodies specifically recognizing recombinant 2019-nCoV N protein were detected in the culture by indirect ELISA. The cross reactivity of the cell culture supernatant is identified by adopting an indirect ELISA method, a 2019-nCoV N protein recombinant antigen is used for coating a 96-well plate, the 96-well plate is closed, hybridoma cell culture supernatant is added for incubation, secondary antibodies are added, the reaction value at 405nm is measured, and cell strains with higher reaction values (more than 0.5) are selected to prepare antibodies for the next screening experiment. The following antibodies with better reactivity were obtained by screening.
EXAMPLE 2 identification of antibody binding fragments
The 2019-nCoV N protein recombinant antigens of different fragments are respectively coated on micropores, PBS+20% NBS (N-bromosuccinimide) is used as diluent, diluted monoclonal antibodies are used as primary antibodies with the concentration of 1 mug/ml, goat anti-mouse IgG-HRP (horseradish peroxidase) is used as secondary antibodies, and the monoclonal antibody fragments are determined according to the reaction conditions of the monoclonal antibodies on different antigens. The antibodies obtained by screening are respectively aimed at the following fragments by statistics:
TABLE 1
The responses of the eleven antibodies binding to the 44-180aa fragment to the fragment were extracted as shown in Table 2:
TABLE 2
Antibodies to 6I3 9D2 4C9 5C3 1A6 1E5 8E4 6K1 6A5 7R1 5S7
Reactivity of 2.362 2.616 2.491 2.493 2.486 2.528 2.941 2.949 2.444 2.552 2.778
The reactivity, OD, in Table 2 405 Reaction value.
Example 3 paired screening
The antibodies were used for coating and labeling, respectively, and the experimental procedure was as follows:
1. new crown N antibody labeling: adding 30-40 μl of 0.2. 0.2M K into 5ml of colloidal gold with concentration of 4/ten thousand 2 CO 3 Stirring for 5min, adding new crown N-labeled antibody (added antibody volume=50 μg/antibody concentration), stirring for 5min, and adding 50 μl of 10% bsa (bovine serum albumin) to block the stop label; centrifugal 10000rpm,7min, removing supernatant, re-dissolving precipitate with gold seed re-dissolving solution, and finally fixing volume with gold seed re-dissolving solution to 0.5ml (i.e. 1/10 colloidal gold solution volume).
2. Preparing a working solution of the golden seeds: the gold seed complex solution is used for preparing gold seed working solution from concentrated gold of the novel crown N marked antibody according to a dilution ratio of 20 percent, and the gold seed working solution is paved on glass fibers.
3. Preparing dried golden seeds: the paved golden seeds are put into a freeze dryer for freeze drying (1-2 h) or are put into a drying room for drying overnight at 37 ℃.
4. New crown N antibody coating: assembling the nitrocellulose membrane and the PVC base plate for standby; diluting the new crown N coated antibody to 1.0-1.5mg/ml, uniformly scribing on NC film by using a metal spraying film drawing instrument, and placing in a 37 ℃ incubator for 60min.
5. Preparing a gold mark strip: and cutting the gold standard strips according to the required width by using a strip cutting machine, and adding samples for detection after assembly.
6. Detection of
(1) Quality control product: 2019-nCoV N protein recombinant antigen, diluting to 1ng/ml by using PBS, and carrying out antibody pairing primary screening;
(2) The detection method comprises the following steps: and judging the color development reading value by naked eyes according to the color card comparison.
7. Results (detection results for one antibody per fragment):
TABLE 3-1 3B8 detection results as coated antibody
Labeled antibodies 6I3 9D2 4C9 5C3 1A6 1E5 8E4 6K1 6A5 7R1
Quality control product C8 C8 C7 C8 C8 C8 C8 C8 C8 C7
PBS C9 B C8 C9 C9 B C9 C9 B B
Labeled antibodies 5S7 6D9 4B7 1B5 2F4 7C5 5D9 4D1 6D5 4C6
Quality control product C7 C8 C8 C8 C8 C8 C8 C8 C7 C7
PBS B C9 C9 B C9 B C9 C9 C8 C8
TABLE 3-2 5C3 detection results of coated antibodies
Labeled antibodies 6I3 9D2 4C9 3B8 1A6 1E5 8E4 6K1 6A5 7R1
Quality control product C4 C4 C4 C8 C4 C3 C5 C5 C4 C3
PBS B B B C9 B B B B B B
Labeled antibodies 5S7 6D9 4B7 1B5 2F4 7C5 5D9 4D1 6D5 4C6
Quality control product C3 C7 C7 C8 C8 C8 C7 C7 C8 C8
PBS B B B B C9 B B B C9 B
Tables 3-3 4B7 detection results as coated antibodies
Labeled antibodies 6I3 9D2 4C9 5C3 1A6 1E5 8E4 6K1 6A5 7R1
Quality control product C7 C7 C7 C7 C8 C7 C7 C7 C7 C7
PBS C9 C9 C9 B C9 C9 C9 C9 C9 B
Labeled antibodies 5S7 6D9 3B8 1B5 2F4 7C5 5D9 4D1 6D5 4C6
Quality control product C7 C8 C9 C9 C9 C9 C8 C9 C9 C9
PBS B B C9 B C9 B B C9 C9 B
Tables 3-4 1B5 detection results as coated antibodies
Labeled antibodies 6I3 9D2 4C9 5C3 1A6 1E5 8E4 6K1 6A5 7R1
Quality control product C8 C6 C7 C6 C8 C6 C7 C9 C9 C6
PBS C9 B C9 B C9 B C9 C9 C9 B
Labeled antibodies 5S7 6D9 4B7 3B8 2F4 7C5 5D9 4D1 6D5 4C6
Quality control product C6 C7 C7 C7 C7 C8 C7 C7 C8 C8
PBS B C8 C9 C7 C8 C9 B C9 B B
Tables 3-5 detection results of 4C6 as coating antibody
Labeled antibodies 6I3 9D2 4C9 5C3 1A6 1E5 8E4 6K1 6A5 7R1
Quality control product C8 C7 C7 C7 C8 C9 C7 C9 C9 C8
PBS B C9 C9 B B C9 B C9 B B
Labeled antibodies 5S7 6D9 4B7 1B5 2F4 7C5 5D9 4D1 6D5 3B8
Quality control product C9 C7 C8 C8 C7 C8 C7 C7 C8 C8
PBS B C8 C9 C9 C8 C9 B C9 B B
In tables 3-1, 3-2, 3-3, 3-4, 3-5, letter B represents no color development (undetected), and the number following letter C represents color development, the larger the number, the weaker the color development (the lower the activity).
From the detection results, the antibodies binding to the 44-180aa segment showed higher activity in terms of detection performance, so that further fragment identification was performed on the antibodies within the segment.
Example 4 dominant antibody further fragment identification
The 44-180aa fragment of the 2019-nCoV N protein recombinant antigen is expressed in small fragments, the 2019-nCoV N protein recombinant antigen of different fragments is respectively coated on micropores, PBS+20% NBS is used as diluent, diluted monoclonal antibody is used as primary antibody concentration to 1 mug/ml, goat anti-mouse IgG-HRP is used as secondary antibody, the monoclonal antibody fragment is determined according to the reaction condition of each monoclonal antibody to different antigens, and the specific data are as follows:
TABLE 4-1
Antibody numbering 6I3 9D2 4C9 5C3 1A6 1E5
44-180 2.362 2.616 2.491 2.493 2.486 2.528
44-84 0.034 0.032 0.070 0.059 0.020 0.018
65-103 0.067 2.819 0.037 0.035 0.017 0.019
96-136 0.012 0.015 0.017 0.016 0.008 0.014
130-180 0.028 0.012 0.064 0.044 0.013 0.015
Antibody recognition segments 44-180 65-103 44-180 44-180 44-180 44-180
TABLE 4-2
From the above results, the three 9D2, 7R1, 5S7 antibodies further recognized the binding of 65-103aa, and the remaining antibodies recognized only 44-180aa.
Example 5 further validation of dominant antibody pairing property
The preparation process of antibody labeling, coating, assembly and the like is described in example 3, steps 1-5.
Detection of
(1) Quality control product: 2019-nCoV N protein recombinant antigen, using PBS diluted to 25ng/ml, 2ng/ml, 500pg/ml, 100pg/ml, 25pg/ml, 10pg/ml;
(2) The detection method comprises the following steps: and judging the color development reading value by naked eyes according to the color card comparison.
Results
1. Activity: the following example pair showed significant detection of recombinant antigen expressed in E.coli at 100pg/ml (C7+ development) and 25pg/ml (C8 or C8+ development), with a minimum detection limit of 10pg/ml.
TABLE 5
2. Specificity:
the three example pairs respectively carry out colloidal gold chromogenic tests on 300 collected normal human nasal swabs, 300 normal human pharyngeal swabs and 300 normal human saliva specimens, and the specificity is 100%.
3. Cross reaction:
the three examples were paired to test for the presence of cross-reactivity by diluting various pathogens such as adenovirus, cytomegalovirus, epstein barr virus, measles virus, mumps virus, mycoplasma pneumoniae, parainfluenza virus, respiratory and cytoviruses, rotaviruses, varicella-zoster virus, influenza a/B virus, etc. at different concentrations.
4. And (5) positive and negative pairing:
the three examples above were paired for the exchange of labeled antibodies with coated antibodies, with the pairing performance remaining comparable.
Among them, 5C3, 1E5, 7R1 were sequenced as follows.
5C3
Heavy chain variable region SEQ No.1: QIQLVQSGPELKKPGETVKISCKASGYTFTNYGMNWVKQAPGKGLKWMGWINTYTGEPTYADDFKGRFAFSLETSASTAYLQINNLKNEDTATYFCARSALLRSYFDYWGQGTTLTVSS.
Light chain variable region SEQ No.2: DIVMTQSHKFMSTSVGDRVSITCKASQDVSTAVAWCQQKPGQSPKLLIYWASTRHTGVPDRFTGIRSGTDYTLTISSVQAEDLALYYCQQHYSTPLTFGAGTKLELKR.
Heavy chain SEQ NO.3: QIQLVQSGPELKKPGETVKISCKASGYTFTNYGMNWVKQAPGKGLKWMGWINTYTGEPTYADDFKGRFAFSLETSASTAYLQINNLKNEDTATYFCARSALLRSYFDYWGQGTTLTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK.
Light chain SEQ NO.4: DIVMTQSHKFMSTSVGDRVSITCKASQDVSTAVAWCQQKPGQSPKLLIYWASTRHTGVPDRFTGIRSGTDYTLTISSVQAEDLALYYCQQHYSTPLTFGAGTKLELKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC.
1E5
Heavy chain variable region SEQ No.5: QIQLVQSGPELKKPGETVKISCKASGYTFTNYGMNWVKQAPGKGLKWMGWINTYTGEPTYADDFKGRFAFSLETSASTAYLQINNLKNEDTATYFCARKGNWDEENAMDYWGQGTSVTVSS.
Light chain variable region SEQ No.6: DIQMTQSPASLSVSVGETVTITCRASENIYSNLAWYQQKQGKSPQLLVYTATDLPDGVPSRFSGSGSGTQYSLKINSLQSEDFGTYYCQHFWGTPWTFGGGTKLEIK.
Heavy chain SEQ NO.7: QIQLVQSGPELKKPGETVKISCKASGYTFTNYGMNWVKQAPGKGLKWMGWINTYTGEPTYADDFKGRFAFSLETSASTAYLQINNLKNEDTATYFCARKGNWDEENAMDYWGQGTSVTVSSAKTTPPSVYPLPPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGAHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPG.
Light chain SEQ NO.8: DIQMTQSPASLSVSVGETVTITCRASENIYSNLAWYQQKQGKSPQLLVYTATDLPDGVPSRFSGSGSGTQYSLKINSLQSEDFGTYYCQHFWGTPWTFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC
7R1
Heavy chain variable region SEQ No.9: DVQLVESGGGLVQPGGSRKLSCAASGFTFSTFGMHWVRQAPEKGLEWVAYINSGSNIIYYADTVKGRFTISRDNPKNTLFLQMTSLRSEDTAMYYCARHAMDYWGQGTSVTVSS.
Light chain variable region SEQ NO.10: DIVLTQSPASLAVSLGQRATISCKASQSVDYDGDSYMNWFQQKPGQPPKLLIYAASNLESGIPARFSGSGSGTDFTLNIHPVEEEDAATYYCQQSNEDPYTFGGGTKLEIK.
Heavy chain SEQ NO.11: DVQLVESGGGLVQPGGSRKLSCAASGFTFSTFGMHWVRQAPEKGLEWVAYINSGSNIIYYADTVKGRFTISRDNPKNTLFLQMTSLRSEDTAMYYCARHAMDYWGQGTSVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK.
Light chain SEQ NO.12: DIVLTQSPASLAVSLGQRATISCKASQSVDYDGDSYMNWFQQKPGQPPKLLIYDASNLESGIPARFSGSGSGTDFTLNIHPVEEEDAATYYCQQSNEDPYTFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC.
5. Verification of antibody detection Performance binding to the same epitope as 5C3, 1E5 or 7R1
Single point mutation of the 5C3 heavy chain variable region to give variant 5C3-1 which binds to the same epitope as 5C 3; single point mutation of the heavy chain variable region and the light chain variable region of 1E5 to obtain a variant 1E5-1 binding to the same epitope as 1E 5; single point mutation of the heavy chain variable region and the light chain variable region of 7R1 to obtain variant 7R1-1 which binds to the same epitope as 7R 1; the 5C3-1, 1E5-1 or 7R1-1 is used for correspondingly replacing the 5C3, 1E5 or 7R1 in the original pairing, the detection effect on the novel crown recombination antigen is equivalent, and the replaced antibody is the same as the original antibody epitope through epitope experiments. The variant sequences are as follows.
5C3-1
Heavy chain variable region SEQ No.13: QIQLVQSGPELKKPGETVKISCKASGYTFTNYGWNWVKQAPGKGLKWMGWINTYTGEPTYADDFKGRFAFSLETSASTAYLQINNLKNEDTATYFCARSGLLRSYFDYWGQGTTLTVSS.
Heavy chain SEQ NO.14: QIQLVQSGPELKKPGETVKISCKASGYTFTNYGWNWVKQAPGKGLKWMGWINTYTGEPTYADDFKGRFAFSLETSASTAYLQINNLKNEDTATYFCARSGLLRSYFDYWGQGTTLTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK.
1E5-1
Heavy chain variable region SEQ No.15: QIQLVQSGPELKKPGETVKISCKASGYTFTNYGWNWVKQAPGKGLKWMGWINTYTGEPTYGDDFKGRFAFSLETSASTAYLQINNLKNEDTATYFCARKGNWDEENAMDYWGQGTSVTVSS.
Light chain variable region SEQ NO.16: DIQMTQSPASLSVSVGETVTITCRGSENIYSNLAWYQQKQGKSPQLLVYTATDLPDGVPSRFSGSGSGTQYSLKINSLQSEDFGTYYCQHFWGTPWTFGGGTKLEIK.
Heavy chain SEQ NO.17: QIQLVQSGPELKKPGETVKISCKASGYTFTNYGWNWVKQAPGKGLKWMGWINTYTGEPTYGDDFKGRFAFSLETSASTAYLQINNLKNEDTATYFCARKGNWDEENAMDYWGQGTSVTVSSAKTTPPSVYPLPPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGAHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPG.
Light chain SEQ NO.18: DIQMTQSPASLSVSVGETVTITCRGSENIYSNLAWYQQKQGKSPQLLVYTATDLPDGVPSRFSGSGSGTQYSLKINSLQSEDFGTYYCQHFWGTPWTFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC.
7R1-1
Heavy chain variable region SEQ No.19: DVQLVESGGGLVQPGGSRKLSCAASGFTFSTFGMHWVRQAPEKGLEWVAYINSASNIIYYADTVKGRFTISRDNPKNTLFLQMTSLRSEDTAMYYCARHAMDYWGQGTSVTVSS.
Light chain variable region SEQ NO.20: DIVLTQSPASLAVSLGQRATISCKASQSVDYDGDSYMNWFQQKPGQPPKLLIYDASNLESGIPARFSGSGSGTDFTLNIHPVEEEDAATYYCQQSNEDPYTFGGGTKLEIK.
Heavy chain SEQ NO.21: QIQLVQSGPELKKPGETVKISCKASGYTFTNYGWNWVKQAPGKGLKWMGWINTYTGEPTYGDDFKGRFAFSLETSASTAYLQINNLKNEDTATYFCARKGNWDEENAMDYWGQGTSVTVSSAKTTPPSVYPLPPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGAHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPG.
Light chain SEQ NO.22: DIQMTQSPASLSVSVGETVTITCRGSENIYSNLAWYQQKQGKSPQLLVYTATDLPDGVPSRFSGSGSGTQYSLKINSLQSEDFGTYYCQHFWGTPWTFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC.
Example 6 advantageous antibody preparation
The present disclosure also provides antibodies that bind to the 44-180aa fragment, but do not bind to the 44-84aa fragment, the 65-103aa fragment, the 96-136aa fragment, and the 130-180aa fragment, e.g., by reactive screening for ELISA to assess OD, using the amino acid fragments at positions 44-180 of SARS-CoV-2 nucleocapsid protein as an immunogen, using the antibody preparation protocol described above 405 Antibodies 1COV19-8 and 3COV19-17 of ∈ 0.5, and the like, the performance also satisfies the requirements of high sensitivity, good specificity and no cross reaction.
The foregoing embodiments are alternative embodiments of the present disclosure, but the embodiments of the present disclosure are not limited by the foregoing embodiments, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principles of the present disclosure should be made as equivalent substitutions and modifications within the scope of the present disclosure.

Claims (14)

1. An antibody, wherein said antibody binds to amino acid fragments 44-180 of SARS-CoV-2 nucleocapsid protein.
2. The antibody of claim 1, wherein the antibody does not bind to amino acid fragments 44-84, 65-103, 96-136, and 130-180 of the SARS-CoV-2 nucleocapsid protein.
3. The antibody of claim 1 or 2, wherein the antibody binds to an epitope that is identical to an epitope comprising SEQ ID NO:1 and the heavy chain variable region of SEQ ID NO:2, or the epitope bound by the antibody of the light chain variable region is the same, or
And a polypeptide comprising SEQ ID NO:5 and SEQ ID NO:6, the epitope to which the antibody of the light chain variable region binds is identical.
4. An antibody combination comprising antibody 1 and antibody 2, wherein said antibody 1 binds to an epitope that is identical to an epitope comprising SEQ ID NO:1 and the heavy chain variable region of SEQ ID NO:2, and the epitope to which the antibody of the light chain variable region binds is the same, and
the antibody 2 binds to an epitope that is identical to an epitope comprising SEQ ID NO:5 and SEQ ID NO:6, the epitope to which the antibody of the light chain variable region binds is identical.
5. An antibody combination comprising antibody 3 and antibody 4, wherein antibody 3 is an antibody according to any one of claims 2-3 and antibody 4 is an antibody that binds to amino acid fragments 65-103 of SARS-CoV-2 nucleocapsid protein.
6. The antibody combination of claim 5, wherein the antibody 4 binds to an epitope that is identical to an epitope comprising SEQ ID NO:9 and SEQ ID NO:10, and the epitope to which the antibody of the light chain variable region binds is the same.
7. A kit comprising the antibody of any one of claims 1-3, or the antibody combination of any one of claims 4-6.
8. Use of an antibody according to any one of claims 1-3, or a combination of antibodies according to any one of claims 4-6, in the preparation of a kit.
9. A method of making the antibody of any one of claims 1-3, comprising: 1) Immunizing an animal with an antigen or hapten comprising an amino acid fragment of SARS-CoV-2 nucleocapsid protein at positions 44-180; and 2) obtaining antibodies binding to amino acid fragments 44-180 of SARS-CoV-2 nucleocapsid protein from ascites of said animal.
10. A method for preparing the antibody of any one of claims 1-3, comprising preparing the antibody by hybridoma technique or phage display technique using the amino acid fragment of SARS-CoV-2 nucleocapsid protein at positions 44-180 as antigen.
11. Use of the antibody of any one of claims 1-3, or the antibody combination of any one of claims 4-6, or the kit of claim 7, for detecting SARS-CoV-2.
12. A method of detecting SARS-Cov-2, comprising:
a) Contacting the antibody of any one of claims 1-3, or the antibody combination of any one of claims 4-6, or the kit of claim 7, with a sample under conditions sufficient for a binding reaction to occur; and
b) Detecting immune complexes generated by the binding reaction.
13. A method of diagnosing whether a subject is infected with SARS-Cov-2 or has a disease associated with SARS-Cov-2 infection, comprising:
a) Contacting the antibody of any one of claims 1-3, or the antibody combination of any one of claims 4-6, or the kit of claim 7 with a sample from the subject under conditions sufficient for a binding reaction to occur; and
b) Detecting immune complexes generated by the binding reaction.
14. The method of claim 13, wherein the disease associated with SARS-Cov-2 infection comprises at least one of respiratory symptoms, fever, cough, breathlessness, dyspnea, pneumonia, severe acute respiratory syndrome, renal failure.
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