WO2022089045A1 - 抗新型冠状病毒的抗体和检测新型冠状病毒的试剂盒 - Google Patents
抗新型冠状病毒的抗体和检测新型冠状病毒的试剂盒 Download PDFInfo
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- C07K2317/51—Complete heavy chain or Fd fragment, i.e. VH + CH1
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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
- C07K2317/515—Complete light chain, i.e. VL + CL
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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
- C07K2317/52—Constant or Fc region; Isotype
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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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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
- C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
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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
- G01N2333/08—RNA viruses
- G01N2333/165—Coronaviridae, e.g. avian infectious bronchitis virus
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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
Definitions
- the present disclosure relates to the technical field of antibodies, and in particular, to an antibody against novel coronavirus and a kit for detecting novel coronavirus.
- the structural proteins of the new coronavirus 2019-nCoV are divided into: spike glycoprotein (S protein), envelope glycoprotein (E protein), membrane glycoprotein (M protein) and nucleocapsid protein (N protein). These proteins include multiple antigenic epitopes.
- S protein spike glycoprotein
- E protein envelope glycoprotein
- M protein membrane glycoprotein
- N protein nucleocapsid protein
- the N protein and the viral genome RNA are intertwined to form the viral nucleocapsid, which plays an important role in the synthesis of viral RNA.
- the N protein is relatively conservative and occupies the largest proportion in the structural proteins of the virus.
- the body can produce high-level antibodies against the N protein in the early stage of infection.
- the N protein is an important marker protein of the new coronavirus. Using the principle of specific binding of antigens and antibodies, the presence of the antigen can be detected by the N protein monoclonal antibody, thereby directly proving that the sample contains the new coronavirus and realizing the detection
- the detected antibodies are mainly divided into two categories: IgM and IgG.
- IgM antibodies are produced early, and once infected, they are rapidly produced, maintained for a short time, and disappear quickly.
- a positive test in the blood can reflect that the body is in an acute infection state and can be used as an indicator of early infection.
- antibody detection samples are serum or plasma, which are less affected by sample sampling, which is conducive to early diagnosis and exclusion of suspicious cases. At the same time, the detection is fast, convenient, and suitable for large-scale screening.
- 2019-nCoV Since the outbreak of the novel coronavirus 2019-nCoV pneumonia, it has spread around the world, posing a serious threat to human life and health. Respiratory droplets and close contact transmission are the main transmission routes of novel coronavirus pneumonia, and there is the possibility of aerosol transmission in the case of prolonged exposure to high concentrations of aerosols in a relatively closed environment. 2019-nCoV is highly contagious, and most patients develop clinical symptoms after infection, but some patients are asymptomatic. Common signs of people infected with coronavirus include respiratory symptoms, fever, cough, shortness of breath, and difficulty breathing. In more severe cases, the infection can lead to pneumonia, severe acute respiratory syndrome, kidney failure, and even death. Although there is currently no specific treatment for the disease caused by the new coronavirus, treatment for mild or asymptomatic patients can greatly improve the cure rate and shorten the treatment time. Therefore, the detection or identification of patients becomes particularly important.
- nucleic acid detection and viral gene sequencing are mainly used as etiological confirmation evidence. Due to various factors such as sampling, operation, and reagents, nucleic acid detection will produce false negative results.
- the positive detection rate of viral nucleic acid in patients with highly suspected 2019 novel coronavirus (2019-nCoV) infection is only 30% to 50%.
- nucleic acid testing has high requirements on equipment, testing sites and environmental conditions, and has shortcomings such as long testing time and low throughput, which is not convenient for large-scale testing of people under the current epidemic situation. Therefore, there is an urgent need to develop a rapid and convenient detection kit for clinical detection, so as to isolate the infected population as soon as possible to block the spread of the virus. Therefore, antibody detection kits have become more important.
- the present disclosure provides an antibody against a novel coronavirus or its N protein or a functional fragment thereof, the antibody or functional fragment thereof comprising the following complementarity determining regions:
- CDR-VH1 G-X1-T-F-T-X2-Y-X3-M-N; where: X1 is Y or F; X2 is D or N; X3 is A or G;
- CDR-VH2 W-X1-N-T-Y-X2-G-E-P-T-Y-A-X3-X4-F; where: X1 is L or I; X2 is T or S; X3 is D or E; X4 is D or E;
- CDR-VH3 A-R-X1-A-X2-X3-R-S-Y; wherein: X1 is S or T; X2 is I or L; X3 is I or L;
- CDR-VL1 K-A-S-X1-D-X2-S-T-A-X3-A; wherein: X1 is Q or E; X2 is I, V or L; X3 is I, V or L;
- CDR-VL2 W-X1-S-T-R-H-X2; where: X1 is A or G; X2 is T or S;
- CDR-VL3 Q-Q-H-X1-S-T-P-X2; wherein: X1 is Y or W; X2 is L, V or I.
- X1 is Y
- X1 is I
- X1 is S
- X1 is Q
- X1 is A.
- X2 is D.
- X2 is N.
- X3 is A.
- X3 is G.
- X2 is T.
- X2 is S.
- X3 is D in the CDR-VH2.
- X3 is E.
- X4 is D.
- X4 is E.
- X2 is 1.
- X2 is L.
- X3 is 1.
- X3 is L.
- X2 is 1 in CDR-VL1.
- X2 is V.
- X2 is L.
- X3 is 1.
- X3 is V.
- X3 is L.
- X2 is T.
- X2 is S.
- X1 is Y.
- X1 is W.
- X2 is L.
- X2 is V.
- X2 is 1.
- each complementarity determining region of the antibody or functional fragment thereof is selected from any one of the following mutation combinations 1-42:
- the antibody or its functional fragment binds to the N protein of the novel coronavirus with an affinity of K D ⁇ 8 ⁇ 10 -9 mol/L. In some embodiments, K D ⁇ 4 ⁇ 10 ⁇ 10 mol/L.
- X1 is F
- X1 is L
- X1 is T
- X1 is E
- X1 is G.
- each complementarity determining region of the antibody or functional fragment thereof is selected from any one of the following mutation combinations 43-48:
- the antibody comprises the light chain framework regions FR1-L, FR2-L, FR3-L and FR4-L of the sequence shown in SEQ ID NO: 1-4, and/or the sequence of Heavy chain framework regions FR1-H, FR2-H, FR3-H and FR4-H shown in SEQ ID NOs: 5-8.
- the antibody further comprises a constant region.
- the constant region is selected from the constant region of any one of IgGl, IgG2, IgG3, IgG4, IgA, IgM, IgE, and IgD.
- the species source of the constant region is bovine, horse, dairy cow, pig, sheep, goat, rat, mouse, dog, cat, rabbit, camel, donkey, deer, mink, chicken, duck , goose, turkey, cockfight or man.
- the constant region is derived from a mouse.
- the light chain constant region sequence of the constant region is set forth in SEQ ID NO:9
- the heavy chain constant region sequence of the constant region is set forth in SEQ ID NO:10.
- the functional fragment is selected from any one of VHH, F(ab')2, Fab', Fab, Fv and scFv of the antibody.
- the antibody comprises light chain framework regions FR1-L, FR2-L, FR3-L and FR1-L, FR2-L, FR3-L, and FR4-L, and/or, heavy chain framework regions FR1-H, FR2-H, FR3-H and FR4-H, FR2-H, FR3-H, and FR4-H having at least 80% homology with sequences of SEQ ID NOs: 5, 6, 7, 8, respectively H.
- the present disclosure provides a reagent or kit for detecting a novel coronavirus or its N protein, comprising any of the antibodies or functional fragments thereof described above.
- the antibody or functional fragment thereof is labeled with a detectable label.
- the detectable label is selected from the group consisting of fluorescent dyes, enzymes that catalyze color development of substrates, radioisotopes, chemiluminescent reagents, and nanoparticle-based labels.
- the fluorescent dye is selected from fluorescein dyes and derivatives thereof, rhodamine dyes and derivatives thereof, Cy series dyes and derivatives thereof, Alexa series dyes and derivatives thereof, and protein dyes and its derivatives.
- the enzyme that catalyzes coloration of the substrate is selected from the group consisting of horseradish peroxidase, alkaline phosphatase, beta-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and 6- Phosphoglucose deoxygenase.
- the radioisotope is selected from212Bi , 131I , 111In , 90Y , 186Re , 211At , 125I , 188Re , 153Sm , 213Bi , 32P , 94mTc ,99mTc , 203 Pb, 67 Ga, 68 Ga, 43 Sc, 47 Sc, 110 mIn, 97 Ru, 62 Cu, 64 Cu, 67 Cu, 68 Cu, 86 Y, 88 Y, 121 Sn, 161 Tb, 166 Ho, 105 Rh, 177 Lu, 172 Lu, and 18 F.
- the chemiluminescent reagent is selected from the group consisting of luminol and its derivatives, lucigenin, crustacean fluorescein and its derivatives, ruthenium bipyridine and its derivatives, acridine esters and its derivatives, Oxetane and its derivatives, Lofenine and its derivatives, and peroxyoxalate and its derivatives.
- the nanoparticle-based label is selected from the group consisting of nanoparticles, colloids, organic nanoparticles, magnetic nanoparticles, quantum dot nanoparticles, and rare earth complex nanoparticles.
- the colloid is selected from the group consisting of colloidal metals, disperse dyes, dye-labeled microspheres, and latex.
- the colloidal metal is selected from colloidal gold, colloidal silver, and colloidal selenium.
- the present disclosure provides a nucleic acid molecule encoding any one of the above-mentioned antibodies or functional fragments thereof.
- the present disclosure provides a vector containing a nucleic acid fragment encoding any of the above-described antibodies or functional fragments thereof.
- the present disclosure provides a recombinant cell containing the vector.
- the present disclosure provides the use of the antibody or its functional fragment, the reagent or the kit in detecting novel coronavirus.
- the present disclosure provides the use of the antibody or its functional fragment, the reagent or the kit for detecting novel coronavirus.
- the present disclosure provides a method for detecting a novel coronavirus, comprising:
- the present disclosure provides a method for diagnosing a subject infected with a novel coronavirus or a disease associated with the novel coronavirus infection, comprising:
- the disease associated with novel coronavirus infection includes respiratory symptoms, fever, cough, shortness of breath, dyspnea, pneumonia, severe acute respiratory syndrome, renal failure.
- the subjects infected with the novel coronavirus include asymptomatic infected persons, asymptomatic infected persons, and symptomatic infected persons.
- the present disclosure provides a method for preparing any one of the above-mentioned antibodies or functional fragments thereof, comprising: culturing the recombinant cells, and separating and purifying the antibodies or functional fragments thereof from the cultured product.
- FIG. 1 shows the results of reducing SDS-PAGE of the anti-novel coronavirus antibody of Example 1.
- the term "complementarity determining regions” means: an intact or complete antibody comprises two heavy chains and two light chains; each heavy chain comprises a variable region (VH) and a constant region (CH); each The light chain contains a variable region (VL) and a constant region (CL); the antibody has a "Y" shape with the stem of the Y consisting of the second and third of the two heavy chains held together by disulfide bonds
- the constant region consists of; each arm of Y includes the variable region and the first constant region of a single heavy chain combined with the variable region and constant region of a single light chain; the variable region and heavy chain of the light chain
- the variable regions of the are responsible for antigen binding; the variable regions in both chains typically contain three hypervariable regions, called complementarity determining regions.
- the term "functional fragment” is intended to refer to a portion of an antibody comprising a heavy or light chain polypeptide that retains some or all of the binding activity of the antibody from which the fragment was derived.
- These functional fragments may include, for example, Fd, Fv, Fab, F(ab'), F(ab)2, F(ab')2, single chain Fv (scFv), diabodies, triple chain Antibodies (triabodies), tetrabodies (tetrabodies) and minibodies (minibodies).
- Other functional fragments may include, for example, heavy or light chain polypeptides, variable region polypeptides or CDR polypeptides or portions thereof, so long as these functional fragments retain binding activity.
- constant region refers to a relatively stable region of the light and heavy chains of an antibody molecule near the C-terminal amino acid sequence.
- variable region refers to the region of the light and heavy chains of an antibody molecule that varies greatly in amino acid sequence near the N-terminus.
- naked anti-stable refers to an antibody or functional fragment thereof that is not labeled, eg, an antibody or functional fragment thereof that is not labeled with a detectable label.
- Some embodiments of the present disclosure provide an antibody against the novel coronavirus and a kit for detecting the novel coronavirus, the antibody can specifically bind to the N protein of the novel coronavirus, and has a high affinity for it, and the antibody can be used to detect the novel coronavirus
- the virus has good sensitivity and specificity.
- the present disclosure provides a richer selection of antibodies for the detection of novel coronaviruses.
- One embodiment of the present disclosure provides an antibody against a novel coronavirus or its N protein or a functional fragment thereof, wherein the antibody or functional fragment thereof has the following complementarity determining regions:
- CDR-VH1 G-X1-T-F-T-X2-Y-X3-M-N; where: X1 is Y or F; X2 is D or N; X3 is A or G;
- CDR-VH2 W-X1-N-T-Y-X2-G-E-P-T-Y-A-X3-X4-F; where: X1 is L or I; X2 is T or S; X3 is D or E; X4 is D or E;
- CDR-VH3 A-R-X1-A-X2-X3-R-S-Y; wherein: X1 is S or T; X2 is I or L; X3 is I or L;
- CDR-VL1 K-A-S-X1-D-X2-S-T-A-X3-A; wherein: X1 is Q or E; X2 is I, V or L; X3 is I, V or L;
- CDR-VL2 W-X1-S-T-R-H-X2; where: X1 is A or G; X2 is T or S;
- CDR-VL3 Q-Q-H-X1-S-T-P-X2; wherein: X1 is Y or W; X2 is L, V or I.
- the anti-novel coronavirus antibody or its functional fragment provided by the present disclosure has the above-mentioned complementarity determining region structure, and the above-mentioned complementarity determining region structure enables the antibody or its functional fragment to specifically bind to the anti-novel coronavirus N protein, and its It has good affinity, and the antibody or its functional fragment is used to detect novel coronavirus with good specificity and sensitivity.
- the present disclosure provides a richer selection of antibodies for the detection of novel coronaviruses.
- X1 in CDR-VH1, X1 is Y; in CDR-VH2, X1 is I; in CDR-VH3, X1 is S; in CDR-VL1, X1 is Q; in CDR-VL2, X1 is A.
- X2 is D in CDR-VH1.
- X2 is N in CDR-VH1.
- X3 is A in CDR-VH1.
- X3 is G.
- X2 is T.
- X2 is S.
- X3 is D.
- X3 is E.
- X4 is D.
- X4 is E.
- X2 is 1.
- X2 is L.
- X3 is 1.
- X3 is L.
- X2 is 1.
- X2 is V.
- X2 is L.
- X3 is 1.
- X3 is V.
- X3 is L.
- X2 is T.
- X2 is S.
- X1 is Y.
- X1 is W.
- X2 is L.
- X2 is V.
- X2 is 1.
- each complementarity determining region of the antibody or functional fragment thereof is selected from any one of the following mutation combinations 1-42:
- the antibody or its functional fragment binds to the anti-novel coronavirus N protein with an affinity of K D ⁇ 8 ⁇ 10 ⁇ 9 mol / L.
- K D ⁇ 4 ⁇ 10 ⁇ 10 mol/L.
- K D The detection of K D is performed with reference to the methods in the embodiments of the present disclosure.
- X1 in CDR-VH1, X1 is F; in CDR-VH2, X1 is L; in CDR-VH3, X1 is T; in CDR-VL1, X1 is E; in CDR-VL2, X1 is G.
- each complementarity determining region of the antibody or functional fragment thereof is selected from any one of the following mutation combinations 43-48:
- the antibody comprises the light chain framework regions FR1-L, FR2-L, FR3-L and FR4-L, and/or the sequences of the light chain framework regions shown in SEQ ID NOs: 1-4 in sequence.
- variable region (VH) of the heavy chain and the variable region (VL) of the light chain can be obtained by linking the following numbered CDRs and FRs in the following combinations: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.
- the antibody comprises light chain framework regions FR1-L, FR2-L, FR3-L and FR4-L, FR2-L, FR3-L and FR4- L, and/or, heavy chain framework regions FR1-H, FR2-H, FR3-H and FR4-H having at least 80% homology with sequences of SEQ ID NOs: 5, 6, 7, 8, respectively.
- the antibody comprises light chain framework regions FR1-L, FR2-L, FR3-L, and FR4-L that are at least 80% identical to the sequences of SEQ ID NOs: 1, 2, 3, 4, respectively, in order , and/or, heavy chain framework regions FR1-H, FR2-H, FR3-H and FR4-H having at least 80% identity to the sequences of SEQ ID NOs: 5, 6, 7, 8, respectively.
- each framework region of the antibody or its functional fragment provided by the present disclosure may be the same as the above-mentioned corresponding framework region (SEQ ID NO: 1, 2, 3, 4, 5, 6, 7 or 8) may have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology.
- the antibody further comprises a constant region.
- the constant region is selected from the constant region of any one of IgGl, IgG2, IgG3, IgG4, IgA, IgM, IgE and IgD.
- the species source of the constant region is mammalian or poultry.
- mammals include cows, horses, dairy cows, pigs, sheep, goats, rats, mice, dogs, cats, rabbits, camels, donkeys, deer, minks, or humans.
- poultry animals include chickens, ducks, geese, turkeys or fighting cocks.
- the species source of the constant region is bovine, equine, dairy cattle, pig, sheep, goat, rat, mouse, dog, cat, rabbit, camel, donkey, deer, mink, chicken , duck, goose, turkey, cockfight or man.
- the constant region is derived from a mouse.
- the light chain constant region sequence of the constant region is shown in SEQ ID NO:9
- the heavy chain constant region sequence of the constant region is shown in SEQ ID NO:10.
- the functional fragment is selected from any one of VHH, F(ab')2, Fab', Fab, Fv and scFv of the antibody.
- Functional fragments of the above antibodies generally have the same binding specificity as the antibody from which they were derived.
- the functional fragments of the above-mentioned antibodies can be cleaved by methods including but not limited to enzymatic digestion (including but not limited to pepsin or papain) and/or by chemical reduction Sulfur bond method.
- enzymatic digestion including but not limited to pepsin or papain
- chemical reduction Sulfur bond method On the basis of the structure of the complete antibody provided in the present disclosure, those skilled in the art can easily obtain the above-mentioned functional fragments.
- Functional fragments of the above-described antibodies can also be obtained by recombinant genetic techniques, also known to those skilled in the art, or by, for example, automated peptide synthesizers such as those sold by including, but not limited to, Applied BioSystems and the like.
- One embodiment of the present disclosure provides a reagent or kit for detecting a novel coronavirus or its N protein, which includes the antibody or its functional fragment as described in any of the above.
- the antibody or functional fragment thereof in the above reagent or kit is labeled with a detectable label.
- detectable label refers to a class of substances having properties that can be directly observed by the naked eye or detected or detected by instruments, such as luminescence, coloration, radioactivity, etc., by which the corresponding Qualitative or quantitative detection of a target.
- the detectable labels include, but are not limited to, fluorescent dyes, enzymes that catalyze color development of substrates, radioisotopes, chemiluminescent reagents, and nanoparticle-based labels.
- the fluorescent dyes include, but are not limited to, fluorescein dyes and derivatives thereof (for example, including but not limited to fluorescein isothiocyanate (FITC) hydroxyfluorescein (FAM), tetrachlorofluorescein (TET), etc. or its analogs), rhodamine dyes and their derivatives (such as, but not limited to, red rhodamine (RBITC), tetramethylrhodamine (TAMRA), rhodamine B (TRITC), etc. or the like compounds), Cy series dyes and their derivatives (such as but not limited to Cy2, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, Cy3, etc.
- fluorescein dyes and derivatives thereof for example, including but not limited to fluorescein isothiocyanate (FITC) hydroxyfluorescein (FAM), tetrachlorofluorescein (TET), etc. or its analogs
- Alexa series dyes and their derivatives such as including But not limited to AlexaFluor350, 405, 430, 488, 532, 546, 555, 568, 594, 610, 33, 647, 680, 700, 750, etc. or their analogs
- protein dyes and their derivatives for example, including but Not limited to phycoerythrin (PE), phycocyanin (PC), allophycocyanin (APC), polydinoxanthin-chlorophyll protein (preCP), etc.
- the enzymes that catalyze the coloration of the substrate include but are not limited to horseradish peroxidase, alkaline phosphatase, ⁇ -galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase enzyme and glucose 6-phosphate deoxygenase.
- the radioisotopes include but are not limited to 212Bi , 131I , 111In , 90Y , 186Re , 211At , 125I , 188Re , 153Sm , 213Bi , 32P , 94 mTc, 99 mTc, 203 Pb, 67 Ga, 68 Ga, 43 Sc, 47 Sc, 110 mIn, 97 Ru, 62 Cu, 64 Cu, 67 Cu, 68 Cu, 86 Y, 88 Y, 121 Sn, 161 Tb, 166 Ho, 105 Rh, 177 Lu, 172 Lu and 18 F.
- the chemiluminescent reagents include, but are not limited to, luminol and its derivatives, lucigenin, crustacean fluorescein and its derivatives, ruthenium bipyridine and its derivatives, acridine esters and its derivatives Derivatives, Dioxetane and its Derivatives, Lopine and its Derivatives and Peroxyoxalate and its Derivatives.
- the nanoparticle-based labels include, but are not limited to, nanoparticles, colloids, organic nanoparticles, magnetic nanoparticles, quantum dot nanoparticles, and rare earth complex nanoparticles.
- the colloids include, but are not limited to, colloidal metals, disperse dyes, dye-labeled microspheres, and latex.
- the colloidal metals include, but are not limited to, colloidal gold, colloidal silver, and colloidal selenium.
- An embodiment of the present disclosure provides a nucleic acid molecule encoding the above-mentioned antibody or a functional fragment thereof.
- One embodiment of the present disclosure provides a vector containing the above-mentioned nucleic acid molecule.
- One embodiment of the present disclosure provides a recombinant cell containing the above-mentioned vector.
- Some embodiments of the present disclosure provide the use of the above-mentioned antibodies or functional fragments thereof, reagents or kits for detecting novel coronavirus.
- Some embodiments of the present disclosure provide methods for detecting novel coronaviruses, including:
- a method of diagnosing a subject being infected with a novel coronavirus or a disease associated with the novel coronavirus infection comprising:
- the disease associated with novel coronavirus infection includes respiratory symptoms, fever, cough, shortness of breath, dyspnea, pneumonia, severe acute respiratory syndrome, renal failure.
- the subjects infected with the novel coronavirus include asymptomatic infected persons, asymptomatic infected persons, and symptomatic infected persons.
- One embodiment of the present disclosure provides a method for preparing an antibody or a functional fragment thereof, comprising: culturing the above-mentioned recombinant cells, and separating and purifying the antibody or its functional fragment from the cultured product.
- restriction endonuclease and Prime Star DNA polymerase were purchased from Takara Company.
- MagExtractor-RNA extraction kit was purchased from TOYOBO Company.
- BD SMART TM RACE cDNA Amplification Kit was purchased from Takara Company.
- the pMD-18T vector was purchased from Takara Company.
- Plasmid extraction kit was purchased from Tiangen Company. Primer synthesis and gene sequencing were performed by Invitrogen.
- the mRNA was extracted from the hybridoma cell line that secreted the antibody against the new coronavirus N protein, and the DNA product was obtained by RT-PCR method.
- the heavy chain (Heavy Chain) and the light chain (Light Chain) gene clones were taken after the colonies grew, and 4 clones were sent to a gene sequencing company for sequencing.
- the gene sequences obtained by the above sequencing were placed in the IMGT antibody database (IMGT antibody database from: http://www.imgt.org) for analysis, and the VNTI11.5 software was used for analysis to determine the amplification of heavy chain and light chain primer pairs.
- the added genes are all correct.
- the VL gene sequence is 324bp, belonging to the VkII gene family, and there is a 57bp leader peptide sequence in front of it;
- the gene fragment amplified by the heavy chain primer pair Among them, the VH gene sequence is 357bp, belonging to the VH1 gene family, and there is a 57bp leader peptide sequence in front of it.
- pcDNATM 3.4 vector is the constructed recombinant antibody eukaryotic expression vector.
- the expression vector has introduced polyclonal restriction sites such as HindIII, BamHI and EcoRI, and is named pcDNA3.4A expression vector, hereinafter referred to as 3.4A expression vector; according to the above pMD-18T
- 3.4A expression vector pcDNA3.4A expression vector
- the VL and VH gene-specific primers of the antibody were designed, with HindIII and EcoRI restriction sites and protective bases on both ends, respectively, and a 0.73kb light chain was amplified by PCR amplification method. Gene fragment and 1.42kb heavy chain gene fragment.
- the heavy chain and light chain gene fragments were digested with HindIII/EcoRI double enzymes respectively, and the 3.4A vector was digested with HindIII/EcoRI double enzymes. After the fragments and vectors were purified and recovered, the heavy chain genes and light chain genes were connected to the 3.4A expression vector to obtain the heavy chain. Chain and light chain recombinant expression plasmids.
- the recombinant antibody expression plasmid was transiently transfected into CHO cells to determine the activity of the expression plasmid
- plasmid obtained in step 1-(3) Dilute the plasmid obtained in step 1-(3) to 400ng/ml with ultrapure water, adjust CHO cells to 1.43 ⁇ 10 7 cells/ml in a centrifuge tube, mix 100 ⁇ L of plasmid with 700 ⁇ L of cells, transfer into an electroporation cup, and electroporate, The samples were counted on the 3rd, 5th, and 7th days, and the samples were collected and tested on the 7th day.
- the coating solution (the main component NaHCO 3 ) diluted the 2019-nCoV N protein antigen to 1 ⁇ g/ml, 100 ⁇ L per well, overnight at 4°C; the next day, the washing solution (the main component Na 2 HPO 4 +NaCl) was washed twice and patted dry ;Add blocking solution (20%BSA+80%PBS), 120 ⁇ L per well, 37°C, 1h, pat dry; add diluted CHO cell supernatant, 100 ⁇ L/well, 37°C, 30min (partial supernatant for 1h); Wash 5 times with washing solution and pat dry; add goat anti-mouse IgG-HRP (where HPR is labeled with horseradish peroxidase), 100 ⁇ L per well, 37°C, 30 min; wash with washing solution 5 times, pat dry; add color developing Solution A (50 ⁇ L/well, containing citric acid + sodium acetate + acetanilide + carbamide peroxide), add color developing solution B (50 ⁇ L
- reaction OD was still greater than 1.0 after the cell supernatant was diluted 1000 times, and the reaction OD of the unadded cell supernatant was less than 0.1, indicating that the antibody generated after transient transfection of the plasmid was active against the 2019-nCoV N protein antigen.
- step 2-(2) Dilute the plasmid obtained in step 2-(2) with ultrapure water to 400ng/ml, adjust CHO cells to 1.43 ⁇ 10 7 cells/ml in a centrifuge tube, mix 100 ⁇ L of plasmid with 700 ⁇ L of cells, transfer into an electroporation cup, and electroporate, Count the next day; 25umol/L MSX 96-well pressurized culture for about 25 days.
- the cells obtained in step 2-(3) were recovered by passage, they were first cultured in a 125ml shake flask, the inoculation volume was 30ml, and the medium was 100% Dynamis medium, placed at a rotating speed of 120r/min and a temperature of 37 °C in a shaker with 8% carbon dioxide. After culturing for 72 hours, inoculate and expand the culture at an inoculation density of 500,000 cells/ml, and the expansion volume is calculated according to the production demand, and the medium is 100% Dynamis medium. After that, the culture was expanded every 72h. When the cell volume meets the production requirements, the seeding density is strictly controlled to be about 500,000 cells/ml for production.
- Shaking flask parameters rotating speed 120r/min, temperature 37°C, carbon dioxide 8%.
- Feed feeding start feeding every day after culturing in the shake flask for 72 hours.
- HyClone Cell Boost Feed 7a is fed 3% of the initial culture volume every day, and Feed 7b is fed daily at 1/1000 of the initial culture volume. Supplement until the 12th day (feeding on the 12th day).
- Glucose was supplemented with 3 g/L on the sixth day. Samples were collected on the 13th day.
- Affinity purification was performed using a protein A affinity chromatography column. Take 4 ⁇ g of purified antibody for reducing SDS-PAGE, and 4 ⁇ g foreign control antibody as control.
- the electropherogram is shown in Figure 1 below. After reducing SDS-PAGE, two bands are displayed, and one Mr is 50KD (heavy chain, SEQ ID NO: 14), the other Mr is 28KD (light chain, SEQ ID NO: 13).
- the antibody (WT) sequence of Example 1 was analyzed, and its heavy chain variable region was shown in SEQ ID NO: 12, wherein the amino acid sequence of each complementarity determining region on the heavy chain variable region was as follows:
- CDR-VH1 G-F(X1)-T-F-T-D(X2)-Y-A(X3)-M-N;
- CDR-VH2 W-L(X1)-N-T-Y-S(X2)-G-E-P-T-Y-A-E(X3)-D(X4)-F;
- CDR-VH3 A-R-T(X1)-A-I(X2)-L(X3)-R-S-Y;
- CDR1-VL K-A-S-E(X1)-D-L(X2)-S-T-A-L(X3)-A;
- CDR-VL2 W-G(X1)-S-T-R-H-S(X2);
- CDR-VL3 Q-Q-H-W(X1)-S-T-P-L(X2).
- the coating solution (the main component NaHCO 3 ) diluted the 2019-nCoV N protein antigen to 1 ⁇ g/ml for microplate coating, 100 ⁇ l per well, overnight at 4°C; the next day, the washing solution was washed twice and patted dry; the blocking solution was added (20%BSA+80%PBS), 120 ⁇ l per well, 37°C, 1h, pat dry; add the diluted monoclonal antibody in Table 1, 100 ⁇ l/well, 37°C, 30min-60min; wash 5 times with washing solution , pat dry; add goat anti-mouse IgG-HRP, 100 ⁇ l per well, 37°C, 30 min; wash 5 times with washing solution (PBS), pat dry; add chromogenic solution A (50 ⁇ l/well, containing 2.1 g/L lemon acid, 12.25g/L citric acid, 0.07g/L acetanilide and 0.5g/L carbamide peroxide), add color developing solution B (50 ⁇ l/well, containing 1.05g/L
- Antibody concentration 5000 1000 500 250 125 0.00 WT 0.712 0.731 0.351 0.152 0.52 0.090 Mutation 1 0.952 0.825 0.625 0.364 0.204 0.025 Mutation 2 0.912 0.831 0.631 0.402 0.214 0.028 Mutation 3 0.825 0.841 0.594 0.387 0.198 0.022 Mutation 4 0.847 0.918 0.542 0.362 0.148 0.023 Mutation 5 0.168 0.021 - - - - Mutation 6 0.146 0.02 - - - - - -
- the purified antibody was diluted to 10 ⁇ g/mL with PBST (phosphate Tween buffer, the main component Na 2 HPO 4 +NaCl+TW-20), and the 2019-nCoV N protein antigen was serially diluted with PBST: 1.41 ⁇ g/mL, 0.70 ⁇ g/mL, 0.35 ⁇ g/mL, 0.18 ⁇ g/mL, 0.09 ⁇ g/mL, 0.04 ⁇ g/mL.
- PBST phosphate Tween buffer, the main component Na 2 HPO 4 +NaCl+TW-20
- Running process equilibrate in buffer 1 (PBST) for 60s, immobilize antibody in antibody solution for 300s, incubate in buffer 2 (PBST) for 180s, bind in antigen solution for 420s, dissociate in buffer 2 for 1200s, use 10mM pH 1.69 GLY solution and buffer 3 to regenerate the sensor and output data.
- K D is the equilibrium dissociation constant or affinity; kon is the association rate; kdis is the dissociation rate.
- the above antibodies were placed at 4°C (refrigerator), -80°C (refrigerator), and 37°C (incubator) for 21 days, and the 7-day, 14-day, and 21-day samples were taken for state observation, and the 21-day sample was tested for activity. , the results showed that there was no obvious change in the protein state of the antibodies under the three test conditions for 21 days, and the activity did not show a downward trend with the increase of the test temperature, indicating that the above antibodies were stable.
- the following table 7 mutation 1 antibody is the OD results of the enzyme immunoassay activity detection for 21 days.
- the present disclosure provides an antibody against the novel coronavirus and a kit for detecting the novel coronavirus.
- the antibody can specifically bind to the N protein of the novel coronavirus and has a high affinity for it, and the antibody can be used to detect the novel coronavirus better. sensitivity and specificity.
- the detection kit provided by the present disclosure also has the same technical effect as the antibody, and has broad application prospects and high market value.
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Abstract
Description
CDR-VH1 | CDR- | CDR- | CDR- | CDR- | |
WT | F | L | T | E | G |
突变1 | Y | I | S | Q | A |
突变2 | Y | L | T | E | A |
突变3 | F | I | S | E | A |
突变4 | Y | I | S | Q | G |
突变5 | Y | H | S | Q | A |
突变6 | Y | L | T | F | G |
抗体浓度(ng/ml) | 5000 | 1000 | 500 | 250 | 125 | 0.00 |
WT | 0.712 | 0.731 | 0.351 | 0.152 | 0.52 | 0.090 |
突变1 | 0.952 | 0.825 | 0.625 | 0.364 | 0.204 | 0.025 |
突变2 | 0.912 | 0.831 | 0.631 | 0.402 | 0.214 | 0.028 |
突变3 | 0.825 | 0.841 | 0.594 | 0.387 | 0.198 | 0.022 |
突变4 | 0.847 | 0.918 | 0.542 | 0.362 | 0.148 | 0.023 |
突变5 | 0.168 | 0.021 | - | - | - | - |
突变6 | 0.146 | 0.02 | - | - | - | - |
K D(M) | kon(1/Ms) | kdis(1/s) | |
突变1 | 1.21E-10 | 3.51E+06 | 4.24E-04 |
突变1-1 | 1.03E-10 | 4.21E+06 | 4.35E-04 |
突变1-2 | 1.29E-10 | 2.42E+06 | 3.12E-04 |
突变1-3 | 1.49E-10 | 3.19E+06 | 4.76E-04 |
突变1-4 | 1.22E-10 | 2.58E+06 | 3.14E-04 |
突变1-5 | 3.28E-10 | 2.07E+06 | 6.78E-04 |
突变1-6 | 2.47E-10 | 3.03E+06 | 7.48E-04 |
突变1-7 | 1.67E-10 | 3.78E+06 | 6.32E-04 |
突变1-8 | 3.15E-10 | 2.26E+06 | 7.12E-04 |
突变1-9 | 1.13E-10 | 4.68E+06 | 5.31E-04 |
突变1-10 | 1.43E-10 | 4.18E+06 | 5.98E-04 |
突变1-11 | 1.07E-10 | 4.98E+06 | 5.35E-04 |
突变1-12 | 1.46E-10 | 4.72E+06 | 6.89E-04 |
突变1-13 | 1.59E-10 | 4.68E+06 | 7.43E-04 |
突变1-14 | 3.17E-10 | 2.21E+06 | 7.01E-04 |
突变1-15 | 7.54E-11 | 5.00E+06 | 3.77E-04 |
突变1-16 | 1.78E-10 | 2.06E+06 | 3.67E-04 |
突变1-17 | 1.27E-10 | 4.88E+06 | 6.21E-04 |
突变1-18 | 1.93E-10 | 3.41E+06 | 6.59E-04 |
突变1-19 | 9.97E-11 | 3.93E+06 | 3.92E-04 |
突变1-20 | 8.94E-11 | 5.00E+06 | 4.47E-04 |
突变1-21 | 1.55E-10 | 4.50E+06 | 6.96E-04 |
突变1-22 | 1.42E-10 | 2.76E+06 | 3.91E-04 |
突变1-23 | 8.79E-11 | 4.78E+06 | 4.20E-04 |
突变1-24 | 1.50E-10 | 3.65E+06 | 5.48E-04 |
突变1-25 | 1.79E-10 | 3.12E+06 | 5.59E-04 |
突变1-26 | 1.01E-10 | 4.75E+06 | 4.79E-04 |
突变1-27 | 1.64E-10 | 2.38E+06 | 3.91E-04 |
突变1-28 | 1.66E-10 | 2.66E+06 | 4.42E-04 |
突变1-29 | 2.24E-10 | 3.48E+06 | 7.80E-04 |
突变1-30 | 1.74E-10 | 4.57E+06 | 7.97E-04 |
突变1-31 | 1.30E-10 | 4.31E+06 | 5.59E-04 |
突变1-32 | 1.41E-10 | 4.17E+06 | 5.90E-04 |
突变1-33 | 3.03E-10 | 2.32E+06 | 7.04E-04 |
突变1-34 | 1.61E-10 | 2.81E+06 | 4.53E-04 |
突变1-35 | 1.35E-10 | 2.42E+06 | 3.27E-04 |
突变1-36 | 1.41E-10 | 2.22E+06 | 3.12E-04 |
突变1-37 | 8.89E-11 | 4.95E+06 | 4.40E-04 |
突变1-38 | 1.05E-10 | 3.38E+06 | 3.55E-04 |
突变1-39 | 1.26E-10 | 4.31E+06 | 5.42E-04 |
突变1-40 | 1.09E-10 | 4.36E+06 | 4.75E-04 |
突变1-41 | 1.31E-10 | 3.10E+06 | 4.06E-04 |
K D(M) | kon(1/Ms) | kdis(1/s) | |
WT | 7.25E-09 | 1.80E+05 | 1.31E-03 |
WT 1 | 2.65E-09 | 2.28E+05 | 6.05E-04 |
WT 2 | 2.48E-09 | 3.10E+05 | 7.69E-04 |
WT 3 | 7.08E-09 | 1.36E+05 | 9.63E-04 |
WT 4 | 4.62E-09 | 1.15E+05 | 5.31E-04 |
WT 5 | 2.20E-09 | 3.56E+05 | 7.82E-04 |
样品浓度(ng/ml) | 500 | 250 | 0 |
4℃,21天样品 | 0.708 | 0.414 | 0.034 |
-80℃,21天样品 | 0.752 | 0.521 | 0.032 |
37℃,21天样品 | 0.731 | 0.551 | 0.037 |
Claims (18)
- 一种抗新型冠状病毒或其N蛋白的抗体或其功能性片段,其特征在于,所述抗体或其功能性片段包括如下互补决定区:CDR-VH1:G-X1-T-F-T-X2-Y-X3-M-N;其中:X1是Y或F;X2是D或N;X3是A或G;CDR-VH2:W-X1-N-T-Y-X2-G-E-P-T-Y-A-X3-X4-F;其中:X1是L或I;X2是T或S;X3是D或E;X4是D或E;CDR-VH3:A-R-X1-A-X2-X3-R-S-Y;其中:X1是S或T;X2是I或L;X3是I或L;CDR-VL1:K-A-S-X1-D-X2-S-T-A-X3-A;其中:X1是Q或E;X2是I、V或L;X3是I、V或L;CDR-VL2:W-X1-S-T-R-H-X2;其中:X1是A或G;X2是T或S;CDR-VL3:Q-Q-H-X1-S-T-P-X2;其中:X1是Y或W;X2是L、V或I。
- 根据权利要求1所述的抗体或其功能性片段,其特征在于,CDR-VH1中,X1是Y;CDR-VH2中,X1是I;CDR-VH3中,X1是S;CDR-VL1中,X1是Q;CDR-VL2中,X1是A;优选的,CDR-VH1中,X2是D;优选的,CDR-VH1中,X2是N;优选的,CDR-VH1中,X3是A;优选的,CDR-VH1中,X3是G;优选的,CDR-VH2中,X2是T;优选的,CDR-VH2中,X2是S;优选的,CDR-VH2中,X3是D;优选的,CDR-VH2中,X3是E;优选的,CDR-VH2中,X4是D;优选的,CDR-VH2中,X4是E;优选的,CDR-VH3中,X2是I;优选的,CDR-VH3中,X2是L;优选的,CDR-VH3中,X3是I;优选的,CDR-VH3中,X3是L;优选的,CDR-VL1中,X2是I;优选的,CDR-VL1中,X2是V;优选的,CDR-VL1中,X2是L;优选的,CDR-VL1中,X3是I;优选的,CDR-VL1中,X3是V;优选的,CDR-VL1中,X3是L;优选的,CDR-VL2中,X2是T;优选的,CDR-VL2中,X2是S;优选的,CDR-VL3中,X1是Y;优选的,CDR-VL3中,X1是W;优选的,CDR-VL3中,X2是L;优选的,CDR-VL3中,X2是V;优选的,CDR-VL3中,X2是I;优选的,所述抗体或其功能性片段的各互补决定区选自如下突变组合1-42中的任意一种:
- 根据权利要求2所述的抗体或其功能性片段,其特征在于,所述抗体或其功能性片段与新型冠状病毒的N蛋白以K D≤8×10 -9mol/L的亲和力结合;优选的,K D≤4×10 -10mol/L。
- 根据权利要求1-4任一项所述的抗体或其功能性片段,其特征在于,所述抗体包括序列依次如SEQ ID NO:1-4所示的轻链骨架区FR1-L、FR2-L、FR3-L及FR4-L,和/或,序列依次如SEQ ID NO:5-8所示的重链骨架区FR1-H、FR2-H、FR3-H及FR4-H;优选的,所述抗体还包含恒定区;优选的,所述恒定区选自IgG1、IgG2、IgG3、IgG4、IgA、IgM、IgE和IgD中的任意一者的恒定区;优选的,所述恒定区的种属来源为牛、马、乳牛、猪、绵羊、山羊、大鼠、小鼠、狗、猫、兔、骆驼、驴、鹿、貂、鸡、鸭、鹅、火鸡、斗鸡或人;优选的,所述恒定区来源于小鼠;优选的,所述恒定区的轻链恒定区序列如SEQ ID NO:9所示,所述恒定区的重链恒定区序列如SEQ ID NO:10所示;优选的,所述功能性片段选自所述抗体的VHH、F(ab’)2、Fab’、Fab、Fv和scFv中的任意一种。
- 根据权利要求1-4中任一项所述的抗新型冠状病毒的抗体或其功能性片段,其特征在于,所述抗体包括分别依次与序列SEQ ID NO:1、2、3、4具有至少80%的同源性的轻链骨架区FR1-L、FR2-L、FR3-L及FR4-L,和/或,分别依次与序列SEQ ID NO:5、6、7、8具有至少80%的同源性 的重链骨架区FR1-H、FR2-H、FR3-H及FR4-H。
- 一种检测新型冠状病毒或其N蛋白的试剂或试剂盒,其特征在于,其包括如权利要求1-6任一项所述的抗体或其功能性片段。
- 根据权利要求7所述的试剂或试剂盒,其特征在于,所述抗体或其功能性片段标记有可被检测的标记物;优选的,所述可被检测的标记物选自荧光染料、催化底物显色的酶、放射性同位素、化学发光试剂和纳米颗粒类标记物;优选的,所述荧光染料选自荧光素类染料及其衍生物、罗丹明类染料及其衍生物、Cy系列染料及其衍生物、Alexa系列染料及其衍生物和蛋白类染料及其衍生物;优选的,所述催化底物显色的酶选自辣根过氧化物酶、碱性磷酸酶、β-半乳糖苷酶、葡萄糖氧化酶、碳酸酐酶、乙酰胆碱酯酶以及6-磷酸葡萄糖脱氧酶;优选的,所述放射性同位素选自 212Bi、 131I、 111In、 90Y、 186Re、 211At、 125I、 188Re、 153Sm、 213Bi、 32P、 94mTc、 99mTc、 203Pb、 67Ga、 68Ga、 43Sc、 47Sc、 110mIn、 97Ru、 62Cu、 64Cu、 67Cu、 68Cu、 86Y、 88Y、 121Sn、 161Tb、 166Ho、 105Rh、 177Lu、 172Lu和 18F;优选的,所述化学发光试剂选自鲁米诺及其衍生物、光泽精、甲壳动物荧光素及其衍生物、联吡啶钌及其衍生物、吖啶酯及其衍生物、二氧环乙烷及其衍生物、洛粉碱及其衍生物和过氧草酸盐及其衍生物;优选的,所述纳米颗粒类标记物选自纳米颗粒、胶体、有机纳米颗粒、磁性纳米颗粒、量子点纳米颗粒和稀土络合物纳米颗粒;优选的,所述胶体选自胶体金属、分散型染料、染料标记的微球和乳胶;优选的,所述胶体金属选自胶体金、胶体银和胶体硒。
- 一种核酸分子,所述核酸分子编码根据权利要求1-6任一项所述的抗体或其功能性片段。
- 一种载体,其特征在于,其含有编码如权利要求1-6任一项所述的抗体或其功能性片段的核酸片段。
- 一种重组细胞,其特征在于,其含有权利要求10所述的载体。
- 如权利要求1-6任一项所述的抗体或其功能性片段或者如权利要求7或8所述的试剂或试剂盒在检测新型冠状病毒中的用途。
- 如权利要求1-6任一项所述的抗体或其功能性片段或者如权利要求7或8所述的试剂或试剂盒,用于检测新型冠状病毒的用途。
- 一种检测新型冠状病毒的方法,包括:A)在足以发生结合反应的条件下,使权利要求1-6任一项所述的抗体或其功能性片段与样品接触以进行结合反应;以及B)检测结合反应产生的免疫复合物。
- 一种诊断受试者在感染新型冠状病毒或与新型冠状病毒感染相关疾病中的方法,包括:A)在足以发生结合反应的条件下,使权利要求1-6任一项所述的抗体或其功能性片段与来自所述受试者的样品接触以进行结合反应;以及B)检测结合反应产生的免疫复合物。
- 根据权利要求15所述的方法,其中,所述与新型冠状病毒感染相关的疾病包括呼吸道症状、发热、咳嗽、气促、呼吸困难、肺炎、严重急性呼吸综合征、肾衰竭。
- 根据权利要求15所述的方法,其中,所述感染新型冠状病毒的受试者包括无症状感染者、无明显症状感染者、有症状感染者。
- 一种制备如权利要求1-6任一项所述的抗体或其功能性片段的方法,其特征在于,其包括:培养权利要求11所述的重组细胞,从培养产物中分离纯化得到所述抗体或其功能性片段。
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CN114426575B (zh) * | 2020-10-29 | 2023-10-03 | 东莞市朋志生物科技有限公司 | 抗新型冠状病毒的抗体和检测新型冠状病毒的试剂盒 |
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CN114409767A (zh) * | 2021-12-08 | 2022-04-29 | 广东菲鹏生物有限公司 | 一种鉴别新冠突变型抗原的抗体、试剂及方法 |
CN116444656B (zh) * | 2022-01-10 | 2023-09-22 | 东莞市朋志生物科技有限公司 | 一种鉴别新冠突变型抗原的抗体、试剂及方法 |
CN116444657B (zh) * | 2022-01-10 | 2023-10-31 | 东莞市朋志生物科技有限公司 | 抗新型冠状病毒的抗体、检测新型冠状病毒试剂和试剂盒 |
CN117402239B (zh) * | 2022-07-07 | 2024-05-10 | 东莞市朋志生物科技有限公司 | 一种抗糖化血红蛋白抗体、检测糖化血红蛋白的试剂和试剂盒 |
CN117487010A (zh) * | 2022-08-02 | 2024-02-02 | 东莞市朋志生物科技有限公司 | 抗四碘甲状腺素抗体或其功能性片段、检测四碘甲状腺素的试剂和试剂盒 |
CN117659180A (zh) * | 2022-09-06 | 2024-03-08 | 东莞市朋志生物科技有限公司 | 抗新型冠状病毒抗体或其功能性片段、检测新型冠状病毒的试剂和试剂盒 |
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