CN114516913B - Antibody against N-terminal brain natriuretic peptide precursor and detection kit - Google Patents

Antibody against N-terminal brain natriuretic peptide precursor and detection kit Download PDF

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CN114516913B
CN114516913B CN202011301274.6A CN202011301274A CN114516913B CN 114516913 B CN114516913 B CN 114516913B CN 202011301274 A CN202011301274 A CN 202011301274A CN 114516913 B CN114516913 B CN 114516913B
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孟媛
钟冬梅
叶庆妮
姜瑢瑢
王晨
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Dongguan Pengzhi Biotechnology Co Ltd
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Abstract

The invention discloses an antibody of an N-terminal brain natriuretic peptide precursor and a detection kit, and relates to the technical field of antibodies. The antibodies disclosed herein against N-terminal brain natriuretic peptide precursors comprise a heavy chain complementarity determining region and a light chain complementarity determining region. The antibody has better affinity to the N-terminal brain natriuretic peptide precursor, and the detection of the N-terminal brain natriuretic peptide precursor by using the antibody has better sensitivity and specificity.

Description

Antibody against N-terminal brain natriuretic peptide precursor and detection kit
Technical Field
The invention relates to the technical field of antibodies, in particular to an antibody for resisting an N-terminal brain natriuretic peptide precursor and a detection kit.
Background
Sudoh, a Japanese scholars in 1988, isolated from pig brains for the first time to obtain a polypeptide with potent diuretic, vasodilatory and antihypertensive effects, designated brain natriuretic peptide (Brain Natriuretic Peptide, BNP). BNP is distributed at the highest heart content, but the first to be synthesized by cardiomyocytes is proBNP (BNP precursor) containing 108 amino acids, which, when stimulated by the action of endoenzymes, lyses into 76 amino acids, biologically inactive N-terminal pro-B-natriuretic peptide (NT-proBNP) and 32 amino acids, active B-type natriuretic peptide (BNP), both of the same origin and released by equimolar secretion into the blood circulation.
When the cardiac capacity load is increased or the cardiac function is impaired, the index concentration of N-terminal brain natriuretic peptide precursor (NT-proBNP) and BNP is abnormally increased, wherein the NT-proBNP has better biological stability relative to the BNP, has longer half-life (120 min), has relatively stable concentration, has long effective detection time, has the content in blood which is about 16-20 times higher than that of the BNP, is relatively easy to detect, and has long stability (48 h) of a plasma specimen in vitro, and is the optimal cardiac marker for diagnosing heart failure and evaluating cardiac function.
Normal human blood typically has a NT-proBNP level of less than 0.3ng/mL. When the cardiac function is impaired and the myocardium dilates, NT-proBNP is rapidly synthesized and secreted in large quantities into the human blood. When some related early symptoms are found, the amount of NT-proBNP in blood can be accurately, sensitively, efficiently and stably measured, and a rapid and accurate early diagnosis basis can be provided for the aspects of treatment of heart failure, heart failure and dyspnea of the early stage, prognosis monitoring, grading of acute coronary syndrome and the like. Current methods for detecting NT-proBNP content mainly include gold-calibration assays, fluorescent immunoassays, enzyme-linked immunosorbent assays (ELISA) and magnetic particle Chemiluminescence (CMIA), but these measurement methods all require monoclonal antibodies specific for NT-proBNP. At present, specific antibodies aiming at N-terminal brain natriuretic peptide precursors in the market have certain defects in specificity and sensitivity.
In view of this, the present invention has been made.
Disclosure of Invention
The invention aims to provide an antibody for resisting an N-terminal brain natriuretic peptide precursor and a detection kit, wherein the antibody has better affinity for the N-terminal brain natriuretic peptide precursor, and the detection of the N-terminal brain natriuretic peptide precursor by using the antibody has better sensitivity and specificity.
The invention is realized in the following way:
in one aspect, the invention provides an antibody or functional fragment thereof against an N-terminal brain natriuretic peptide precursor, said antibody or functional fragment thereof having the following complementarity determining regions:
CDR-VH1: G-Y-X1-F-T-X2-Y-X3-M-H; wherein: x1 is S or T; x2 is N or D; x3 is E, D or N;
CDR-VH2: A-X1-D-P-X2-T-G-G-T-A-Y-S-X3-K-F-K-G; wherein: x1 is L or I; x2 is E, Q or N; x3 is Q or E;
CDR-VH3: X1-R-E-G-D-Y-X2-Y-G-T-X3-D; wherein: x1 is A or T; x2 is F or Y; x3 is I, V or L;
CDR-VL1: R-S-S-Q-T-X1-X2-Y-S-X3-G-N-T-Y-L-E; wherein: x1 is I, V or L; x2 is I, V or L; x3 is D or N;
CDR-VL2: K-X1-S-N-R-X2-S; wherein: x1 is I, V or A; x2 is F or Y;
CDR-VL3: F-Q-X1-S-H-X2-P-P; in (a): x1 is G or A; x2 is L, V or I.
The antibody or the functional fragment thereof for resisting the N-terminal brain natriuretic peptide precursor provided by the invention has the complementarity determining region structure, can specifically bind to the N-terminal brain natriuretic peptide precursor antigen, has better affinity to the N-terminal brain natriuretic peptide precursor antigen, and has better specificity and sensitivity when the antibody or the functional fragment thereof is used for detecting the N-terminal brain natriuretic peptide precursor. The invention provides a richer antibody choice for detecting N-terminal brain natriuretic peptide precursors and diagnosis of related diseases thereof.
In an alternative embodiment, CDR-VH1, X1 is T; in CDR-VH2, X1 is I; in CDR-VH3, X1 is T; in CDR-VL1, X3 is D; in CDR-VL2, X2 is F; in CDR-VL3, X1 is G.
The inventors of the present invention found that when the above mutation site in each complementarity determining region is the above amino acid residue, the antibody exhibits a better affinity for the N-terminal brain natriuretic peptide precursor.
In an alternative embodiment, in CDR-VH1, X2 is N.
In an alternative embodiment, in CDR-VH1, X2 is D.
In an alternative embodiment, in CDR-VH1, X3 is E.
In an alternative embodiment, in CDR-VH1, X3 is D.
In an alternative embodiment, in CDR-VH1, X3 is N.
In an alternative embodiment, in CDR-VH2, X2 is E.
In an alternative embodiment, in CDR-VH2, X2 is Q.
In an alternative embodiment, in CDR-VH2, X2 is N.
In an alternative embodiment, in CDR-VH2, X3 is Q.
In an alternative embodiment, in CDR-VH2, X3 is E.
In an alternative embodiment, in CDR-VH3, X2 is F.
In an alternative embodiment, in CDR-VH3, X2 is Y.
In an alternative embodiment, in CDR-VH3, X3 is I.
In an alternative embodiment, in CDR-VH3, X3 is V.
In an alternative embodiment, in CDR-VH3, X3 is L.
In an alternative embodiment, in CDR-VL1, X1 is I.
In an alternative embodiment, in CDR-VL1, X1 is V.
In an alternative embodiment, in CDR-VL1, X1 is L.
In an alternative embodiment, in CDR-VL1, X2 is I.
In an alternative embodiment, in CDR-VL1, X2 is V.
In an alternative embodiment, in CDR-VL1, X2 is L.
In an alternative embodiment, in CDR-VL2, X1 is I.
In an alternative embodiment, in CDR-VL2, X1 is V.
In an alternative embodiment, in CDR-VL2, X1 is A.
In an alternative embodiment, in CDR-VL3, X2 is L.
In an alternative embodiment, in CDR-VL3, X2 is V.
In an alternative embodiment, in CDR-VL3, X2 is I.
In alternative embodiments, each complementarity determining region of the antibody or functional fragment thereof is selected from any one of the following combinations of mutations 1-44:
Figure BDA0002786979740000021
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Figure BDA0002786979740000031
in an alternative embodiment, the antibody or functional fragment thereof is administered with an N-terminal brain natriuretic peptide precursor in the form of K D ≤9.2×10 -8 Affinity binding in mol/L.
In an alternative embodiment, K D ≤9×10 -8 mol/L、K D ≤8×10 -8 mol/L、K D ≤7×10 -8 mol/L、K D ≤6×10 -8 mol/L、K D ≤5×10 -8 mol/L、K D ≤4×10 -8 mol/L、K D ≤3×10 -8 mol/L、K D ≤2×10 -8 mol/L、K D ≤1×10 -8 mol/L、K D ≤9×10 -9 mol/L、K D ≤8×10 -9 mol/L、K D ≤7×10 -9 mol/L、K D ≤6×10 - 9 mol/L、K D ≤5×10 -9 mol/L、K D ≤4×10 -9 mol/L、K D ≤3×10 -9 mol/L、K D ≤2×10 -9 mol/L、K D ≤1×10 -9 mol/L、K D ≤9×10 -10 mol/L、K D ≤8×10 -10 mol/L、K D ≤7×10 -10 mol/L、K D ≤6×10 -10 mol/L、K D ≤5×10 -10 mol/L、K D ≤4×10 -10 mol/L、K D ≤3×10 -10 mol/L、K D ≤2×10 -10 mol/L or K D ≤1×10 -10 mol/L。
In an alternative embodiment, K D ≤1.2×10 -9 mol/L。
In an alternative embodiment, 1.45×10 -10 mol/L≤K D ≤1.21×10 -9 mol/L。
K D Reference is made to the method in the embodiment of the invention.
In an alternative embodiment, in CDR-VH1, X1 is S; in CDR-VH2, X1 is L; in CDR-VH3, X1 is A; in CDR-VL1, X3 is N; in CDR-VL2, X2 is Y; in CDR-VL3, X1 is A.
In alternative embodiments, each complementarity determining region of the antibody or functional fragment thereof is selected from any one of the following combinations of mutations 45-50:
Figure BDA0002786979740000041
in alternative embodiments, the antibody comprises light chain framework regions FR1-L, FR2-L, FR3-L and FR4-L, which are shown in sequence SEQ ID NO. 1-4, and/or heavy chain framework regions FR1-H, FR2-H, FR3-H and FR4-H, which are shown in sequence SEQ ID NO. 5-8.
Typically, the heavy chain variable region (VH) and the light chain variable region (VL) are obtained by joining CDRs of the following numbering with FRs in a combined arrangement: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.
In other embodiments, each framework region amino acid sequence of an antibody or functional fragment thereof provided herein 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 to the corresponding framework region (SEQ ID NO:1, 2, 3, 4, 5, 6, 7 or 8) described above.
In alternative embodiments, the antibody further comprises a constant region.
In alternative embodiments, the constant region is selected from the group consisting of the constant region of any one of IgG1, igG2, igG3, igG4, igA, igM, igE and IgD.
In alternative embodiments, the constant region is of species origin of cow, horse, cow, pig, sheep, goat, rat, mouse, dog, cat, rabbit, camel, donkey, deer, mink, chicken, duck, goose, turkey, cock or human.
In alternative embodiments, the constant region is derived from a mouse.
In an alternative embodiment, the constant region has a light chain constant region sequence as shown in SEQ ID NO. 9 and a heavy chain constant region sequence as shown in SEQ ID NO. 10.
In alternative embodiments, the functional fragment is selected from any one of VHH, F (ab ') 2, fab', fab, fv and scFv of the antibody.
The functional fragments of the above antibodies generally have the same binding specificity as the antibody from which they were derived. It will be readily appreciated by those skilled in the art from the disclosure herein that functional fragments of the above antibodies may be obtained by methods such as enzymatic digestion (including pepsin or papain) and/or by methods of chemical reduction cleavage of disulfide bonds. The above functional fragments are readily available to those skilled in the art based on the disclosure of the structure of the intact antibodies.
Functional fragments of the above antibodies may also be synthesized by recombinant genetic techniques also known to those skilled in the art or by, for example, automated peptide synthesizers such as those sold by Applied BioSystems and the like.
In another aspect, the invention provides a reagent or kit for detecting an N-terminal brain natriuretic peptide precursor comprising an antibody or functional fragment thereof as described in any one of the above.
In an alternative embodiment, the antibody or functional fragment thereof in the above-described reagent or kit is labeled with a detectable label.
A detectable label refers to a substance of a type having properties such as luminescence, color development, radioactivity, etc., that can be directly observed by the naked eye or detected by an instrument, by which a qualitative or quantitative detection of the corresponding target can be achieved.
In alternative embodiments, the detectable label includes, but is not limited to, fluorescent dyes, enzymes that catalyze the development of substrates, radioisotopes, chemiluminescent reagents, and nanoparticle-based labels.
In the actual use process, a person skilled in the art can select a suitable marker according to the detection conditions or actual needs, and no matter what marker is used, the marker belongs to the protection scope of the invention.
In alternative embodiments, the fluorescent dyes include, but are not limited to, fluorescein-based dyes and derivatives thereof (including, but not limited to, fluorescein Isothiocyanate (FITC) hydroxy-light (FAM), tetrachlorolight (TET), and the like, or analogs thereof), rhodamine-based dyes and derivatives thereof (including, but not limited to, red Rhodamine (RBITC), tetramethyl rhodamine (TAMRA), rhodamine B (TRITC), and the like, or analogs thereof), cy-based dyes and derivatives thereof (including, but not limited to, cy2, cy3B, cy3.5, cy5, cy5.5, cy3, and the like, or analogs thereof), alexa-based dyes and derivatives thereof (including, but not limited to, alexa fluor350, 405, 430, 488, 532, 546, 555, 568, 594, 610, 33, 647, 680, 700, 750, and the like, or analogs thereof), and protein-based dyes and derivatives thereof (including, but not limited to, for example, phycoerythrin (PE), phycocyanin (PC), allophycocyanin (APC), polyazosin (chlorophyll), and the like).
In alternative embodiments, the enzymes that catalyze the development of a substrate include, but are not limited to, horseradish peroxidase, alkaline phosphatase, beta-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and 6-phosphoglucose deoxygenase.
In alternative embodiments, the radioisotope includes, but is not limited to 212 Bi、 131 I、 111 In、 90 Y、 186 Re、 211 At、 125 I、 188 Re、 153 Sm、 213 Bi、 32 P、 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。
in alternative embodiments, the chemiluminescent reagents include, but are not limited to, luminol and its derivatives, lucigenin, crustacean fluorescein and its derivatives, ruthenium bipyridine and its derivatives, acridinium esters and its derivatives, dioxane and its derivatives, lomustine and its derivatives, and peroxyoxalate and its derivatives.
In alternative embodiments, the nanoparticle-based labels include, but are not limited to, nanoparticles, colloids, organic nanoparticles, magnetic nanoparticles, quantum dot nanoparticles, and rare earth complex nanoparticles.
In alternative embodiments, the colloids include, but are not limited to, colloidal metals, disperse dyes, dye-labeled microspheres, and latex.
In alternative embodiments, the colloidal metals include, but are not limited to, colloidal gold, colloidal silver, and colloidal selenium.
In another aspect, the invention provides a nucleic acid molecule encoding an antibody or functional fragment thereof as described above.
In another aspect, the invention provides a vector comprising the nucleic acid molecule described above.
In another aspect, the present invention provides recombinant cells comprising the above vector.
In another aspect, the invention provides a method of making an antibody or functional fragment thereof comprising: culturing the recombinant cells as described above, and separating and purifying the culture product to obtain the antibody or the functional fragment thereof.
Based on the present disclosure of the amino acid sequence of an antibody or functional fragment thereof, it is readily apparent to a person skilled in the art that the preparation of the antibody or functional fragment thereof by genetic engineering techniques or other techniques (chemical synthesis, hybridoma cells), e.g., isolation and purification from a culture product of recombinant cells capable of recombinantly expressing an antibody or functional fragment thereof as described in any of the above, is within the scope of the present disclosure, irrespective of the technique used to prepare the antibody or functional fragment thereof.
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In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the embodiments will be briefly described below, it being understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and other related drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 shows the result of reducing SDS-PAGE of the anti-N-terminal brain natriuretic peptide precursor antibody of example 1.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
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 disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of formulations or unit doses herein, some methods and materials are now described. Unless otherwise indicated, techniques employed or contemplated herein are standard methods. The materials, methods, and examples are illustrative only and not intended to be limiting.
Unless otherwise indicated, practice of the present invention will employ conventional techniques of cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry and immunology, which are within the ability of a person skilled in the art. This technique is well explained in the literature, as is the case for molecular cloning: laboratory Manual (Molecular Cloning: A Laboratory Manual), second edition (Sambrook et al, 1989); oligonucleotide Synthesis (Oligonucleotide Synthesis) (M.J.Gait et al, 1984); animal cell culture (Animal Cell Culture) (r.i. freshney, 1987); methods of enzymology (Methods in Enzymology) (Academic Press, inc.), experimental immunology handbook (Handbook of Experimental Immunology) (D.M.Weir and C.C.Blackwell, inc.), gene transfer vectors for mammalian cells (Gene Transfer Vectors for Mammalian Cells) (J.M.Miller and M.P.calos, inc., 1987), methods of contemporary molecular biology (Current Protocols in Molecular Biology) (F.M.Ausubel et al, inc., 1987), PCR: polymerase chain reaction (PCR: the Polymerase Chain Reaction, inc., 1994), and methods of contemporary immunology (Current Protocols in Immunology) (J.E.Coligan et al, 1991), each of which is expressly incorporated herein by reference.
The features and capabilities of the present invention are described in further detail below in connection with the examples.
The restriction enzymes, prime Star DNA polymerase in the examples were purchased from Takara. MagExtractor-RNA extraction kit was purchased from TOYOBO company. BD SMART TM RACE cDNA Amplification Kit kit was purchased from Takara. pMD-18T vector was purchased from Takara. Plasmid extraction kits were purchased from Tiangen. Primer synthesis and gene sequencing were accomplished by Invitrogen corporation.
Example 1
1 construction of recombinant plasmid
(1) Antibody Gene production
mRNA is extracted from hybridoma cell strains secreting anti-N-terminal brain natriuretic peptide precursor antibodies, DNA products are obtained through an RT-PCR method, the products are inserted into a pMD-18T vector after an A adding reaction by rTaq DNA polymerase, and are transformed into DH5 alpha competent cells, after colonies grow out, the Heavy Chain and Light Chain gene clones are respectively taken for 4 clones to send to a gene sequencing company for sequencing.
(2) Sequence analysis of antibody variable region genes
The gene sequence obtained by sequencing is placed in an IMGT antibody database for analysis, and VNTI11.5 software is utilized for analysis to determine that the amplified genes of the heavy Chain primer pair and the Light Chain primer pair are correct, wherein in the gene fragment amplified by the Light Chain, the VL gene sequence is 339bp, belongs to the VkII gene family, and the front part of the VL gene sequence is 57bp of leader peptide sequence; in the gene fragment amplified by the Heavy Chain primer pair, the VH gene sequence is 360bp, belongs to the VH1 gene family, and a 57bp leader peptide sequence is arranged in front of the VH gene family.
(3) Construction of recombinant antibody expression plasmids
pcDNA TM 3.4
Figure BDA0002786979740000061
vector is a constructed eukaryotic expression vector of the recombinant antibody, and the expression vector is introduced into a HindIII, bamHI, ecoRI polyclonal enzyme cutting site, named pcDNA3.4A expression vector and is hereinafter abbreviated as 3.4A expression vector; according to the result of the antibody variable region gene sequencing in pMD-18T, VL and VH gene specific primers of the antibody are designed, hindIII, ecoRI restriction sites and protective bases are respectively arranged at two ends, and a 0.74kb Light Chain gene fragment and a 1.41kb Heavy Chain gene fragment are amplified by a PCR amplification method.
The Heavy Chain gene fragment and the Light Chain gene fragment are respectively cut by HindIII/EcoRI double enzyme, the 3.4A vector is cut by HindIII/EcoRI double enzyme, and the Heavy Chain gene fragment and the Light Chain gene fragment after the fragment and the vector are purified and recovered are respectively connected into the 3.4A expression vector to respectively obtain recombinant expression plasmids of the Heavy Chain gene fragment and the Light Chain gene fragment.
2 stable cell line selection
(1) Recombinant antibody expression plasmid transient transfection CHO cells, determination of expression plasmid activity
The plasmid was diluted to 40 ug/100. Mu.L with ultrapure water, and CHO cells were regulated to 1.43X 10 7 100. Mu.L of plasmid was mixed with 700. Mu.L of cells in a centrifuge tube, transferred to an electrocuvette, electroblotted, sample counted on days 3, 5, 7, and harvested on day 7.
The coating solution was diluted to 3. Mu.g/ml of NT-proBNP antigen, 100. Mu.L per well, overnight at 4 ℃; the next day, washing with washing liquid for 2 times, and drying; blocking solution (20% BSA+80% PBS) was added, 120. Mu.L per well, 37℃for 1h, and the mixture was dried by shaking; adding diluted cell supernatant at 37deg.C for 30min at 100 μl/well; washing with washing liquid for 5 times, and drying; adding goat anti-mouse IgG-HRP, 100 mu L of each hole, and 30min at 37 ℃; washing with washing liquid for 5 times, and drying; adding a color development solution A (50 mu L/hole, containing citric acid, sodium acetate, acetanilide and carbamide peroxide), and adding a color development solution B (50 mu L/hole, containing citric acid, EDTA, 2Na+TMB and concentrated HCl) for 10min; adding stop solution (50. Mu.L/well, EDTA. 2Na+ concentrated H) 2 SO 4 ) The method comprises the steps of carrying out a first treatment on the surface of the OD was read on the microplate reader at 450nm (reference 630 nm). The results showed that the OD of the reaction was still greater than 1.0 after 1000-fold dilution of the cell supernatant, without addition of cell supernatant wellsThe reaction OD was less than 0.1, indicating that antibodies generated after transient transformation of the plasmid were active on NT-proBNP.
(2) Linearization of recombinant antibody expression plasmids
The following reagents were prepared: buffer 50 mu L, DNA mu g/tube, puv I enzyme 10 mu L, sterile water to 500 mu L, water bath at 37 ℃ for enzyme digestion overnight; firstly, extracting with equal volume of phenol/chloroform/isoamyl alcohol (lower layer) 25:24:1, and then sequentially extracting with chloroform (water phase); precipitating 0.1 times volume (water phase) of 3M sodium acetate and 2 times volume of ethanol on ice, rinsing the precipitate with 70% ethanol, removing organic solvent, completely volatilizing ethanol, re-thawing with appropriate amount of sterilized water, and measuring concentration.
(3) Stable transfection of recombinant antibody expression plasmid and pressure screening of stable cell strain
The plasmid was diluted to 40 ug/100. Mu.L with ultrapure water, and CHO cells were regulated to 1.43X 10 7 Placing cells/ml in a centrifuge tube, mixing 100 μl of plasmid with 700 μl of cells, transferring into an electrorotor, electrorotating, and counting the next day; 25umol/L MSX 96-well pressure culture for about 25 days.
Observing the clone holes with the cells under a microscope, and recording the confluency; taking culture supernatant, and carrying out sample feeding detection; selecting cell strains with high antibody concentration and relative concentration, turning 24 holes, and turning 6 holes about 3 days; 3 days later, seed preservation and batch culture are carried out, the cell density is regulated to be 0.5 multiplied by 106cells/ml, batch culture is carried out by 2.2ml, and seed preservation is carried out by 2ml, wherein the cell density is 0.3 multiplied by 106 cells/ml; and (3) carrying out sample feeding detection on the culture supernatant of the 6-hole batch culture for 7 days, and selecting cell strains with smaller antibody concentration and smaller cell diameter to transfer TPP for seed preservation and passage.
3 recombinant antibody production
(1) Cell expansion culture
After cell recovery, the cells were first cultured in 125ml shake flasks with an inoculation volume of 30ml and a medium of 100% dynamis and placed in a shaker at a speed of 120r/min at 37℃and with 8% carbon dioxide. Culturing for 72h, inoculating and expanding culture at 50 ten thousand cells/ml inoculating density, and calculating the expanding culture volume according to production requirements, wherein the culture medium is 100% Dynamis culture medium. After that, the culture was spread every 72 hours. When the cell quantity meets the production requirement, the inoculation density is strictly controlled to be about 50 ten thousand cells/ml for production.
(2) Shake flask production and purification
Shake flask parameters: the rotating speed is 120r/min, the temperature is 37 ℃, and the carbon dioxide is 8%. Feeding: feeding was started every day until 72h of culture in shake flasks, hyCloneTM Cell BoostTM Feed a fed-batch was 3% of the initial culture volume every day, feed 7b fed-batch was one thousandth of the initial culture volume every day, and fed-batch was continued until day 12 (day 12 Feed). Glucose was fed at 3g/L on day six. Samples were collected on day 13. Affinity purification was performed using a proteona affinity column. 4. Mu.g of the purified antibody was subjected to reducing SDS-PAGE, and 4. Mu.g of an external control antibody was used as a control, and the electrophoretogram is shown in FIG. 1 below, showing two bands after reducing SDS-PAGE, 1 Mr of 50KD (heavy chain, SEQ ID NO: 14) and the other Mr of 28KD (light chain, SEQ ID NO: 13).
Example 2
Performance detection of antibodies
(1) Example 1 Activity detection of antibodies and mutants thereof
The antibody (WT) sequence of example 1 was analyzed and its heavy chain variable region is shown in SEQ ID NO. 12, wherein the amino acid sequence of each complementarity determining region on the heavy chain variable region is as follows:
CDR-VH1:G-Y-S(X1)-F-T-D(X2)-Y-D(X3)-M-H;
CDR-VH2:A-L(X1)-D-P-N(X2)-T-G-G-T-A-Y-S-Q(X3)-K-F-K-G;
CDR-VH3:A(X1)-R-E-G-D-Y-F(X2)-Y-G-T-I(X3)-D;
the light chain variable region is shown as SEQ ID NO. 11, wherein the amino acid sequence of each complementarity determining region on the light chain variable region is as follows:
CDR1-VL:R-S-S-Q-T-L(X1)-L(X2)-Y-S-N(X3)-G-N-T-Y-L-E;
CDR-VL2:K-V(X1)-S-N-R-Y(X2)-S;
CDR-VL3:F-Q-A(X1)-S-H-I(X2)-P-P。
on the basis of the anti-N-terminal brain natriuretic peptide precursor antibody (WT) of example 1, mutations were made at sites related to antibody activity in the complementarity determining regions, wherein X1, X2, X3 are mutation sites. See table 1 below.
TABLE 1 mutation sites related to antibody Activity
Figure BDA0002786979740000081
Antibody binding Activity assay in Table 1:
the coating solution (main component NaHCO 3) dilutes the NT-proBNP antigen to 3. Mu.g/ml, 100. Mu.l per well, overnight at 4 ℃; the next day, washing with washing liquid for 2 times, and drying; blocking solution (20% BSA+80% PBS) was added, 120. Mu.l per well, 37℃for 1h, and the mixture was dried by pipetting; adding the diluted purified antibody into the mixture at the temperature of 37 ℃ for 30-60 min at the concentration of 100 mu l/hole; washing with washing liquid for 5 times, and drying; adding goat anti-mouse IgG-HRP, 100 μl per well, 37deg.C, 30min; washing with washing solution (PBS) for 5 times, and drying; adding a color development solution A (50. Mu.l/hole, containing 2.1g/L citric acid, 12.25g/L citric acid, 0.07g/L acetanilide and 0.5g/L carbamide peroxide), and adding a color development solution B (50. Mu.l/hole, containing 1.05g/L citric acid, 0.186 g/LEDTA.2Na, 0.45g/L TMB and 0.2ml/L concentrated HCl) for 10min; adding stop solution (50. Mu.l/well, 0.75 g/EDTA.2Na and 10.2ml/L of concentrated H) 2 SO 4 ) The method comprises the steps of carrying out a first treatment on the surface of the OD was read on the microplate reader at 450nm (reference 630 nm). The results are shown in Table 2 below.
Table 2 Activity data for WT antibodies and mutants thereof
Antibody concentration (ng/ml) 15.63 7.81 3.91 1.95 0.98 0.00
WT 1.568 0.764 0.351 0.102 0.092 0.031
Mutation 1 1.694 0.996 0.550 0.292 0.169 0.014
Mutation 2 1.637 0.972 0.534 0.278 0.160 0.010
Mutation 3 1.633 0.970 0.513 0.268 0.158 0.013
Mutation 4 1.601 1.034 0.591 0.318 0.177 0.007
Mutation 5 0.432 0.276 0.156 - - -
Mutation 6 0.511 0.265 0.159 - - -
The data in Table 2 show that the activity of WT, mutation 1 to mutation 3 is higher compared to mutations 5 and 6, with the activity of mutation 1 performing optimally.
(2) Affinity detection of antibodies and mutants thereof
(a) Other sites were mutated based on mutation 1, and the sequence of each mutation is shown in Table 3 below.
TABLE 3 mutation sites related to antibody affinity
Figure BDA0002786979740000091
/>
Figure BDA0002786979740000101
Affinity analysis
The purified antibody was diluted to 10ug/ml with PBST using AMC sensor, and NT-proBNP was gradient diluted with PBST;
the operation flow is as follows: equilibration for 60s in buffer 1 (PBST), antibody 300s in antibody solution, incubation for 180s in buffer 2 (PBST), binding for 420s in antigen solution, dissociation for 1200s in buffer 2, sensor regeneration with 10mM pH 1.69GLY solution and buffer 3, and data output. The results are shown in Table 4.K (K) D Representing equilibrium dissociation constant, i.e. affinity; kon represents the binding rate; kdis represents the dissociation rate.
Table 4 affinity assay data
Figure BDA0002786979740000102
/>
Figure BDA0002786979740000111
As can be seen from the data in Table 4, the mutant 1 and its series of mutant antibodies have a higher affinity for the NT-proBNP antigen, indicating that all antibodies obtained by mutation in the manner of the mutation in Table 3 have a higher affinity on the basis of the mutant 1.
(b) Other sites were mutated on the WT basis and the affinity of each mutant was examined, the sequence of each mutation being shown in table 5 below, and the corresponding affinity data being shown in table 6.
TABLE 5 mutation with WT as backbone
Figure BDA0002786979740000112
Table 6 affinity detection results of WT antibodies and mutants thereof
K D (M) kon(1/Ms) kdis(1/s)
WT 9.18E-08 9.07E+04 8.33E-03
WT 1 8.22E-08 8.87E+04 7.29E-03
WT2 8.25E-08 9.38E+04 7.74E-03
WT 3 8.69E-08 9.07E+04 7.88E-03
WT 4 8.03E-08 1.08E+05 8.67E-03
WT5 8.82E-08 8.63E+04 7.61E-03
As can be seen from Table 6, WT and its series of mutant antibodies also had good affinity, indicating that on a WT basis, the mutant antibodies obtained by the mutation in Table 5 had good affinity for the antigen.
(3) Bare stability assessment
Placing the antibody at 4 ℃ (refrigerator), 80 ℃ (refrigerator) and 37 ℃ (incubator) for 21 days, taking 7 days, 14 days and 21 days samples for state observation, and detecting the activity of the 21 days samples, wherein the result shows that no obvious protein state change is seen for the antibody placed for 21 days under three examination conditions, and the activity is not in a descending trend along with the increase of the examination temperature, thus indicating that the antibody is stable. The mutant 1 antibodies of Table 7 below were tested for OD results for 21 days of enzyme-free activity.
TABLE 7
Sample concentration (ng/ml) 10 4 0
4 ℃,21 days sample 1.582 0.582 0.008
Sample at-80℃for 21 days 1.554 0.545 0.038
37 ℃ and 21 days of sample 1.549 0.508 0.03
(4) Evaluation of Performance
The antibodies in the above examples were used as coating antibodies, respectively, in combination with another NT-proBNP labeled antibody (obtainable from fepeng organism) which was paired, and the performance differences between the mutant antibodies and the WT antibodies were compared on a fluorescent rapid diagnosis platform using a double antibody sandwich method, the performance levels of which are shown in table 8 below.
TABLE 8
Conditions (conditions) Specificity (specificity) Sensitivity of Consistency of Correlation of Linearity of
WT 96.0% 92.0% 91.6% 0.94 0.92
Mutation 1 99.0% 94.5% 94.7% 0.98 0.94
In addition, the specificity of mutation 1-1 to mutation 1-43 ranged from 99.2% to 99.7%, and the sensitivity ranged from 94.3% to 95.9%.
The results in Table 8 show that the antibody provided by the embodiment of the invention has higher sensitivity and specificity when applied to chemiluminescent platform detection.
The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Sequence listing
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Claims (27)

1. An antibody or functional fragment thereof directed against an N-terminal brain natriuretic peptide precursor, wherein the antibody or functional fragment thereof comprises the complementarity determining regions:
CDR-VH1: G-Y-X1-F-T-X2-Y-X3-M-H; wherein: x1 is T; x2 is N or D; x3 is E, D or N;
CDR-VH2: A-X1-D-P-X2-T-G-G-T-A-Y-S-X3-K-F-K-G; wherein: x1 is I; x2 is E, Q or N; x3 is Q or E;
CDR-VH3: X1-R-E-G-D-Y-X2-Y-G-T-X3-D; wherein: x1 is T; x2 is F or Y; x3 is I, V or L;
CDR-VL1: R-S-S-Q-T-X1-X2-Y-S-X3-G-N-T-Y-L-E; wherein: x1 is I, V or L; x2 is I, V or L; x3 is D;
CDR-VL2: K-X1-S-N-R-X2-S; wherein: x1 is I, V or A; x2 is F;
CDR-VL3: F-Q-X1-S-H-X2-P-P; in (a): x1 is G; x2 is L, V or I;
the antibody or functional fragment thereof and N-terminal brain natriuretic peptide precursor antigen are used as a vaccine D ≤9.2×10 -8 Affinity binding in mol/L.
2. An antibody or functional fragment thereof directed against an N-terminal brain natriuretic peptide precursor, wherein the antibody or functional fragment thereof comprises the complementarity determining regions:
CDR-VH1: G-Y-X1-F-T-X2-Y-X3-M-H; wherein: x1 is T;
CDR-VH2: A-X1-D-P-X2-T-G-G-T-A-Y-S-X3-K-F-K-G; wherein: x1 is I;
CDR-VH3: X1-R-E-G-D-Y-X2-Y-G-T-X3-D; wherein: x1 is T;
CDR-VL1: R-S-S-Q-T-X1-X2-Y-S-X3-G-N-T-Y-L-E; wherein: x3 is D;
CDR-VL2: K-X1-S-N-R-X2-S; wherein: x2 is F;
CDR-VL3: F-Q-X1-S-H-X2-P-P; in (a): x1 is G;
each complementarity determining region of the antibody or functional fragment thereof is selected from any one of the following combinations of mutations 1-44:
Figure FDF0000019873810000011
/>
Figure FDF0000019873810000021
3. an antibody or functionality thereof against an N-terminal brain natriuretic peptide precursor according to any one of claims 1-2Fragments, wherein the antibody or functional fragment thereof and the N-terminal brain natriuretic peptide precursor antigen are administered in the form of K D ≤1.2×10 -9 Affinity binding in mol/L.
4. The antibody or functional fragment thereof against an N-terminal brain natriuretic peptide precursor according to any one of claims 1-2, wherein the antibody comprises the light chain framework regions FR1-L, FR2-L, FR3-L and FR4-L and/or the heavy chain framework regions FR1-H, FR2-H, FR3-H and FR4-H and/or the heavy chain framework regions FR1-H, FR2-H, FR-H and FR4-H, respectively, having the sequences shown in SEQ ID NOs 5-8.
5. The antibody or functional fragment thereof against an N-terminal brain natriuretic peptide precursor according to claim 4, wherein the antibody further comprises a constant region.
6. The antibody or functional fragment thereof against an N-terminal brain natriuretic peptide precursor according to claim 5, wherein the constant region is selected from the group consisting of the constant region of any one of IgG1, igG2, igG3, igG4, igA, igM, igE and IgD.
7. The antibody or functional fragment thereof against an N-terminal brain natriuretic peptide precursor according to claim 5, wherein the constant region is of bovine, equine, porcine, ovine, caprine, rat, mouse, canine, feline, rabbit, donkey, deer, mink, chicken, duck, goose, or human origin.
8. The antibody or functional fragment thereof against an N-terminal brain natriuretic peptide precursor according to claim 5, wherein the constant region is of bovine species origin.
9. The antibody or functional fragment thereof against an N-terminal brain natriuretic peptide precursor according to claim 5, wherein the constant region is of a species origin of turkey or chicken.
10. The antibody or functional fragment thereof against an N-terminal brain natriuretic peptide precursor according to claim 5, wherein the species source of the constant region is mouse.
11. The antibody or functional fragment thereof against an N-terminal brain natriuretic peptide precursor according to claim 5, wherein the light chain constant region sequence of the constant region is shown in SEQ ID No. 9 and the heavy chain constant region sequence of the constant region is shown in SEQ ID No. 10.
12. The antibody or functional fragment thereof against an N-terminal brain natriuretic peptide precursor according to any one of claims 1-2, wherein the functional fragment is selected from any one of F (ab ') 2, fab', fab, fv and scFv of the antibody.
13. A reagent or kit for detecting an N-terminal brain natriuretic peptide precursor, comprising an antibody or functional fragment thereof according to any one of claims 1-12.
14. The reagent or kit according to claim 13, wherein the antibody or functional fragment thereof is labelled with a detectable label.
15. The reagent or kit of claim 14, wherein the detectable label is selected from the group consisting of fluorescent dyes, enzymes that catalyze the development of substrates, radioisotopes, chemiluminescent reagents, and nanoparticle-based labels.
16. The reagent or kit according to claim 15, wherein the fluorescent dye is selected from the group consisting of fluorescein-based dyes and derivatives thereof, and protein-based dyes and derivatives thereof.
17. The reagent or kit according to claim 16, wherein the fluorescein-based dye and its derivative are selected from rhodamine-based dye and its derivative, cy-based dye and its derivative, alexa-based dye and its derivative.
18. The reagent or kit according to claim 15, wherein the enzyme catalyzing the development of a substrate is selected from horseradish peroxidase, alkaline phosphatase, β -galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and glucose-6-phosphate deoxygenase.
19. The reagent or kit according to claim 15, wherein the radioisotope is selected from the group consisting of 212 Bi、 131 I、 111 In、 90 Y、 186 Re、 211 At、 125 I、 188 Re、 153 Sm、 213 Bi、 32 P、 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。
20. the reagent or kit according to claim 15, wherein the chemiluminescent reagent is selected from luminol and its derivatives, lucigenin, crustacean fluorescein and its derivatives, ruthenium bipyridine and its derivatives, acridine esters and its derivatives, dioxane and its derivatives, lotensine and its derivatives, and peroxyoxalate and its derivatives.
21. The reagent or kit according to claim 15, wherein the nanoparticle-based label is selected from the group consisting of nanoparticles and colloids.
22. The reagent or kit of claim 21, wherein the nanoparticles are selected from the group consisting of organic nanoparticles, magnetic nanoparticles, quantum dot nanoparticles, and rare earth complex nanoparticles.
23. The reagent or kit of claim 21, wherein the colloid is selected from the group consisting of colloidal metals, disperse dyes, dye-labeled microspheres, latex, and colloidal selenium.
24. The reagent or kit of claim 23, wherein the colloidal metal is selected from the group consisting of colloidal gold and colloidal silver.
25. A vector comprising a nucleic acid fragment encoding the antibody or functional fragment thereof according to any one of claims 1-12.
26. A recombinant cell comprising the vector of claim 25.
27. A method of preparing an antibody or functional fragment thereof according to any one of claims 1 to 12, comprising: culturing the recombinant cell of claim 26, and isolating and purifying the antibody or functional fragment thereof from the culture product.
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