CN113999311A

CN113999311A - Antibody capable of specifically binding 25-hydroxyvitamin D, application thereof and diagnostic kit

Info

Publication number: CN113999311A
Application number: CN202010739828.4A
Authority: CN
Inventors: 崔鹏; 何志强; 孟媛; 钟冬梅; 季红斌; 游辉
Original assignee: Dongguan Pengzhi Biotechnology Co Ltd
Current assignee: Dongguan Pengzhi Biotechnology Co Ltd
Priority date: 2020-07-28
Filing date: 2020-07-28
Publication date: 2022-02-01
Anticipated expiration: 2040-07-28
Also published as: WO2022022532A1; CN113999311B

Abstract

The invention discloses an antibody capable of specifically binding 25-hydroxyvitamin D, application thereof and a diagnostic kit, and relates to the technical field of antibodies. The antibodies or functional fragments thereof disclosed herein have complementarity determining regions CDR-VH1-3 and CDR-VL 1-3. The polypeptide can be specifically combined with 25-hydroxyvitamin D, and has better sensitivity, affinity and specificity.

Description

Antibody capable of specifically binding 25-hydroxyvitamin D, application thereof and diagnostic kit

Technical Field

The invention relates to the technical field of antibodies, in particular to an antibody capable of specifically binding 25-hydroxyvitamin D, application thereof and a diagnostic kit.

Background

Rickets were discovered by Francis Glisson in 1650, and this disease is widely prevalent in the affluent population. During the industrial revolution of the 18 th century, the incidence of rickets is increased by 40-60% due to crowding and pollution in cities. In 1822, snidecki found a correlation between rickets and lack of sunlight. With the development of science, further research shows that the vitamin D content in a human body is lower than the normal level after sunshine deficiency, so that rickets and other diseases are caused. Thus, vitamin D is widely used as an essential nutrient for the human body and is widely used in the public.

Vitamin d (vitamin d) is both a class of fat-soluble vitamins and a steroid hormone, an important nutrient essential for the life of higher animals including humans. Vitamin D mainly comprises five compounds, namely vitamin D1, D2, D3, D4 and D5, of which the most important are D2 and D3, and the two forms are referred to as vitamin D. Vitamin D, which is synthesized by itself and is food-derived, is absorbed in the jejunum and ileum, then combined with chylomicron or vitamin D transporters, transported to the liver by the lymphatic system or the blood system, converted into 25-hydroxyvitamin D [25- (OH) D ] by the action of vitamin D-25-hydroxylase, and stored in the liver. When human vitamin D is deficient, 25-hydroxyvitamin D is transported by the liver and is further metabolized in the kidney under the action of thyroxine to the active metabolite 1,25 dihydroxyvitamin D. Although the 1,25 dihydroxy vitamin D has the strongest biological activity, the half-life is short and is only 4 hours. The half-life of 25-hydroxyvitamin D is 3 weeks, and is the main existence form of vitamin D in the metabolic cycle of a human body, so that the half-life of 25-hydroxyvitamin D is often used as an index for evaluating the nutrient level of vitamin D in the body.

The current standard for judging the adult vitamin D nutrition status is as follows: vitamin D deficiency is a serum 25-hydroxyvitamin D level <50nmol/L (1nmol/L ═ 0.4ng/ml), less than 50-75 nmol/L, more than 75 nmol/L. Increasing epidemiological and laboratory evidence suggests that serum 25-hydroxyvitamin D levels are associated with the development of rickets, diabetes, chronic kidney disease, hepatitis, immune dysfunction (sjogren's syndrome, multiple sclerosis, rheumatoid arthritis), asthma, osteoporosis, parkinson's disease, cardiovascular disease, hypertension, type 2 diabetes, tumors (prostate, colon, breast, etc.), and other diseases in children. Therefore, the detection of 25-hydroxyvitamin D is clinically significant for the diagnosis and prevention of diseases.

The detection method of 25-hydroxyvitamin D can be classified into a chromatography method and an immunological method according to the principle. The chromatography mainly comprises High Performance Liquid Chromatography (HPLC), ultra-high performance liquid chromatography and liquid chromatography-mass spectrometry (LCMS/MS). The chromatography has the advantages of high sensitivity, specificity and accuracy, but the pretreatment of the sample is complex, the time is long, and the instrument and equipment are expensive, so that the method is limited to be widely applied clinically. The immunological method mainly comprises Radioimmunoassay (RIA), chemiluminescence immunoassay (CLIA), electrochemiluminescence immunoassay (ECLIA), enzyme-linked immunosorbent assay (ELISA) and full-automatic biochemical method. The immunoassay method has the advantages of sensitivity, rapidness, simplicity and the like, and is widely applied to clinical diagnosis. Different immunoassays have advantages and disadvantages, but all require antibodies that bind to 25-hydroxyvitamin D.

At present, monoclonal antibodies aiming at 25-hydroxyvitamin D have few sources, can only depend on the high import price, and have defects of sensitivity, affinity and specificity,

in view of this, the invention is particularly proposed.

Disclosure of Invention

The purpose of the present invention is to provide an antibody that specifically binds to 25-hydroxyvitamin D, its use, and a diagnostic kit.

The invention is realized by the following steps:

in one aspect, the present invention provides an antibody or functional fragment thereof that specifically binds to 25-hydroxyvitamin D, said antibody or functional fragment thereof having the following complementarity determining regions:

CDR-VH1 (heavy chain complementarity determining region 1): G-F-X1-F-D-X2-Y-G-X3-G, wherein: x1 is S or T; x2 is N or D; x3 is L, V or I;

CDR-VH2 (heavy chain complementarity determining region 2): G-X1-D-X2-H-G-X3-R-G-Y-N-R-X4-L-K-S, wherein: x1 is L, V or I; x2 is K or R; x3 is V, A or I; x4 is V, A or I;

CDR-VH3 (heavy chain complementarity determining region 3): X1-R-X2-W-Y-S-G-X3-G-F-X4-F, wherein: x1 is T or A; x2 is L, V or I; x3 is N or Q; x4 is E or D;

CDR-VL1 (light chain complementarity determining region 1): S-G-S-X1-S-N-X2-G-Y-G-N-Y-X3-S, wherein: x1 is S or T; x2 is I, V or L; x3 is I, V or L;

CDR-VL2 (light chain complementarity determining region 2): D-S-X1-T-R-X2-S-G-X3-P, wherein: x1 is I, V or L; x2 is G or A; x3 is I, V or L;

CDR-VL3 (light chain complementarity determining region 3): A-S-X1-D-S-S-X2-G, wherein: x1 is W, Y or F; x2 is E or D.

The antibody or the functional fragment thereof provided by the invention has the complementarity determining region structure, can be specifically combined with 25-hydroxyvitamin D, and has better sensitivity, affinity and specificity. The antibody or the functional fragment thereof has wide application, can be used for detecting the level of 25-hydroxyvitamin D, can also be prepared into related diagnostic reagents, is used for diagnosing diseases related to vitamin D metabolism, and provides more antibody selection schemes for the current immunoassay of 25-hydroxyvitamin D.

In an alternative embodiment of the method of the present invention,

in CDR-VH1, X1 is S;

in CDR-VH2, X2 is K;

in CDR-VH3, X1 is A;

in CDR-VL1, X1 is S;

in CDR-VL2, X2 is A;

in CDR-VL3, X2 is D.

The present inventors have found that when the mutation site in each complementarity determining region is the amino acid residue, the antibody exhibits a better affinity for 25-hydroxyvitamin D.

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 L.

In an alternative embodiment, in CDR-VH1, X3 is V.

In an alternative embodiment, in CDR-VH1, X3 is I.

In an alternative embodiment, in CDR-VH2, X1 is L.

In an alternative embodiment, in CDR-VH2, X1 is V.

In an alternative embodiment, in CDR-VH2, X1 is I.

In an alternative embodiment, in CDR-VH2, X3 is V.

In an alternative embodiment, in CDR-VH2, X3 is a.

In an alternative embodiment, in CDR-VH2, X3 is I.

In an alternative embodiment, in CDR-VH2, X4 is V.

In an alternative embodiment, in CDR-VH2, X4 is a.

In an alternative embodiment, in CDR-VH2, X4 is I.

In an alternative embodiment, in CDR-VH3, X2 is L.

In an alternative embodiment, in CDR-VH3, X2 is V.

In an alternative embodiment, in CDR-VH3, X2 is I.

In an alternative embodiment, in CDR-VH3, X3 is N.

In an alternative embodiment, in CDR-VH3, X3 is Q.

In an alternative embodiment, in CDR-VH3, X4 is E.

In an alternative embodiment, in CDR-VH3, X4 is Q.

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-VL1, X3 is I.

In an alternative embodiment, in CDR-VL1, X3 is V.

In an alternative embodiment, in CDR-VL1, X3 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 L.

In an alternative embodiment, in CDR-VL2, X3 is I.

In an alternative embodiment, in CDR-VL2, X3 is V.

In an alternative embodiment, in CDR-VL2, X3 is L.

In an alternative embodiment, in CDR-VL1, X1 is W.

In an alternative embodiment, in CDR-VL1, X1 is Y.

In an alternative embodiment, in CDR-VL1, X1 is F.

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 1-62:

in alternative embodiments, the antibody or functional fragment thereof binds 25-hydroxyvitamin D with K_D≤3.08×10^-6Affinity binding of mol/L, preferablyOptionally, K_D≤7.38×10^-7mol/L。

In an alternative embodiment, K_D≤3×10^-6mol/L、K_D≤2×10^-6mol/L、K_D≤2×10^-6mol/L、K_D≤9×10⁷mol/L、K_D≤8×10⁷mol/L、K_D≤7×10⁷mol/L、K_D≤6×10⁷mol/L、K_D≤5×10⁷mol/L、K_D≤4×10⁷mol/L、K_D≤3×10⁷mol/L、K_D≤2×10⁷mol/L or K_D≤1×10⁷mol/L。

In an alternative embodiment, 1.20 × 10^-7mol/L≤K_D≤7.38×10^-7mol/L。

K_DThe detection of (2) is carried out with reference to the method in the examples of the present invention.

In alternative embodiments, in CDR-VH1, X1 is T;

in CDR-VH2, X2 is R;

in CDR-VH3, X1 is T;

in CDR-VL1, X1 is T;

in CDR-VL2, X2 is G;

in CDR-VL3, X2 is E.

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 63-70:

in alternative embodiments, the antibody comprises the light chain framework regions FR1-L, FR2-L, FR3-L and FR4-L in sequence as set forth in SEQ ID NOS: 1-4, and/or the heavy chain framework regions FR1-H, FR2-H, FR3-H and FR4-H in sequence as set forth in SEQ ID NOS: 5-8.

In general, the variable regions of the heavy chain (VH) and light chain (VL) can be obtained by linking the CDRs and FRs numbered below in a combined arrangement as follows: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR 4.

It is noted that in other embodiments, each framework region amino acid sequence of an antibody or functional fragment thereof provided herein can 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 described above (SEQ ID NO:1, 2, 3, 4, 5, 6, 7, or 8).

In alternative embodiments, the antibody further comprises a constant region.

In alternative embodiments, the constant region is selected from the constant regions of any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, and IgD.

In alternative embodiments, the species of the constant region is derived from a cow, horse, dairy cow, pig, sheep, goat, rat, mouse, dog, cat, rabbit, camel, donkey, deer, mink, chicken, duck, goose, turkey, chicken fight, or human.

In alternative embodiments, the constant region is derived from sheep.

In alternative embodiments, the light chain constant region sequence of the constant region is set forth in SEQ ID NO. 9 and the heavy chain constant region sequence of the constant region is set forth 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.

Functional fragments of the above antibodies typically have the same binding specificity as the antibody from which they are derived. It will be readily understood by those skilled in the art from the disclosure of the present invention that functional fragments of the above antibodies can be obtained by methods such as enzymatic digestion (including pepsin or papain) and/or by chemical reduction cleavage of disulfide bonds. Based on the disclosure of the structure of the intact antibody, the above-described functional fragments are readily available to those skilled in the art.

Functional fragments of the above antibodies can also be obtained by recombinant genetic techniques also known to those skilled in the art or synthesized by, for example, automated peptide synthesizers, such as those sold by Applied BioSystems and the like.

In another aspect, the present invention provides the use of the antibody or functional fragment thereof according to any one of the above aspects in the preparation of a diagnostic reagent or a diagnostic kit for a disease associated with vitamin D metabolism.

In alternative embodiments, the disease includes, but is not limited to rickets in children, diabetes, chronic kidney disease, hepatitis, sjogren's syndrome, multiple sclerosis, rheumatoid arthritis, asthma, osteoporosis, parkinson's disease, cardiovascular disease, hypertension, type 2 diabetes, and tumors.

In another aspect, the present invention provides a diagnostic reagent or an antibody or functional fragment kit for a disease associated with vitamin D metabolism, comprising the antibody or functional fragment thereof according to any one of the above.

In another aspect, the present invention provides a reagent or a kit for detecting 25-hydroxyvitamin D, comprising the antibody or the functional fragment thereof according to 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.

Detectable labels are substances having properties, such as luminescence, color development, radioactivity, etc., which can be observed directly by the naked eye or detected by an instrument, by which qualitative or quantitative detection of the respective target substance can be achieved.

In alternative embodiments, the detectable labels include, but are not limited to, fluorescent dyes, enzymes that catalyze the development of a substrate, radioisotopes, chemiluminescent reagents, and nanoparticle-based labels.

In the actual use process, one skilled in the art can select a suitable marker according to the detection condition or actual requirement, and whatever marker is used belongs to the protection scope of the present invention.

In alternative embodiments, the fluorescent dyes include, but are not limited to, fluorescein-based dyes and derivatives thereof (e.g., including, but not limited to, Fluorescein Isothiocyanate (FITC) hydroxyphoton (FAM), tetrachlorofluorescein (TET), etc. or analogs thereof), rhodamine-based dyes and derivatives thereof (e.g., including, but not limited to, red Rhodamine (RBITC), Tetramethylrhodamine (TAMRA), rhodamine b (tritc), etc. or analogs thereof), Cy-series dyes and derivatives thereof (e.g., including, but not limited to, Cy2, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, Cy3, etc. or analogs thereof), Alexa-series dyes and derivatives thereof (e.g., including, but not limited to, Alexa fluor350, 405, 430, 488, 532, 546, 555, 568, 594, 610, 33, 647, 680, 700, 750, etc. or analogs thereof), and protein-based dyes and derivatives thereof (e.g., including, but not limited to, Phycoerythrin (PE), Phycocyanin (PC), phycocyanin (e.g., including, pyrenochrome, and analogs thereof), Allophycocyanin (APC), polymethacrylic flavin-chlorophyll protein (precP), etc.).

In alternative embodiments, the enzyme that catalyzes the color development of the substrate includes, but is not limited to, horseradish peroxidase, alkaline phosphatase, beta-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and glucose-6-phosphate deoxyenzyme.

In alternative embodiments, the radioisotope includes, but is not limited to²¹²Bi、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 and¹⁸F。

in alternative embodiments, the chemiluminescent reagent includes, but is not limited to, luminol and its derivatives, lucigenin, crustacean fluorescein and its derivatives, bipyridyl ruthenium and its derivatives, acridinium ester and its derivatives, dioxane and its derivatives, lotrine 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 colloid includes, but is 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 present invention provides a nucleic acid molecule encoding the above antibody or functional fragment thereof.

In another aspect, the invention provides a vector comprising the nucleic acid molecule described above.

In another aspect, the present invention provides a recombinant cell comprising the vector described above.

In another aspect, the present invention provides a method of preparing an antibody or functional fragment thereof, comprising: culturing a recombinant cell capable of recombinantly expressing the antibody or the functional fragment thereof as described in any one of the above, and isolating and purifying the antibody or the functional fragment thereof from the culture product.

Based on the disclosure of the amino acid sequence of the antibody or its functional fragment, it is easy for those skilled in the art to think that the antibody or its functional fragment can be prepared by genetic engineering techniques or other techniques (chemical synthesis, hybridoma cells), for example, by separating and purifying the antibody or its functional fragment from the culture product of recombinant cells capable of recombinantly expressing the antibody or its functional fragment as described above, and this is within the scope of the present invention, regardless of the technique used to prepare the antibody or its functional fragment.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.

FIG. 1 is a reducing SDS-PAGE result of the anti-25-hydroxyvitamin D antibody of example 1.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

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 the formulations or unit dosages herein, some are now described. Unless otherwise indicated, the techniques employed or contemplated herein are standard methods. The materials, methods, and examples are illustrative only and not intended to be limiting.

The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry and immunology, which are within the skill of the art. Such techniques are well explained in the literature, e.g. "molecular cloning: a Laboratory Manual, second edition (Sambrook et al, 1989); oligonucleotide Synthesis (oligo Synthesis) (eds. m.j. goal, 1984); animal Cell Culture (Animal Cell Culture), ed.r.i. freshney, 1987; methods in Enzymology (Methods in Enzymology), Handbook of Experimental Immunology (Handbook of Experimental Immunology) (ed. D.M.Weir and C.C.Black well), Gene Transfer Vectors for Mammalian Cells (ed. J.M.Miller and M.P.Calos) (ed. J.M.and M.P.Calos) (ed. 1987), Methods in Current Generation (Current Protocols in Molecular Biology) (ed. F.M.Ausubel.et al, 1987), PCR, Polymerase Chain Reaction (ed. PCR: The Polymerase Chain Reaction) (ed. Mullis et al, 1994), and Methods in Current Immunology (ed. J.1991).

The features and properties of the present invention are described in further detail below with reference to examples.

Example 1

1 construction of recombinant plasmid

(1) Antibody Gene preparation

mRNA is extracted from a hybridoma cell strain secreting an anti-25-hydroxyvitamin D antibody, a DNA product is obtained by an RT-PCR method, the product is added with A by rTaq DNA polymerase for reaction and then inserted into a pMD-18T vector, the product is transformed into DH5 alpha competent cells, after colonies are grown out, 4 clones of the Heavy Chain and Light Chain gene clones are respectively taken and sent to a gene sequencing company for sequencing.

(2) Sequence analysis of antibody variable region genes

Putting the gene sequence obtained by sequencing in an IMGT antibody database for analysis, and analyzing by using VNTI11.5 software to determine that the genes amplified by 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 345bp, belongs to the VkII gene family, and a leader peptide sequence of 57bp is arranged in front of the VL gene sequence; in the gene fragment amplified by the Heavy Chain primer pair, the VH gene sequence is 360bp, belongs to a VH1 gene family, and has a leader peptide sequence of 57bp in front.

(3) Construction of recombinant antibody expression plasmid

pcDNA^TM 3.4

vector is a constructed recombinant antibody eukaryotic expression vector, and multiple cloning enzyme cutting sites such as HindIII, BamHI, EcoRI and the like are introduced into the expression vector and named as pcDNA3.4A expression vector, and the vector is called as 3.4A expression vector for short in the following; root of herbaceous plantAccording to the sequencing result of the antibody variable region gene in the pMD-18T, VL and VH gene specific primers of the antibody are designed, two ends of the VL and VH gene specific primers are respectively provided with HindIII and EcoRI restriction sites and protective bases, and a Light Chain gene fragment of 0.73KB and a Heavy Chain gene fragment of 1.42KB are amplified by a PCR amplification method.

The gene fragments of the Heavy Chain and the Light Chain are subjected to double enzyme digestion by HindIII/EcoRI respectively, the 3.4A vector is subjected to double enzyme digestion by HindIII/EcoRI, the Heavy Chain gene and the Light Chain gene are respectively connected into the 3.4A expression vector after the fragments and the vector are purified and recovered, and recombinant expression plasmids of the Heavy Chain and the Light Chain are respectively obtained.

2 Stable cell line selection

(1) Transient transfection of recombinant antibody expression plasmid into CHO cells and determination of expression plasmid activity

Plasmid was diluted to 400ng/ml with ultrapure water and CHO cells were conditioned at 1.43X 10⁷cells/ml are put into a centrifuge tube, 100 mu L of plasmid is mixed with 700 mu L of cells, the mixture is transferred into an electric rotating cup and is electrically rotated, the sampling counting is carried out on days 3, 5 and 7, and the sampling detection is carried out on day 7.

Coating liquid (main component NaHCO)₃) Diluting 25 hydroxyl VD-BSA to 3. mu.g/ml, 100. mu.L per well, and standing overnight at 4 ℃; the next day, washing liquid (main component Na)₂HPO₄NaCl) for 2 times, patting dry; adding blocking solution (20% BSA + 80% PBS), and drying at 37 deg.C for 1 hr in each well; adding diluted cell supernatant at 100 μ L/well, 37 deg.C for 30min (partial supernatant for 1 h); washing with washing solution for 5 times, and drying; adding rabbit anti-sheep-HRP (horse radish peroxidase) with the volume of 100 mu L per hole, the temperature of 37 ℃ and the time of 30 min; washing with washing solution for 5 times, and drying; adding a developing solution A (50 muL/hole, containing citric acid, sodium acetate, acetanilide and carbamide peroxide), and adding a developing solution B (50 muL/hole, containing citric acid, EDTA-2 Na, TMB and concentrated HCl) for 10 min; adding stop solution (50 μ L/hole, EDTA-2 Na + concentrated H2SO 4); OD readings were taken at 450nm (reference 630nm) on the microplate reader. The results show that the OD of the reaction after the cell supernatant is diluted 1000 times is still larger than 1.0, and the OD of the reaction without the cell supernatant is smaller than 0.1, which indicates that the antibody generated after the plasmid is transiently transformed has activity on the myoglobin.

(2) Linearization of recombinant antibody expression plasmids

The following reagents were prepared: buffer 50 mu L, DNA100 mu g/tube, Puv I enzyme 10 mu L, sterile water to 500 mu L, 37 ℃ water bath enzyme digestion overnight; sequentially extracting with equal volume of phenol/chloroform/isoamyl alcohol (lower layer) 25:24:1 and then chloroform (water phase); precipitating with 0.1 volume (water phase) of 3M sodium acetate and 2 volumes of ethanol on ice, rinsing with 70% ethanol, removing organic solvent, re-melting with appropriate amount of sterilized water after ethanol is completely volatilized, and finally measuring concentration.

(3) Stable transfection of recombinant antibody expression plasmid, pressurized screening of stable cell lines

Plasmid was diluted to 400ng/ml with ultrapure water and CHO cells were conditioned at 1.43X 10⁷cells/ml are put into a centrifuge tube, 100 mu L of plasmid is mixed with 700 mu L of cells, and the mixture is transferred into an electric rotating cup and is electrically rotated, and the next day is counted; 25umol/L MSX 96-well pressure culture for about 25 days.

Observing the marked clone holes with cells under a microscope, and recording the confluence degree; taking culture supernatant, and sending the culture supernatant to a sample for detection; selecting cell strains with high antibody concentration and relative concentration, transferring the cell strains into 24 holes, and transferring the cell strains into 6 holes after 3 days; after 3 days, the seeds were kept and cultured in batch with cell density of 0.5X 106cells/ml and 2.2ml adjusted, cell density of 0.3X 10⁶cell/ml, 2ml for seed preservation; and (4) 7 days, carrying out batch culture supernatant sample sending detection in 6 holes, and selecting cell strains with small antibody concentration and cell diameter to transfer TPP for seed preservation and passage.

3 recombinant antibody production

(1) Cell expanding culture

After the cells are recovered, the cells are cultured in a shaking flask with the specification of 125ml, the inoculation volume is 30ml, the culture medium is 100% Dynamis culture medium, and the cells are placed in a shaking table with the rotation speed of 120r/min, the temperature of 37 ℃ and the carbon dioxide of 8%. Culturing for 72h, inoculating and expanding culture at an inoculation density of 50 ten thousand cells/ml, wherein the expanding culture volume is calculated according to production requirements, and the culture medium is 100% Dynamis culture medium. Then the culture is expanded every 72 h. When the cell amount meets the production requirement, the production is carried out by strictly controlling the inoculation density to be about 50 ten thousand cells/ml.

(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 percent. Feeding in a flowing mode: daily feeding was started when the culture was carried out for 72h in a shake flask, 3% of the initial culture volume was fed daily to HyCloneTM Cell BoostTM Feed 7a, and one thousandth of the initial culture volume was fed daily to Feed 7b, up to day 12 (day 12 feeding). Glucose was supplemented with 3g/L on the sixth day. Samples were collected on day 13. Affinity purification was performed using a proteinA affinity column. 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 showed two bands, 1 of which Mr was 50KD (heavy chain, SEQ ID NO:14) and the other Mr was 28KD (light chain, SEQ ID NO:13), as shown in FIG. 1 below, after the reducing SDS-PAGE.

Example 2

Detection of antibody Performance

(1) Example 1 Activity assay of antibodies and mutants thereof

The antibody (WT) of example 1 was further analyzed, and the heavy chain variable region is represented by SEQ ID NO:12, in which the amino acid sequences of the respective complementarity determining regions are as follows:

CDR1-VH：G-F-T(X1)-F-D-D(X2)-Y-G-L(X3)-G；

CDR2-VH：G-L(X1)-D-R(X2)-H-G-V(X3)-R-G-Y-N-R-V(X4)-L-K-S

CDR3-VH：T(X1)-R-V(X2)-W-Y-S-G-Q(X3)-G-F-E(X4)-F

the light chain variable region is shown as SEQ ID NO. 11, wherein the amino acid sequences of the complementarity determining regions of the light chain are as follows:

CDR1-VL：S-G-S-T(X1)-S-N-I(X2)-G-Y-G-N-Y-L(X3)-S，

CDR-VL2：D-S-V(X1)-T-R-G(X2)-S-G-I(X3)-P；

CDR-VL3：A-S-W(X1)-D-S-S-E(X2)-G。

based on the anti-25-hydroxyvitamin D antibody (WT) of example 1, mutations were made in the complementarity determining regions at sites involved in the activity of the antibody, wherein X1, X2, X3, and X4 were all mutated sites. See table 1 below.

TABLE 1 mutant sites associated with antibody Activity

CDR-VH1 X1

CDR-VH2 X2

CDR-VH3 X1

CDR-VL1 X1

CDR-VL2 X2

CDR-VL3 X2

WT

T

R

T

G

E

Mutation 1

S

K

A

S

A

D

Mutation 2

S

R

A

T

A

D

Mutation 3

S

R

A

T

G

E

Mutation 4

S

K

T

A

D

Mutation 5

S

W

A

T

A

D

Mutation 6

S

R

A

S

L

D

Mutation 7

T

R

H

S

G

D

Mutation 8

S

R

T

A

G

E

Antibody binding activity assay in table 1:

coating liquid (main component NaHCO)₃) Diluting 25 hydroxyl VD-BSA to 3ug/ml, 100uL per well, and standing overnight at 4 ℃; the next day, washing liquid (main component Na)₂HPO₄NaCl) for 2 times, patting dry; adding blocking solution (20% BSA + 80% PBS), beating to dry at 37 deg.C for 1h and 120uL per well; adding diluted purified antibody and control antibody at 100 uL/well, 37 deg.C for 30 min; washing with washing solution for 5 times, and drying; adding rabbit anti-sheep-HRP (horse radish peroxidase), wherein each hole is 100uL, the temperature is 37 ℃ and the time is 30 min; washing with washing solution for 5 times, and drying; adding a developing solution A (50 uL/hole), adding a developing solution B (50 uL/hole), and carrying out 10 min; adding stop solution into the mixture, wherein the concentration of the stop solution is 50 uL/hole; OD readings were taken at 450nm (reference 630nm) on the microplate reader. The results are shown in Table 2 below.

TABLE 2 Activity data of WT antibodies and mutants thereof

Antibody concentration (ng/ml)	500	250	125	62.5	31.25	0
							WT	2.004	1.581	1.139	0.650	0.372	0.098
Mutation 1	2.223	1.674	1.256	0.842	0.548	0.09
							Mutation 2	2.156	1.626	1.225	0.896	0.512	0.075
Mutation 3	2.138	1.615	1.258	0.827	0.533	0.079
							Mutation 4	2.176	1.634	1.257	0.836	0.544	0.085
Mutation 5	0.813	0.524	0.19	-	-	-
							Mutation 6	0.818	0.575	0.085	-	-	-
Mutation 7	0.852	0.533	0.123	-	-	-
							Mutation 8	0.845	0.524	0.079	-	-	-

As can be seen from the data in Table 2, WT and mutations 1 to 4 had better binding activity compared to mutations 5 to 8, with the binding activity being better with mutation 1.

(2) Affinity detection of antibodies and mutants thereof

(a) Based on mutation 1, other sites were mutated, and the sequence of each mutation is shown in table 3 below.

TABLE 3 mutation sites related to antibody affinity

Affinity assay

Using AMC sensors, purified antibodies were diluted to 10ug/ml with PBST, 25 hydroxy VD-BSA with PBST gradient dilution: 20ug/ml, 6.66ug/ml, 2.22ug/ml, 0.74ug/ml, 0.24ug/ml, 0.082ug/ml, 0.027ug/ml, 0.0091 ug/ml;

the operation flow is as follows: buffer 1(PBST, main component Na)₂HPO₄+ NaCl + TW-20), immobilized antibody in antibody solution for 300s, incubation in buffer 2(PBST) for 180s, binding in antigen solution for 420s, dissociation in buffer 2 for 1200s, sensor regeneration with 10mM pH 1.69GLY solution and buffer 3, and data output. K_DRepresents the equilibrium dissociation constant, i.e., affinity; kon denotes the binding rate; kdis denotes the off-rate. The results are shown in Table 4 below.

Table 4 affinity assay data

As can be seen from the data in Table 4, based on the mutation 1, the mutation is performed in the way of the mutation in Table 3, and the obtained mutant antibody has higher affinity to 25 hydroxyVD.

(b) Based on WT, mutation is carried out on other sites, and the affinity of each mutant is detected, the sequence of each mutation is shown in Table 5, and the corresponding affinity data is shown in Table 6.

TABLE 5 mutations with WT as backbone

TABLE 6 affinity assay results for WT antibodies and their mutants

	K_D(M)	kon(1/Ms)	kdis(1/s)
				WT	2.11E-06	6.34E+03	1.34E-02
WT 1-1	1.61E-06	7.00E+03	1.13E-02
				WT 1-2	3.08E-06	6.21E+03	1.91E-02
WT 1-3	1.36E-06	8.00E+03	1.09E-02
				WT 1-4	1.78E-06	6.70E+03	1.19E-02
WT 1-5	2.51E-06	6.13E+03	1.54E-02
				WT 1-6	1.64E-06	7.91E+03	1.30E-02
WT1-7	2.27E-06	7.52E+03	1.71E-02

As can be seen from the data in Table 6, on the basis of WT, better affinity can also be achieved by mutating in the manner shown in Table 5.

(3) Evaluation of stability against naked antibody

Placing the antibody in a temperature range of 4 ℃ (refrigerator), -80 ℃ (refrigerator) and 37 ℃ (thermostat) for 21 days, taking samples in 7 days, 14 days and 21 days for state observation, and performing activity detection on the samples in 21 days, wherein the result shows that under three examination conditions, no obvious protein state change is seen in 21 days of placing the antibody, and the activity does not show a descending trend along with the rise of the examination temperature, which indicates that the antibody is stable. The following table 7 shows the results of the detection of OD by the antibody of mutant 1 in the enzyme immunity activity test for 21 days.

TABLE 7

Sample concentration (ng/ml)	250	62.5	0
				Samples at 4 ℃ for 21 days	1.686	0.856	0.088
21 days samples at-80 deg.C	1.658	0.868	0.076
				21 day samples at 37 deg.C	1.604	0.823	0.087

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Sequence listing

<110> Dongguan City of Pengzhi Biotech Co., Ltd

<120> antibody capable of specifically binding 25-hydroxyvitamin D, use thereof and diagnostic kit

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Ala Ser Thr Thr Pro Pro Lys Val Tyr Pro Leu Thr Ser Cys Cys Gly

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Gly Val His Thr Phe Pro Ala Ile Leu Gln Ser Ser Gly Leu Tyr Ser

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Leu Ser Ser Val Val Thr Val Pro Ala Ser Thr Ser Gly Ala Gln Thr

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Phe Ile Cys Asn Val Ala His Pro Ala Ser Ser Thr Lys Val Asp Lys

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Arg Val Glu Pro Gly Cys Pro Asp Pro Cys Lys His Cys Arg Cys Pro

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130 135 140

Val Val Asp Val Gly Gln Asp Asp Pro Glu Val Gln Phe Ser Trp Phe

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Val Asp Asn Val Glu Val Arg Thr Ala Arg Thr Lys Pro Arg Glu Glu

165 170 175

Gln Phe Asn Ser Thr Phe Arg Val Val Ser Ala Leu Pro Ile Gln His

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Phe Leu Tyr Ser Arg Leu Arg Val Asp Lys Asn Ser Trp Gln Glu Gly

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Asp Thr Tyr Ala Cys Val Val Met His Glu Ala Leu His Asn His Tyr

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Thr Gln Lys Ser Ile Ser Lys Pro Pro Gly Lys

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Gln Ala Ala Leu Thr Gln Pro Ser Ser Thr Ser Lys Ala Leu Gly Gln

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Glu Gly Gly Ile Phe Gly Ser Gly Thr Arg Ile Thr Val Leu Gly Gln

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Trp Gly Pro Gly Leu Leu Val Thr Val Ala Ser

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Leu Ser Thr Asn Lys Ala Thr Val Val Cys Leu Ile Asn Asp Phe Tyr

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Gln Asn Val Lys Thr Thr Gln Ala Ser Lys Gln Ser Asn Ser Lys Tyr

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Ala Ala Ser Ser Tyr Leu Thr Leu Thr Gly Ser Glu Trp Lys Ser Lys

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Ser Ser Tyr Thr Cys Glu Val Thr His Glu Gly Ser Thr Val Thr Lys

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Thr Val Lys Pro Ser Glu Cys Ser

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Gly Gly Leu Asp Arg His Gly Val Arg Gly Tyr Asn Arg Val Leu Lys

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Pro Lys Val Tyr Pro Leu Thr Ser Cys Cys Gly Asp Thr Ser Ser Ser

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Ala His Pro Ala Ser Ser Thr Lys Val Asp Lys Arg Val Glu Pro Gly

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Gly Gly Pro Ser Val Phe Ile Phe Pro Pro Lys Pro Lys Asp Thr Leu

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Thr Ile Ser Gly Thr Pro Glu Val Thr Cys Val Val Val Asp Val Gly

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Gln Asp Asp Pro Glu Val Gln Phe Ser Trp Phe Val Asp Asn Val Glu

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Val Arg Thr Ala Arg Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr

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Phe Arg Val Val Ser Ala Leu Pro Ile Gln His Gln Asp Trp Thr Gly

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Gly Lys Glu Phe Lys Cys Lys Val His Asn Glu Gly Leu Pro Ala Pro

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Ile Val Arg Thr Ile Ser Arg Thr Lys Gly Gln Ala Arg Glu Pro Gln

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Val Tyr Val Leu Ala Pro Pro Gln Glu Glu Leu Ser Lys Ser Thr Leu

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Ser Val Thr Cys Leu Val Thr Gly Phe Tyr Pro Asp Tyr Ile Ala Val

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Glu Trp Gln Lys Asn Gly Gln Pro Glu Ser Glu Asp Lys Tyr Gly Thr

385 390 395 400

Thr Thr Ser Gln Leu Asp Ala Asp Gly Ser Tyr Phe Leu Tyr Ser Arg

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Leu Arg Val Asp Lys Asn Ser Trp Gln Glu Gly Asp Thr Tyr Ala Cys

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Val Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Ile

435 440 445

Ser Lys Pro Pro Gly Lys

450

Claims

1. An antibody or functional fragment thereof that specifically binds 25-hydroxyvitamin D, wherein said antibody or functional fragment thereof has complementarity determining regions:

CDR-VH 1: G-F-X1-F-D-X2-Y-G-X3-G, wherein: x1 is S or T; x2 is N or D; x3 is L, V or I;

CDR-VH 2: G-X1-D-X2-H-G-X3-R-G-Y-N-R-X4-L-K-S, wherein: x1 is L, V or I; x2 is K or R; x3 is V, A or I; x4 is V, A or I;

CDR-VH 3: X1-R-X2-W-Y-S-G-X3-G-F-X4-F, wherein: x1 is T or A; x2 is L, V or I; x3 is N or Q; x4 is E or D;

CDR-VL 1: S-G-S-X1-S-N-X2-G-Y-G-N-Y-X3-S, wherein: x1 is S or T; x2 is I, V or L; x3 is I, V or L;

CDR-VL 2: D-S-X1-T-R-X2-S-G-X3-P, wherein: x1 is I, V or L; x2 is G or A; x3 is I, V or L;

CDR-VL 3: A-S-X1-D-S-S-X2-G, wherein: x1 is W, Y or F; x2 is E or D.

2. The antibody or functional fragment thereof that specifically binds 25-hydroxyvitamin D according to claim 1,

in CDR-VH1, X1 is S;

in CDR-VH2, X2 is K;

in CDR-VH3, X1 is A;

in CDR-VL1, X1 is S;

in CDR-VL2, X2 is A;

in CDR-VL3, X2 is D;

preferably, in CDR-VH1, X2 is N;

preferably, in CDR-VH1, X2 is D;

preferably, in CDR-VH1, X3 is L;

preferably, in CDR-VH1, X3 is V;

preferably, in CDR-VH1, X3 is I;

preferably, in CDR-VH2, X1 is L;

preferably, in CDR-VH2, X1 is V;

preferably, in CDR-VH2, X1 is I;

preferably, in CDR-VH2, X3 is V;

preferably, in CDR-VH2, X3 is A;

preferably, in CDR-VH2, X3 is I;

preferably, in CDR-VH2, X4 is V;

preferably, in CDR-VH2, X4 is A;

preferably, in CDR-VH2, X4 is I;

preferably, in CDR-VH3, X2 is L;

preferably, in CDR-VH3, X2 is V;

preferably, in CDR-VH3, X2 is I;

preferably, in CDR-VH3, X3 is N;

preferably, in CDR-VH3, X3 is Q;

preferably, in CDR-VH3, X4 is E;

preferably, in CDR-VH3, X4 is Q;

preferably, in CDR-VL1, X2 is I;

preferably, in CDR-VL1, X2 is V;

preferably, in CDR-VL1, X2 is L;

preferably, in CDR-VL1, X3 is I;

preferably, in CDR-VL1, X3 is V;

preferably, in CDR-VL1, X3 is L;

preferably, in CDR-VL2, X1 is I;

preferably, in CDR-VL2, X1 is V;

preferably, in CDR-VL2, X1 is L;

preferably, in CDR-VL2, X3 is I;

preferably, in CDR-VL2, X3 is V;

preferably, in CDR-VL2, X3 is L;

preferably, in CDR-VL1, X1 is W;

preferably, in CDR-VL1, X1 is Y;

preferably, in CDR-VL1, X1 is F.

Preferably, each complementarity determining region of the antibody or functional fragment thereof is selected from any one of the following combinations of mutations 1-62:

3. the antibody or functional fragment thereof that specifically binds 25-hydroxyvitamin D according to claim 2, wherein the antibody or functional fragment thereof binds 25-hydroxyvitamin DBiotin D with K_D≤3.08×10^-6Affinity binding of mol/L, preferably, K_D≤7.38×10^-7mol/L。

4. The antibody or functional fragment thereof that specifically binds 25-hydroxyvitamin D according to claim 1,

in CDR-VH1, X1 is T;

in CDR-VH2, X2 is R;

in CDR-VH3, X1 is T;

in CDR-VL1, X1 is T;

in CDR-VL2, X2 is G;

in CDR-VL3, X2 is E;

preferably, each complementarity determining region of the antibody or functional fragment thereof is selected from any one of the following combinations of mutations 63-70:

5. the antibody or functional fragment thereof capable of specifically binding to 25-hydroxyvitamin D according to any one of claims 1 to 4, wherein said antibody comprises the light chain framework regions FR1-L, FR2-L, FR3-L and FR4-L in sequence as shown in SEQ ID Nos. 1 to 4, and/or the heavy chain framework regions FR1-H, FR2-H, FR3-H and FR4-H in sequence as shown in SEQ ID Nos. 5 to 8;

preferably, the antibody further comprises a constant region;

preferably, the constant region is selected from the constant regions of any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE and IgD;

preferably, the species of the constant region is from a cow, horse, cow, pig, sheep, goat, rat, mouse, dog, cat, rabbit, camel, donkey, deer, mink, chicken, duck, goose, turkey, chicken fighting or human;

preferably, the constant region is derived from sheep;

preferably, the light chain constant region sequence of the constant region is shown as SEQ ID NO. 9, and the heavy chain constant region sequence of the constant region is shown as SEQ ID NO. 10;

preferably, the functional fragment is selected from any one of VHH, F (ab ') 2, Fab', Fab, Fv and scFv of the antibody.

6. Use of the antibody or functional fragment thereof according to any one of claims 1 to 5 for the preparation of a diagnostic agent or a diagnostic kit for a disease associated with vitamin D metabolism;

preferably, the disease is selected from rickets in children, diabetes, chronic kidney disease, hepatitis, sjogren's syndrome, multiple sclerosis, rheumatoid arthritis, asthma, osteoporosis, parkinson's disease, cardiovascular disease, hypertension, type 2 diabetes and tumors.

7. A diagnostic reagent for a disease associated with vitamin D metabolism or an antibody or functional fragment thereof, comprising the antibody or functional fragment thereof according to any one of claims 1 to 5.

8. A reagent or kit for the detection of 25-hydroxyvitamin D comprising an antibody or functional fragment thereof according to any one of claims 1 to 5.

9. The reagent or kit according to claim 7 or 8, wherein the antibody or functional fragment thereof is labeled with a detectable label;

preferably, the detectable label is selected from the group consisting of a fluorescent dye, an enzyme that catalyzes the color development of a substrate, a radioisotope, a chemiluminescent reagent, and a nanoparticle-based label;

preferably, the fluorescent dye is selected from fluorescein dyes and derivatives thereof, rhodamine dyes and derivatives thereof, Cy dyes and derivatives thereof, Alexa dyes and derivatives thereof, and protein dyes and derivatives thereof;

preferably, the enzyme catalyzing the color development of the substrate is selected from the group consisting of horseradish peroxidase, alkaline phosphatase, beta-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and glucose-6-phosphate deoxyenzyme;

preferably, the radioisotope is selected from the group consisting of²¹²Bi、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 and¹⁸F；

preferably, the chemiluminescent reagent is selected from luminol and its derivatives, lucigenin, crustacean fluorescein and its derivatives, bipyridine ruthenium and its derivatives, acridinium ester and its derivatives, dioxetane and its derivatives, lowhine and its derivatives, and peroxyoxalate and its derivatives;

preferably, 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;

preferably, the colloid is selected from the group consisting of colloidal metals, disperse dyes, dye-labeled microspheres, and latexes;

preferably, the colloidal metal is selected from the group consisting of colloidal gold, colloidal silver and colloidal selenium.

10. A method of producing an antibody or functional fragment thereof, comprising: culturing a recombinant cell capable of recombinantly expressing the antibody or functional fragment thereof according to any one of claims 1 to 5, and isolating and purifying the antibody or functional fragment thereof from the culture product.