CN117285638B

CN117285638B - Anti-buprenorphine antibodies, reagents and kits for the detection of buprenorphine

Info

Publication number: CN117285638B
Application number: CN202210684495.9A
Authority: CN
Inventors: 孟媛; 钟冬梅; 覃文新; 游辉; 曹慧方
Original assignee: Dongguan Pengzhi Biotechnology Co Ltd
Current assignee: Dongguan Pengzhi Biotechnology Co Ltd
Priority date: 2022-06-17
Filing date: 2022-06-17
Publication date: 2024-05-10
Anticipated expiration: 2042-06-17
Also published as: CN117285638A

Abstract

The invention discloses an anti-buprenorphine antibody, a reagent and a kit for detecting buprenorphine, and relates to the technical field of antibodies. The anti-buprenorphine antibodies disclosed herein comprise a heavy chain complementarity determining region and a light chain complementarity determining region. The antibody provides an important raw material source for buprenorphine detection and has improved affinity and activity.

Description

Anti-buprenorphine antibodies, reagents and kits for the detection of buprenorphine

Technical Field

The invention relates to the technical field of antibodies, in particular to an anti-buprenorphine antibody, a reagent for detecting buprenorphine and a kit.

Background

Buprenorphine (Buprenorphine ) is a drug preparation of Ding Nanuo-hydrochloride, commercial names of sand phenanthrene tablet, buprenorphine, shu Meifen and the like, chemical names of N-cyclopropylmethyl-7 a [ I- (S) -1-hydroxy-1, 2, 3-trimethyl ] -6, 14-ridge ethane-6, 7,8, 14-tetrahydronorPapaverine is, and the Buprenorphine is a drug with opioid receptor agonistic and antagonistic activities. The medicine is a high-lipophilicity medicine, the injection or sublingual buccal effect has relatively quick effect, the low dosage can show the agonistic activity of opioid receptors, and the high dosage can show the antagonistic effect of opioid receptors. Buprenorphine is currently available in injection and tablet form, and buprenorphine is also used in many countries and in many places in China for withdrawal treatment of heroin addicts. However, because of the characteristic of being very easy to form dependence, the control of the use condition of the medicine is very strict in various countries, and the medicine is classified as class 1 psychotropic medicine for control in China.

Buprenorphine has, in addition to its medicinal effect, relatively serious side effects, similar to other opioid drugs, most commonly: is easy to be addicted, the nervous system is damaged, the memory is reduced, insomnia, sleepiness, sweating, constipation, headache, nausea, and the like, and can cause hypertension and induce hypertension, respiratory depression and the like. Therefore, the drug addict should not misuse various soft drugs such as buprenorphine while keeping away from heroin drugs.

There are many detection methods of Guan Dingbing norrphines, and at present, the detection method with the advantages of rapidness, portability and the like mainly comprises a colloidal gold immunochromatography method, which is an immunological detection method based on the specific reaction of an antibody and an antigen, and simultaneously amplifying and displaying a detected signal by using colloidal gold. And the preparation of antibodies against buprenorphine is key to developing colloidal gold immunochromatographic test papers. Thus, there is a strong need in the art for antibodies that are effective and bind to and detect buprenorphine.

In view of this, the present invention has been made.

Disclosure of Invention

The application provides an anti-buprenorphine antibody with improved affinity and/or activity to improve buprenorphine detection and provide an important source of raw materials for buprenorphine detection.

In order to achieve the above object, according to one aspect of the present invention, there is provided an antibody or a functional fragment thereof comprising HCDR1 having an amino acid sequence shown in SEQ ID NO. 1, HCDR2 having an amino acid sequence shown in SEQ ID NO. 2, HCDR3 having an amino acid sequence shown in SEQ ID NO. 3, and LCDR1 having an amino acid sequence shown in SEQ ID NO. 4, LCDR2 having an amino acid sequence shown in SEQ ID NO. 5, LCDR2 having an amino acid sequence shown in SEQ ID NO. 6 or LCDR3 having an amino acid sequence shown in SEQ ID NO. 15.

In order to achieve the above object, according to a second aspect of the present invention, there is provided an anti-buprenorphine antibody or a functional fragment thereof, the antibody or functional fragment thereof comprising a heavy chain variable region comprising the sequence structure of HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4 and/or a light chain variable region comprising the sequence structure of LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4, the amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3 being the amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3 described above.

To achieve the above object, according to a third aspect of the present invention there is provided an anti-buprenorphine antibody comprising a heavy chain variable region as described above and/or a light chain comprising a light chain variable region as described above.

In order to achieve the above object, according to a fourth aspect of the present invention, there is provided an antibody conjugate comprising the above antibody or a functional fragment thereof.

In order to achieve the above object, according to a fifth aspect of the present invention, there is provided a reagent or kit for detecting buprenorphine, the reagent or kit comprising an antibody or functional fragment thereof as described above or an antibody conjugate as described above.

In order to achieve the above object, the present invention also provides a vector, a cell and a method for preparing the above antibody or a functional fragment thereof.

Drawings

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 results of reducing SDS-PAGE of Anti-BUP-53G9mut1 to Anti-BUP-53G9mut 4.

Detailed Description

The invention provides an antibody or a functional fragment thereof, which comprises HCDR1 with an amino acid sequence shown as SEQ ID NO.1, HCDR2 with an amino acid sequence shown as SEQ ID NO.2, HCDR3 with an amino acid sequence shown as SEQ ID NO.3, and LCDR1 with an amino acid sequence shown as SEQ ID NO. 4, LCDR2 with an amino acid sequence shown as SEQ ID NO. 5, LCDR 6 or LCDR3 with an amino acid sequence shown as SEQ ID NO. 15. The antibodies have improved affinity and/or activity.

In the present invention, the term "antibody" is used in the broadest sense and may include full length monoclonal antibodies, bispecific or multispecific antibodies, and chimeric antibodies so long as they exhibit the desired biological activity.

In the present invention, the terms "complementarity determining regions", "CDRs" or "CDRs" refer to the highly variable regions of the heavy and light chains of immunoglobulins, and refer to regions comprising one or more or even all of the major amino acid residues responsible for the binding of an antibody or antigen-binding fragment to the antigen or epitope recognized by it. In a specific embodiment of the invention, CDRs refer to the highly variable regions of the heavy and light chains of the antibody.

In the present invention, the heavy chain complementarity determining region is represented by HCDR, which includes HCDR1, HCDR2 and HCDR3; the light chain complementarity determining regions are denoted by LCDR and include LCDR1, LCDR2 and LCDR3. CDR labeling methods commonly used in the art include: the Kabat numbering scheme, the IMGT numbering scheme, the Chothia and Lesk numbering schemes, 1997 Lefranc et al, all protein sequences of the immunoglobulin superfamily. Kabat et al were the first to propose a standardized numbering scheme for immunoglobulin variable regions. Over the past few decades, the accumulation of sequences has led to the creation of KABATMAN databases, and the Kabat numbering scheme is generally considered as a widely adopted standard for numbering antibody residues. The invention adopts Kabat annotation standard to mark CDR regions, but other methods to mark CDR regions also belong to the protection scope of the invention.

In the present invention, a "framework region" or "FR" region includes a heavy chain framework region and a light chain framework region, and refers to regions other than CDRs in an antibody heavy chain variable region and a light chain variable region; wherein the heavy chain framework regions can be further subdivided into contiguous regions separated by CDRs comprising HFR1, HFR2, HFR3 and HFR4 framework regions; the light chain framework regions may be further subdivided into contiguous regions separated by CDRs comprising LFR1, LFR2, LFR3 and LFR4 framework regions.

In the present invention, the heavy chain variable region is obtained by connecting the following numbered CDRs with FRs in the following combination arrangement: HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4; the light chain variable region is obtained by ligating the following numbered CDRs with the FR in the following combination arrangement: LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4.

In alternative embodiments, the antibody or functional fragment thereof further comprises HFR1, HFR2, HFR3, HFR4 having amino acid sequences shown in SEQ ID NO. 7 through SEQ ID NO. 10, and LFR1, LFR2, LFR3 and LFR4 having amino acid sequences shown in SEQ ID NO. 11 through SEQ ID NO. 14.

In other embodiments, each of the framework region amino acid sequences of the antibodies or functional fragments 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% identity to the corresponding framework region (SEQ ID NO:7, 8, 9, 10, 11, 12, 13 or 14) described above.

In an alternative embodiment, the amino acid sequence of HFR3 of the antibody or functional fragment thereof is set forth in SEQ ID NO. 16.

In an alternative embodiment, the amino acid sequence of LFR3 of said antibody or functional fragment thereof is shown in SEQ ID NO. 17.

In alternative embodiments, the antibody or functional fragment thereof binds buprenorphine with an affinity of KD < 9.53 x 10 ^-9 M.

In alternative embodiments, the antibody or functional fragment thereof binds buprenorphine with an affinity of KD.ltoreq.10 10 ^-9M、KD≤10^-10 M or KD.ltoreq.10 10 ^-11 M.

In an alternative embodiment, the antibody or functional fragment thereof binds buprenorphine with an affinity of KD.ltoreq.6.97X10 ^-10 M.

In another aspect, embodiments of the present invention provide an anti-buprenorphine antibody or functional fragment thereof, comprising a heavy chain variable region comprising the sequence structure of HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4 and/or a light chain variable region comprising the sequence structure of LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4, wherein the amino acid sequence of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3 is the amino acid sequence of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3 described above, and the amino acid sequence of HFR1, HFR2, HFR3, HFR4, LFR1, HFR2, LFR3, r4 is the amino acid sequence of HFR1, HFR2, HFR3, LFR4, LFR2, LFR 4.

In an alternative embodiment, the heavy chain variable region amino acid sequence is as set forth in any one of SEQ ID NOs 20 to 21.

In an alternative embodiment, the light chain variable region amino acid sequence is set forth in any one of SEQ ID NOs 22 to 25.

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

In alternative embodiments, the constant region comprises a heavy chain constant region and/or a light chain constant region.

In alternative embodiments, the heavy chain constant region is selected from the group consisting of an IgG1, igG2, igG3, igG4, igA, igM, igE, or IgD heavy chain constant region, and the light chain constant region is selected from the group consisting of kappa-type or lambda-type light chain constant regions.

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

In an alternative embodiment, the constant region is of murine species origin.

In an alternative embodiment, the heavy chain constant region sequence (CH) is shown in SEQ ID NO. 18 and the light chain constant region (CL) sequence is shown in SEQ ID NO. 19.

In other embodiments, the constant region sequence 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% identity to the constant region (SEQ ID NO:18 or 19) described above.

In alternative embodiments, the functional fragment is selected from any one of 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 an antibody against buprenorphine comprising a heavy chain and/or a light chain, said heavy chain comprising a heavy chain variable region as described above and a heavy chain constant region as described above; the light chain comprises the light chain variable region described above and the light chain constant region described above.

In an alternative embodiment, the amino acid sequence of the heavy chain is as shown in any one of SEQ ID NOs 26 to 27.

In an alternative embodiment, the amino acid sequence of the light chain is as shown in any one of SEQ ID NOs 28 to 31.

In another aspect, the invention provides an antibody conjugate comprising an antibody as described above. Wherein the antibody is directly or indirectly covalently coupled to the conjugate. Or the antibody is coupled to the conjugate to be conjugated in a non-covalent adsorption manner.

In an alternative embodiment, the antibody in the above antibody conjugate is conjugated to biotin or a biotin derivative.

In an alternative embodiment, the antibody in the above antibody conjugate is conjugated to a label.

In an alternative embodiment, the above-mentioned marker refers to a substance having a property such as luminescence, color development, radioactivity, etc., which can be directly observed by naked eyes or detected by an instrument, by which qualitative or quantitative detection of the corresponding target can be achieved.

In alternative embodiments, the labels include, but are not limited to, fluorescent dyes, enzymes, 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 (preCP), and the like).

In alternative embodiments, the enzymes include, but are not limited to, horseradish peroxidase, alkaline phosphatase, beta-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and glucose 6-phosphate deoxygenase.

In alternative embodiments, the radioisotope includes, but is not limited to 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 and 18F.

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 an alternative embodiment, the colloidal metal is colloidal gold.

In an alternative embodiment, the antibody in the above antibody conjugate is conjugated to a solid phase.

In alternative embodiments, the solid phase is selected from the group consisting of microspheres, plates, and membranes.

In alternative embodiments, the solid phase includes, but is not limited to, magnetic microspheres, plastic microparticles, microplates, glass, capillaries, nylon, and nitrocellulose membranes.

In an alternative embodiment, the solid phase is a nitrocellulose membrane.

In another aspect, the invention provides a reagent or kit for detecting buprenorphine, the reagent or kit comprising an antibody or functional fragment thereof as described above or an antibody conjugate as described above.

In another aspect, the invention provides the use of an antibody as described above or a functional fragment thereof, an antibody conjugate or a reagent or kit as described above in the detection of buprenorphine.

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 invention provides a cell comprising the vector described above.

In another aspect, the invention provides a method of making an antibody or functional fragment thereof comprising: the cells as described above were cultured.

On the basis of the present invention, which discloses the amino acid sequence of an antibody or a functional fragment thereof, it is easy for a person skilled in the art to prepare the antibody or the functional fragment thereof by genetic engineering techniques or other techniques (chemical synthesis, recombinant expression), for example, by separating and purifying the antibody or the functional fragment thereof from a culture product of recombinant cells capable of recombinantly expressing the antibody or the functional fragment thereof according to any one of the above, and on the basis of this, it is within the scope of the present invention to prepare the antibody or the functional fragment thereof by any technique.

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, eds., 1984); animal cell Culture (ANIMAL CELL Culture) (r.i. freshney, 1987); methods of enzymology (Methods in Enzymology) (academic Press Co., ltd. (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 vector 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, 1987), polymerase chain reaction (PCR: the Polymerase Chain Reaction) (Mullis et al, 1994), and methods of contemporary immunology (Current Protocols in Immunology) (J.E.Coligan et al, 1991), each of which are expressly incorporated herein by reference.

The features and capabilities of the present invention are described in further detail below in connection with the examples.

EXAMPLE 1 preparation of Anti-BUP-53G9 monoclonal antibody

Restriction enzymes, PRIME STAR DNA polymerase in this example 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.

1 Construction of recombinant plasmid

(1) Antibody Gene production

MRNA is extracted from hybridoma cell strain secreting monoclonal antibody against buprenorphine, DNA product is obtained through RT-PCR method, the product is inserted into pMD-18T vector after adding A reaction by rTaq DNA polymerase, and is transformed into DH5 alpha competent cells, HEAVY CHAIN and LIGHT CHAIN gene clones are respectively taken after colony growth, and each 4 clones are sent to gene sequencing company for sequencing.

(2) Sequence analysis of Anti-BUP-53G9 antibody variable region Gene

The gene sequence obtained by sequencing is placed in a Kabat antibody database for analysis, and VNTI 11.5.5 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 LIGHT CHAIN amplified gene fragment, the VL gene sequence is 321bp, and the front of the VL gene fragment is 57bp leader peptide sequence; the gene fragment amplified by HEAVY CHAIN primer pair has 354bp VH gene sequence, belonging to VH1 gene family, and the front of the gene fragment has 57bp leader peptide sequence.

(3) Construction of recombinant antibody expression plasmids

pcDNA^TM 3.4Vector is a constructed eukaryotic expression vector of the recombinant antibody, and the expression vector is introduced with HindIII, bamHI, ecoRI and other polyclonal enzyme cutting sites and named pcDNA3.4A expression vector, and is hereinafter abbreviated as 3.4A expression vector; according to the result of the gene sequencing of the antibody variable region in pMD-18T, VL and VH gene specific primers of the antibody are designed, and the two ends of the VL and VH gene specific primers are respectively provided with HindIII, ecoRI digestion sites and protective bases, and a LIGHT CHAIN gene fragment of 0.73kb and a HEAVY CHAIN gene fragment of 1.40kb are amplified by a PCR amplification method.

The HEAVY CHAIN and LIGHT CHAIN gene fragments are respectively cut by HindIII/EcoRI double enzyme, the 3.4A vector is cut by HindIII/EcoRI double enzyme, the HEAVY CHAIN gene and LIGHT CHAIN gene after the fragments and the vector are purified and recovered are respectively connected with the 3.4A expression vector, and recombinant expression plasmids of HEAVY CHAIN and LIGHT CHAIN are respectively obtained.

2 Stable cell line selection

(1) Recombinant antibody expression plasmid transient transfection CHO cells, determination of expression plasmid activity

The plasmid was diluted to 40ug/100ul with ultrapure water, CHO cells were adjusted to 1.43X10 ⁷ cells/ml in centrifuge tubes, 100. Mu.L of plasmid was mixed with 700. Mu.L of cells, transferred to an electrotransfer cup, electrotransferred, sampled and counted on days 3, 5, 7, and collected on day 7.

BUP-BSA (Handson, inc. of HDS-A4018) was diluted to 2ug/ml with coating solution (NaHCO 3 as main ingredient) 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 100. Mu.L/well, 37℃for 30min (1 h for part of supernatant); 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/hole, EDTA.2Na+ concentrated H ₂SO₄); OD was read on the microplate reader at 450nm (reference 630 nm). The results showed that the reaction OD after 1000-fold dilution of the cell supernatant was still greater than 1.0, and that the reaction OD without cell supernatant was less than 0.1, indicating that the antibodies produced after transient transformation of the plasmid were active on BUP-BSA.

The reactivity of the cell supernatant with BSA was examined by the same method as described above, and the result showed that the cell supernatant was diluted 1000-fold and the OD of the reaction was less than 0.1, and that the cell supernatant was not added and the OD of the reaction was less than 0.1, indicating that the antibody produced after transient transformation of the plasmid was inactive against BSA, and further demonstrating that the cell supernatant was active against BUP (buprenorphine).

(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

Diluting the plasmid to 40ug/100ul with ultrapure water, regulating CHO cells to 1.43X10 ⁷ cells/ml in a centrifuge tube, mixing 100 mu L of plasmid with 700 mu L of cells, transferring into an electrorotating cup, 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; seed preservation batch culture is carried out after 3 days, cell density is adjusted to be 0.5X10 ⁶ cells/ml, batch culture is carried out by 2.2ml, and seed preservation is carried out by 2ml, wherein the cell density is 0.3X10 ⁶ 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 is started every day when the culture is carried out in a shake flask for 72 hours, hyCloneTM Cell BoostTM Feed a is fed with 3% of the initial culture volume every day, and the feeding amount of Feed 7b is one thousandth of the initial culture volume every day until the 12 th day (feeding on 12 th day). Glucose was fed at 3g/L on day six. Samples were collected on day 13. Affinity purification was performed using a proteona affinity column. 3 μg of purified antibody was subjected to reducing SDS-PAGE, and the electrophoretogram shows two bands after reducing SDS-PAGE, 1 Mr at 50KD and the other Mr at 28KD.

Example 2 affinity and Activity optimization

The Anti-BUP-53G9 monoclonal antibody obtained in example 1 had a binding ability to buprenorphine, but was not satisfactory in affinity and antibody activity, and thus the applicant had performed directed mutation on the light chain CDR and the heavy chain CDR of the antibody. The method comprises the steps of performing structural simulation of an antibody variable region, structural simulation of an antigen-antibody variable region acting complex, analysis of key amino acids of an antibody and mutation design by using a computer, designing and synthesizing a two-way primer covering a mutation site according to a mutation scheme, synthesizing primers at two ends of target DNA, performing high-fidelity PCR reaction, cloning a PCR product to a vector, and preparing the mutant antibody according to the method described in the example 1. Monoclonal antibodies with remarkably improved affinity and antibody activity are obtained through screening and are named as: anti-BUP-53G9mut1 to Anti-BUP-53G9mut4. The amino acid sequences of the respective monoclonal antibodies are shown in the following table:

TABLE 1 antibody sequences

Example 3 detection of Performance of antibodies

1 Affinity assay

Using the AMC sensor, the purified antibody was diluted to 10ug/ml with PBST, and BUP-BSA (Handson, cat. HDS-A4018) was diluted gradient 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 1.69 GLY solution and buffer 3 (PBST), output data. (KD represents equilibrium dissociation constant, i.e., affinity; kon represents binding rate; kdis represents dissociation rate. PBST major component Na2 HPO4+NaCl+TW-20).

Table 2 affinity data

Sample name	KD(M)	kon(1/Ms)	kdis(1/s)
				Control	9.53E-09	4.43E+04	4.22E-04
Anti-BUP-53G9mut1	3.41E-10	4.26E+04	1.45E-05
				Anti-BUP-53G9mut2	6.97E-10	3.09E+04	2.15E-05
Anti-BUP-53G9mut3	2.42E-10	4.11E+04	9.97E-06
				Anti-BUP-53G9mut4	3.11E-10	3.14E+05	9.75E-05

2 Activity assay

BUP-BSA (Handson, inc. of HDS-A4018) was diluted to 2ug/ml with coating solution (NaHCO 3 as main ingredient) 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 monoclonal 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; stop solution (50. Mu.l/well, 0.75 g/EDTA.2Na and 10.2ml/L concentrated H ₂SO₄) was added; OD was read on the microplate reader at 450nm (reference 630 nm). The results are shown in the following table.

TABLE 3 Activity data

3 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. Table 4 below shows the results of the detection of OD after 21 days of antibody examination.

Table 4 stability data

Sample concentration (ng/ml)	500	125	0
				4 ℃,21 Days sample	1.011	0.574	0.031
Sample at-80℃for 21 days	1.043	0.598	0.021
				37 ℃ And 21 days of sample	0.992	0.552	0.033

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.

The partial amino acid sequence related to the application is as follows:

Sequence numbering	Sequence fragments
		SEQ ID NO:1	DYAMS
SEQ ID NO:2	SISSGGYTYYPDNVKG
		SEQ ID NO:3	VYFDYDGF
SEQ ID NO:4	RASQDISHFLN
		SEQ ID NO:5	YTSRLHS
SEQ ID NO:6	QGGNTLP
		SEQ ID NO:15	QGGNTIP

SEQUENCE LISTING

<110> Dongguan City, pengzhi biotechnology Co., ltd

<120> Anti-buprenorphine antibodies, reagents and kits for the detection of buprenorphine

<130> P2022054CN01

<160> 31

<170> PatentIn version 3.5

<210> 1

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Asp Tyr Ala Met Ser

1 5

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Ser Ile Ser Ser Gly Gly Tyr Thr Tyr Tyr Pro Asp Asn Val Lys Gly

1 5 10 15

<210> 3

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Val Tyr Phe Asp Tyr Asp Gly Phe

1 5

<210> 4

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Arg Ala Ser Gln Asp Ile Ser His Phe Leu Asn

1 5 10

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Tyr Thr Ser Arg Leu His Ser

1 5

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Gln Gly Gly Asn Thr Leu Pro

1 5

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Glu Val Lys Leu Val Glu Ser Gly Gly Asp Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser

20 25 30

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Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Ala Ala

1 5 10

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Arg Phe Thr Val Ser Arg Asp Asn Val Arg Asn Ile Leu Tyr Leu Gln

1 5 10 15

Met Asn Ser Leu Arg Ser Glu Asp Thr Ala Ile Tyr Tyr Cys Ala Arg

20 25 30

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Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser

1 5 10

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Asp Ile Gln Leu Thr Gln Thr Pro Ser Ser Leu Ser Ala Ser Leu Gly

1 5 10 15

Asp Arg Val Thr Ile Ser Cys

20

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Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Met Tyr

1 5 10 15

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Gly Val Pro Ser Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Ser

1 5 10 15

Leu Thr Ile Arg Asn Leu Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys

20 25 30

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Pro Pro Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys

1 5 10

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Gln Gly Gly Asn Thr Ile Pro

1 5

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Arg Phe Thr Val Ser Arg Asp Asn Val Arg Asn Ile Leu Tyr Leu Gln

1 5 10 15

Met Asn Ser Leu Arg Ser Glu Asp Thr Ala Leu Tyr Tyr Cys Ala Arg

20 25 30

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Gly Val Pro Ser Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Ser

1 5 10 15

Leu Thr Ile Arg Asn Leu Glu Gln Glu Asp Leu Ala Thr Tyr Phe Cys

20 25 30

<210> 18

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

1 5 10 15

Ala Gln Thr Asn Ser Met Val Thr Leu Gly Cys Leu Val Lys Gly Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Thr Trp Asn Ser Gly Ser Leu Ser Ser

35 40 45

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

50 55 60

Ser Ser Ser Val Thr Val Pro Ser Ser Thr Trp Pro Ser Glu Thr Val

65 70 75 80

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

85 90 95

Ile Val Pro Arg Asp Cys Gly Cys Lys Pro Cys Ile Cys Thr Val Pro

100 105 110

Glu Val Ser Ser Val Phe Ile Phe Pro Pro Lys Pro Lys Asp Val Leu

115 120 125

Thr Ile Thr Leu Thr Pro Lys Val Thr Cys Val Val Val Asp Ile Ser

130 135 140

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

145 150 155 160

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

165 170 175

Phe Arg Ser Val Ser Glu Leu Pro Ile Met His Gln Asp Trp Leu Asn

180 185 190

Gly Lys Glu Phe Lys Cys Arg Val Asn Ser Ala Ala Phe Pro Ala Pro

195 200 205

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

210 215 220

Val Tyr Thr Ile Pro Pro Pro Lys Glu Gln Met Ala Lys Asp Lys Val

225 230 235 240

Ser Leu Thr Cys Met Ile Thr Asp Phe Phe Pro Glu Asp Ile Thr Val

245 250 255

Glu Trp Gln Trp Asn Gly Gln Pro Ala Glu Asn Tyr Lys Asn Thr Gln

260 265 270

Pro Ile Met Asp Thr Asp Gly Ser Tyr Phe Val Tyr Ser Lys Leu Asn

275 280 285

Val Gln Lys Ser Asn Trp Glu Ala Gly Asn Thr Phe Thr Cys Ser Val

290 295 300

Leu His Glu Gly Leu His Asn His His Thr Glu Lys Ser Leu Ser His

305 310 315 320

Ser Pro Gly

<210> 19

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Arg Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu

1 5 10 15

Gln Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe

20 25 30

Tyr Pro Lys Asp Ile Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg

35 40 45

Gln Asn Gly Val Leu Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser

50 55 60

Thr Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu

65 70 75 80

Arg His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser

85 90 95

Pro Ile Val Lys Ser Phe Asn Arg Asn Glu Cys

100 105

<210> 20

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Glu Val Lys Leu Val Glu Ser Gly Gly Asp Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Ala

35 40 45

Ala Ser Ile Ser Ser Gly Gly Tyr Thr Tyr Tyr Pro Asp Asn Val Lys

50 55 60

Gly Arg Phe Thr Val Ser Arg Asp Asn Val Arg Asn Ile Leu Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Arg Ser Glu Asp Thr Ala Ile Tyr Tyr Cys Ala

85 90 95

Arg Val Tyr Phe Asp Tyr Asp Gly Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Thr Leu Thr Val Ser Ser

115

<210> 21

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Glu Val Lys Leu Val Glu Ser Gly Gly Asp Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Ala

35 40 45

Ala Ser Ile Ser Ser Gly Gly Tyr Thr Tyr Tyr Pro Asp Asn Val Lys

50 55 60

Gly Arg Phe Thr Val Ser Arg Asp Asn Val Arg Asn Ile Leu Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Arg Ser Glu Asp Thr Ala Leu Tyr Tyr Cys Ala

85 90 95

Arg Val Tyr Phe Asp Tyr Asp Gly Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Thr Leu Thr Val Ser Ser

115

<210> 22

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Asp Ile Gln Leu Thr Gln Thr Pro Ser Ser Leu Ser Ala Ser Leu Gly

1 5 10 15

Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser His Phe

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Met

35 40 45

Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Thr Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Ser Leu Thr Ile Arg Asn Leu Glu Gln

65 70 75 80

Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gly Gly Asn Thr Leu Pro Pro

85 90 95

Pro Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 23

<211> 107

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Asp Ile Gln Leu Thr Gln Thr Pro Ser Ser Leu Ser Ala Ser Leu Gly

1 5 10 15

Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser His Phe

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Met

35 40 45

Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Thr Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Ser Leu Thr Ile Arg Asn Leu Glu Gln

65 70 75 80

Glu Asp Leu Ala Thr Tyr Phe Cys Gln Gly Gly Asn Thr Leu Pro Pro

85 90 95

Pro Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 24

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<400> 24

Asp Ile Gln Leu Thr Gln Thr Pro Ser Ser Leu Ser Ala Ser Leu Gly

1 5 10 15

Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser His Phe

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Met

35 40 45

Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Thr Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Ser Leu Thr Ile Arg Asn Leu Glu Gln

65 70 75 80

Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gly Gly Asn Thr Ile Pro Pro

85 90 95

Pro Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 25

<211> 107

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<400> 25

Asp Ile Gln Leu Thr Gln Thr Pro Ser Ser Leu Ser Ala Ser Leu Gly

1 5 10 15

Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser His Phe

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Met

35 40 45

Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Thr Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Ser Leu Thr Ile Arg Asn Leu Glu Gln

65 70 75 80

Glu Asp Leu Ala Thr Tyr Phe Cys Gln Gly Gly Asn Thr Ile Pro Pro

85 90 95

Pro Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 26

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Glu Val Lys Leu Val Glu Ser Gly Gly Asp Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Ala

35 40 45

Ala Ser Ile Ser Ser Gly Gly Tyr Thr Tyr Tyr Pro Asp Asn Val Lys

50 55 60

Gly Arg Phe Thr Val Ser Arg Asp Asn Val Arg Asn Ile Leu Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Arg Ser Glu Asp Thr Ala Ile Tyr Tyr Cys Ala

85 90 95

Arg Val Tyr Phe Asp Tyr Asp Gly Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

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

115 120 125

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

130 135 140

Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Val Thr Trp Asn

145 150 155 160

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

165 170 175

Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Pro Ser Ser Thr

180 185 190

Trp Pro Ser Glu Thr Val Thr Cys Asn Val Ala His Pro Ala Ser Ser

195 200 205

Thr Lys Val Asp Lys Lys Ile Val Pro Arg Asp Cys Gly Cys Lys Pro

210 215 220

Cys Ile Cys Thr Val Pro Glu Val Ser Ser Val Phe Ile Phe Pro Pro

225 230 235 240

Lys Pro Lys Asp Val Leu Thr Ile Thr Leu Thr Pro Lys Val Thr Cys

245 250 255

Val Val Val Asp Ile Ser Lys Asp Asp Pro Glu Val Gln Phe Ser Trp

260 265 270

Phe Val Asp Asp Val Glu Val His Thr Ala Gln Thr Gln Pro Arg Glu

275 280 285

Glu Gln Phe Asn Ser Thr Phe Arg Ser Val Ser Glu Leu Pro Ile Met

290 295 300

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

305 310 315 320

Ala Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly

325 330 335

Arg Pro Lys Ala Pro Gln Val Tyr Thr Ile Pro Pro Pro Lys Glu Gln

340 345 350

Met Ala Lys Asp Lys Val Ser Leu Thr Cys Met Ile Thr Asp Phe Phe

355 360 365

Pro Glu Asp Ile Thr Val Glu Trp Gln Trp Asn Gly Gln Pro Ala Glu

370 375 380

Asn Tyr Lys Asn Thr Gln Pro Ile Met Asp Thr Asp Gly Ser Tyr Phe

385 390 395 400

Val Tyr Ser Lys Leu Asn Val Gln Lys Ser Asn Trp Glu Ala Gly Asn

405 410 415

Thr Phe Thr Cys Ser Val Leu His Glu Gly Leu His Asn His His Thr

420 425 430

Glu Lys Ser Leu Ser His Ser Pro Gly

435 440

<210> 27

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<400> 27

Glu Val Lys Leu Val Glu Ser Gly Gly Asp Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Ala

35 40 45

Ala Ser Ile Ser Ser Gly Gly Tyr Thr Tyr Tyr Pro Asp Asn Val Lys

50 55 60

Gly Arg Phe Thr Val Ser Arg Asp Asn Val Arg Asn Ile Leu Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Arg Ser Glu Asp Thr Ala Leu Tyr Tyr Cys Ala

85 90 95

Arg Val Tyr Phe Asp Tyr Asp Gly Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

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

115 120 125

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

130 135 140

Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Val Thr Trp Asn

145 150 155 160

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

165 170 175

Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Pro Ser Ser Thr

180 185 190

Trp Pro Ser Glu Thr Val Thr Cys Asn Val Ala His Pro Ala Ser Ser

195 200 205

Thr Lys Val Asp Lys Lys Ile Val Pro Arg Asp Cys Gly Cys Lys Pro

210 215 220

Cys Ile Cys Thr Val Pro Glu Val Ser Ser Val Phe Ile Phe Pro Pro

225 230 235 240

Lys Pro Lys Asp Val Leu Thr Ile Thr Leu Thr Pro Lys Val Thr Cys

245 250 255

Val Val Val Asp Ile Ser Lys Asp Asp Pro Glu Val Gln Phe Ser Trp

260 265 270

Phe Val Asp Asp Val Glu Val His Thr Ala Gln Thr Gln Pro Arg Glu

275 280 285

Glu Gln Phe Asn Ser Thr Phe Arg Ser Val Ser Glu Leu Pro Ile Met

290 295 300

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

305 310 315 320

Ala Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly

325 330 335

Arg Pro Lys Ala Pro Gln Val Tyr Thr Ile Pro Pro Pro Lys Glu Gln

340 345 350

Met Ala Lys Asp Lys Val Ser Leu Thr Cys Met Ile Thr Asp Phe Phe

355 360 365

Pro Glu Asp Ile Thr Val Glu Trp Gln Trp Asn Gly Gln Pro Ala Glu

370 375 380

Asn Tyr Lys Asn Thr Gln Pro Ile Met Asp Thr Asp Gly Ser Tyr Phe

385 390 395 400

Val Tyr Ser Lys Leu Asn Val Gln Lys Ser Asn Trp Glu Ala Gly Asn

405 410 415

Thr Phe Thr Cys Ser Val Leu His Glu Gly Leu His Asn His His Thr

420 425 430

Glu Lys Ser Leu Ser His Ser Pro Gly

435 440

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Asp Ile Gln Leu Thr Gln Thr Pro Ser Ser Leu Ser Ala Ser Leu Gly

1 5 10 15

Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser His Phe

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Met

35 40 45

Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Thr Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Ser Leu Thr Ile Arg Asn Leu Glu Gln

65 70 75 80

Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gly Gly Asn Thr Leu Pro Pro

85 90 95

Pro Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys Arg Ala Asp Ala Ala

100 105 110

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

115 120 125

Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe Tyr Pro Lys Asp Ile

130 135 140

Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln Asn Gly Val Leu

145 150 155 160

Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr Tyr Ser Met Ser

165 170 175

Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg His Asn Ser Tyr

180 185 190

Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro Ile Val Lys Ser

195 200 205

Phe Asn Arg Asn Glu Cys

210

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Asp Ile Gln Leu Thr Gln Thr Pro Ser Ser Leu Ser Ala Ser Leu Gly

1 5 10 15

Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser His Phe

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Met

35 40 45

Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Thr Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Ser Leu Thr Ile Arg Asn Leu Glu Gln

65 70 75 80

Glu Asp Leu Ala Thr Tyr Phe Cys Gln Gly Gly Asn Thr Leu Pro Pro

85 90 95

Pro Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys Arg Ala Asp Ala Ala

100 105 110

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

115 120 125

Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe Tyr Pro Lys Asp Ile

130 135 140

Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln Asn Gly Val Leu

145 150 155 160

Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr Tyr Ser Met Ser

165 170 175

Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg His Asn Ser Tyr

180 185 190

Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro Ile Val Lys Ser

195 200 205

Phe Asn Arg Asn Glu Cys

210

<210> 30

<211> 214

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<400> 30

Asp Ile Gln Leu Thr Gln Thr Pro Ser Ser Leu Ser Ala Ser Leu Gly

1 5 10 15

Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser His Phe

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Met

35 40 45

Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Thr Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Ser Leu Thr Ile Arg Asn Leu Glu Gln

65 70 75 80

Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gly Gly Asn Thr Ile Pro Pro

85 90 95

Pro Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys Arg Ala Asp Ala Ala

100 105 110

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

115 120 125

Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe Tyr Pro Lys Asp Ile

130 135 140

Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln Asn Gly Val Leu

145 150 155 160

Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr Tyr Ser Met Ser

165 170 175

Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg His Asn Ser Tyr

180 185 190

Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro Ile Val Lys Ser

195 200 205

Phe Asn Arg Asn Glu Cys

210

<210> 31

<211> 214

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<400> 31

Asp Ile Gln Leu Thr Gln Thr Pro Ser Ser Leu Ser Ala Ser Leu Gly

1 5 10 15

Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser His Phe

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Met

35 40 45

Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Thr Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Ser Leu Thr Ile Arg Asn Leu Glu Gln

65 70 75 80

Glu Asp Leu Ala Thr Tyr Phe Cys Gln Gly Gly Asn Thr Ile Pro Pro

85 90 95

Pro Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys Arg Ala Asp Ala Ala

100 105 110

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

115 120 125

Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe Tyr Pro Lys Asp Ile

130 135 140

Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln Asn Gly Val Leu

145 150 155 160

Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr Tyr Ser Met Ser

165 170 175

Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg His Asn Ser Tyr

180 185 190

Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro Ile Val Lys Ser

195 200 205

Phe Asn Arg Asn Glu Cys

210

Claims

1. An anti-buprenorphine antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof comprises an HCDR1 having an amino acid sequence shown as SEQ ID No.1, an HCDR2 having an amino acid sequence shown as SEQ ID No. 2, an HCDR3 having an amino acid sequence shown as SEQ ID No. 3, and an LCDR1 having an amino acid sequence shown as SEQ ID No. 4, an LCDR2 having an amino acid sequence shown as SEQ ID No. 5, an LCDR 6 or an LCDR3 having an amino acid sequence shown as SEQ ID No. 15.

2. The antibody or antigen-binding fragment thereof according to claim 1, further comprising an amino acid sequence of HFR1, HFR2, HFR3, HFR4, shown in sequence as SEQ ID No. 7 to SEQ ID No. 10, and an amino acid sequence of LFR1, LFR2, LFR3, and LFR4, shown in sequence as SEQ ID No. 11 to SEQ ID No. 14, or an amino acid sequence having at least 80% identity to each of said HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3, LFR 4.

3. The antibody or antigen-binding fragment thereof according to claim 2, wherein the amino acid sequence of HFR3 of the antibody or antigen-binding fragment thereof is shown in SEQ ID No. 16.

4. The antibody or antigen-binding fragment thereof of claim 2, wherein LFR3 of the antibody or antigen-binding fragment thereof has the amino acid sequence shown in SEQ ID No. 17.

5. The antibody or antigen-binding fragment thereof of claim 1, wherein the antibody or antigen-binding fragment thereof binds buprenorphine with an affinity of KD < 9.53 x 10 ^-9 M.

6. An anti-buprenorphine antibody or antigen-binding fragment thereof, comprising a heavy chain variable region comprising the sequence structure of HFR1-HCDR 2-HFR3-HCDR3-HFR4 and a light chain variable region comprising the sequence structure of LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4, wherein the amino acid sequence of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3 is the amino acid sequence of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3, HFR1, HFR2, HFR4, HFR1, LFR2, LFR3, LFR4 of the amino acid sequence of HFR1, HFR2, HFR3, LCDR3, LFR4 of the amino acid sequence of claim 1, HFR2, HFR3, LFR4, LFR3, LFR 4.

7. An anti-buprenorphine antibody or antigen-binding fragment thereof, wherein said antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region having amino acid sequences as set forth in SEQ ID No. 20 and SEQ ID No. 22, respectively; or the amino acid sequences of the heavy chain variable region and the light chain variable region are respectively shown as SEQ ID NO. 21 and SEQ ID NO. 22; or the amino acid sequences of the heavy chain variable region and the light chain variable region are respectively shown as SEQ ID NO. 20 and SEQ ID NO. 23; or the amino acid sequences of the heavy chain variable region and the light chain variable region are respectively shown as SEQ ID NO. 20 and SEQ ID NO. 24.

8. The antibody or antigen-binding fragment thereof of any one of claims 1 to 7, wherein the antibody or antigen-binding fragment thereof further comprises a constant region.

9. The antibody or antigen-binding fragment thereof of claim 8, wherein the constant regions comprise a heavy chain constant region and a light chain constant region.

10. The antibody or antigen-binding fragment thereof of claim 9, wherein the heavy chain constant region is selected from the group consisting of the heavy chain constant region of IgG1, igG2, igG3, igG4, igA, igM, igE, or IgD; the light chain constant region is selected from kappa-type or lambda-type light chain constant regions.

11. The antibody or antigen-binding fragment thereof of claim 8, wherein the constant region is of a species origin of bovine, equine, porcine, ovine, caprine, rat, mouse, canine, feline, rabbit, donkey, deer, mink, chicken, duck, goose, or human.

12. The antibody or antigen-binding fragment thereof of claim 8, wherein the constant region is of murine species origin.

13. The antibody or antigen-binding fragment thereof of claim 9, wherein the heavy chain constant region sequence is as shown in SEQ ID No. 18 or has at least 80% identity thereto and the light chain constant region sequence is as shown in SEQ ID No. 19 or has at least 80% identity thereto.

14. The antibody or antigen-binding fragment thereof of any one of claims 1 to 7, wherein the antigen-binding fragment is selected from any one of F (ab ') 2, fab', fab, fv, and scFv of the antibody.

15. An anti-buprenorphine antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises the heavy chain variable region of any one of claims 6 to 7 and the heavy chain constant region of any one of claims 9, 10 or 13; the light chain comprises the light chain variable region of any one of claims 6 to 7 and the light chain constant region of any one of claims 9, 10 or 13.

16. An anti-buprenorphine antibody, wherein the amino acid sequences of the heavy and light chains are shown in SEQ ID NO. 26 and SEQ ID NO. 28, respectively; or the amino acid sequences of the heavy chain variable region and the light chain variable region are respectively shown as SEQ ID NO. 27 and SEQ ID NO. 28; or the amino acid sequences of the heavy chain variable region and the light chain variable region are respectively shown as SEQ ID NO. 26 and SEQ ID NO. 29; or the amino acid sequences of the heavy chain variable region and the light chain variable region are respectively shown as SEQ ID NO. 26 and SEQ ID NO. 30.

17. An antibody conjugate comprising the antibody or antigen-binding fragment thereof of any one of claims 1 to 14 or the antibody of any one of claims 15 to 16.

18. The antibody conjugate of claim 17, wherein the antibody is conjugated to biotin.

19. The antibody conjugate of claim 17, wherein the antibody is conjugated to a solid phase.

20. The antibody conjugate of claim 17, wherein the antibody is conjugated to a label.

21. The antibody conjugate of claim 20, wherein the label is selected from the group consisting of a fluorescent dye, an enzyme, a radioisotope, a chemiluminescent reagent, and a nanoparticle-based label.

22. The antibody conjugate of claim 20, wherein the label is colloidal gold.

23. A reagent for the detection of buprenorphine, comprising an antibody or antigen-binding fragment thereof according to any one of claims 1 to 14 or an antibody according to any one of claims 15 to 16 or an antibody conjugate according to any one of claims 17 to 22.

24. A nucleic acid encoding the antibody or antigen binding fragment thereof of any one of claims 1 to 14 or the antibody of any one of claims 15 to 16.

25. A vector comprising the nucleic acid of claim 24.

26. A cell comprising the nucleic acid of claim 24 or the vector of claim 25.

27. A method of preparing the antibody or antigen-binding fragment thereof of any one of claims 1 to 14, comprising: culturing the cell of claim 26.