Anti-digoxin antibodies or antigen-binding fragments thereof and uses thereof

Abstract

The application provides an anti-digoxin antibody or antigen binding fragment thereof and application thereof, and relates to the technical field of antibodies. The anti-digoxin antibody or antigen binding fragment thereof comprises a heavy chain variable region and a light chain variable region; the heavy chain variable region comprises VHCDR1 of the 17th-24th amino acid residues at the N terminus of the amino acid sequence shown in SEQ ID NO. 1, VHCDR2 of the 42nd-49th amino acid residues, and VHCDR3 of the 88th-98th amino acid residues; and the light chain variable region comprises VLCDR1 of the 27th-37th amino acid residues at the N terminus of the amino acid sequence shown in SEQ ID NO. 2, VLCDR2 of the 55th-57th amino acid residues, and VLCDR3 of the 94th-102th amino acid residues. The antibody or antigen binding fragment thereof can be used for immunological detection, and has been verified to have good specific binding capacity.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of antibody technology, in particular to an anti-digoxin antibody or antigen binding fragment thereof and application thereof. BACKGROUND

[0002] Digoxin is the most widely used drug in cardiac glycosides, and has been clinically used for more than 200 years. It has high affinity and selectivity for the heart. At therapeutic doses, it can slightly inhibit Na + / K + -ATPase, disrupt the active transport of sodium on the myocardial cell membrane, increase the intracellular Na + concentration, and reduce the K+concentration. Subsequently, through the Na + and Ca 2+ bidirectional exchange mechanism, the intracellular Ca 2+ concentration is increased, the myocardial contractility is enhanced, and a positive inotropic effect is produced, which is used to treat congestive heart failure and certain arrhythmias.

[0003] Digoxin is a cardiac glycoside extracted from the leaves of Digitalis lanata, also known as digoxigenin. The molecular formula of digoxin is C 41 H 64 O 14 , and the relative molecular mass is 780.95. It is formed by dehydration condensation of a 5-membered unsaturated lactone ring on the C17 of the steroidal nucleus and the C3 of three D-digitalis toxic sugars. Digoxin has one more hydroxyl group than digitoxin, which increases the polarity of digoxin and reduces the lipophilicity. Correspondingly, the oral absorption of digoxin is lower than that of digitoxin. Digoxin is a white crystalline or crystalline powder with a bitter taste, easily soluble in pyridine, insoluble in water and diethyl ether, and easily hydrolyzed in acidic or basic conditions, but stable in a sealed and light-proof state.

[0004] Digoxin is mainly used to treat various heart diseases with heart failure, and is particularly effective for congestive heart failure with edema, premature beats, atrial fibrillation, and supraventricular tachycardia. With the widespread use of digoxin in clinical practice, its therapeutic safety index and toxicity have always been a concern. The therapeutic safety range of digoxin is small, and the effective blood drug concentration range is 0.8-2.0 μg·L -1 . Once the dosage is too large, it can cause cardiac Ca 2+ overload, and obvious cardiotoxicity can occur. In addition, in the treatment environment of universal and routine drug combination, the influence of drug interaction on the blood drug concentration of digoxin cannot be ignored.

[0005] Digeon can be detected by detection methods based on immunological principles, such as immunofluorescence, enzyme-linked immunosorbent assay, colloidal gold immunochromatography and the like. Antibodies are the main reagents in immunological detection methods, and providing more high-quality antibodies combined with digoxin can provide stable and reliable raw material sources for detection methods based on immunological principles, reduce the dependence on imported raw materials of domestic products, and reduce product costs. At the same time, the detection rate and sensitivity of digoxin rapid diagnostic products such as gold standard test strips and ELISA kits are improved, the false positive rate is reduced, and the competitiveness of self-produced products is improved.

[0006] Therefore, the present application is provided. SUMMARY

[0007] The purpose of the present application is to provide an anti-digoxin antibody or antigen-binding fragment thereof, a monoclonal antibody obtained by screening with digoxin as an antigen, and the CDR region sequence thereof determined by cloning, identification and gene structure analysis. Another purpose of the present application is to provide biological materials related to the antibody or antigen-binding fragment thereof and their applications.

[0008] To solve the above technical problems, the present application adopts the following technical solutions:

[0009] In a first aspect, an anti-digoxin antibody or antigen-binding fragment thereof is provided, which comprises a heavy chain variable region and a light chain variable region; the heavy chain variable region comprises VHCDR1 of the N-terminal 17th-24th amino acid residues, VHCDR2 of the 42nd-49th amino acid residues and VHCDR3 of the 88th-98th amino acid residues of the amino acid sequence shown in SEQ ID NO. 1;

[0010] The light chain variable region comprises VLCDR1 of the N-terminal 27th-37th amino acid residues, VLCDR2 of the 55th-57th amino acid residues and VLCDR3 of the 94th-102th amino acid residues of the amino acid sequence shown in SEQ ID NO. 2.

[0011] In a second aspect, a biological material is provided, which is selected from any one of (i)-(iii):

[0012] (i) a polynucleotide comprising a nucleotide sequence encoding the anti-digoxin antibody or antigen-binding fragment thereof described above;

[0013] (ii) a vector carrying the polynucleotide of (i) above.

[0014] (iii) a cell carrying the polynucleotide of (i) above, or containing the vector of (ii) above, or expressing the anti-digoxin antibody or antigen-binding fragment thereof described above.

[0015] In a third aspect, there is provided use of the anti-digoxin antibody or antigen-binding fragment thereof described above, or the biological material described above, in detecting digoxin for non-diagnostic and therapeutic purposes, or in preparing a product for detecting digoxin.

[0016] In a fourth aspect, there is provided a reagent or kit for detecting digoxin, comprising the anti-digoxin antibody or antigen-binding fragment thereof described above.

[0017] Compared with the prior art, the present application has the following beneficial effects:

[0018] The present application uses digoxin to immunize Balb / c mice, fuses the mouse spleen cells with myeloma cells, and obtains hybridoma cells with high specificity through specific high-throughput screening. A large amount of mouse ascites is obtained through culture and re-immunization, and high-purity, high-sensitivity and high-specificity anti-digoxin monoclonal antibody anti-DGX-mab1 is obtained through multi-step separation and purification, which provides the required raw material for developing an immunological test strip for detecting digoxin. The anti-digoxin monoclonal antibody anti-DGX-mab1 of the present application can be used for immunoblotting, immunofluorescence and other immunological detection, and the obtained antibody has been verified to have good specific binding capacity. DETAILED DESCRIPTION

[0019] The technical solutions of the present application will be described clearly and completely in combination with the embodiments below. Obviously, the described embodiments are part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0020] Generally, the nomenclature used in connection with, and the techniques of, cell and tissue culture, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are those well-known and commonly used in the art. Unless otherwise indicated, the methods and techniques of the present application are generally performed according to conventional methods in the art, and such methods and techniques are described in various general and more specific references that are cited throughout the present specification. Enzymatic reactions and purification techniques are performed according to manufacturer's specifications, as commonly accomplished in the art, or as described herein. The nomenclature used in connection with, and the techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well-known and commonly used in the art.

[0021] "Antibody or antigen-binding fragment thereof" as used herein refers to a protein that binds a specific antigen, and refers broadly to all proteins and protein fragments that comprise complementarity determining regions (CDR regions). In addition, "antibody or antigen-binding fragment thereof" includes naturally occurring antibodies as well as non-naturally occurring antibodies. "Antigen-binding fragment" as used herein refers to a substance that comprises CDRs of an antibody, which lacks at least some of the amino acids present in a full-length chain but is still capable of specifically binding to an antigen. Such fragments are biologically active in that they bind to a target antigen and can compete with other antigen-binding molecules, including intact antibodies, for binding to a given epitope. The antigen-binding fragments of the present application have the effect of specifically recognizing and binding to digoxin.

[0022] The "variable region" or "variable domain" of an antibody or antigen-binding fragment thereof refers to the amino-terminal antigen recognition and binding domain of antibody heavy or light chain, the sequence and arrangement of amino acids in this segment determine the specificity of the antibody for its antigen. The heavy chain variable region can be referred to as "VH". The light chain variable region can be referred to as "VL". These domains are generally the most variable parts of an antibody and contain the antigen binding site. Each of the variable regions of the heavy and light chains is composed of three complementarity determining regions (CDRs) (also known as hypervariable regions) connected by four framework (FR) regions. The extent of the framework and CDRs have been precisely defined, for example, in Kabat (see Sequences of Proteins of Immunological Interest, E. Kabat et al.) and Chothia, any CDR-determining method known in the art, including combinations of methods, can identify the CDRs of the variable domain. The CDRs in each chain are held together in a variable region by the FRs. Generally, the variable regions of the heavy and light chains, VL / VH, can be obtained by arranging the CDRs and FRs in the following combinations: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0023] The term "polynucleotide" as used herein refers to a polymeric form of nucleotides of any length, and includes ribonucleotides and / or deoxyribonucleotides. Examples of polynucleotides include, but are not limited to, single-, double-, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or a polymer comprising purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases. A polynucleotide comprises a portion of the above-described antibody or antigen-binding fragment thereof, optionally encoding a sense or antisense strand. The polynucleotide can be naturally occurring, synthetic, recombinant, or any combination thereof.

[0024] The term "vector" refers to a nucleic acid vehicle into which a polynucleotide can be inserted. When the vector is capable of directing the expression of a polynucleotide inserted into it, the vector is said to be an expression vector. The vector can be introduced into a host cell by transformation, transduction or transfection, and the vector's genetic material is then expressed in the host cell.

[0025] The vectors are well known to those skilled in the art and include, but are not limited to, plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1 -derived artificial chromosomes (PACs); bacteriophages such as lambda phage or M13 phage; and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papova viruses. In some embodiments, the vectors of the present application comprise regulatory elements commonly used in genetic engineering, such as enhancers, promoters, internal ribosome entry sites (IRES), and other expression control elements (e.g., transcription termination signals, or polyadenylation signals and poly-U sequences).

[0026] As used herein, the expressions "cell", "cell line", and "cell culture" are used interchangeably and all such designations include the progeny. The progeny can not necessarily be completely identical to the parent cell, e.g., in morphological or genetic composition, as a result of natural, accidental, or deliberate mutation. "Transformants" and "transformed cells" include the primary subject cell and cultures derived from it.

[0027] Unless otherwise clearly indicated, the term "comprise", or variations such as "comprises" or "comprising", will be understood to embrace the elements or components that the term describes, including equivalents thereof, but not to exclude other elements or components.

[0028] According to one aspect of the present application, there is provided an anti-digoxin antibody or an antigen-binding fragment thereof, which comprises a heavy chain variable region and a light chain variable region; the heavy chain variable region comprises a VHCDR1 (GYTFTSYT) of the 17th-24th amino acid residues, a VHCDR2 (INPYSVYT) of the 42nd-49th amino acid residues, and a VHCDR3 (VREYYGSGSDY) of the 88th-98th amino acid residues at the N-terminus of the amino acid sequence shown in SEQ ID NO. 1.

[0029] The light chain variable region comprises a VL CDR1 (QSLVHSNGNTY) of the amino acid sequence set forth in SEQ ID NO. 2 at amino acid residues 27-37 of the N-terminus, a VL CDR2 (KVS) of the amino acid sequence set forth in SEQ ID NO. 2 at amino acid residues 55-57, and a VL CDR3 (SQTTHVPPT) of the amino acid sequence set forth in SEQ ID NO. 2 at amino acid residues 94-102.

[0030] In an alternative embodiment, the heavy chain variable region of the anti-digoxin antibody or antigen-binding fragment thereof has the structure:

[0031] VHFR1-VHCDR1-VHFR2-VHCDR2-VHFR3-VHCDR3-VHFR4; wherein the amino acid sequence of VHFR1 is set forth in SEQ ID NO. 3 (TDWHTGASVQMSCKAS), and / or the amino acid sequence of VHFR2 is set forth in SEQ ID NO. 4 (IHWVKQMPGQGLEWIGS), and / or the amino acid sequence of VHFR3 is set forth in SEQ ID NO. 5 (SYNQNFMDKATLTADKSSSTAYMQLSSLTSEDSAVYYC), and / or the amino acid sequence of VHFR4 is set forth in SEQ ID NO. 6 (WGQGTTLIVSSA).

[0032] In an alternative embodiment, the heavy chain variable region of the anti-digoxin antibody or antigen-binding fragment thereof has the amino acid sequence set forth in SEQ IN NO. 1.

[0033] In an alternative embodiment, the light chain variable region of the anti-digoxin antibody or antigen-binding fragment thereof has the structure:

[0034] VLFR1-VLCDR1-VLFR2-VLCDR2-VLFR3-VLCDR3-VLFR4; wherein the amino acid sequence of VLFR1 is set forth in SEQ ID NO. 7 (DIVITQTPLSLPVSLGDQASISCRSS), and / or the amino acid sequence of VLFR2 is set forth in SEQ ID NO. 8 (LHWYLQKPGQSPKLLIY), and / or the amino acid sequence of VLFR3 is set forth in SEQ ID NO. 9 (NRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFC), and / or the amino acid sequence of VLFR4 is set forth in SEQ ID NO. 10 (FGGGTPSV).

[0035] In an alternative embodiment, the light chain variable region of the anti-digoxin antibody or antigen-binding fragment thereof has the amino acid sequence set forth in SEQ IN NO. 2.

[0036] In an alternative embodiment, the amino acid sequence of the heavy chain variable region of the anti-digoxin antibody or antigen-binding fragment thereof is as set forth in SEQ ID NO. 1 and the amino acid sequence of the light chain variable region is as set forth in SEQ ID NO. 2.

[0037] In an alternative embodiment, the antibody or antigen-binding fragment thereof further comprises a sequence of a portion or all of the constant region of any one of IgGl, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD.

[0038] In an alternative embodiment, the antigen-binding fragment comprises one or more of F(ab')2, Fab', Fab, Fv, scFv, dsFv, diabody and minimal recognition unit of an antibody.

[0039] In an alternative embodiment, the antibody or antigen-binding fragment thereof, except for the CDR regions, the remaining sequence is derived from one or more of mouse, rat, guinea pig, hamster, rabbit, ferret, cat, dog, goat, sheep, cow, pig, horse, monkey and human.

[0040] In an alternative embodiment, the antibody further comprises a heavy chain constant region and a light chain constant region.

[0041] In an alternative embodiment, the heavy chain constant region and / or the light chain constant region is derived from mouse.

[0042] In an alternative embodiment, the light chain of the antibody is kappa chain.

[0043] In an alternative embodiment, the antibody is an IgG antibody.

[0044] According to another aspect of the present application, there is also provided a biological material selected from any one of (i) to (iii):

[0045] (i) a polynucleotide comprising a nucleotide sequence encoding the aforementioned anti-digoxin antibody or antigen-binding fragment thereof; in an alternative embodiment, the polynucleotide encodes the heavy chain variable region, the nucleotide sequence being as set forth in SEQ ID NO. 11; in an alternative embodiment, the polynucleotide encodes the light chain variable region, the nucleotide sequence being as set forth in SEQ ID NO. 12.

[0046] (ii) a vector carrying the aforementioned (i) polynucleotide.

[0047] (iii) a cell carrying the aforementioned (i) polynucleotide, or containing the aforementioned (ii) vector, or expressing the aforementioned anti-digoxin antibody or antigen-binding fragment thereof.

[0048] According to another aspect of the present application, there is also provided use of the anti-digoxin antibody or antigen-binding fragment thereof described above, or the biomaterial described above, in detecting digoxin for non-diagnostic and therapeutic purposes, or in preparing a product for detecting digoxin.

[0049] According to another aspect of the present application, there is also provided a reagent or kit for detecting digoxin, comprising the anti-digoxin antibody or antigen-binding fragment thereof described above.

[0050] In optional embodiments, the label comprises one or more of an enzyme, a fluorescent molecule label, fluorescent microspheres, colored microspheres, colloidal gold, biotin or streptavidin.

[0051] In optional embodiments, the label in the reagent or kit is coupled to the anti-digoxin antibody or antigen-binding fragment thereof in any manner acceptable in the art.

[0052] In optional embodiments, the reagent or kit is used for immunoassay. In optional embodiments, the reagent or kit comprises an immunochromatographic assay reagent or kit, an ELISA assay reagent or kit, an immunomagnetic microparticle assay reagent or kit, an immunofluorescence assay reagent or kit, or an immunoblotting assay reagent or kit.

[0053] In optional embodiments, the reagent or kit further comprises reagents and / or consumables for detection, including but not limited to one or more of primers, probes, buffers, dyes, diluents, washing solutions, developing solutions, lysis solutions, negative controls, positive controls and blank controls; and consumables including but not limited to solid phase carriers such as magnetic beads, enzyme-labeled wells or NC membranes, etc. The skilled person can select the reagent composition of the kit according to the specific detection means, and the present application does not limit this.

[0054] In optional embodiments, the kit comprises an immunochromatographic test strip, which is a competitive immunochromatographic test strip. The competitive immunochromatographic test strip is coated with the antibody or antigen-binding fragment thereof of any of the embodiments described above, and is used to capture digoxin in the sample to be tested.

[0055] In an optional embodiment, the immunochromatography test paper comprises a sample pad, a conjugate pad and a detection pad along the sample flow direction, the detection pad is provided with a detection line and a quality control line; the conjugate pad is coated with the labeled anti-digoxin antibody or antigen-binding fragment thereof; the detection line is coated with digoxin antigen; the quality control line is coated with an antibody specifically binding to the anti-digoxin antibody or antigen-binding fragment thereof; when the sample contains digoxin, the digoxin in the sample competes with the anti-digoxin antibody or antigen-binding fragment thereof on the conjugate pad for binding to the digoxin antigen on the detection line, the higher the content of digoxin in the sample, the less the anti-digoxin antibody or antigen-binding fragment thereof binds to the digoxin antigen on the detection line, and the less the signal detected on the detection line. The labeled anti-digoxin antibody or antigen-binding fragment thereof flows to the quality control line and is captured by the antibody coated on the quality control line, and a measurable signal appears, indicating that the immunochromatography test paper is available.

[0056] In an optional embodiment, the conjugate pad is coated with the colloidal gold-labeled anti-digoxin antibody or antigen-binding fragment thereof. In an optional embodiment, the immunochromatography test paper is coated with a label-labeled antibody binding to digoxin, the heavy chain variable region amino acid sequence of the antibody binding to digoxin is shown in SEQ ID NO. 1, and the light chain variable region amino acid sequence is shown in SEQ ID NO. 2.

[0057] The application will be further described in the following specific examples. It should be understood that these examples are only used for a more detailed description and should not be understood as limiting the application in any form.

[0058] Example 1 Preparation of monoclonal antibody anti-DGX-mab1

[0059] (I) Preparation of digoxin-PHT antigen

[0060] Table 1

[0061] Antigen source Antigen name Batch no. Concentration Total amount Purpose Self-produced antigen Digoxin-Dig-BSA #D230220-1 5.6 mg / ml 5.0 mg For immunization and screening

[0062] The chemically synthesized digoxin hapten Dig-BSA was used to immunize the experimental mice in stages.

[0063] (II) Preparation of monoclonal antibody anti-DGX-mab1

[0064] Healthy female Balb / c mice aged 6-8 weeks were selected and immunized according to the pre-specified immunization scheme. The BALB / c mice were immunized as immunogens, and the spleen lymphocytes of the successfully immunized mice were extracted. The lymphocytes were fused with mouse myeloma cells SP2 / 0 by cell fusion technology, and after two rounds of subcloning screening, a hybridoma cell strain stably secreting anti-digoxin monoclonal antibody was obtained, thereby obtaining the anti-digoxin monoclonal antibody.

[0065] 2.1 The chemically synthesized digoxin hapten Dig-BSA was used to immunize experimental mice in stages. The specific steps of the animal immunization experiment included:

[0066] (1) Balb / c mice with consistent average body weight and age were randomly divided into two groups, an aluminum adjuvant (aluminum hydroxide adjuvant) group and a non-aluminum adjuvant group.

[0067] (2) Before the experiment, the pre-immune serum of each mouse was collected (the pre-immune serum was collected on the fifth day by eye blood collection, and an appropriate amount of blood was taken to ensure the normal state of the mouse). The collected serum was stored at -80°C.

[0068] (3) The aluminum adjuvant (aluminum hydroxide adjuvant) group was prepared as follows: before immunization, each antigen was diluted to the corresponding dose (75 μg per mouse) in 75 μL PBS and mixed with alum adjuvant (1 mg per mouse), according to the volume antigen: adjuvant = 3:1 (i.e. 75 μl of immunogen diluent was added with 25 μl of adjuvant); before use, the adjuvant was shaken well, and the injected adjuvant (25 μl) was slowly added to the immunogen solution; after the adjuvant and the immunogen diluent were mixed well, they were mixed thoroughly for 30 minutes to allow the adjuvant to effectively adsorb the antigen; subsequent animal immunization experiment operations were performed.

[0069] (4) The non-aluminum adjuvant group: the antigen was diluted to the corresponding dose (75 μg per mouse) in 100 μL PBS according to the above table (100 μL of immunogen), and subsequent animal immunization experiment operations were performed.

[0070] (5) Subcutaneous injection at 2-week intervals: the experimental design was 3 immunizations, but eye blood collection was performed 7 days after each immunization, and the supernatant of some mice was obtained by centrifugation, followed by serum titer detection. Seven days after the last immunization, the maximum blood volume was taken from the heart, and the supernatant was obtained by centrifugation and stored at -80°C.

[0071] 2.2 Detection of serum titer:

[0072] Three mice were immunized, and the mouse numbers were A0, A1, and A2 in order. After 3 immunizations, the serum titer was detected. The detection data are shown in Table 2, and the detection method is as follows:

[0073] Digoxin standard was used as antigen for competitive detection, and Dig-BSA was used as coating antigen for indirect ELISA and competitive ELISA to detect the serum titer of immunized mice.

[0074] Indirect ELISA method enzyme-labeled plate per hole plus 1 μg / ml of the use of coating solution diluted coating antigen 50 μl, 4 ℃ coating overnight, then washing plate 3 times (the same below) with washing solution (PBST), each hole added blocking solution 200 μl (5% skim milk powder), placed in 37 ℃ incubator for 2 h, after taking out and washing, each hole added diluted serum 50 μl, again in 37 ℃ incubator for 30 min, after washing, added goat anti-mouse IgG-HRP solution 50 μl, 37 ℃ incubator for 30 min. After washing, added substrate solution 100 μl, 37 ℃ incubator for 10 min, finally added 2 mol / L H2SO4, 50 μL to stop the reaction, read A450 value in enzyme-labeled instrument. The three mice after three immunizations, the orbital blood titer was >62500.

[0075] The process of indirect competitive ELISA is mostly the same as that of indirect ELISA, except that after blocking and washing the enzyme-labeled plate, 50 μl of diluted 50 ng / ml and 300 ng / ml small molecule digoxin standard solution is added, then 50 μl of diluted serum antibody is added, and the remaining steps are the same. The 50 ng / ml and 300 ng / ml small molecule digoxin competition detection can reach more than 50% at 1:12500 for 3 mice, and can be arranged for fusion.

[0076] Table 2 serum titer detection data

[0077]

[0078] 2.3 Immunization of spleen cells and fusion with SP2 / 0 cell line, screening of fused cells by HAT selection medium (HAT selection medium contains hypoxanthine, aminopterin and thymine), and ELISA positive screening and subcloning of fused cells; the positive monoclonal selected is taken from ascites, and the antibody is purified by Protein A / G antibody purification column, the ELISA titer of the purified antibody is >1:128,000, and the purity is >90%.

[0079] (Three) ELISA detection of binding activity of digoxin recognition

[0080] The IgG antibody titer detection method includes the following steps:

[0081] (1) bottom plate coating: the used antigen is diluted to 3 μg / ml with coating diluent, 100 μl of the prepared coating solution is added to each hole, and placed in a 4 ℃ refrigerator for 24 h.

[0082] (2) After 24 h, take out from the refrigerator and place in a 37 ℃ incubator for 30 min, then discard the liquid in the hole; wash the hole with washing solution for 3 times, 3 min each time.

[0083] (3) Close the enzyme label reaction hole: after adding 200 μl 5% calf serum to each hole, close 37°C for 90 min, and then wash the hole with washing solution for 3 times, 3 min each time.

[0084] (4) Add the sample to be detected: dilute the sample according to the required ratio, add the diluted sample to the enzyme label reaction hole, 100 μl per hole, and place it at 37°C for 90 min; wash the hole with washing solution for 3 times, 3 min each time.

[0085] (5) Add enzyme label antibody: add the secondary antibody with appropriate concentration according to the instruction; 37°C, 90 min, add 100 μl to each hole, and wash as before.

[0086] (6) Add substrate solution: add 100 μl of substrate to each hole, and place it at 37°C for 15-30 min in the dark.

[0087] (7) Stop the reaction: add 50 μl of stop solution to each hole to stop the reaction, and measure the experimental results within 20 min.

[0088] (Four) Detection of the binding activity of the monoclonal antibody to digoxin

[0089] 4.1 Cell fusion and cloning screening data

[0090] The mouse numbers are A0, A1, A2 in turn, and four rounds of fusion are completed.

[0091] A0 mouse fusion screening selected 22 positive holes, subcloned, and finally completed 3 cell strains. After fusion screening, 43 positive clones with OD450 value > 2.2 were selected for detection of titer by doubling dilution, and then second and third subcloning screening was performed. Three cell strains were obtained, which were named A0-1 to A0-3.

[0092] A1 mouse fusion screening selected 18 positive holes, subcloned, and after fusion screening, 30 positive holes with OD450 value > 2.1 were selected for detection of titer by doubling dilution, and then second and third subcloning screening was performed. Finally, 3 cell strains were completed, which were named A1-1 and A1-3.

[0093] A2 mouse fusion screening selected 25 positive holes, subcloned, and then second and third subcloning screening was performed. Finally, 4 cell strains were completed, which were named A2-1 to A2-4.

[0094] After four cell fusions, a total of 10 cell strains were obtained.

[0095] 4.2 Ascites preparation and detection data

[0096] Each complete cell strain was injected into 3 F1 mice, and 10 ascites were prepared, and the titer data of all ascites were shown in Table 3.

[0097] Table 3

[0098]

[0099]

[0100] 4.3 Antibody purification condition exploration and detection data

[0101] The above ascites were purified by 3.3% n-octanoic acid-thiamine precipitation method, and 10 antibodies were obtained, and the titer detection data of all antibodies were shown in Table 4.

[0102] Table 4

[0103]

[0104] The above data showed that the monoclonal antibodies of the 10 cell strains had good specific binding ability to digoxin antigen. The digoxin colloidal gold product detected urine samples, so the standard digoxin was used for competition experiment, and the results showed that the A0-3, A1-1, A1-2, A1-3, A2-1, A2-2 and A2-3 antibodies had competition effect on small molecule digoxin. Therefore, the A0-3 and A1-1 antibodies were selected for testing of the digoxin colloidal gold product.

[0105] Example 2 Application of monoclonal antibody anti-DGX-mab1 in product

[0106] The digoxin antibodies A0-3 and A1-1 were verified by the immunocolloidal gold platform.

[0107] Specifically, the immunochromatography test paper comprises a sample pad, a conjugate pad and a detection pad, and the detection pad is provided with a detection line and a quality control line. The conjugate pad is coated with a colloidal gold-labeled selected monoclonal antibody, and the coating concentration is 1 mg / ml. The detection line is coated with a digoxin standard, and the quality control line is coated with a goat anti-mouse antibody. The experimental group uses the digoxin antibodies A0-3 and A1-1 obtained by the above experiment to detect the digoxin standard of Hangzhou Anxu Biological Technology Co., Ltd. The negative control is a urine sample. The above reagents are added, and then the POCT detection instrument ACG1000 (ID-A003) of Hangzhou Anxu Biological Technology Co., Ltd. is used to detect the results, and the experimental data results are shown in Table 5.

[0108] Table 5

[0109]

[0110] Note: G4-G8 means the grade of the color of the strip, the higher the value, the darker the color, + / - means slightly darker or lighter than the color of the grade. The addition of digoxin standard value is lower, indicating that the small molecule has a competitive effect, and the antibody can be combined with digoxin standard.

[0111] The results show that A0-3 and A1-1 antibodies have good gradient and can be used in digoxin products, with a cut-off value of 1000 ng / ml. The stability of A0-3 antibody was evaluated, and three batches of antibody samples were prepared, and the evaluation results are as follows:

[0112] Table 6

[0113]

[0114] According to the evaluation results of the product, the A0-3 antibody is named anti-DGX-mab1, which can be used for detection of digoxin antigen detection reagent kit.

[0115] Example 3

[0116] Sequence analysis of the heavy chain V region (VH) and light chain V region (VL) of the monoclonal antibody anti-DGX-mab1:

[0117] (I) Analysis method:

[0118] (1) Design primers for amplifying heavy chain V region (VH) and light chain V region (VL) genes, primers as follows:

[0119] Heavy chain variable region forward primer (VH-FOR, SEQ ID NO. 13);

[0120] GGGAATTCGAGGTGCAGCTGCAGGAGTCTGG;

[0121] Heavy chain variable region reverse primer (VH-BACK, SEQ ID NO. 14);

[0122] GGAAGGTGTGCACACCGCTGGAC;

[0123] Light chain variable region forward primer (VL-FOR, SEQ ID NO. 15);

[0124] CACGCTAGGGGCGGCCACTGTGGATCCGGATACAGTTGGTGCAGCATC;

[0125] Light chain variable region reverse primer (VL-BACK, SEQ ID NO. 16);

[0126] GGCTGAGCGGGGCTAGATGCCTCGAGGATATTGTGATAACCCAG

[0127] (2) Take anti-DGX-mab1 hybridoma cell strain (about 10 7 cells) in logarithmic growth phase, extract total RNA of cells according to the instruction of Trizol RNA extraction kit, synthesize cDNA first strand with total RNA as template, and take the above amplification product as template to amplify VH / VL gene of antibody by PCR.

[0128] (3) Recover heavy chain VH (about 360 bp) and light chain VL (about 300 bp) fragments of anti-DGX-mab1, and send to company for sequencing.

[0129] (4) Then analyze VH / VL gene sequence.

[0130] (II) Analysis result:

[0131] (1) Heavy chain variable region (anti-DGX-VH):

[0132] The amino acid sequence of heavy chain variable region is:

[0133] TDWHTGASVQMSCKAS GYTFTSYT IHWVKQMPGQGLEWIGS INPYSVYT SYNQNFMDKATLTADKSSSTAYMQLSSLTSEDSAVYYC VREYYGSGSDY WGQGTTLIVSSA (SEQ ID NO. 1, wherein the underlined parts represent VHCDR1-3 in turn).

[0134] The nucleotide sequence of heavy chain variable region is:

[0135] ACTGACTGGCACACTGGAGCCTCAGTGCAGATGTCCTGCAAGGCTTCT GGCTACACCTTTACTAGCTA CACG ATACACTGGGTAAAACAGATGCCTGGACAGGGTCTGGAATGGATTGGATCC ATTAATCCTTACAGTGTTTAT ACT AGTTACAATCAAAATTTCATGGACAAGGCCACATTGACTGCAGACAAATCCTCCAGCACAGCCTACATGCAACTGAGCAGCCTGACATCTGAGGACTCTGCGGTCTATTACTGT GTAAGAGAATACTACGGTAGTGGCTCAGACTACTGGGGCCAAGGCACCACTCTCATAGTCTCCTCAGCC (SEQ ID NO. 11, wherein the underlined parts represent VHCDR1-3 in sequence, respectively).

[0136] (2) Light chain variable region (anti-DGX-mab1 LVK):

[0137] The amino acid sequence of the light chain variable region is:

[0138] DIVITQTPLSLPVSLGDQASISCRSS QSLVHSNGNTY LHWYLQKPGQSPKLLIY KVS NR FSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQTTHVPPTFGGGTPSV (SEQ ID NO. 2, wherein the underlined parts represent VLCDR1-3 in sequence, respectively).

[0139] The nucleotide sequence of the light chain variable region is:

[0140] GATATTGTGATAACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGCAGATCTAGT CAGAGCCTTGTACACAGTAATGGAAACACCTAT TTACATTGGTACCTGCAGAAGCCAGGCCAGTCTCCAAAGCTCCTGATCTAC AAAGTTTCC AACCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAGCAGAGTGGAGGCTGAGGATCTGGGAGTTTATTTCTGC TCTCAAACTACACATGT TCCTCCGACG TTCGGTGGAGGCACTCCAAGCGTA (SEQ ID NO. 12, wherein the underlined parts represent VLCDR1-3 in sequence, respectively).

[0141] Finally, it should be noted that: the above embodiments are only used to illustrate the technical solutions of the present application, and not to limit them; although the present application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that: it can still modify the technical solutions recorded in the foregoing embodiments, or make equivalent replacement for part or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present application.

Claims

1. An anti-digoxin antibody or its antigen-binding fragment, characterized in that, It includes a heavy chain variable region and a light chain variable region; the heavy chain variable region contains VHCDR1 of amino acid residues 17-24 at the N-terminus of the amino acid sequence shown in SEQ ID NO.1, VHCDR2 of amino acid residues 42-49 at the N-terminus, and VHCDR3 of amino acid residues 88-98 at the N-terminus. The light chain variable region contains VLCDR1 of amino acid residues 27-37 at the N-terminus of the amino acid sequence shown in SEQ ID NO.2, VLCDR2 of amino acid residues 55-57, and VLCDR3 of amino acid residues 94-102.

2. The antibody or its antigen-binding fragment according to claim 1, characterized in that, The amino acid sequence of VHFR1 in the heavy chain variable region is shown in SEQ ID NO.3, and / or the amino acid sequence of VHFR2 is shown in SEQ ID NO.4, and / or the amino acid sequence of VHFR3 is shown in SEQ ID NO.5, and / or the amino acid sequence of VHFR4 is shown in SEQ ID NO.6; And / or, the amino acid sequence of VLFR1 in the variable region of the light chain is shown in SEQ ID NO.7, and / or the amino acid sequence of VLFR2 is shown in SEQ ID NO.8, and / or the amino acid sequence of VLFR3 is shown in SEQ ID NO.9, and / or the amino acid sequence of VLFR4 is shown in SEQ ID NO.

10.

3. The antibody or its antigen-binding fragment according to claim 2, characterized in that, The amino acid sequence of the heavy chain variable region is shown in SEQ IN NO.1; and / or, the amino acid sequence of the light chain variable region is shown in SEQ IN NO.

2.

4. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, characterized in that, The antibody or its antigen-binding fragment further comprises a sequence of part or all of the constant region of any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD.

5. The antibody or its antigen-binding fragment according to claim 4, characterized in that, The antigen-binding fragments include one or more of F(ab')2, Fab', Fab, Fv, scFv, and dsFv.

6. The antibody or its antigen-binding fragment according to claim 4, characterized in that, The antibody or its antigen-binding fragment, excluding the CDR region, is derived from any of the following species: mouse, rat, guinea pig, hamster, rabbit, ferret, cat, dog, goat, sheep, cow, pig, horse, monkey, and human.

7. The antibody or its antigen-binding fragment according to claim 4, characterized in that, The antibody also includes a heavy chain constant region and a light chain constant region.

8. The antibody or its antigen-binding fragment according to claim 7, characterized in that, The light chain of an antibody is a κ chain.

9. The antibody or its antigen-binding fragment according to claim 7, characterized in that, The antibody is an IgG antibody.

10. A biomaterial, characterized in that, The biomaterial is selected from any one of (i) to (iii): (i) Polynucleotides, including nucleotide sequences encoding the anti-digoxigenin antibody or its antigen-binding fragment as claimed in any one of claims 1 to 9; (ii) A vector carrying the aforementioned (i) polynucleotide; (iii) Cells carrying the polynucleotide in (i), or containing the carrier in (ii), or expressing the aforementioned anti-digoxin antibody or its antigen-binding fragment.

11. The biomaterial according to claim 10, characterized in that, The nucleotide sequence encoding the heavy chain variable region in the polynucleotide is shown in SEQ ID NO.

11.

12. The biomaterial according to claim 10, characterized in that, The nucleotide sequence encoding the light chain variable region in the polynucleotide is shown in SEQ ID NO.

12.

13. The anti-digoxin antibody or its antigen-binding fragment according to any one of claims 1 to 9, or the biomaterial according to any one of claims 10 to 12, in the detection of digoxin for non-diagnostic and therapeutic purposes, or in the preparation of products for the detection of digoxin.

14. A reagent or kit for detecting digoxin, characterized in that, It includes the anti-digoxin antibody or its antigen-binding fragment as described in any one of claims 1 to 9.

15. The reagent or kit according to claim 14, characterized in that, It also includes markers, which include one or more of enzymes, fluorescent molecular markers, fluorescent microspheres, colored microspheres, colloidal gold, biotin, or streptavidin.

16. The reagent or kit according to claim 14, characterized in that, The reagents or kits include those used for immunoassay.

17. The reagent or kit according to claim 16, characterized in that, The reagents or kits include immunochromatographic detection reagents or kits, ELISA detection reagents or kits, immunomagnetic particle detection reagents or kits, immunofluorescence detection reagents or kits, or immunoblotting detection reagents or kits.

18. The reagent or kit according to any one of claims 14 to 17, characterized in that, The kit includes immunochromatographic strips, which are competitive immunochromatographic strips.

19. The reagent or kit according to claim 18, characterized in that, The immunochromatographic test strip is coated with a labeled antibody that binds to digoxigenin. The amino acid sequence of the heavy chain variable region of the antibody that binds to digoxigenin is shown in SEQ ID NO.1, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.2.

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