CN117229410A

CN117229410A - Anti-digoxin antibody or antigen-binding fragment thereof and application thereof

Info

Publication number: CN117229410A
Application number: CN202311368608.5A
Authority: CN
Inventors: 上官科科; 魏文涛; 成含锋
Original assignee: Sure Biotech (hangzhou) Ltd
Current assignee: Sure Biotech (hangzhou) Ltd
Priority date: 2023-10-20
Filing date: 2023-10-20
Publication date: 2023-12-15

Abstract

The invention provides an anti-digoxin antibody or an antigen binding fragment thereof and application thereof, and relates to the technical field of antibodies. An anti-digoxin antibody or antigen-binding fragment thereof includes a heavy chain variable region and a light chain variable region; the heavy chain variable region comprises VHCDR1 of amino acid residues 17 to 24, VHCDR2 of amino acid residues 42 to 49 and VHCDR3 of amino acid residues 88 to 98 of the N-terminal of the amino acid sequence shown in SEQ ID NO.1, and the light chain variable region comprises VLCDR1 of amino acid residues 27 to 37, VLCDR2 of amino acid residues 55 to 57 and VLCDR3 of amino acid residues 94 to 102 of the N-terminal of the amino acid sequence shown in SEQ ID NO. 2. The antibody or the antigen binding fragment thereof can be detected by immunology, and has good specific binding capacity through verification.

Description

Anti-digoxin antibody or antigen-binding fragment thereof and application thereof

Technical Field

The invention relates to the technical field of antibodies, in particular to an anti-digoxin antibody or an antigen binding fragment thereof and application thereof.

Background

Digoxin is the most widely used drug in cardiac glycoside drugs, and clinical application has been 200 years old so far, and has high affinity and selectivity to heart. Can slightly inhibit Na at therapeutic dosage ⁺ /K ⁺ ATPase disrupting active transport of sodium on the membrane of the cardiac muscle cell, causing intracellular Na ⁺ The concentration increases and the K+ concentration decreases. Then through Na ⁺ And Ca ²⁺ Bidirectional exchange mechanism leading to intracellular Ca ²⁺ Increased concentration, enhanced myocardial contractility, produces positive inotropic effects, and is useful in treating congestive heart failure and certain arrhythmias.

Digoxin is a cardiac glycoside drug extracted from leaves of digitalis, also known as digoxigenin. Digoxin has a molecular formula of C ₄₁ H ₆₄ O ₁₄ The relative molecular mass 780.95 is formed by dehydration condensation of a 5-membered unsaturated lactone ring on a steroid mother nucleus C17 and C3 positions of 3D-digitoxin saccharides. Digoxin is structurally one hydroxyl group more than digitoxin, the number of hydroxyl groups is increased, so that the polarity of the digoxin is increased, the lipophilicity is weakened, and correspondingly, the oral absorption of the digoxin is lower than that of the digitoxin. Digoxin is white crystal or crystalline powder, bitter in taste, easy to dissolve in pyridine, insoluble in water and diethyl ether, easy to hydrolyze in an acidic or alkaline state, and stable in a sealed and light-proof state.

Digoxin is mainly used for treating various heart diseases accompanied by heart failure, and is particularly effective for congestive heart failure accompanied by edema, premature beat, atrial fibrillation, supraventricular tachycardia and the like. Along with the wide clinical application of digoxin, the therapeutic safety index and toxicity of the digoxin are always important problems. The treatment safety range of digoxin is small, and the effective blood concentration range is 0.8-2.0 mug.L ^-1 Once the dosage is too large, ca in the heart is caused ²⁺ Overload, significant cardiotoxic reactions can occur. In addition, the effect of drug interactions on digoxin blood concentration is also a non-negligible issue in the therapeutic setting where drug combinations are generalized and conventional.

Digoxin can be detected by adopting a detection method based on an immunological principle, such as an immunofluorescence method, an enzyme-linked immunosorbent assay, a colloidal gold immunochromatography technology and the like. The antibody is a main reagent in an immunodetection method, provides more high-quality antibodies combined with digoxin, can provide a stable and reliable raw material source for the detection method based on an immunology principle, reduces the dependence of domestic commodities on imported raw materials, and reduces the product cost. Meanwhile, the detection rate and sensitivity of digoxin rapid diagnosis commodity such as a gold-labeled test strip, an ELISA kit and the like are improved, the false positive rate is reduced, and the competitiveness of self-produced commodity is improved.

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

Disclosure of Invention

The present invention is directed to an anti-digoxin antibody or antigen-binding fragment thereof, a monoclonal antibody obtained by screening with digoxin as an antigen, and CDR region sequences thereof determined by cloning, identification and analysis of gene structure, and a biological material related to the antibody or antigen-binding fragment thereof and uses thereof.

In order to solve the technical problems, the invention adopts the following technical scheme:

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

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

In a second aspect, there is provided a biomaterial selected from any one of (i) to (iii):

a polynucleotide comprising a nucleotide sequence encoding an anti-digoxin antibody or antigen-binding fragment thereof as set forth above;

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

(iii) a cell carrying the polynucleotide of (i), or comprising the vector of (ii), or expressing the anti-digoxin antibody or antigen-binding fragment thereof.

In a third aspect, there is provided the use of an anti-digoxin antibody or antigen-binding fragment thereof as described above, or of a biological material as described above, for the detection of digoxin for non-diagnostic and therapeutic purposes, or for the preparation of a product for the detection of digoxin.

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

Compared with the prior art, the invention has the following beneficial effects:

according to the invention, digoxin is used for immunizing a Balb/c mouse, spleen cells of the mouse are fused with myeloma cells, hybridoma cells with high specificity are obtained through specific high-throughput screening, a large amount of mouse ascites is obtained through culture and re-immunization, and then a high-purity, high-sensitivity and high-specificity anti-digoxin monoclonal antibody anti-DGX-mab1 is obtained through multi-step separation and purification, so that a required raw material is provided for developing an immune test strip for detecting digoxin. The anti-digoxin monoclonal antibody anti-DGX-mab1 can be used for immunoblotting, immunofluorescence and other immunological detection, and the obtained antibody has good specific binding capacity through verification.

Detailed Description

The technical solutions of the present invention will be clearly and completely described in connection with the embodiments, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

Generally, the nomenclature used in connection with the cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization described herein and the techniques thereof are those well known and commonly employed in the art. Unless otherwise indicated, the methods and techniques of the present invention are generally well known in the art and are performed according to conventional methods as described in various general and more specific references cited and discussed throughout the present specification. Enzymatic reactions and purification techniques are performed according to manufacturer's instructions, as commonly accomplished in the art, or as described herein. Nomenclature used in connection with the analytical chemistry, synthetic organic chemistry, and medical and pharmaceutical chemistry described herein, and the laboratory procedures and techniques therefor, are those well known and commonly employed in the art.

An "antibody or antigen-binding fragment thereof" as used herein refers to a protein that binds a particular antigen, which broadly refers to all proteins and protein fragments that comprise complementarity determining regions (CDR regions). In addition, "antibody or antigen-binding fragment thereof" also includes naturally occurring antibodies as well as non-naturally occurring antibodies. An "antigen binding fragment" in this context is a substance comprising the CDRs of an antibody which lacks at least some of the amino acids present in the 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 invention have the effect of specifically recognizing and binding digoxin.

"variable region" or "variable domain" of an antibody or antigen binding fragment thereof refers to the domain of an antibody that recognizes and binds to an antigen at the amino terminus of the heavy or light chain of the antibody, the composition and arrangement of the amino acids of the segment determining the specificity of the antibody for recognizing the antigen. The heavy chain variable region may be referred to as "VH". The light chain variable region may be referred to as "VL". These domains are typically the most variable parts of an antibody and contain antigen binding sites. The variable regions of the heavy and light chains each consist of 3 complementarity-determining region (CDRs) (also known as hypervariable regions) connected by 4 Frameworks (FR). The framework and CDR ranges have been precisely defined, for example in Kabat (see sequence of immunologically important proteins ((Sequences of Proteins of Immunological Interest), E.Kabat et al) and Chothia), any CDR determination method well known in the art, including combinations of methods, can identify the CDRs of the variable domain the CDRs in each chain are held closely together by the FRs to form the variable region, typically the variable regions VL/VH of the heavy and light chains are obtained by ligating the CDRs numbered FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 in a combination arrangement with the FRs.

The term "polynucleotide" herein refers to a polymeric form of nucleotides of any length, including 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 polymers comprising purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural or derivatized nucleotide bases. The polynucleotide comprises a portion encoding the antibody or antigen binding fragment thereof described above, optionally encoding the sense or antisense strand. The polynucleotide may be naturally occurring, synthetic, recombinant, or any combination thereof.

The term "vector" refers to a nucleic acid vehicle into which a polynucleotide may be inserted. When a vector enables expression of a protein encoded by an inserted polynucleotide, the vector is referred to as an expression vector. The vector may be introduced into a host cell by transformation, transduction or transfection such that the genetic material elements carried thereby are expressed in the host cell.

Such vectors are well known to those skilled in the art and include, but are not limited to: a plasmid; phagemid; a cosmid; artificial chromosomes, such as Yeast Artificial Chromosome (YAC), bacterial Artificial Chromosome (BAC), or P1-derived artificial chromosome (PAC); phages such as lambda phage or M13 phage, animal viruses, etc. Animal viruses that may be used as vectors include, but are not limited to, retrovirus (including lentivirus), adenovirus, adeno-associated virus, herpes virus (e.g., herpes simplex virus), poxvirus, baculovirus, papilloma virus, and papilloma virus. In some embodiments, the vectors of the invention 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, etc.).

The expressions "cell", "cell line" and "cell culture" are used interchangeably herein and all such designations include progeny. The offspring may not necessarily be identical to the primary cells due to natural, accidental or deliberate mutation, e.g. are morphologically and/or differ from the primary cells in genomic DNA. "transformant" and "transformed cell" include primary test cells and cultures derived therefrom.

Throughout the specification and claims, unless explicitly stated otherwise, the term "comprise" or variations thereof such as "comprises" or "comprising" or the like will be understood to include the stated element or component without excluding other elements or components.

According to one aspect of the present invention there is provided an anti-digoxin antibody or antigen-binding fragment thereof, the antibody or antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region; the heavy chain variable region comprises VHCDR1 (GYTFTSYT) of amino acid residues 17 to 24, VHCDR2 (INPYSYT) of amino acid residues 42 to 49 and VHCDR3 (VREYYGSGSDY) of amino acid residues 88 to 98 of the N-terminal amino acid sequence shown in SEQ ID NO. 1.

The light chain variable region comprises VLCDR1 (QSLVHSNGNTY) of amino acid residues 27-37, VLCDR2 (KVS) of amino acid residues 55-57 and VLCDR3 (SQTTHVPPT) of amino acid residues 94-102 of the amino acid sequence N-terminal to the amino acid sequence shown in SEQ ID NO. 2.

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

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

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 shown IN SEQ ID NO. 1.

In alternative embodiments, the light chain variable region of the anti-digoxin antibody or antigen-binding fragment thereof has the structure:

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

IN an alternative embodiment, the amino acid sequence of the light chain variable region of the anti-digoxin antibody or antigen-binding fragment thereof is shown IN SEQ ID NO. 2.

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 shown IN SEQ ID NO.1 and the amino acid sequence of the light chain variable region is shown IN SEQ ID NO. 2.

In alternative embodiments, the antibody or antigen binding fragment thereof further comprises the sequence of a portion or all of the constant region of any one of IgG1, igG2, igG3, igG4, igA, igM, igE or IgD.

In alternative embodiments, the antigen binding fragment comprises F (ab') ₂ One or more of Fab', fab, fv, scFv, dsFv, bispecific antibodies, and antibody minimal recognition units.

In alternative embodiments, the antibody or antigen binding fragment thereof removes CDR regions, and the remaining sequence-derived species include, but are not limited to, one or more of mouse, rat, guinea pig, hamster, rabbit, ferret, cat, dog, goat, sheep, cow, pig, horse, monkey, and human.

In alternative embodiments, the antibody further comprises a heavy chain constant region and a light chain constant region.

In alternative embodiments, the heavy chain constant region and/or the light chain constant region are derived from mice.

In alternative embodiments, the light chain of the antibody is a kappa chain;

in an alternative embodiment, the antibody is an IgG antibody.

According to another aspect of the present invention, there is also provided a biomaterial selected from any one of (i) to (iii):

a polynucleotide comprising a nucleotide sequence encoding the aforementioned antibody or antigen binding fragment thereof; in an alternative embodiment, the polynucleotide encodes the heavy chain variable region and the nucleotide sequence is shown in SEQ ID NO. 11; in an alternative embodiment, the polynucleotide encodes the light chain variable region and the nucleotide sequence is set forth in SEQ ID NO. 12.

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

(iii) a cell carrying said polynucleotide (i), or comprising said vector (ii), or expressing an anti-digoxin antibody or antigen-binding fragment thereof as hereinbefore described.

According to another aspect of the present invention there is also provided the use of an anti-digoxin antibody or antigen-binding fragment thereof as described above, or of a biological material as described above, for the detection of digoxin for non-diagnostic and therapeutic purposes, or for the preparation of a product for the detection of digoxin.

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

In alternative embodiments, a label comprising one or more of an enzyme, a fluorescent molecular label, a fluorescent microsphere, a colored microsphere, colloidal gold, biotin, or streptavidin is also included.

In alternative 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.

In alternative embodiments, the reagent or kit is used for immunodetection. In alternative embodiments, the reagent or kit comprises an immunochromatographic detection reagent or kit, an ELISA detection reagent or kit, an immunomagnetic microparticle detection reagent or kit, an immunofluorescent detection reagent or kit, or an immunoblotting detection reagent or kit.

In alternative embodiments, the reagent or kit further contains reagents and/or consumables for detection, including, but not limited to, one or more of primers, probes, buffers, dyes, dilutions, washes, color-developing solutions, lysates, negative controls, positive controls, and blank controls; consumables include, but are not limited to, solid supports such as magnetic beads, enzyme-labeled wells, or NC membranes, and the like. The reagent composition of the kit can be selected by those skilled in the art according to the specific detection means, and the present invention is not limited thereto.

In an alternative embodiment, the kit comprises an immunochromatographic test strip, which is a competitive immunochromatographic test strip. The competitive immunochromatography test paper is coated with the antibody or the antigen binding fragment thereof in any one of the above embodiments, and is used for capturing digoxin in a sample to be detected.

In an alternative embodiment, the immunochromatographic test paper comprises a sample pad, a combination pad and a detection pad along the flowing direction of the sample, wherein the detection pad is provided with a detection line and a quality control line; the binding pad is coated with the anti-digoxin antibody or antigen-binding fragment thereof labeled by a label; the detection line is coated with digoxin antigen; coating the quality control line with an antibody specifically combined with the anti-digoxin antibody or antigen-binding fragment thereof; when the sample contains digoxin, the digoxin in the sample competitively binds with the digoxin antigen coated on the detection line and the anti-digoxin antibody or the antigen binding fragment thereof on the binding pad, and the higher the digoxin content in the sample, the less the anti-digoxin antibody or the antigen binding fragment thereof bound with the digoxin antigen on the detection line, and the less the detectable signal on the detection line. The anti-digoxin antibody or antigen binding fragment thereof marked by the marker flows through the quality control line to be captured by the antibody coated by the quality control line, and a measurable signal appears, which indicates that the immunochromatographic test paper can be used.

In an alternative embodiment, the binding pad is coated with the anti-digoxin antibody or antigen-binding fragment thereof that is labeled with colloidal gold. In an alternative embodiment, the immunochromatographic test paper is coated with a label-labeled digoxin-binding antibody, the heavy chain variable region amino acid sequence of the digoxin-binding antibody is shown as SEQ ID NO.1, and the light chain variable region amino acid sequence is shown as SEQ ID NO. 2.

The invention is further illustrated by the following specific examples, however, it should be understood that these examples are for the purpose of illustration only in greater detail and are not to be construed as limiting the invention in any way.

EXAMPLE 1 preparation of monoclonal antibody anti-DGX-mab1

Preparation of digoxin-PHT antigen

TABLE 1

Antigen sources	Antigen name	Lot number	Concentration of	Total amount of	Use of the same
						Self-producing antigens	digoxin-Dig-BSA	#D230220-1	5.6mg/ml	5.0mg	For immunization and screening

The chemically synthesized digoxin hapten Dig-BSA was used for stepwise immunization of experimental mice.

Preparation of monoclonal antibody anti-DGX-mab1

Balb/c healthy female mice of 6-8 weeks of age were selected for immunization according to a pre-specified immunization protocol. As immunogen, immunizing BALB/c mouse, extracting spleen lymphocyte of the immunized mouse, fusing lymphocyte with myeloma cell SP2/0 of the mouse by cell fusion technology, and obtaining hybridoma cell strain for stably secreting digoxin-resistant monoclonal antibody after two rounds of subcloning and screening, thereby obtaining digoxin-resistant monoclonal antibody.

2.1 step-by-step immunization of the experimental mice with the chemically synthesized digoxin hapten Dig-BSA, wherein the animal immunization experiment comprises the following specific steps:

(1) Balb/c mice with consistent weight and age average were randomly divided into 2 groups, with aluminum adjuvant (aluminum hydroxide adjuvant) and without aluminum adjuvant.

(2) Before the experiment starts, mice are respectively collected with preimmune serum (preimmune serum is collected on the fifth day, blood is taken through eyeballs, a proper amount of blood is taken, the normal state of the mice is ensured), and the collected serum is stored at-80 ℃.

(3) The preparation mode of the aluminum adjuvant (aluminum hydroxide adjuvant) group comprises the following steps: prior to immunization, each antigen was diluted to the corresponding dose (75 μg/mouse) in 75 μl PBS, respectively, and mixed with alum adjuvant (1 mg/mouse) at a volume antigen: adjuvant=3:1 (i.e., 25 μl adjuvant was added to 75 μl immunogen dilution); shaking the adjuvant before use, and slowly dripping the injected adjuvant (25 μl) into the immunogen solution; after the adjuvant and the immunogen dilution were thoroughly mixed, the two were thoroughly mixed for 30 minutes. Allowing the adjuvant to effectively adsorb the antigen; the subsequent experiments were performed according to the experimental procedure of immunized animals.

(4) Group without aluminum adjuvant: the antigens were diluted in 100 μl PBS at the corresponding doses (75 μg/mouse) in the above table, 100 μl of immunogen, followed by the following immunization animal experimental procedure.

(5) Subcutaneous injections at 2 week intervals: the experiment is designed into a 3-time immunization mode, but partial mouse supernatant is obtained by taking blood from eyeballs after 7 days of each immunization injection respectively and by a centrifugation method, serum titer is detected firstly, the heart takes blood to obtain the maximum blood volume after 7 days of the last immunization, and the supernatant is obtained by centrifugation and stored at-80 ℃.

2.2 detection of serum titers:

3 mice were immunized and the mice were numbered A0, A1, A2 in this order. Serum titers were measured after 3 immunizations were completed. The test data are shown in Table 2 below, and the test method is as follows:

and respectively using digoxin standard as antigen competition detection, and using digoxin hapten Dig-BSA as coating antigen to perform indirect ELISA and competition ELISA detection on serum titers of immunized mice.

The indirect ELISA method comprises the steps of adding 1 mu g/ml of coating antigen diluted by coating liquid into each hole of an ELISA plate, coating the plate for 3 times at 4 ℃ after coating, adding 200 mu l of sealing liquid (5% skimmed milk powder) into each hole, standing for 2 hours in a 37 ℃ incubator, taking out the plate, adding 50 mu l of diluted serum into each hole, reacting for 30 minutes in the 37 ℃ incubator, adding 50 mu l of goat anti-mouse IgG-HRP solution after washing, and reacting for 30 minutes in the 37 ℃ incubator. After washing, 100. Mu.l of substrate solution was added, the reaction was developed in a 37℃incubator in the absence of light for 10min, and finally, 2mol/L H SO4 and 50. Mu.L of the reaction were added to terminate the reaction, and the A450 value was read by an ELISA reader. The three post-exemption orbital blood titers of 3 mice were >62500.

The indirect competition ELISA procedure was largely identical to the indirect ELISA procedure except that 50. Mu.l of diluted 50ng/ml and 300ng/ml small molecule digoxin standard solution were added after blocking with blocking solution and washing the ELISA plate, and then 50. Mu.l of diluted serum antibody was added, with the remaining steps being identical. The competition detection of 50ng/ml and 300ng/ml small molecular digoxin can reach more than 50% only when 3 mice are 1:12500, and fusion can be arranged.

TABLE 2 serum potency assay data

2.3 fusion of immune splenocytes with myeloma cell line SP2/0 cells, screening the fused cells by HAT selection medium (HAT selection medium contains hypoxanthine, aminopterin and thymidine), and performing ELISA positive screening and subcloning on the fused cells; the screened positive monoclonal is taken as ascites, protein A/G antibody purification column is used for purifying antibody, and ELISA titer of the purified antibody is more than 1:128,000, and purity is more than 90%.

(III) ELISA detection of digoxin-recognizing binding Activity

The IgG antibody titer detection method comprises the following steps:

(1) And (3) coating a bottom plate: the antigen used was diluted to 3. Mu.g/ml with coating dilution, 100. Mu.l of the prepared coating was added to each well, and the mixture was placed in a refrigerator at 4℃for 24 hours.

(2) After 24h, taking out from the refrigerator, balancing at 37 ℃ for 30min, and then discarding the liquid in the holes; washing the wells with washing liquid for 3 times and 3min each time.

(3) Closing the enzyme-labeled reaction hole: 200 μl of 5% calf serum is added into each well, and the wells are sealed for 90min at 37 ℃, and after sealing, the wells are washed 3 times with washing liquid for 3min each time.

(4) Adding a sample to be detected: diluting the sample according to the required proportion, adding the diluted sample into enzyme-labeled reaction holes, placing 100 mu l of each hole at 37 ℃ for 90min; washing the wells with washing liquid for 3 times and 3min each time.

(5) Adding enzyme-labeled antibody: adding secondary antibody with proper concentration according to the specification; 100 μl of wash was applied to each well at 37deg.C for 90 min.

(6) Adding a substrate solution: the substrate is added in an amount of 100 mu l per hole, and the mixture is placed at 37 ℃ and protected from light for 15-30 min.

(7) Terminating the reaction: the reaction was stopped by adding 50. Mu.l of stop solution to each well, and the experimental results were measured within 20 min.

(IV) detecting the binding Activity of monoclonal antibody to recognize digoxin

4.1 cell fusion and cloning screening data

The serial numbers of the mice are A0, A1 and A2 in sequence, and four rounds of fusion are completed.

And (3) carrying out fusion screening on 43 positive clones with OD450 value more than 2.2, carrying out multiple dilution detection titer, and carrying out secondary subcloning screening. 3 cell lines were obtained, designated A0-1 to A0-3, respectively.

And (3) carrying out fusion screening on the A1 mice, picking 18 positive holes in total, carrying out subcloning, carrying out fusion screening on the positive holes with the OD450 value of more than 30 and the positive holes with the OD450 value of more than 2.1 in total, carrying out multiple ratio dilution detection titer, carrying out secondary and tertiary subcloning screening, and finally, completely obtaining 3 cell strains which are named as A1-1 and A1-3 respectively.

A2 mice were screened for fusion to pick 25 positive wells, subcloned, and screened for second and third subcloning, and finally 4 cell lines were completed, designated A2-1 to A2-4, respectively.

A total of 10 cell lines were obtained after four cell fusions.

4.2 ascites preparation and detection data

3F 1 mice were challenged with each complete cell line, 10 ascites were prepared in total, and all ascites test titers were as shown in Table 3.

TABLE 3 Table 3

4.3 antibody purification Condition fumbling and detection data

The above ascites was purified by 3.3% n-octanoic acid-thiamine precipitation to obtain 10 antibodies in total, and the potency detection data of all antibodies are shown in Table 4.

TABLE 4 Table 4

The data show that the monoclonal antibodies of 10 cell lines have good specific binding capacity to digoxin antigens, and the digoxin colloidal gold products are urine samples, so that competition experiments are carried out by using standard digoxin, and 6 antibodies such as A0-3, A1-1, A1-2, A1-3, A2-1, A2-2 and A2-3 have competition effects on small molecular digoxin, so that the antibodies A0-3 and A1-1 are selected to be used for testing the digoxin colloidal gold products.

EXAMPLE 2 application of monoclonal antibody anti-DGX-mab1 to product

Digoxin antibodies A0-3 and A1-1 were validated by an immune colloidal gold platform.

Specifically, the immunochromatography test paper comprises a sample pad, a combination pad and a detection pad, wherein the detection pad is provided with a detection line and a quality control line. The binding pad is coated with the screened monoclonal antibody marked by colloidal gold, the coating concentration is 1mg/ml, the detection line is coated with digoxin standard substance, and the quality control line is coated with goat anti-mouse antibody. The experimental group is to use the digoxin antibodies A0-3 and A1-1 obtained by the experiment to detect the digoxin standard of the Hangzhou An Xu biotechnology limited company. The negative control is a urine sample. The above reagents were applied, and then the results were detected using POCT detection instrument ACG1000 (ID-A003) from Hangzhou An Xu Biotechnology Co., ltd, and the results of the experimental data are shown in Table 5, and the experimental results are shown in Table 5.

TABLE 5

Note that: G4-G8 represent the level of the strip color shade of the test strip, with higher values representing darker colors and +/-representing a slightly darker or lighter color than that level. The low value of the added digoxin standard substance indicates that the small molecules have competitive action, and the antibody can be combined with the digoxin standard substance.

The results show that the A0-3 and A1-1 antibodies have better gradient, can be used in digoxin products, the cut-off value is 1000ng/ml, then the stability evaluation of the A0-3 antibodies is carried out, 3 batches of antibody samples are prepared, and the evaluation results are shown in the following table:

TABLE 6

The antibody A0-3 is named as anti-DGX-mab1 according to the evaluation result of the product, and can be used for detecting the digoxin antigen detection kit.

Example 3

Monoclonal antibody anti-DGX-mab1 heavy chain V region (VH) and light chain V region (VL) sequence analysis:

the analysis method comprises the following steps:

(1) Primers for amplifying heavy chain V region (VH) and light chain V region (VL) genes were designed as follows:

heavy chain variable region forward primer (VH-FOR, SEQ ID NO. 13);

GGGAATTCGAGGTGCAGCTGCAGGAGTCTGG；

heavy chain variable region reverse primer (VH-BACK, SEQ ID No. 14);

GGAAGGTGTGCACACCGCTGGAC；

light chain variable region forward primer (VL-FOR, SEQ ID NO. 15);

CACGCTAGGGGCGGCCACTGTGGATCCGGATACAGTTGGTGCAGCATC；

a light chain variable region reverse primer (VL-BACK, SEQ ID NO. 16);

GGCTGAGCGGGGCTAGATGCCTCGAGGATATTGTGATAACCCAG

(2) Hybridoma cell line (about 10) in logarithmic growth phase of anti-DGX-mab1 was obtained ⁷ And (3) extracting total RNA of the cells according to the instruction of the Trizol RNA extraction kit, performing reverse transcription to synthesize a first strand of cDNA by taking the total RNA as a template, and amplifying the VH/VL genes of the antibody by taking the amplified product as the template through PCR.

(3) The heavy chain VH (about 360 bp) and light chain VL (about 300 bp) fragments of anti-DGX-mab1 were recovered and sequenced by the company.

(4) The VH/VL gene sequences were then analyzed.

(II) analysis results:

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

the heavy chain variable region amino acid sequence is:

TDWHTGASVQMSCKASGYTFTSYTIHWVKQMPGQGLEWIGSINPYSVYTSYNQNFMDKATLTADKSSSTAYMQLSSLTSEDSAVYYCVREYYGSGSDYWGQGTTLIVSSA (SEQ ID NO.1, wherein the underlined parts represent VHCDRs 1 to 3, respectively, in sequence).

The heavy chain variable region nucleotide sequence is:

ACTGACTGGCACACTGGAGCCTCAGTGCAGATGTCCTGCAAGGCTTCTGGCTACACCTTTACTAGCTA CACGATACACTGGGTAAAACAGATGCCTGGACAGGGTCTGGAATGGATTGGATCCATTAATCCTTACAGTGTTTAT ACTAGTTACAATCAAAATTTCATGGACAAGGCCACATTGACTGCAGACAAATCCTCCAGCACAGCCTACATGCAACTGAGCAGCCTGACATCTGAGGACTCTGCGGTCTATTACTGTGTAAGAGAATACTACGGTAGTGGCTCAGACTACTGGGGCCAAGGCACCACTCTCATAGTCTCCTCAGCC (SEQ ID NO.11, wherein the underlined parts represent VHCDRs 1 to 3, respectively, in sequence).

(2) Light chain variable region (anti-DGX-mab 1 LVκ):

the amino acid sequence of the light chain variable region is as follows:

DIVITQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLHWYLQKPGQSPKLLIYKVSNR FSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQTTHVPPTFGGGTPSV (SEQ ID NO.2, wherein the underlined parts represent VLCDRs 1 to 3, respectively, in sequence).

The light chain variable region nucleotide sequence is:

GATATTGTGATAACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGCAGATCTAGTCAGAGCCTTGTACACAGTAATGGAAACACCTATTTACATTGGTACCTGCAGAAGCCAGGCCAGTCTCCAAAGCTCCTGATCTACAAAGTTTCCAACCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAGCAGAGTGGAGGCTGAGGATCTGGGAGTTTATTTCTGCTCTCAAACTACACATGT TCCTCCGACGTTCGGTGGAGGCACTCCAAGCGTA (SEQ ID NO. 12), wherein the underlined parts represent VLCDRs 1 to 3, respectively, in sequence.

Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and not for limiting the same; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the invention.

Claims

1. An anti-digoxin antibody or antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region; the heavy chain variable region comprises VHCDR1 of 17 th to 24 th amino acid residues, VHCDR2 of 42 th to 49 th amino acid residues and VHCDR3 of 88 th to 98 th amino acid residues of the N-terminal of the amino acid sequence shown in SEQ ID NO. 1;

2. The antibody or antigen-binding fragment thereof according to claim 1, wherein the amino acid sequence of VHFR1 in the heavy chain variable region is shown as SEQ ID No.3 and/or the amino acid sequence of VHFR2 is shown as SEQ ID No.4 and/or the amino acid sequence of VHFR3 is shown as SEQ ID No.5 and/or the amino acid sequence of VHFR4 is shown as SEQ ID No. 6;

and/or the amino acid sequence of VLFR1 in the light chain variable region is shown as SEQ ID NO.7, and/or the amino acid sequence of VLFR2 is shown as SEQ ID NO.8, and/or the amino acid sequence of VLFR3 is shown as SEQ ID NO.9, and/or the amino acid sequence of VLFR4 is shown as SEQ ID NO. 10.

3. The antibody or antigen-binding fragment thereof of claim 2, wherein the amino acid sequence of the heavy chain variable region is set forth IN SEQ IN No. 1; and/or the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 2.

4. The antibody or antigen-binding fragment thereof of any one of claims 1 to 3, further comprising the sequence of a portion or all of the constant region of any one of IgG1, igG2, igG3, igG4, igA, igM, igE, or IgD;

preferably, the antigen binding fragment comprises F (ab') ₂ One or more of Fab', fab, fv, scFv, dsFv, bispecific antibodies, and antibody minimal recognition units;

preferably, the antibody or antigen binding fragment thereof removes CDR regions, and the remaining sequence-derived species include one or more of mouse, rat, guinea pig, hamster, rabbit, ferret, cat, dog, goat, sheep, cow, pig, horse, monkey, and human;

preferably, the antibody further comprises a heavy chain constant region and a light chain constant region;

preferably, the light chain of the antibody is a kappa chain;

preferably, the antibody is an IgG antibody.

5. A biomaterial, characterized in that the biomaterial is selected from any one of (i) to (iii):

a polynucleotide comprising a nucleotide sequence encoding the anti-digoxin antibody or antigen-binding fragment thereof of any one of claims 1-4;

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

(iii) a cell carrying the polynucleotide of (i), or comprising the vector of (ii), or expressing the anti-digoxin antibody or antigen-binding fragment thereof;

preferably, the polynucleotide encodes the heavy chain variable region, and the nucleotide sequence is shown in SEQ ID NO. 11;

preferably, the polynucleotide encodes the light chain variable region and the nucleotide sequence is shown in SEQ ID NO. 12.

6. Use of an anti-digoxin antibody or antigen-binding fragment thereof as set forth in any one of claims 1-4, or a biological material as set forth in claim 5 for the detection of digoxin for non-diagnostic and therapeutic purposes, or for the preparation of a product for the detection of digoxin.

7. A reagent or kit for detecting digoxin, characterized in that it comprises an anti-digoxin antibody or antigen-binding fragment thereof as set forth in any one of claims 1-4.

8. The reagent or kit of claim 7, further comprising a label comprising one or more of an enzyme, a fluorescent molecular label, a fluorescent microsphere, a colored microsphere, colloidal gold, biotin, or streptavidin.

9. The reagent or kit of claim 8, wherein the reagent or kit comprises a reagent or kit for immunodetection;

preferably, the reagent or kit comprises an immunochromatographic detection reagent or kit, an ELISA detection reagent or kit, an immunomagnetic particle detection reagent or kit, an immunofluorescent detection reagent or kit, or an immunoblotting detection reagent or kit.

10. The reagent or kit according to claim 9, wherein the kit comprises an immunochromatographic test strip, which is a competitive immunochromatographic test strip;

preferably, the immunochromatography test paper is coated with a digoxin-binding antibody marked by a marker, the heavy chain variable region amino acid sequence of the digoxin-binding antibody is shown as SEQ ID NO.1, and the light chain variable region amino acid sequence is shown as SEQ ID NO. 2.