CN116396392A - Antibody specific to digoxigenin and related application thereof - Google Patents

Antibody specific to digoxigenin and related application thereof Download PDF

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CN116396392A
CN116396392A CN202310087302.6A CN202310087302A CN116396392A CN 116396392 A CN116396392 A CN 116396392A CN 202310087302 A CN202310087302 A CN 202310087302A CN 116396392 A CN116396392 A CN 116396392A
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antibody
dig
monoclonal antibody
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CN116396392B (en
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李燕飞
袁丽
欧阳海桥
龚春喜
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Zhuhai Heavy Chain Biotechnology Co ltd
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Abstract

The invention relates to a novel monoclonal antibody, a preparation method of the antibody and related application of the antibody in immunodetection. The monoclonal antibody provided by the invention can be highly sensitive and highly specific to Digoxigenin (DIG) or digoxigenin (digoxin), so that the monoclonal antibody can be used for detecting target molecules marked by DIG, such as nucleic acid molecules, and detecting medicinal molecules digoxin, and can be helpful for judging digoxin poisoning and guiding clinical medication, and has important significance for guaranteeing the therapeutic effectiveness and safety of digoxin.

Description

Antibody specific to digoxigenin and related application thereof
Technical Field
The invention relates to the field of immunodetection, in particular to a monoclonal antibody specific to Digoxigenin (DIG) or digoxin, a preparation method thereof and related application of the monoclonal antibody in immunodetection.
Background
Digoxigenin, digoxigenin (DIG), is a steroid hapten molecule which can be used to label nucleic acids and detection systems, and DIG-labeled nucleic acid probes are less hazardous, have a long shelf life, have higher sensitivity than radioactive probes, and can be detected more quickly.
Figure SMS_1
DIG can form a hapten-anti-hapten detection system with a DIG antibody, the detection sensitivity is high, and because the mammalian cells do not contain endogenous DIG and DIG binding proteins, the DIG-labeled nucleic acid probe cannot generate nonspecific binding, and the detection system has high specificity, is accepted by people and is widely applied.
Thus, highly specific and sensitive antibodies against Digoxigenin (DIG) are a continuing need in the art.
Disclosure of Invention
As previously mentioned, there is a need in the art for a high affinity, high specificity and high sensitivity antibody to Digoxigenin (DIG).
The inventor uses Digoxigenin (DIG) coupled with macromolecular carrier protein such as Bovine Serum Albumin (BSA) as immunogen to induce mice to generate immune response, takes spleen cells of the mice to fuse with myeloma cells, screens hybridoma cell strains with antibody titer more than 1000K, and obtains monoclonal antibodies with high affinity, high sensitivity and high specificity. Thus, the present invention has been achieved.
In a first aspect, the present invention provides a monoclonal antibody comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a polypeptide having a sequence consisting of, respectively Heavy chain complementarity determining region V of the amino acid sequences shown in SEQ ID NOS.1-3 H CDR1、V H CDR2 and V H CDR3, the light chain variable region including light chain complementarity determining region V having the amino acid sequences shown in SEQ ID NOS 4-6, respectively L CDR1、V L CDR2 and V L CDR3。
In a second aspect, the invention provides a nucleic acid molecule encoding a monoclonal antibody of the first aspect of the invention.
In a third aspect, the invention provides a vector comprising a nucleic acid molecule of the second aspect of the invention.
In a fourth aspect, the invention provides an expression cell comprising the nucleic acid molecule of the second aspect of the invention or the vector of the third aspect.
In a fifth aspect, the present invention provides a method for detecting Digoxigenin (DIG), comprising the step of using a monoclonal antibody according to the first aspect of the invention.
In a sixth aspect, the present invention provides a method of detecting digoxigenin (digoxin) for non-diagnostic or diagnostic purposes comprising the step of using a monoclonal antibody according to the first aspect of the invention.
In a seventh aspect, the present invention provides the use of a monoclonal antibody according to the first aspect for the preparation of a reagent for detecting digoxigenin or digoxin.
In an eighth aspect, the invention provides a kit for detecting Digoxigenin (DIG) or digoxigenin (digoxin), the kit comprising a monoclonal antibody according to the first aspect of the invention and instructions for use in directing how to detect using the monoclonal antibody.
The present invention provides a monoclonal antibody exhibiting high affinity, high sensitivity and high specificity for Digoxigenin (DIG) and digoxigenin (digoxin), which can be used to detect the presence of DIG or digoxin in a sample. Thus, in one aspect, the monoclonal antibodies of the invention are capable of specifically binding to DIG and thus can be used to directly detect DIG itself or via DIG-labeled nucleic acid probes, or to indirectly detect target nucleic acid molecules capable of specifically interacting with nucleic acid probes via DIG-labeled. On the other hand, the monoclonal antibody can be used for measuring the concentration of digoxin in serum, is beneficial to judging the poisoning of the digoxin and guiding clinical medication, and has important significance for ensuring the treatment effectiveness and safety of the digoxin. The monoclonal antibody and the related detection method thereof have high specificity, low hazard and long shelf life when used for detecting nucleic acid, have higher sensitivity than a radioactive probe, and can detect more quickly.
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In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.
FIG. 1 shows the competitive inhibition profile of the monoclonal antibody DIG2H1 of the invention.
FIG. 2 shows the competitive inhibition profile of the monoclonal antibody DIG2H1-C of the invention.
Detailed Description
Hereinafter, the present invention will be described in detail with reference to the accompanying drawings. It is to be understood that the following description is intended to illustrate the invention by way of example only, and is not intended to limit the scope of the invention as defined by the appended claims. And, it is understood by those skilled in the art that modifications may be made to the technical scheme of the present invention without departing from the spirit and gist of the present invention. The technical means used in the examples are conventional means well known to those skilled in the art unless otherwise indicated.
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 the subject matter described herein belongs. Before describing the present invention in detail, the following definitions are provided to better understand the present invention.
Where a range of values is provided, such as a range of concentrations, a range of percentages, or a range of ratios, it is to be understood that each intervening value, to the tenth of the unit of the lower limit, between the upper and lower limit of the range, and any other stated or intervening value in that stated range, is encompassed within the subject matter unless the context clearly dictates otherwise. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and such embodiments are also included in the subject matter, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the subject matter.
In the context of the present invention, many embodiments use the expression "comprising", "including" or "consisting essentially/mainly of … …". The expression "comprising," "including," or "consisting essentially of … …" is generally understood to mean an open-ended expression that includes not only the individual elements, components, assemblies, method steps, etc., specifically listed thereafter, but also other elements, components, assemblies, method steps. In addition, the expression "comprising," "including," or "consisting essentially of … …" is also to be understood in some instances as a closed-form expression, meaning that only the elements, components, assemblies, and method steps specifically listed thereafter are included, and no other elements, components, assemblies, and method steps are included. At this time, the expression is equivalent to the expression "consisting of … …".
For a better understanding of the present teachings and without limiting the scope of the present teachings, all numbers expressing quantities, percentages or proportions used in the specification and claims, and other numerical values, are to be understood as being modified in all instances by the term "about" unless otherwise indicated. Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained. At the very least, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.
As used herein, the term "antibody" refers to an immunoglobulin molecule that is typically composed of two pairs of polypeptide chains, each pair having one "light" (L) chain and one "heavy" (H) chain. Antibody light chains can be classified as kappa and lambda light chains. Heavy chains can be classified as μ, δ, γ, α or ε, and the isotypes of antibodies can be defined accordingly as IgM, igD, igG, igA and IgE, respectively. Within the light and heavy chains, the variable and constant regions are linked by a "J" region (hinge region) of about 12 or more amino acids, and the heavy chain also comprises a "D" region of about 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (V H ) And a heavy chain constant region (C) H ) Composition is prepared. The heavy chain constant region consists of 3 domains (C H1 、C H2 And C H3 ) Composition is prepared. Each light chain consists of a light chain variable region (V L ) And a light chain constant region (C L ) Composition is prepared. The light chain constant region consists of one domain C L Composition is prepared. The constant region of an antibody may mediate the binding of an immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (C1 q). V (V) H And V L The region can also be subdivided into regions of high variability, termed Complementarity Determining Regions (CDRs), interspersed with regions that are more conserved, termed Framework Regions (FR). For each heavy or light chain, its variable region comprises three CDRs, CDR1, CDR2 and CDR3, respectively. Thus, each V H And V L By the following sequence: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 consist of 3 CDRs and 4 FRs arranged from amino-terminus to carboxy-terminus. The variable region (V H And V L ) Respectively forming antigen binding sites.
The rules of allocation of amino acids to regions or domains give the relevant definitions in several documents: kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, bethesda m.d. (1987 and 1991)); chothia & Lesk j.mol.biol.1987;196:901-917; chothia et al, nature 1989;342:878-883; ehrenmann, francois, quentin Kaas, and Marie-Paule Lefranc. "IMGT/3Dstructure-DB and IMGT/DomainGapAlign: a database and a tool for immunoglobulins or antibodies, T cell acceptors, MHC, igSF and MhcSF." Nucleic acids research 2009;38 (suppl_1): D301-D307.
The exact boundaries of CDRs have been defined differently from system to system, and the Kabat system provides not only a clear residue numbering system applicable to any variable region of an antibody, but also precise residue boundaries defining 3 CDRs, referred to as Kabat CDRs; chothia found that some subfractions within the CDRs of the Kabat system, which are termed Chothia CDRs with boundaries overlapping the Kabat CDRs, have almost identical peptide backbone conformations, despite great diversity at the amino acid sequence level. The overlapping boundaries, in turn, are described by Padlan and MacCallum, and CDR boundary definitions may not strictly adhere to the above system, such as the AbM definition. In this context, the CDRs may be defined according to any of these systems, although the preferred embodiment uses the antibody numbering system of Chothia et al.
As used herein, the term "mab" or "monoclonal antibody" refers to an antibody or a fragment of an antibody from a population of highly homologous antibody molecules, i.e., a population of identical antibody molecules except for natural mutations that may occur spontaneously. The antibody molecule is an immunoglobulin, whether it be a natural immunoglobulin or an immunoglobulin obtained partially or wholly by synthetic means. The antibody molecules also include all polypeptides or proteins having antibody domains, antibody fragments having antibody domains are molecules such as Fab, scFv, fv, dAb, fd, and bifunctional antibodies. Monoclonal antibodies have a high specificity for a single epitope on an antigen. Polyclonal antibodies are relative to monoclonal antibodies, which typically comprise at least 2 or more different antibodies, which typically recognize different epitopes on an antigen. Monoclonal antibodies are generally obtainable by the hybridoma technique first reported by Kohler et al
Figure SMS_2
Milstein C.Continuous cultures of fused cells secreting antibody of predefined specificity[J]Natural, 1975;256 495) but may also be obtained using recombinant DNA techniques (see, e.g., U.S. patent 4,816,567). As used hereinThe terms "monoclonal antibody" and "mab" have the same meaning and are used interchangeably; the terms "polyclonal antibody" and "polyclonal antibody" have the same meaning and are used interchangeably; the terms "polypeptide" and "protein" have the same meaning and are used interchangeably. And in the present invention, amino acids are generally indicated by single-letter and three-letter abbreviations well known in the art. For example, alanine can be represented by A or Ala.
As used herein, the term "recombinant antibody" refers to an antibody that is expressed by cloning an antibody gene into an expression vector by molecular biological techniques and then transfecting the expression vector into a suitable host cell line. The recombinant antibody-encoding gene may or may not be identical to the naturally derived antibody-encoding gene. For example, the whole coding gene of an antibody obtained by immunizing an animal may be cloned into an expression vector to express, thereby obtaining an antibody identical to the antibody obtained by immunizing an animal, or a gene coding for a variable region (including a heavy chain variable region and a light chain variable region) of an antibody obtained by immunizing an animal may be cloned into an expression vector together with a gene coding for a constant region of an antibody derived from another species (e.g., human), thereby obtaining an antibody comprising a heavy chain and a light chain variable region sequence from one species and a constant region sequence from another species, e.g., an antibody having a mouse heavy chain and a light chain variable region linked to a human constant region. Such antibodies are commonly referred to in the art as "chimeric antibodies".
As described above, the present invention aims to provide a monoclonal antibody against Digoxigenin (DIG) or digoxigenin (digoxin) with high affinity, high sensitivity and high specificity.
DIG, which is a small molecule hapten, does not produce good immunoreactivity by itself, and therefore requires conjugation with a macromolecular carrier protein to induce an immune response in an animal. DIG can be coupled to any carrier protein including, but not limited to, hemocyanin KLH, bovine serum albumin BSA, ovalbumin OVA, or human serum albumin HSA. The corresponding anti-DIG antibodies can be produced by immunizing a suitable host (e.g., vertebrates, including human, mouse, rat, rabbit, sheep, goat, pig, cow, horse, reptile, fish, amphibian, and eggs of birds, reptiles, and fish). The animals may be immunized by any method known in the art, and methods for immunizing non-human animals such as mice, rats, rabbits, and the like are known in the art.
Thus, in a first aspect, the present invention provides a monoclonal antibody comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a heavy chain complementarity determining region V having the amino acid sequences set forth in SEQ ID NOS 1-3, respectively H CDR1、V H CDR2 and V H CDR3, the light chain variable region including light chain complementarity determining region V having the amino acid sequences shown in SEQ ID NOS 4-6, respectively L CDR1、V L CDR2 and V L CDR3。
In some embodiments, the antibody comprises at least six CDRs.
In some preferred embodiments, the antibody comprises:
1) A heavy chain variable region comprising or consisting of:
SEQ ID NO:7, or an amino acid sequence as set forth in SEQ ID NO:7, preferably a sequence having 80% or more, 85% or more, 90% or more, or 95% or more identity to each other, preferably 95% or more identity to each other;
2) A light chain variable region comprising or consisting of:
SEQ ID NO:8, or an amino acid sequence as set forth in SEQ ID NO:8, preferably a sequence having 80% or more, 85% or more, 90% or more, or 95% or more identity to each other, preferably 95% or more identity to each other.
In a specific embodiment, the antibody may be any one selected from the group consisting of a bifunctional antibody, fab, F (ab ') 2, fab ', fd, fv, (dsFv) 2, dsFv-dsFv ', disulfide stabilized bifunctional antibody, scFv dimer, multispecific antibody, nanobody, domain antibody, or bivalent domain antibody.
In one placeIn some embodiments, the antibody may be an intact antibody comprising a variable region and a constant region. For the antibodies of the invention, any Framework Region (FR) as well as any constant region may be used. The amino acid sequence of the FR or constant region used in the antibody of the invention may be the amino acid sequence of the original FR or constant region from which it is derived, or may be a different amino acid sequence obtained by substituting, deleting, adding and/or inserting 1 or more amino acids into the amino acid sequence of the original FR or constant region. The structure used to support the CDRs or sets of CDRs of the invention, which are located at naturally occurring V encoded by rearranged immunoglobulin genes, typically belongs to the antibody heavy or light chain sequences or major parts thereof H And V L The CDRs or sets of CDRs of the antibody variable domains are located at corresponding positions.
In some embodiments, the antibody further comprises a constant region sequence, such as one selected from any of IgG, lgA, igM, igE, igD, which can be selected by one of skill in the art based on need, and is not particularly limited herein. In addition, the constant region sequence may be derived from a species such as, but not limited to, rat, mouse, rabbit, goat, sheep, horse, dog, cow, pig, chicken, duck, goose, or human.
Although the monoclonal antibodies of the invention are obtained by immunizing mice with conjugates of DIG and carrier protein, it is noted that DIG is a steroid aglycone of digoxin, both of which have partially identical cyclic structures, the antibodies of the invention have high specificity not only for DIG but also for digoxin.
The monoclonal antibodies of the invention exhibit high affinity, high specificity and high sensitivity to DIG and digoxin. The term IC is used herein 50 Represents the concentration of competitor (i.e., free DIG) competing with coated DIG for binding to the antibody of the invention at an inhibition of 50%, which concentration characterizes the binding affinity of the antibody of the invention to DIG. The inhibited IC can be measured using any method known in the art, such as competitive ELISA methods 50 . In one embodiment, the monoclonal antibody DIG2H1 of the invention is a DIG-to-DIG IC 50 Only 0.34ppb, IC for digoxin 50 0.351ppb.
In a second aspect, the invention provides a nucleic acid molecule encoding a monoclonal antibody of the first aspect of the invention.
It will be apparent to those skilled in the art that the determination of the nucleic acid coding sequence is well within the ability of a protein, such as the amino acid sequence of a monoclonal antibody of the invention. In addition, in order to obtain a monoclonal recombinant antibody by recombinant means, the nucleic acid molecule may be cloned into an expression vector and the expression vector further introduced into a host cell to express the antibody protein using the host cell.
In a third aspect, the invention provides a vector comprising a nucleic acid molecule of the second aspect of the invention.
In a specific embodiment, the vector may be a plasmid vector, such as pEE12, pCAGGS, pTOPO, pcDNA, pTT, pTT3, pEFBOS, pBV, pJV and pBJ.
In a more specific embodiment, the vector may be pcdna3.1.
In a fourth aspect, the invention provides an expression cell comprising the nucleic acid molecule of the second aspect or the vector of the third aspect.
As described above, the present inventors have conjugated DIG with a macromolecular carrier protein such as Bovine Serum Albumin (BSA) and induced an immune response in mice as an immunogen, then obtained hybridoma cell lines capable of secreting anti-DIG monoclonal antibodies by using hybridoma technology, and screened to obtain monoclonal anti-DIG antibodies with high affinity, high sensitivity and high specificity. After screening a monoclonal cell line secreting the antibody of interest, heavy and light chain variable region cdnas may be recovered from the cell line by reverse transcriptase-PCR, and appropriate immunoglobulin constant regions (e.g., human constant regions) are selected, and then the heavy and light chain variable region cdnas and the constant region cdnas are transferred into host cells such as COS or CHO cells, thereby obtaining the expression cells expressing the antibody of interest of the present invention. Other antibodies or chimeric molecules may be produced that retain the specificity of the original antibody using monoclonal and other antibody and recombinant DNA techniques, which may include introducing DNA encoding the immunoglobulin variable or Complementarity Determining Regions (CDRs) of the antibody into the constant regions or constant region plus framework regions of different immunoglobulins.
In one embodiment, the expression cell may be a protozoan cell, a plant cell, an animal cell, or a fungal cell. The animal cells may be mammalian cells, such as chinese hamster ovary Cells (CHO), hamster kidney cells, monkey kidney cells, mouse thymoma cells, human embryonic kidney cells, COS cells, HEK293 cells.
In a more specific embodiment, the expression cell may be, for example, a monkey kidney cell transformed with SV40 (COS-7, ATCC CRL 1651), a human embryonic kidney cell (HEK 293 or HEK293 cell subcloned for growth in suspension culture, graham et al, 1977,J.Gen Virol.36:59), baby hamster kidney cell (BHK, ATCC CCL 10), chinese hamster ovary cell/-DHFR 1 (CHO, urlaub et al, 1980, proc.Natl. Acad.sci.usa77:4216; such as DG 44), mouse thymoma cells (NSO), mouse testis support cells (TM 4, mather,1980, biol. Reprod. 23:243-251), monkey kidney cells (CV-1, ATCC CCL 70), african green monkey kidney cells (VERO-76, ATCC CRL-1587), human cervical cancer cells (HELA, ATCC CCL 2), canine kidney cells (MDCK, ATCC CCL 34), buffalo rat liver cells (BRL 3A, ATCC CRL 1442), human lung cells (W138, ATCC CCL 75), human liver cells (HepG 2, HB 8065), mouse mammary tumors (MMT 060562, ATCC CCL 51), TR1 cells (Mather et al, 1982,Annals N.Y.Acad.Sci.383:44-68), MRC5 cells, FS4 cells, etc., but are not limited thereto.
In a fifth aspect, the present invention provides a method of detecting Digoxigenin (DIG), comprising the step of using a monoclonal antibody of the first aspect.
In a specific embodiment, the monoclonal antibodies of the invention are labeled directly or indirectly with a detectable substance. Herein, a "detectable substance" may be a displayable signaling indicator. Those skilled in the art will appreciate that detection of a target molecule can be accomplished by detecting a signal indicative of a signal indicator. Such detection may be performed by, for example, any one or several of immunochromatography such as Western Blot, enzyme-linked immunosorbent assay (ELISA), chemiluminescence, electrochemiluminescence, radioimmunoassay (RIA) or in situ hybridization.
In a preferred embodiment, the detectable substance comprises colloidal gold, an enzyme, a fluorescent substance, a luminescent substance, or a radioactive substance.
In a more preferred embodiment, the enzyme may be an enzyme that catalyzes the development of a substrate, such as horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase, but is not limited thereto; the fluorescent substance may include umbelliferone, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin, but is not limited thereto; the luminescent material may be a chemiluminescent reagent such as aminobenzene dihydrazide, but is not limited thereto; the radioactive material may include 3 H、 14 C、 35 S、 90 Y、 99 Tc、 111 In、 125 I、 131 I、 177 Lu、 166 Ho or 153 Sm, but is not limited thereto.
The method of the invention for detecting Digoxigenin (DIG) refers not only to the detection of DIG itself, but also to the detection of DIG-labeled target molecules such as nucleic acid amplification products, or to the detection of nucleic acid molecules capable of specific interaction with DIG-labeled nucleic acid probes in combination with such nucleic acid probes.
DIG is a highly sensitive non-radioactive nucleic acid label that can be used to label target molecules, such as nucleic acid amplification products, nucleic acid probes, and the like. The DIG-labeled nucleic acid probe has the advantages of less hazard, long shelf life, higher sensitivity than the radioactive probe and faster detection. DIG detection sensitivity is close to that of isotope without radioactivity hazard, compared with fluorescence, special detection equipment is not needed, compared with biotin, the DIG detection sensitivity is free from endogenous interference of a sample, and the DIG detection sensitivity is suitable for non-radioactive labeling detection of nucleic acid. Furthermore, since the endogenous DIG and DIG-binding proteins are not contained in mammalian cells, non-specific binding of DIG-labeled nucleic acid probes does not occur. Therefore, the detection system has high specificity and sensitivity and has been widely used.
In one exemplary embodiment, DIG is linked to deoxyuridine triphosphate (dUTP) to form DIG-11-dUTP, thereby labeling DNA or RNA. Labelling may be carried out by methods well known in the art, for example by incorporating DIG-11-dUTP into DNA amplification products or probes by random primer methods, PCR methods and nick translation methods. For another example, DIG-11-dUTP is incorporated into an RNA probe by transcription using RNA polymerase. For another example, oligonucleotide probes may be labeled by methods such as end transferase catalysis, 3' end labeling, 5' end labeling, and addition of DIG-11-dUTP tail at the 3' end.
DIG antibodies labeled with a detectable substance can thus detect DNA or RNA labeled with DIG, and can also detect additional DNA or RNA that specifically binds to the DNA or RNA labeled with DIG.
In a specific embodiment, DIG-labeled nucleic acid amplification products are detected using the methods of the invention. More specifically, the DNA template is amplified by a PCR method using DIG-labeled amplification primers, thereby obtaining a DIG-labeled amplification product. The amplification product thus obtained, because of carrying DIG, can specifically interact with the antibody of the present invention, and in the case where the antibody of the present invention is labeled with a detectable substance, the amplification product can be detected accordingly.
For example, when using a colloidal gold immunochromatographic assay for DNA detection, the monoclonal antibody of the present invention can be labeled with colloidal gold, and a double-sandwich chromatography system can be constructed using streptavidin as a coating, to detect the amplification product of the double labeling of DIG and biotin. The sample amplification primers are DIG-labeled on one hand and biotin-labeled on the other hand, so that the amplified products are DIG and biotin double-labeled. And assembling according to the preparation mode of the immune test strip to obtain the colloidal gold test paper. When the detection is carried out, the amount of DNA can be obtained by comparing the readings of the colorimetric card.
In another embodiment, the method of the invention and DIG-labeled nucleic acid probes specific for a target nucleic acid molecule are used to detect the target nucleic acid molecule. For example, as described above, nucleic acid probes are labeled with DIG and antibodies of the invention are labeled with a detectable substance by techniques in the art. In the detection process, the DIG-labeled nucleic acid probe is contacted with a sample which possibly contains target nucleic acid molecules, hybridization reaction is carried out on the DIG-labeled nucleic acid probe and the sample, then the antibody is added to enable the DIG-labeled nucleic acid probe to specifically interact with DIG on the nucleic acid probe in the sample, and the detectable substance on the antibody is utilized for final detection.
In a sixth aspect, the present invention provides a method for detecting digoxigenin (digoxin) for non-diagnostic or diagnostic purposes, comprising the step of using the monoclonal antibody of the first aspect.
Digoxin (Digoxin), also known as Digoxin digoxigenin, DIG is a steroid aglycone of Digoxin, both of which have partially identical cyclic structures (shown in block parts), and the Digoxin structure is shown in the following formula:
Figure SMS_3
digoxin is a beta-suppressant extracted from digitalis and is widely used as a medicament for treating acute and chronic cardiac insufficiency such as hypertension, valvular heart disease, congenital heart disease and the like. Because of the narrow therapeutic index of digoxin, the symptoms of excessive medication and insufficient medication are similar, and the individual difference is large, the occurrence rate of poisoning is high, and the digoxin is one of the most difficult to master medicines clinically. The most effective method for removing digoxin toxicity at present is to monitor serum/plasma digoxin concentration by adopting an immunoassay method of anti-digoxin antibody, provide information on bioavailability and pharmacodynamics difference among different individuals, judge digoxin toxicity and guide clinical medication, which is a model of current therapeutic drug monitoring.
Since the monoclonal antibody has a partially identical ring structure with DIG, the inventor discovers that the monoclonal antibody can specifically bind to digoxin, thereby realizing the immunodetection of the digoxin.
Monoclonal antibodies of the inventionAlso shows high affinity, high specificity and high sensitivity to digoxin. For example, the monoclonal antibody DIG2H1 of the invention is directed against digoxin IC 50 Only 0.351ppb.
In a specific embodiment, the antibody is labeled directly or indirectly with a detectable substance. Those skilled in the art will appreciate that after the specific binding of the antibody of the present invention to digoxin, the quantitative detection of digoxin can be achieved by detecting the detectable substance of the labeled antibody, so that the concentration of digoxin in serum/plasma can be monitored, which is of great importance for ensuring the effectiveness and safety of digoxin treatment.
In a preferred embodiment, the detectable substance comprises colloidal gold, an enzyme, a fluorescent substance, a luminescent substance, or a radioactive substance.
In a more preferred embodiment, the enzyme may be an enzyme that catalyzes the development of a substrate, such as horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase, but is not limited thereto; the fluorescent substance may include umbelliferone, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin, but is not limited thereto; the luminescent material may be a chemiluminescent reagent such as aminobenzene dihydrazide, but is not limited thereto; the radioactive material may include 3 H、 14 C、 35 S、 90 Y、 99 Tc、 111 In、 125 I、 131 I、 177 Lu、 166 Ho or 153 Sm, but is not limited thereto.
In a seventh aspect, the present invention provides the use of a monoclonal antibody according to the first aspect for the preparation of a reagent for detecting Digoxigenin (DIG) -labeled target molecules or digoxigenin (digoxin).
In the context of the present invention, the detection may be, but is not limited to, western Blot (Western Blot), enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA) or immunohistochemistry. The detection method can be selected by those skilled in the art as needed, and the present invention is not particularly limited.
As described above, the monoclonal antibody of the present invention can bind DIG-labeled nucleic acid with high specificity and high sensitivity, thereby realizing detection of nucleic acid molecules. In one aspect, since the mammalian cells do not contain endogenous DIG and DIG-binding proteins, non-specific binding of DIG-tagged nucleic acid molecules does not occur. On the other hand, DIG-labeled nucleic acid molecules are less hazardous, have a long shelf life, have higher sensitivity than radioactive probes, and can be detected more quickly.
Thus, in a preferred embodiment, the target molecule may be a nucleic acid molecule.
As described above, the specific binding of the monoclonal antibody of the present invention to digoxin enables monitoring of the concentration of digoxin in serum/plasma, which is of great importance for ensuring the effectiveness and safety of digoxin as a β inhibitor drug for cardiovascular disease treatment.
In an eighth aspect, the present invention provides a kit for detecting a Digoxigenin (DIG) -labeled target molecule or digoxigenin (digoxin), the kit comprising a monoclonal antibody of the first aspect and instructions for directing how the monoclonal antibody is to be detected.
In a specific embodiment, the kit can be used to detect DIG-labeled nucleic acid molecules.
Examples
In the following examples, the methods of preparation of the antibodies of the invention and characterization of relevant properties are shown. Unless otherwise indicated, all test procedures used herein were conventional, and all test materials used in the examples described below were purchased from a conventional reagent store, unless otherwise indicated. 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 invention belongs.
It should be noted that the terminology used in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The foregoing summary of the invention and the following detailed description are only for the purpose of illustrating the invention and are not intended to limit the invention in any way. The scope of the invention is determined by the appended claims without departing from the spirit and scope of the invention.
Example 1: preparation of monoclonal antibodies
Coupling and immunization of antigen: DIG was conjugated to Bovine Serum Albumin (BSA) as an immunogen (DIG-BSA) and mice were immunized. The mice were selected from female BALB/c mice of 6-8 weeks of age. The total immunization was 4 times, each time was 2 weeks apart, and the immunization dose was 100. Mu.g/mouse. The first immunization mixes the BSA-coupled DIG with equal volumes of Freund's complete adjuvant (Sigma-Aldrich Co.), the back subcutaneous multiple injections, and the last three immunizations mix the BSA-coupled DIG with equal volumes of Freund's incomplete adjuvant (Sigma-Aldrich Co.), and the intraperitoneal injections. The tail of the fourth immunized mouse is cut off for blood collection 7 days, serum is separated, the antibody titer level of the immune mouse antiserum is detected by adopting an indirect ELISA method to observe the immune response effect, the mouse with the serum antibody titer higher than 1:10000 is selected for cell fusion experiment, and 3 days before the cell fusion experiment, the immune is enhanced by intraperitoneal injection of DIG-BSA without adjuvant (100 mug/mouse).
Establishment of hybridoma cells: on the day of fusion, the spleen of immunized mice was removed under sterile conditions and the organs were made into single cell suspensions. Mouse myeloma cells (SP 2/0) were taken with the above immunized BALB/c mouse spleen cells at a ratio of 1:5, and washing the cells twice before fusing with PEG. The cells were washed with pre-warmed PEG1500, gently shaken, and pre-warmed serum-free RPMI-1640 medium, and resuspended in HAT selective medium. The cell suspension was plated at 200 μl per well into 96-well plates and at 37 ℃ with 5% co 2 The cells are cultured under conditions. After 4-7d of culture, HT medium is used for culture, and when fused cells grow to 1/10-1/5 of the bottom area of the 96-well plate, the supernatant is taken for antibody detection.
Screening of positive hybridoma cells: diluting OVA-coupled DIG (DIG-OVA) with coating buffer (0.05 mol/L pH9.6 PBS) to a final concentration of 1 μg/ml, adding 100 μl/well into 96-well plate, coating overnight at 4deg.C, removing coating solution, and adding PWashing BST for 3 times, and beating to dry; blocking with PBST containing 3% skimmed milk, incubating at 37deg.C for 2h at 150 μl/well, washing with PBST 3 times, and drying; adding the supernatant of the fusion cells, the immune mouse positive serum diluted by 1:1000 and the mouse negative serum diluted by 1:1000 into corresponding holes, incubating for 1h at 37 ℃, washing by PBST for 3 times, and drying by beating; horseradish peroxidase (HRP) -labeled goat anti-mouse IgG (from Sigma Co.) at 1:4000 dilution was added, 100. Mu.L/well, incubated for 1h at 37℃and PBST washed 3 times, and patted dry; adding TMB substrate, 100 mu L/hole, and developing at room temperature in dark place for 10min; the reaction was terminated by adding 50. Mu.L of 2mol/L sulfuric acid per well. Detecting OD of all holes of the ELISA plate under 450nm wavelength of the ELISA plate 450nm Values. OD when negative serum 450nm At a value of 0.1 or less, the OD of the well (DIG-BSA) was measured 450nm Values greater than the OD of the negative wells 450nm The value is more than 2.1 times positive as a judgment standard. Positive hybridoma cells were subjected to the next cloning.
Cloning of positive cell lines: sampling and counting positive cell holes secreting antibody, diluting to 100 cells/10 mL culture medium, adding diluted cell suspension into 96-well cell culture plate, placing 100 μl of each well at 37deg.C, and 5% CO 2 Culturing in a cell culture incubator. After 6-7 days, the formation of cloned cells was observed under a microscope, a single gram of Long Sheng long holes were marked, and the cell supernatant was taken out, and ELISA detection (the same as the fusion detection described above) was performed to select positive monoclonal cells. Limiting dilution is carried out on positive hole cells, ELISA values are measured 5-6 days after each limiting dilution, and ELISA detection OD is selected 450nm The monoclonal well with higher positive value is subjected to limiting dilution until the result of ELISA measurement of the whole 96-well plate is positive. And selecting monoclonal fixed strains with high positive values. Finally, a cell strain which stably secretes the anti-DIG is obtained and named as a hybridoma cell strain DIG2H1.
Preparation and purification of cell-on-list antibody: the cell line was cultured in a 10cm dish with 15% serum in RPMI-1640 medium and expanded to about 4X 10 7 At each dish, centrifugation was carried out at 800rpm for 5min, the supernatant was discarded and the cells were transferred to a 2L roller bottle, and serum-free medium was added to give a cell density of about 3X 10 5 And (3) culturing in a rotary bottle with the concentration of each mL. After the culture is continued for 1 to 2 weeks, when the cells dieWhen the ratio reaches 80% -90% (at this time, the cell density is approximately 1-2X 10) 6 And (3) collecting a cell suspension, centrifuging at 6000rpm for 20min, collecting a supernatant, and purifying the supernatant by using a Protein A immunochromatography method. The monoclonal antibody prepared by the hybridoma cell DIG2H1 is named as DIG2H1; the concentration of the monoclonal DIG2H1 antibody was 2.014mg/mL as identified by a micro-spectrophotometer. Measuring the concentration of the purified monoclonal antibody, sub-packaging (100 mu L/tube, concentration is 1 mg/mL), and storing at 4-8 ℃. The antibody was identified by SDS-PAGE electrophoresis, and a heavy chain band of 51KD and a light chain band of 26KD were present.
And (3) purity detection: the SEC-HPLC is used for analyzing the monoclonal antibody, under the condition that all components in the sample to be detected are ensured to generate peaks, the purity percentage of the main peak is calculated by using a peak area normalization method, and the purity reaches more than 98 percent.
Example 2: indirect ELISA method for detecting antibody titer
In this example, the titers of the antibodies obtained in example 1 were detected by enzyme-linked immunosorbent assay (ELISA) for screening.
Diluting OVA-coupled DIG with 0.05mol/L carbonate buffer solution with pH of 9.6 to a concentration of 1 mug/mL, adding 96-well ELISA plates into 100 mug/well, coating overnight at 4 ℃, washing 3 times with PBST on an automatic plate washer, and beating to dry; blocking with 3% nonfat milk powder in PBST, 100. Mu.L/well, incubation at 37℃for 2h, PBST washing 3 times, and patting dry. anti-DIG monoclonal antibodies were subjected to gradient dilution with PBS buffer, pH7.4,0.02M, to obtain monoclonal antibody samples of different concentrations. Adding the diluted monoclonal antibody sample into the ELISA plate, incubating for 1h at 37 ℃ with 100 mu L/hole, washing for 3 times, and drying by beating; horseradish peroxidase (HRP) -labeled goat anti-mouse IgG (purchased from Sigma) at 1:4000 dilution, 100. Mu.L/well, incubated for 1h at 37℃and washed 3 times with PBST, and patted dry; adding TMB substrate, 100 mu L/hole, and developing at room temperature in dark place for 10min; the reaction was terminated by adding 50. Mu.L of 2mol/L sulfuric acid per well. OD determination by means of an ELISA reader 450nm The values were taken as the titers of the antibodies with the highest dilution of the positive reaction forming a distinct gradient, as shown in table 1 below.
Table 1: results of monoclonal antibody titer detection
Figure SMS_4
As can be seen from Table 1, the monoclonal antibodies of the present invention have a potency of more than 1000K and a high potency, and thus have a good affinity.
Example 3: cloning and sequencing of anti-DIG hybridoma cell line antibody variable region sequences
Total RNA was isolated from DIG2H1 cell clones, cDNA clone immunoglobulin sequences were prepared by reverse transcription, and the variable region sequences of the hybridoma cell lines described above were determined.
Extraction of RNA: the total RNA was extracted and immediately reverse transcribed from the hybridoma cell line described above, with reference to the instructions of the total RNA M5 extraction kit (from Beijing polymeric Biotech Co., ltd.).
Reverse transcription of RNA into cDNA: the total RNA extracted in the previous step was reverse transcribed with reference to M5 First Strand cDNA Synthesis Kit Polymei (available from Beijing polymeric Biotechnology Co., ltd.) to prepare cDNA, which was frozen at-20℃for use.
c. PCR amplification and recovery of variable region sequences: amplifying immunoglobulin heavy chain (IgH) cDNA by using the cDNA obtained in the previous step as a template and using a heavy chain primer PCR; similarly, immunoglobulin light chain (IgK) cDNA was PCR amplified using light chain primers, and PCR products were recovered; the PCR reaction used a thermostable pfu dna polymerase throughout.
d. Cloning and sequencing of variable region sequences: according to the specification of cloning vector pTOPO-Blunt Clo ning kit (purchased from Beijing polymeric Biotechnology Co., ltd.), the heavy chain and light chain variable region genes were respectively linked to pTOPO vector, E.coli DH 5. Alpha. Was transformed, positive clones were picked up, and submitted to Beijing Rui Biotechnology Co., ltd for sequencing.
The heavy chain variable region gene sequence and the light chain variable region gene sequence of the hybridoma cell strain DIG2H1 were obtained by sequencing, and analyzed, the complementarity determining regions of the heavy chain and the complementarity determining region sequences of the light chain are shown in Table 2 below.
Table 2: the complementarity determining region sequences (according to the Chothia numbering system) of the heavy and light chains of the monoclonal antibody DIG2H1 of the invention
Figure SMS_5
In addition, the amino acid sequences of the heavy chain variable region and the light chain variable region of the antibody are shown in SEQ ID NO. 7 and SEQ ID NO. 8, respectively.
Example 4: preparation and purification of recombinant antibodies
Recombinant antibodies are constructed, cell lines for stably expressing the antibodies are prepared through eukaryotic expression, and large-scale culture and purification are carried out on the cell lines.
The heavy and light chain variable region gene (V) of the mouse monoclonal antibody was amplified by PCR using cDNA obtained by reverse transcription as a template H And V L ) And sequence was determined by sequencing. V to antibody L And V H The gene is used for constructing a recombinant antibody eukaryotic expression vector by using a plasmid pCDNA3.1 by a molecular cloning method. The heavy chain and light chain gene expression plasmids of the antibodies are electrically transduced into CHO host cells, the cells are added into a pressure screening culture medium (50 mu M MSX) after being electrically transduced, and after 20 days of culture, the supernatant is taken for ELISA detection (horseradish peroxidase (HRP) is used for marking goat anti-mouse IgG as secondary antibody for screening, and the method is the same as the above), and recombinant antibody cell strains with stable expression are screened.
And (3) carrying out large-scale cell culture on the screened stable transgenic cell strain by adopting a cell roller bottle culture technology, and preparing a recombinant antibody. Cells were grown in (0.2-0.3). Times.10 with medium (Vega CHO) 6 The cells/mL were inoculated into roller bottles, 1L roller bottles contained 300mL of medium (Vega CHO), the number of inoculated bottles was determined according to the production requirements, and the roller bottles inoculated with cells were placed into a cell-transfer bottle machine and cultured in a cell incubator. The culture conditions were 900 rpm, the temperature was 37℃and the carbon dioxide was 5%. After 7-9 days of culture, observing under a sampling microscope, and centrifuging to collect samples when the cell activity rate is less than 50%. And carrying out affinity purification on the sample by using a protein A affinity chromatography column to obtain an antibody, namely a recombinant monoclonal antibody DIG2H1-C.
Example 5: monoclonal antibody specificity analysis
The ELISA plate was coated with 1. Mu.g/mL Ovalbumin (OVA), bovine Serum Albumin (BSA), DIG-OVA, digoxin-OVA and two other steroids, respectively, 100. Mu.L/well, and the hybridoma cell culture supernatants were assayed for cross-reactive OD of the antibodies to these proteins 450nm The specificity of the antibodies was judged by adding 100. Mu.L of diluted monoclonal antibody per well at a dilution of 1:1000K, and after development, the results are shown in Table 3 below:
Table 3: cross-reaction results of monoclonal antibodies of the invention against multiple proteins
Figure SMS_6
The results in table 3 show that the monoclonal antibodies of the invention not only have the activity of specifically recognizing DIG, but also bind to digoxin without cross-reacting with OVA, BSA, and with other steroids (e.g., human estrogens and androgens). The above results demonstrate that the monoclonal antibodies of the present invention have high specificity.
Example 6: competition ELISA method for determining antibody
The monoclonal antibody is further tested by competition ELISA method, which includes sample dilution, antigen coating, blocking, adding monoclonal antibody, adding enzyme-labeled antibody, developing and stopping, and OD measurement 450nm Reading and data processing. The competition ELISA detection method comprises the following steps: the coating antigen is DIG-OVA, 100 mu L of DIG-OVA with the concentration of 1 mu g/mL is added into a 96-well ELISA plate small hole, and the mixture is coated overnight. Then washing the DIG-OVA coated 96-well ELISA plate twice with washing liquid (PBS buffer solution containing 0.05% Tween), adding sealing liquid (3% skimmed milk powder/washing liquid) and sealing at room temperature for 2 hours, and pouring out the sealing liquid to obtain a sealed ELISA plate; the monoclonal antibodies DIG2H1 and DIG2H1-C are subjected to 1000K dilution to obtain a sample to be detected; adding the supernatant to be detected into a sealed ELISA plate, adding 100 mu L of the sample into each hole, adding 50 mu L of a DIG standard substance of 100ppb, uniformly mixing, incubating for 1 hour at room temperature, pouring out the supernatant, and washing for 2 times by a plate washing machine; mu.l of HRP-labeled goat anti-mouse Ig was added G antibody, dilution concentration of enzyme-labeled antibody is 1:4000, 50 mu L of each hole is incubated for 1 hour at room temperature, washing is carried out 3 times by using washing liquid, and the mixture is dried by beating on absorbent paper; add freshly prepared 100. Mu.l TMB chromogenic substrate, develop color at room temperature for 30 minutes and then 2mol/L H 2 SO 4 Terminating, absorbance A at a wavelength of 450nm 450nm 。
The data processing method comprises the following steps: by means of the absorbance A measured 450nm Percent absorbance values (%) = (B/B0) ×100% were calculated, where B is the OD of the detection wells to which competitor DIG was added 450nm (B0) OD of control wells without competitor 450nm The inhibition rate is 1-percent absorbance value.
Table 4: reaction results of Competition ELISA method
antibody/OD 450nm DIG2H1 Recombinant antibody DIG2H1-C
antibody+PBS (B) 0 ) 1.092 1.112
Antibody +5ppb DIG (B) 0.135 0.136
Antibody +10ppb DIG (B) 0.098 0.107
Percent absorbance value 10％ 9.62％
Inhibition rate 90％ 90.38％
In an indirect competition ELISA, the antibodies of the invention are limited, the free standard DIG added competes with the coated solid phase antigen, binds to the antibody, and the solid phase adsorbed DIG is inversely proportional to the amount of free DIG added. The DIG standard diluted in a gradient competes with the coating antigen, and when the DIG concentration of the added standard is around 10ppb, the percentage absorbance value is less than 10%, i.e. the inhibition rate is >90%.
Further, IC was measured by the following procedure 50 ：
DIG-OVA conjugate coating concentration is 1 mug/mL, 50 mug/hole, the monoclonal antibody is diluted according to 1:1000000 (1 ng/mL), enzyme-labeled secondary antibody is diluted according to 1:4000, DIG free small molecules with gradient concentration are added, ten gradients are added from 0.025ng/mL to 10ng/mL, a competition inhibition curve is made, and the concentration of the competitive DIG free small molecules added at 50% inhibition rate, namely IC, is determined 50 (DIG); meanwhile, in another hole, DIG free small molecules are replaced by other steroids of structural foods, and the concentration gradient is also made to find out the semi-inhibition concentration, namely IC 50 (analogues).
FIGS. 1 and 2 show the competitive inhibition curves of the coated DIG-OVA and the competitor DIG for binding to antibodies DIG2H1 and DIG2H1-C of the invention, plotted on the abscissa of the competitor DIG concentration (log) to the OD of the detection wells to which competitor DIG was added 450nm (B) OD with control wells without competitor 450nm (B0) The ratio (B/B0%, percent absorbance) is on the ordinate. According to the competition inhibition curve shown, the anti-DIG monoclonal antibody of the invention shows better specificity and detection sensitivity to DIG, and DIG2H 1-to-DIG IC 50 IC of DIG2H1-C to DIG of 0.399 ppb 50 0.330ppb。
DIG-based IC 50 Based on this, the cross-reactivity of the monoclonal antibodies of the present invention with some structural analogs was also measured, and the results are shown in table 5 below. The cross-reactivity of the antibodies was calculated by the following formula: CR (%) =ic 50 (DIG)/IC 50 (analog), the cross-reactivity indicates the specificity of the antibody, and the smaller the cross-reactivity value, the lower the cross-reactivity with the analog, indicating a higher antibody and specificity. From the data in Table 5, it can be seen that the monoclonal antibodies of the present invention have a high cross-reactivity to digoxin, while having a very low cross-reactivity to estrogens, progesterone and androgens, thus demonstrating that the monoclonal antibodies of the present invention have a high specificity to DIG and digoxin.
Table 5: cross-reactivity of monoclonal antibodies and certain structural analogs
Figure SMS_7
Figure SMS_8
Example 7: colloidal gold immunoassay
In this example, the monoclonal antibody of the present invention was labeled with colloidal gold, and streptavidin was used as a coating to construct a double-sandwich chromatography system for detection of DIG and biotin double-labeled amplification products. The sample amplification primers are DIG-labeled on one hand and biotin-labeled on the other hand, so that the amplified products are DIG and biotin double-labeled.
Colloidal gold labeled antibody: 200mL of ultrapure water was heated to boiling, 1mL of a 2% chloroauric acid (Sigma-Aldrich Co., product No. 16961-25-4) solution was added, 1mL of a 2% trisodium citrate (Sigma-Aldrich Co., product No. 6132-04-3) aqueous solution was added immediately after boiling, stirring and boiling were continued for 10 minutes to obtain a colloidal gold solution, and cooling was performed for use. 10mL of the above colloidal gold solution was placed in a beaker, and 120. Mu.L of 0.2. 0.2M K was added 2 CO 3 Adjusting the pH to 7.0; 100. Mu.g of the monoclonal antibody of the invention was addedA body; 0.1mL of 10% BSA was added, stirred and centrifuged, and the supernatant was discarded and the precipitate was fixed to 1mL with colloid Jin Xishi solution (10mM PB,150mM NaCl,0.2%BSA,0.1%TritonX-100,3% Sucrose,0.01% Proclin 300) as a monoclonal antibody colloidal gold complex. And (5) diluting the compound by 10 times, treating glass fiber, and freeze-drying to obtain the gold-labeled pad. Streptavidin (heavy chain organism HP 155-2) was diluted to 0.5mg/mL, streaked onto Millipore HF135002 nitrocellulose membranes with a spot film reader, and dried for use. And assembling the gold-labeled pad and the nitrocellulose membrane into a colloidal gold immunoassay test strip, and detecting an amplification product.
The recovery concentration of the DNA amplification product was quantified to 5 ng/. Mu.L, and a series of concentration gradients of the amplification product were set by double dilution, from 1 ng/. Mu.L to 2 pg/. Mu.L, with each concentration gradient being doubled, and each test loading amount was 40. Mu.L, whereby the loading amount of each test DNA was calculated, wherein the DNA amplification fragment length of this example was 200bp.
The test was performed using an assembled colloidal gold immunoassay test strip and the results are shown in table 6 below by comparing the readings from the colorimetric cards. As is clear from the results in Table 6, when the DNA loading was 40ng and the corresponding DIG content was 0.3pmol (the corresponding DIG concentration was 7.5 pmol/mL), the sample was read as 2 by comparison with the colorimetric card, and when the DNA loading was 0.313ng and the corresponding DIG content was 2.4fmol (the corresponding DIG concentration was 0.06 pmol/mL), the sample was read as 8. Taking reading 8 and above as positive standard, the colloidal gold immune test strip of the embodiment can detect the DNA amount of about 0.3ng at the minimum, the corresponding DIG content was 2.4fmol, i.e., the sensitivity of the antibodies of the invention to DIG was 0.06pmol/mL.
Table 6: results of assembled colloidal gold immunoassay test strips
DNA amount 40ng 20ng 10ng 5ng 2.5ng 1.25ng 0.625ng 0.313ng 0.156ng 0.078ng
DIG2H1 2 3 4 4- 5 6 7 8 9 B
DIG2H1-C 2 3 4 4- 5 6 7 8 9 9-
Sequence listing
SEQ ID NO:1(V H CDR1)：GFTFSNY
SEQ ID NO:2(V H CDR2)：TSGAGY
SEQ ID NO:3(V H CDR3)：EGPY
SEQ ID NO:4(V L CDR1)：RSSTGAVTTSNYAT
SEQ ID NO:5(V L CDR2)：GTNNRVP
SEQ ID NO:6(V L CDR3)：ALWFINRSV
SEQ ID NO:7(V H )：
QVKLEESGGDLVTPGGSLTISCTASGFTFSNYTMSWVRQTPDKRLEWVATITSGAGYTYYPDDLQGRFTISRDNAKNNLYLQMSSLKSEDTAMYYCAREGPYWGGGTLVTVSA
SEQ ID NO:8(V L )：
ETVVTQESALTTSPGETVTLACRSSTGAVTTSNYATWVQENPRHLFSGVMGGTNNRVPGVPARFSGSQIGDKAALTVTGGQTEDEALYFCALWFINRSVFGGGTKLTVL。

Claims (10)

1. A monoclonal antibody comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a heavy chain complementarity determining region V having amino acid sequences represented by SEQ ID NOS 1-3, respectively H CDR1、V H CDR2 and V H CDR3, the light chain variable region including light chain complementarity determining region V having the amino acid sequences shown in SEQ ID NOS 4-6, respectively L CDR1、V L CDR2 and V L CDR3；
Preferably, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO. 7 or a sequence identical to SEQ ID NO:7, and the light chain variable region comprises the amino acid sequence shown in SEQ ID No. 8 or a sequence having 80% or more, 85% or more, 90% or more, or 95% or more identity to the amino acid sequence shown in SEQ ID NO:8, has a sequence having 80% or more, 85% or more, 90% or more, or 95% or more identity.
2. The antibody of claim 1, wherein the antibody is any one selected from the group consisting of a bifunctional antibody, fab, F (ab ') 2, fab ', fd, fv, (dsFv) 2, dsFv-dsFv ', disulfide stabilized bifunctional antibody, scFv dimer, multispecific antibody, nanobody, domain antibody, or bivalent domain antibody;
preferably, the antibody further comprises a constant region; more preferably, the constant region is a constant region selected from any one of IgG, igA, igM, igE and IgD; more preferably, the constant region is of a species source of rat, mouse, rabbit, goat, sheep, horse, dog, cow, pig, chicken, duck, goose or human.
3. A nucleic acid molecule encoding the monoclonal antibody of claim 1 or 2.
4. A vector comprising the nucleic acid molecule of claim 3; preferably, the expression vector is a plasmid vector, e.g., pEE12, pCAGGS, pTOPO, pcDNA such as pCDNA3.1, pTT3, pEFBOS, pBV, pJV or pBJ.
5. An expression cell comprising the nucleic acid molecule of claim 3 or the expression vector of claim 4; preferably, the expression cell is a protozoan cell, an animal cell such as a mammalian cell (e.g., chinese hamster ovary cell, human embryonic kidney cell, COS cell) or a fungal cell.
6. A method for detecting Digoxigenin (DIG) comprising the step of using the monoclonal antibody of claim 1 or 2.
7. A method of detecting digoxigenin, said method being for non-diagnostic purposes, comprising the step of using the monoclonal antibody of claim 1 or 2.
8. The method of claim 6 or 7, wherein the monoclonal antibody is directly or indirectly labeled with a detectable substance;
preferably, the detectable substance comprises colloidal gold, an enzyme, a fluorescent substance, a luminescent substance, or a radioactive substance;
More preferably, the enzyme comprises horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase; the fluorescent substance comprises umbelliferone, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; the luminescent material comprises aminobenzene dihydrazide; the radioactive material comprises 3 H、 14 C、 35 S、 90 Y、 99 Tc、 111 In、 125 I、 131 I、 177 Lu、 166 Ho or 153 Sm。
9. Use of a monoclonal antibody according to claim 1 or 2 for the preparation of a reagent for the detection of Digoxigenin (DIG) or digoxigenin.
10. A kit for detecting Digoxigenin (DIG) or digoxigenin, the kit comprising the monoclonal antibody of claim 1 or 2 and instructions for how to use the monoclonal antibody for detection.
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