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

Abstract

The invention provides an anti-citalopram antibody or an antigen binding fragment thereof and application thereof, and relates to the technical field of biology. The antibody or the antigen binding fragment thereof contains a complementarity determining region of a heavy chain variable region and a complementarity determining region of a light chain variable region, wherein the complementarity determining region of the heavy chain variable region comprises amino acid sequences consistent with HCDR1, HCDR2 and HCDR3 of the heavy chain variable region as shown in SEQ ID NO.1; the complementarity determining region of the light chain variable region comprises amino acid sequences consistent with LCDR1, LCDR2 and LCDR3 of the light chain variable region as shown in SEQ ID NO.2. The antibody or the antigen binding fragment thereof can be combined with citalopram or a metabolite thereof, can be used for immunological detection, and provides a stable and reliable raw material source for a method for detecting citalopram based on an immunological principle.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to an anti-citalopram antibody or an antigen-binding fragment thereof and applications thereof. Background Art

[0002] The following statements merely provide background information related to the present disclosure and do not necessarily constitute prior art.

[0003] Depression is a common mental illness in psychiatry, and most patients are accompanied by anxiety symptoms. This comorbidity of depression and anxiety is not only common in young patients, but also more common in elderly patients, and even becomes one of the main symptoms for patients to seek medical treatment. Citalopram has achieved good results in the treatment of elderly patients with depression accompanied by anxiety symptoms. It has mild effects, few drug interactions, good safety, and little effect on other neurotransmitters, especially no inhibitory effect on cholinergic muscarinic receptors, histamine receptors and α-adrenergic receptors. It is particularly suitable for the elderly population.

[0004] The molecular structure of Citalopram is shown in formula (I):

[0005]

[0006] Affected by gene polymorphism, drug concentrations in individuals vary greatly. Blood drug concentrations are closely related to clinical efficacy and adverse reactions. In the era of precision medicine, it is necessary to monitor the patient's blood drug concentration and adjust the drug dosage according to the blood drug concentration to achieve individualized medication. Therefore, measuring the concentration of citalopram in the blood plays an important role in ensuring its efficacy.

[0007] Common methods for determining the blood concentration of citalopram mainly include capillary electrophoresis-electrochemiluminescence, high performance liquid chromatography-fluorescence detection, high performance liquid chromatography-ultraviolet detection, high performance liquid chromatography-mass spectrometry, etc. Among them, the high performance liquid chromatography-mass spectrometry method has high cost, high requirements for personnel and site, and is not suitable for widespread development in small and medium-sized hospitals. Therefore, the method for detecting the concentration of citalopram in human plasma at home and abroad is mainly conventional high performance liquid chromatography, but the blood concentration of citalopram in the human body is low, and the current method has the problems of complex pre-treatment operation, long analysis time, more organic solvent consumption, easy pollution and damage to the chromatographic column. The cost of liquid-mass spectrometry detection is high, different samples, complex method configuration, high system maintenance cost; low recovery rate, inaccurate test, etc. Most of the above methods are limited to laboratory detection, and are less directly used for on-site detection. Realizing on-site detection of citalopram will have great significance for medical monitoring of citalopram blood concentration, so it is necessary to establish a fast, sensitive and accurate detection technology.

[0008] In order to solve the problem of large-scale application on site, immunological methods have attracted much attention due to their fast detection speed, low cost, high throughput, and some immunological detection methods (colloidal gold paper chromatography) are also simple to operate and do not require additional equipment. Therefore, the development of excellent antibodies for citalopram diagnosis has become the key to determining the application prospects of immunological methods.

[0009] In view of this, the present invention is proposed. Summary of the invention

[0010] In a first aspect, an anti-citalopram antibody or an antigen-binding fragment thereof is provided, wherein the antibody or the antigen-binding fragment thereof comprises the complementarity determining regions HCDR1, HCDR2 and HCDR3 of the heavy chain variable region, and the complementarity determining regions LCDR1, LCDR2 and LCDR3 of the light chain variable region;

[0011] The HCDR1, HCDR2 and HCDR3 include amino acid sequences consistent with HCDR1, HCDR2 and HCDR3 of the heavy chain variable region shown in SEQ ID NO.1; the LCDR1, LCDR2 and LCDR3 include amino acid sequences consistent with LCDR1, LCDR2 and LCDR3 of the light chain variable region shown in SEQ ID NO.2.

[0012] In a second aspect, a biomaterial is also provided, wherein the biomaterial is selected from any one of (i) to (iii):

[0013] (i) a polynucleotide encoding the nucleotide sequence of the anti-citalopram antibody or antigen-binding fragment thereof according to the first aspect.

[0014] (ii) a vector carrying the polynucleotide described in (i).

[0015] (iii) a cell, wherein the cell carries the polynucleotide described in (i), or contains the vector described in (ii), or expresses the anti-citalopram antibody or its antigen-binding fragment.

[0016] In a third aspect, a composition containing an antibody is also provided, wherein the composition containing an antibody comprises the following (I) and (II) connected or separately packaged:

[0017] (I) The anti-citalopram antibody or antigen-binding fragment thereof according to the first aspect.

[0018] (II) Tracing marker and / or solid phase carrier.

[0019] In a fourth aspect, a conjugate containing an antibody is also provided, wherein the conjugate comprises the anti-citalopram antibody or its antigen-binding fragment as described in the first aspect and a functional molecule, wherein the functional molecule is covalently linked to the antibody or its antigen-binding fragment, and the functional molecule comprises one or more of sugars, proteins, polypeptides, lipids, nucleic acids and small molecule compounds.

[0020] In the fifth aspect, there is provided the use of the anti-citalopram antibody or antigen-binding fragment thereof described in the first aspect, or the biomaterial described in the second aspect, or the composition containing the antibody described in the third aspect, or the conjugate containing the antibody described in the fourth aspect in any one of the following (I) to (IV):

[0021] (I) Testing of citalopram for non-diagnostic and treatment purposes;

[0022] (II) Detection of citalopram metabolites for non-diagnostic and therapeutic purposes;

[0023] (III) Preparation of products for the detection of citalopram;

[0024] (IV) Preparation of products for detecting citalopram metabolites.

[0025] In the sixth aspect, a kit for detecting citalopram is also provided, the kit comprising the anti-citalopram antibody or its antigen-binding fragment of the first aspect, or the antibody-containing composition of the third aspect, or the antibody-containing conjugate of the fourth aspect.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] The present invention uses citalopram to immunize Balb / c mice, takes mouse spleen cells and myeloma cells to fuse, obtains hybridoma cells with high specificity by specific high-throughput screening, obtains a large amount of mouse ascites by culture and re-immunization, and then obtains high-purity, high-sensitivity and high-specific anti-citalopram monoclonal antibody anti-Citalopram-mab1 through multi-step separation and purification, and provides required raw materials for developing immunoassay products for detecting citalopram. The anti-citalopram antibody or its antigen-binding fragment of the present invention has good specific binding ability after verification, can provide stable and reliable raw material sources for the detection citalopram method based on immunological principles, reduce the dependence of products on purchased raw materials, and reduce product costs. At the same time, the detection rate and sensitivity of citalopram rapid diagnosis products such as gold label test strips, ELISA kits, etc. are improved, and the false positive rate is reduced. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0029] Figure 1 The following are photos of the test results of the colloidal gold immunochromatographic test paper coated with the 25#-2 antibody in Example 3, respectively testing a negative sample, a citalopram metabolite 250 ng / ml, and a citalopram metabolite 750 ng / ml. DETAILED DESCRIPTION

[0030] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0031] Generally, the nomenclature and its technology used in conjunction with cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization described herein are those well-known and commonly used in the art. Unless otherwise indicated, the methods and techniques of the present invention are generally carried out according to conventional methods as well-known in the art, and as described in various general and more specific references, which are cited and discussed throughout this specification. Enzymatic reactions and purification techniques are carried out according to the manufacturer's specifications, as generally achieved in the art, or as described herein. The nomenclature and its laboratory procedures and technology used in conjunction with analytical chemistry, synthetic organic chemistry, and medical and pharmaceutical chemistry described herein are those well-known and commonly used in the art.

[0032] In this article, "anti-citalopram antibody or its antigen-binding fragment" means that the antibody or its antigen-binding fragment can specifically bind to citalopram. Specific binding to citalopram includes but is not limited to specific binding to citalopram or its metabolite or salt as shown in formula (I), or a conjugate of citalopram and other molecules as shown in formula (I).

[0033] Antibodies herein generally refer to all proteins and protein fragments that contain a complementary determining region (CDR region). The terms "antibody" and "full-length antibody" include polyclonal antibodies and monoclonal antibodies. In addition, the term "antibody" includes naturally occurring antibodies as well as non-naturally occurring antibodies, including, for example, chimeric, bifunctional and humanized antibodies, and related synthetic isoforms. Non-naturally occurring antibodies are also referred to herein as "recombinant antibodies," and the term "antibody" can be used interchangeably with "immunoglobulin."

[0034] Antigen binding fragments are fragments that contain part or all of the antibody CDRs, which lack at least some of the amino acids present in the full-length chain but are still able to specifically bind to the antigen. Such fragments are biologically active because they bind to the target antigen and can compete with other antigen binding molecules (including intact antibodies) for binding to a given epitope. Such fragments are selected from, but are not limited to, F(ab') 2 Any one of Fab', Fab, Fv (composed of VH and VL), ScFv (single-chain antibody, VH and VL are connected by a connecting peptide), dsFv (disulfide stabilized Fv fragments, dsFv), bispecific antibodies, nanobodies and antibody minimum recognition units. In addition to the above functional fragments, any fragments with increased half-life are also included.

[0035] The "variable region" or "variable domain" of an antibody refers to the domain at the amino terminus of the heavy chain or light chain of the antibody that recognizes and binds to the antigen. The composition and arrangement of the amino acids in this segment determine the specificity of the antibody in recognizing the antigen. The heavy chain variable domain can be referred to as "VH". The variable domain of the light chain can be referred to as "VL". These domains are usually the most variable parts of the antibody and contain the antigen binding site. The variable regions of the heavy and light chains are each composed of three complementarity-determining regions (CDR) (also called hypervariable regions) connected by four framework regions (FR). The extent of the framework region and CDR has been precisely defined, for example, in Kabat (see Sequences of Proteins of Immunological Interest, E. Kabat et al.) and Chothia. Any CDR determination method well known in the art, including a combination of methods, can identify the CDRs of the variable domain. The CDRs in each chain are held together closely by FRs to form the variable region. Generally, the variable regions VL / VH of the heavy and light chains can be obtained by arranging and connecting the following numbered CDRs and FRs in the following combinations: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0036] The term "constant region" or "constant domain" of an antibody refers to the constant region of an antibody light chain or the constant region of an antibody heavy chain, either alone or in combination. The heavy chain of an antibody has one variable domain (VH), followed by a number of constant domains or regions, such as one or more of a hinge, CH1, CH2, CH3, and CH4, the CH1 domain being adjacent to the VH domain and at the amino terminus of the hinge region of the antibody heavy chain, and not forming part of the Fc region of the antibody; the hinge region includes the portion of the heavy chain molecule that connects the CH1 domain to the CH2 domain; the N-terminus of CH2 is usually the CH3 domain, which usually forms the C-terminal portion of the antibody, and in some antibody types, such as IgM and IgE, the constant region also includes a CH4 domain. The constant region of an antibody can be derived from IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, and IgD, as well as subclasses and mutant forms of the constant regions thereof.

[0037] The present invention does not limit the method of obtaining the anti-citalopram antibody or antigen-binding fragment. In some optional embodiments, the polynucleotide encoding the anti-citalopram antibody or antigen-binding fragment is connected to the vector and then expressed in the cell to obtain the corresponding antibody. The above-mentioned vector can be introduced into eukaryotic cells, especially mammalian cells, to construct and obtain the anti-citalopram antibody or antigen-binding fragment that can express it. In other optional embodiments, the anti-citalopram antibody or antigen-binding fragment can also be obtained by recombinant genetics techniques known to those skilled in the art or by peptide synthesis, such as automatic peptide synthesizers (e.g., automatic peptide synthesizers sold by Applied BioSystems, etc.); the antigen-binding fragment can also be optionally produced by enzymatic cleavage of antigen-binding molecules (including complete antibodies), such as pepsin or papain cleavage; or chemical cleavage, such as chemical reduction to split disulfide bonds to obtain the above-mentioned antigen-binding fragment.

[0038] The terms "specific recognition", "selective binding", "selectively binds" and "specifically binds" or similar expressions refer to the binding of an antibody or its antigen-binding fragment to a predetermined epitope on an antigen. -5 M, for example, less than about 10 -5 M, 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M or 10 -10 The Kd value of an antibody can be determined using methods well established in the art. Other standard assays for evaluating the binding ability of a ligand such as an antibody to a target are known in the art and include, for example, ELISA, Western blot, RIA, and flow cytometry analysis.

[0039] The term "polynucleotide" herein refers to a polymer form of nucleotides of any length, including ribonucleotides and / or deoxyribonucleotides. Examples of polynucleotides include, but are not limited to, single-stranded, double-stranded or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers containing purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural or derived nucleotide bases. The polynucleotide encodes the above-mentioned anti-citalopram antibody or antigen-binding fragment, and the encoding is optionally encoding the sense strand or antisense strand. The polynucleotide may be naturally occurring, synthetic, recombinant, or any combination thereof. The terms "polynucleotide" and "nucleic acid" are used interchangeably herein.

[0040] The term "vector" herein refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted. When a vector can express a protein encoded by the inserted polynucleotide, the vector is called an expression vector. The vector can be introduced into a host cell by transformation, transduction or transfection so that the genetic material elements it carries are expressed in the host cell.

[0041] The vector is well known to those skilled in the art, including but not limited to: plasmid, episomal plasmid, minicircle DNA, phagemid, cosmid; artificial chromosome, such as yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC) or P1-derived artificial chromosome (PAC); bacteriophage such as λ phage or M13 phage and animal virus, etc. Animal viruses that can be used as vectors include but are not limited to retrovirus (including lentivirus), adenovirus, adeno-associated virus, herpes virus (such as herpes simplex virus), poxvirus, baculovirus, papillomavirus, papillomavirus. In some embodiments, the vector of the present invention contains regulatory elements commonly used in genetic engineering, such as enhancers, promoters, internal ribosome entry sites (IRES) and other expression control elements (such as transcription termination signals, or polyadenylation signals and poly-U sequences, etc.).

[0042] As used herein, the expressions "cell", "cell line" and "cell culture" are used interchangeably, and all such designations include progeny. Progeny may not necessarily be identical to the original cell due to natural, accidental or deliberate mutation, and may differ from the original cell in morphology and / or in genomic DNA. "Transformants" and "transformed cells" include the primary subject cell and cultures derived therefrom.

[0043] As used herein, the term "conjugate" refers to a compound formed by covalently linking at least two molecules, and the covalent linking may be direct covalent linking of the two molecules or covalent linking through a linker. Each molecule independently includes, but is not limited to, at least one of sugars, proteins, polypeptides, lipids, nucleic acids, and small molecules. "Antibody-containing conjugate" refers to a conjugate formed by covalently linking an antibody to, but is not limited to, at least one of sugars, proteins, polypeptides, lipids, nucleic acids, and small molecules.

[0044] As used herein, the term "tracing marker" refers to a marker that can provide a detectable signal that can be directly observed by the naked eye or detected by conventional instruments acceptable in the art; the tracing marker can directly provide a signal, such as color (e.g., colloidal gold, colored microspheres), fluorescence (fluorescent molecules), magnetism, radiation, or luminescence; or it can indirectly provide a signal through a subsequent reaction in which the tracing marker participates, such as catalyzing a specific substrate reaction to produce any of the above signals.

[0045] In this document, unless otherwise stated, arbitrary numbering is used to distinguish one entity or behavior from another entity or behavior, and does not necessarily require or imply any actual relationship, order, or importance between these entities or behaviors, such as numbers (I), (II)...(VII).

[0046] As used herein, unless otherwise stated, "optionally", "optional", "optional" or "optional" means that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs or does not occur.

[0047] Herein, the terms "comprise" or "comprising" are intended to include stated elements, integers or steps, but not to exclude any other elements, integers or steps.

[0048] In a first aspect, an anti-citalopram antibody or an antigen-binding fragment thereof is provided, wherein the antibody or the antigen-binding fragment thereof comprises the complementarity determining regions HCDR1, HCDR2 and HCDR3 of the heavy chain variable region, and the complementarity determining regions LCDR1, LCDR2 and LCDR3 of the light chain variable region;

[0049] The HCDR1 includes an amino acid sequence consistent with HCDR1 of the heavy chain variable region shown in SEQ ID NO.1, the HCDR2 includes an amino acid sequence consistent with HCDR2 of the heavy chain variable region shown in SEQ ID NO.1, and the HCDR3 includes an amino acid sequence consistent with HCDR3 of the heavy chain variable region shown in SEQ ID NO.1; and the LCDR1 includes an amino acid sequence consistent with LCDR1 of the light chain variable region shown in SEQID NO.2, the LCDR2 includes an amino acid sequence consistent with LCDR2 of the light chain variable region shown in SEQ ID NO.2, and the LCDR3 includes an amino acid sequence consistent with LCDR3 of the light chain variable region shown in SEQ ID NO.2.

[0050] It is understood that other regions except the CDR regions in the variable regions shown in SEQ ID NO.1 and 2 are not used to limit the anti-citalopram antibody or antigen-binding fragment thereof provided by the present invention. For example, if the antibody or antigen-binding fragment thereof provided in the present application contains a framework region, it may be different from the framework region in the variable regions shown in SEQ ID NO.1 and 2. The CDR regions in the variable regions shown in SEQ ID NO.1 and 2 can be divided according to an optional method known in the art. Optionally, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 or LCDR3 of the variable region are defined by any one of the systems of Kabat, Chothia, IMGT, ABM or Contact or a combination of multiple definition systems. Taking the definition methods of Kabat, Chothia, IMGT, ABM or Contact as examples, the amino acid sequences of HCDR1, HCDR2 and HCDR3 of the heavy chain variable region shown in SEQ ID NO.1 are shown in Table 1, and the amino acid sequences of LCDR1, LCDR2 and LCDR3 of the light chain variable region shown in SEQ ID NO.2 are shown in Table 2:

[0051] Table 1

[0052]

[0053]

[0054] Table 2

[0055]

[0056] In an optional embodiment, the heavy chain variable region further comprises a framework region, and / or the light chain variable region further comprises a framework region. The species origin of the framework region includes, but is not limited to, one or more of rabbit, cattle, horse, dairy cow, pig, sheep, goat, rat, mouse, dog, cat, camel, donkey, deer, mink, chicken, duck, goose, turkey, fighting cock, human and their mutants.

[0057] In an optional embodiment, the heavy chain variable region of the antibody or antigen-binding fragment thereof is shown as SEQ ID NO.1, and the light chain variable region is shown as SEQ ID NO.2.

[0058] In an optional embodiment, the antigen-binding fragment comprises F(ab') 2 , Fab', Fab, Fv, scFv, dsFv and one or more of the minimum recognition unit of antibody.

[0059] In an alternative embodiment, the antibody or antigen-binding fragment thereof comprises a portion or all of the sequence of the constant region.

[0060] In an optional embodiment, the constant region sequence is selected from the sequence of a partial or complete constant region of any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD, and the IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD include their subclasses and mutant forms.

[0061] In an optional embodiment, the species origin of the constant region is one or more of rabbit, cow, horse, dairy cow, pig, sheep, goat, rat, mouse, dog, cat, camel, donkey, deer, mink, chicken, duck, goose, turkey, fighting cock, human and their mutants.

[0062] In an optional embodiment, the antibody comprises a heavy chain constant region and a light chain constant region, is a full-length antibody, the light chain of the antibody is a κ chain, and the antibody is an IgG antibody. In an optional embodiment, the full-length antibody is a mouse antibody.

[0063] In a second aspect, a biomaterial is also provided, wherein the biomaterial is selected from any one of (i) to (iii):

[0064] (i) a polynucleotide encoding the nucleotide sequence of the anti-citalopram antibody or antigen-binding fragment thereof according to the first aspect.

[0065] In an optional embodiment, the polynucleotide contains a nucleotide sequence as shown in SEQ ID NO.3, which encodes the heavy chain variable region shown in SEQ ID NO.1.

[0066] In an optional embodiment, the polynucleotide contains a nucleotide sequence as shown in SEQ ID NO.4, which encodes the light chain variable region shown in SEQ ID NO.2.

[0067] (ii) a vector carrying the polynucleotide described in (i).

[0068] (iii) a cell, wherein the cell carries the polynucleotide described in (i) above, or contains the vector described in (ii) above, or expresses the anti-citalopram antibody or its antigen-binding fragment.

[0069] In a third aspect, a composition containing an antibody is also provided, wherein the composition containing an antibody comprises the following (I) and (II) connected or separately packaged:

[0070] (I) The anti-citalopram antibody or antigen-binding fragment thereof according to the first aspect.

[0071] (II) Tracing marker and / or solid phase carrier.

[0072] The connection includes physical connection and / or chemical connection, for example, it can be but not limited to connecting the anti-citalopram antibody or antigen-binding fragment thereof described in the first aspect with the tracer marker and / or solid phase carrier through chemical bonds or adsorption.

[0073] In an optional embodiment, the tracer marker includes but is not limited to one or more of enzymes, fluorescent molecule markers, fluorescent microspheres, colored microspheres, latex microspheres, colloidal gold, colloidal selenium, colloidal silver, colloidal carbon, quantum dots, biotin, streptavidin, radionuclides, radiocontrast agents, paramagnetic ions, metals and photosensitizers.

[0074] In an optional embodiment, the solid phase carrier includes but is not limited to microtubes, columns, microparticles, nitrocellulose membranes, chromatography matrices or lateral flow devices; more specifically, for example, it can be but is not limited to enzyme-labeled wells, immunochromatographic test strips or magnetic beads.

[0075] In a fourth aspect, a conjugate containing an antibody is also provided, wherein the conjugate comprises the anti-citalopram antibody or its antigen-binding fragment as described in the first aspect and a functional molecule, wherein the functional molecule is covalently linked to the antibody or its antigen-binding fragment, and the functional molecule comprises one or more of sugars, proteins, polypeptides, lipids, nucleic acids and small molecule compounds.

[0076] In the fifth aspect, there is provided the use of the anti-citalopram antibody or antigen-binding fragment thereof described in the first aspect, or the biomaterial described in the second aspect, or the composition containing the antibody described in the third aspect, or the conjugate containing the antibody described in the fourth aspect in any one of the following (I) to (IV):

[0077] (I) Testing of citalopram for non-diagnostic and treatment purposes;

[0078] (II) Detection of citalopram metabolites for non-diagnostic and therapeutic purposes;

[0079] (III) Preparation of products for the detection of citalopram;

[0080] (IV) Preparation of products for detecting citalopram metabolites.

[0081] In a sixth aspect, a kit for detecting citalopram is also provided, the kit comprising the anti-citalopram antibody or antigen-binding fragment thereof of the first aspect, or the composition comprising the antibody of the third aspect, or the conjugate comprising the antibody of the fourth aspect. The kit comprises one or more reagents, and when the kit comprises one reagent, the kit may be equivalent to the reagent.

[0082] In an optional embodiment, the kit includes an immunochromatographic detection kit, an ELISA detection kit, an immunomagnetic particle detection kit, an immunofluorescence detection kit or an immunoblot detection kit.

[0083] In an optional embodiment, the kit further contains reagents and / or consumables for detection, including but not limited to one or more of primers, probes, buffers, dyes, diluents, washing solutions, color developing solutions, lysates, negative controls, positive controls, and blank controls; consumables include but are not limited to solid phase carriers, such as magnetic beads, enzyme-labeled wells, or nitrocellulose membranes, etc. Those skilled in the art can select the reagent composition of the kit according to the specific detection method, and the present invention does not limit this.

[0084] In an optional embodiment, the kit includes an immunochromatographic test paper, which is a competitive immunochromatographic test paper. The competitive immunochromatographic test paper is coated with the antibody or antigen-binding fragment thereof in any of the above embodiments, and is used to capture citalopram in the sample to be tested.

[0085] In an optional embodiment, the detection line of the immunochromatographic test paper is coated with citalopram coupled to a protein carrier, and the conjugate pad is coated with the anti-citalopram antibody or its antigen-binding fragment labeled with a tracer marker. It is understandable that the immunochromatographic test paper has a conventional immunochromatographic test paper structure known in the art, and an exemplary immunochromatographic test paper structure includes a sample pad, a conjugate pad and a detection pad along the sample flow direction, and the detection pad is provided with a detection line and a quality control line. The quality control line is coated with an antibody that specifically binds to the anti-citalopram antibody or its antigen-binding fragment; when the sample contains a citalopram small molecule, the citalopram in the sample and the citalopram antigen coated on the detection line simultaneously compete with the anti-citalopram antibody or its antigen-binding fragment on the conjugate pad, and the citalopram in the sample will preferentially compete with the anti-citalopram antibody or its antigen-binding fragment on the conjugate pad. The higher the citalopram content in the sample, the less anti-citalopram antibodies or antigen-binding fragments thereof that can bind to the citalopram antigen remain on the test line when chromatographed to the test line, and thus the less detectable signal on the test line. The anti-citalopram antibodies or antigen-binding fragments thereof labeled with a tracer marker flow through the quality control line and are captured by the antibodies coated on the quality control line, and a measurable signal appears, indicating that the immunochromatographic test paper is usable.

[0086] In an optional embodiment, the immunochromatographic test paper is coated with an antibody that binds to citalopram and is labeled with a tracer marker, and the amino acid sequence of the heavy chain variable region of the antibody that binds to citalopram is as shown in SEQ ID NO.1, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO.2.

[0087] In an optional embodiment, the anti-citalopram antibody or antigen-binding fragment thereof coated on the conjugate pad is labeled with colloidal gold.

[0088] In an optional embodiment, the protein carrier to which citalopram is coupled includes but is not limited to bovine gamma globulin, bovine serum albumin, ovalbumin, keyhole limpet hemocyanin, human serum albumin, thyroglobulin, tetanus toxoid, synthetic polylysine or purified protein derivatives.

[0089] The present invention is further described below by means of specific examples. However, it should be understood that these examples are only used for more detailed description and should not be construed as limiting the present invention in any form.

[0090] Example 1 Preparation of Citalopram-BGG Antigen

[0091] 1. Prepare 6 mol / L hydrochloric acid solution, dissolve 100 mg of citalopram in the hydrochloric acid solution, and react overnight in an oil bath at 50°C.

[0092] 2. After the reaction is completed, spin dry the solvent and add appropriate amount of water and 4N sodium hydroxide solution to adjust the pH to neutral or weakly acidic.

[0093] 3. Add 20 ml of dichloromethane and extract three times, collect the organic phase, add anhydrous sodium sulfate to dry, filter and spin dry the organic phase to obtain a light yellow viscous liquid - Citalopram hapten.

[0094] 4. Dissolve 20 mg of citalopram hapten, 8 mg of NHS, and 13.4 mg of EDC in DMF and place on a turntable to react overnight at room temperature.

[0095] 5. Weigh 100 mg BGG (bovine gamma globulin) and dissolve it in 10 ml PBS buffer. Stir well to obtain a protein solution with a concentration of 10 mg / ml.

[0096] 6. In an ice bath, slowly add the citalopram hapten solution to the protein solution, and place the resulting mixture on a turntable at room temperature for 8 hours to obtain an artificial antigen mixture;

[0097] 7. The artificial antigen mixture was transferred into a dialysis bag and dialyzed with PBS buffer for 7 times. After the dialysis, the mixture was centrifuged at 10,000 r / min for 10 minutes, the precipitate was discarded, and the supernatant was taken to obtain the Citalopram-BGG antigen. The chemically synthesized Citalopram-BGG antigen was used for phased immunization of experimental mice.

[0098] Example 2 Preparation of monoclonal antibody anti-Citalopram-mab1.

[0099] 1. Preparation of monoclonal antibody anti-Citalopram-mab1:

[0100] Healthy female Balb / c mice aged 6 to 8 weeks were selected and immunized according to a pre-specified immunization scheme. As an immunogen, BALB / c mice were immunized, and spleen lymphocytes of successfully immunized mice were extracted. The lymphocytes were fused with mouse myeloma cells SP2 / 0 by cell fusion technology. After two rounds of subclone screening, a hybridoma cell line that stably secreted anti-citalopram monoclonal antibodies was obtained, thereby obtaining anti-citalopram monoclonal antibodies.

[0101] The citalopram antigen prepared in Example 1 was used to perform phased immunization on experimental mice.

[0102] The specific steps of animal immunization experiments include:

[0103] 1. Balb / c mice with the same mean body weight and age were randomly divided into two groups: the group with aluminum adjuvant (aluminum hydroxide adjuvant) and the group without aluminum adjuvant.

[0104] 2. Before the experiment, the pre-immune serum of each mouse was collected (pre-immune serum was collected on the fifth day, blood was collected through the eyeball, and an appropriate amount of blood was collected to ensure the normal state of the mice), and the collected serum was stored at -80°C.

[0105] 3. Preparation of the aluminum adjuvant (aluminum hydroxide adjuvant) group: Before immunization, each antigen was diluted to the corresponding dose (75 μg / mouse) in 75 μL PBS, and mixed with alum adjuvant (1 mg / mouse), according to the volume of antigen: adjuvant = 3:1 (i.e., 25 μL adjuvant was added to 75 μL immunogen diluent); the adjuvant was shaken before use, and the injection adjuvant (25 μL) was slowly added to the immunogen solution; after the adjuvant and the immunogen diluent were fully mixed, the two were fully mixed for 30 minutes. The adjuvant effectively adsorbed the antigen; the subsequent operation was carried out according to the immunization animal experiment.

[0106] 4. Group without aluminum adjuvant: The antigens were diluted in 100 μL PBS to the corresponding doses in the above table (75 μg / mouse), and 100 μL of immunogen was added. The subsequent operation was carried out according to the animal immunization experiment.

[0107] 5. Subcutaneous injection at intervals of 2 weeks: The experiment was designed for 4 immunizations, but blood was collected from the eyeballs 4 days after each immunization, and part of the mouse supernatant was obtained by centrifugation. The serum titer was first tested. 7 days after the last immunization, blood was collected from the heart to obtain the maximum amount of blood, which was centrifuged to obtain the supernatant and stored at -80°C.

[0108] 6. Detect serum titer.

[0109] (1) Four mice were immunized, and the mice were numbered A0, A1, A2 and A3. After three immunizations, the serum titer was tested. The test data are shown in Table 3 below, and the test method is as follows:

[0110] Citalopram-BGG antigen and Citalopram small molecule were used for antigen competition detection, and Citalopram-BGG antigen was used as coating antigen for indirect ELISA and competitive ELISA to detect the titer of immune mouse serum. 50 μL of coating antigen diluted with coating solution was added to each well of the indirect ELISA enzyme labeling plate at 1 μg / ml. After coating overnight at 4°C, the plate was washed 3 times with washing solution (PBST) (the same below), and 200 μL of blocking solution (5% skim milk powder) was added to each well. The plate was placed in a 37°C incubator for 2 hours. After washing, 50 μL of diluted serum was added to each well, and the plate was reacted in a 37°C incubator for 30 minutes. After washing, 50 μL of goat anti-mouse IgG-HRP solution was added, and the plate was reacted in a 37°C incubator for 30 minutes. After washing, 100 μL of substrate solution was added, and the plate was colorized in a 37°C incubator for 10 minutes in the dark, and finally 2 mol / L H 2 SO 4 , 50 μL was added to terminate the reaction, and the A450 value was read on a microplate reader. The titers of orbital blood of the three mice after three immunizations were all >312,500.

[0111] The process of indirect competitive ELISA is mostly the same as that of indirect ELISA, except that after blocking with blocking solution and washing the ELISA plate, 50 μL of diluted 200 ng / mL small molecule citalopram standard solution is added, and then 50 μL of diluted serum antibody is added to make the final concentration of the small molecule reach 100 ng / mL. The rest of the steps are the same. In the competition test of 100 ng / ml small molecule citalopram, more than 80% can be achieved at 1:62500, and fusion can be arranged.

[0112] Table 3: Serum titer test data

[0113]

[0114]

[0115] The immune spleen cells were fused with myeloma cell line SP2 / 0 cells, and the fused cells were screened by HAT selection medium (HAT selection medium contains hypoxanthine, aminopterin and thymidine), and the fused cells were subjected to ELISA positive screening and subcloning; the screened positive monoclonal antibody was purified from ascites using Protein A / G antibody purification column, and the ELISA titer of the purified antibody was >1:128,000, and the purity was >90%.

[0116] 2. Enzyme-linked immunosorbent assay (ELISA) was used to detect the binding activity of citalopram.

[0117] IgG antibody titer detection method

[0118] 1. Bottom plate coating: dilute the antigen to 3 μg / ml with coating diluent, add 100 μL of the prepared coating solution to each well, and place in a 4°C refrigerator for 24 hours.

[0119] 2. After 24 hours, take out the wells from the refrigerator and equilibrate at 37°C for 30 minutes, then discard the liquid in the wells; wash the wells three times with washing solution, each time for 3 minutes.

[0120] 3. Block the enzyme-labeled reaction wells: add 200 μL of 5% calf serum to each well and block at 37°C for 90 min. After blocking, wash the wells three times with washing solution, each time for 3 min.

[0121] 4. Add the sample to be tested: dilute the sample according to the required ratio, add the diluted sample to the enzyme-labeled reaction well, 100 μL per well, incubate at 37°C for 90 min; wash the well three times with washing solution, 3 min each time.

[0122] 5. Add enzyme-labeled antibody: Add secondary antibody of appropriate concentration according to the instructions; incubate at 37°C for 90 min, add 100 μL per well and wash as before.

[0123] 6. Add substrate solution: 100 μL of substrate is added to each well and placed at 37°C away from light for 15 to 30 minutes.

[0124] 7. Stop reaction: Add 50 μL of stop solution to each well to stop the reaction and measure the experimental results within 20 minutes.

[0125] 3. Detect the binding activity of monoclonal antibodies to citalopram.

[0126] 1. Cell fusion and clone screening data: The mice were numbered A0, A1, A2, and A3, and four rounds of fusion were completed. After cell fusion, a total of 19 complete cell lines were obtained.

[0127] 2. Ascites preparation and detection data: One F1 mouse was injected for each complete cell line, and a total of 19 ascites were prepared.

[0128] 3. Antibody purification conditions and test data: The above ascites was purified by 3.3% octanoic acid-thiamine precipitation method, and a total of 19 antibodies were obtained. The antibody numbers are 1#, 2#, 3#, 4#, 5#, 6#, 9#, 11#, 15#, 16#, 18#, 19#, 20#, 22#, 23#, 24#, 25#, 26#, and 27#. The titer test data of some antibodies are shown in Table 4:

[0129] Table 4: Purified antibody titer test data

[0130]

[0131]

[0132] Antibody titer testing showed that the monoclonal antibodies of 19 cell lines had good specific binding ability to citalopram antigen. Competition experiments with small molecule citalopram showed good competition. Therefore, the 19 antibodies were used to test the citalopram colloidal gold product.

[0133] Example 3 Application of monoclonal antibody anti-Citalopram-mab1 in products

[0134] 19 antibodies against citalopram were verified by the immunocolloidal gold platform. The immunocolloidal gold platform is an immunochromatographic test paper, which includes a sample pad, a conjugation pad and a detection pad according to the chromatography direction, and the detection pad is provided with a detection line and a quality control line. The conjugation pad is coated with a monoclonal antibody selected by colloidal gold labeling, and the detection line is coated with the citalopram-BGG antigen of Example 1.

[0135] Specifically, the experimental group used 19 antibodies from 1# to 27# of citalopram obtained in the above experiment to detect the citalopram standard (citalopram prototype) and citalopram metabolite (norcitalopram) of Hangzhou Anxu Biotechnology Co., Ltd. The above reagents were added, and then the results were tested using the POCT detection instrument ACG1000 (ID-A003) of Hangzhou Anxu Biotechnology Co., Ltd., and the 9# and 25# antibodies were screened out as negative and with good gradients. The experimental data results are shown in the following table:

[0136] Table 5: Preliminary evaluation results of some antibodies

[0137]

[0138] Note: G3 to G10 represent the color depth of the test strip. The higher the value, the darker the color. + / - represents a slightly darker or lighter color than this level. The lower the value after adding citalopram prototype or citalopram metabolite standard, it means that the small molecule has a competitive effect and the antibody can bind to the citalopram standard.

[0139] The results showed that the gradient of 9# and 25# antibodies was good and could be used in products for detecting citalopram and / or citalopram metabolites. The cut-off value was 500ng / ml. After that, the stability of the antibodies was evaluated and 3 batches of antibody samples were prepared. The evaluation results are shown in the following table:

[0140] Table 6: Stability evaluation results of 3 batches of 9# and 25# antibodies

[0141]

[0142] The results showed that the gradient of antibody 25# was better than that of antibody 9#. Based on the evaluation results of the product, antibody 25# was named anti-Citalopram-mab1, and this antibody was preferably used for the detection of the Citalopram antigen detection kit. The colloidal gold immunochromatographic test strips coated with antibody 25#-2 tested negative samples, Citalopram metabolites 250ng / ml, and Citalopram metabolites 750ng / ml, respectively. Figure 1 shown.

[0143] Example 4 Sequence analysis of the heavy chain V region (VH) and light chain V region (VL) of the monoclonal antibody anti-Citalopram-mab1.

[0144] 1. Design primers for amplifying the heavy chain V region (VH) and light chain V region (VL) genes.

[0145] 2. Take the hybridoma cell line of anti-Citalopram-mab1 in the logarithmic growth phase (about 10 7 The total RNA of the cells was extracted according to the instruction of Trizol RNA extraction kit, the first chain of cDNA was synthesized by reverse transcription using the total RNA as a template, and the VH / VL gene of the antibody was amplified by PCR using the above amplified product as a template.

[0146] 3. Recover the heavy chain VH (about 360bp) and light chain VL (about 300bp) fragments of anti-Citalopram-mab1 and send them to the company for sequencing.

[0147] 4. Then analyze the VH / VL gene sequence:

[0148] The resulting sequence is as follows:

[0149] Nucleotide sequence of the variable region of the heavy chain: anti-Citalopram-mab1-VH: 351 bp

[0150] CAGGTCCAGCTGCAGCAGTCTGGACCTGAGCTGGTGAAGCCTGGGGCTTCAGTGAGGATA

[0151] TCCTGCAAGGCTTCTGGCTACACCTTCACATCCTACTATATAGTCTGGGTGAGGCAGAGGCCTG

[0152] GACAGGGACTTGAGTGGATTGGATGGATTTATCCTGGAAATCTTAATACAAAGTACAATGAGAA

[0153] GTTCAAGGGCAAGGCCACACTGACTGCAGACAAATCCTCCAGCACAGCCTACATGCAGCTCAG

[0154] CAGCCTGACCTATGAGGACTCTGCGGTCTATTTCTGTACAAGATACTACTATGGTGCCTCCTTG

[0155] ATTGGGGCCAGGGCACCAGCGTGACCGTGAGCAGC(SEQ ID NO.3)

[0156] The amino acid sequence of the variable region of the heavy chain: anti-Citalopram-mab1protein: 117aa

[0157] QVQLQQSGPELVKPGASVRISCKASGYTFTSYYIVWVRQRPGQGLEWIGWIYPGNLNTKYNE KFKGKATLTADKSSSTAYMQLSSLTYEDSAVYFCTRYYYGASLDWGQGTSVTVSS (SEQ ID NO.1) heavy chain variable region CDR analysis, the results are shown in Table 1:

[0158] Table 1

[0159]

[0160] Variable region nucleotide sequence of light chain: anti-Citalopram-mab1LVκ: 336 bp

[0161] TCTATTGTGCTGACCCAGTCTCCACTCACTTTGTCGGTTACCATTGGACAAGCAGCCTCTATCTCTTGCAAGTCAAGCAGAGCTCTTATATAGTGATGGAAAAATCTATTTGAATTGGTTATTACAGAGGCCAGGCCAGTCTCCAAAACGCCTAATCTATATGGTGTC TAAACTGGACTCTGGAGTCCCTGACAAGTTCGCTGGCAGTGGATCAGGAACAGACTTTACACTGAAAATCAGCAGAATGGAGGCTGAAGATGTTGCCACTTACTACTGCCAGCAGGGTAGTAGTATACCGCTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAA(SEQ ID NO.4)

[0162] The amino acid sequence of the variable region of the light chain: anti-Citalopram-mab1LVκprotein: 112aa

[0163] SIVLTQSPLTLSVTIGQAASISCKSSQSLLYSDGKIYLNWLLQRPGQSPKRLIYMVSKLDSGVPD KFAGSGSGTDFTLKISRMEAEDVATYYCQQGSSIPLTFGAGTKLELK(SEQ ID NO.2)

[0164] The results of light chain variable region CDR analysis are shown in Table 2:

[0165] Table 2

[0166]

[0167] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. An anti-citalopram antibody or an antigen-binding fragment thereof, characterized in that: The antibody or antigen-binding fragment thereof contains the complementarity determining regions HCDR1, HCDR2 and HCDR3 of the heavy chain variable region, and the complementarity determining regions LCDR1, LCDR2 and LCDR3 of the light chain variable region; The HCDR1, HCDR2 and HCDR3 include amino acid sequences consistent with HCDR1, HCDR2 and HCDR3 of the heavy chain variable region shown in SEQ ID NO.1; the LCDR1, LCDR2 and LCDR3 include amino acid sequences consistent with LCDR1, LCDR2 and LCDR3 of the light chain variable region shown in SEQ ID NO.

2.

2. The antibody or antigen-binding fragment thereof according to claim 1, characterized in that: The HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 or LCDR3 of the anti-citalopram antibody or antigen-binding fragment thereof is defined by any one of the systems of Kabat, Chothia, IMGT, ABM or Contact or a combination of multiple definition systems; Optionally, according to the Kabat definition: the amino acid sequence of the HCDR1 is shown as SEQ ID NO.7, the amino acid sequence of the HCDR2 is shown as SEQ ID NO.12, and the amino acid sequence of the HCDR3 is shown as SEQ ID NO.15; the amino acid sequence of the LCDR1 is shown as SEQ ID NO.18, the amino acid sequence of the LCDR2 is shown as SEQ ID NO.21, and the amino acid sequence of the LCDR3 is shown as SEQ ID NO.

23.

3. The antibody or antigen-binding fragment thereof according to claim 1, characterized in that: The heavy chain variable region further comprises a framework region, and / or the light chain variable region further comprises a framework region; Optionally, the heavy chain variable region of the antibody or antigen-binding fragment thereof is as shown in SEQ ID NO.1, and the light chain variable region is as shown in SEQ ID NO.

2.

4. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, characterized in that: The antigen binding fragment is one of F(ab')2, Fab', Fab, Fv, scFv, dsFv, bispecific antibody and antibody minimum recognition unit; Optionally, the antibody is freed of CDR regions, and the remaining sequences are from species including one or more of mice, rats, guinea pigs, hamsters, rabbits, ferrets, cats, dogs, goats, sheep, cows, pigs, horses, monkeys and humans; Optionally, the antibody or antigen-binding fragment thereof further contains part or all of a constant region; Optionally, the constant region sequence is selected from the sequence of a portion or all of the constant region of any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD; Optionally, the antibody further comprises a heavy chain constant region and a light chain constant region, the light chain of the antibody is a κ chain, and the antibody is an IgG antibody.

5. Biomaterial, characterized in that The biological material is selected from any one of (i) to (iii): (i) a polynucleotide comprising a nucleotide sequence encoding the anti-citalopram antibody or antigen-binding fragment thereof according to any one of claims 1 to 4; (ii) a vector carrying the polynucleotide described in (i). (iii) a cell, wherein the cell carries the polynucleotide described in (i), or contains the vector described in (ii), or expresses the anti-citalopram antibody or an antigen-binding fragment thereof; Optionally, the polynucleotide contains a fragment of the nucleotide sequence as shown in SEQ ID NO.3; Optionally, the polynucleotide contains a fragment of the nucleotide sequence shown in SEQ ID NO.

4.

6. A composition containing an antibody, characterized in that Contains the following (I) and (II) connected or separately packaged: (I) the anti-citalopram antibody or antigen-binding fragment thereof according to any one of claims 1 to 4; (II) Tracing marker and / or solid phase carrier.

7. A conjugate containing an antibody, characterized in that The invention comprises the anti-citalopram antibody or antigen-binding fragment thereof and a functional molecule according to any one of claims 1 to 4, wherein the functional molecule is covalently linked to the antibody or antigen-binding fragment thereof, and the functional molecule comprises one or more of sugars, proteins, polypeptides, lipids, nucleic acids and small molecule compounds.

8. Use of the anti-citalopram antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, or the biomaterial according to claim 5, or the antibody-containing composition according to claim 6, or the antibody-containing conjugate according to claim 7 in any one of the following (I) to (IV): (I) Testing of citalopram for non-diagnostic and treatment purposes; (II) Detection of citalopram metabolites for non-diagnostic and therapeutic purposes; (III) Preparation of products for the detection of citalopram; (IV) Preparation of products for detecting citalopram metabolites.

9. A kit for detecting citalopram, characterized in that: A composition comprising the anti-citalopram antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, or the antibody-containing composition according to claim 6, or the antibody-containing conjugate according to claim 7.

10. The kit according to claim 9, characterized in that The kit includes a kit for immunoassay; Optionally, the kit includes an immunochromatographic detection kit, an ELISA detection kit, an immunomagnetic microparticle detection kit, an immunofluorescence detection kit or an immunoblot detection kit; Optionally, the kit includes an immunochromatographic test paper, and the immunochromatographic test paper is a competitive immunochromatographic test paper; Optionally, the detection line of the immunochromatographic test paper is coated with citalopram coupled to a protein carrier, and the conjugate pad is coated with the anti-citalopram antibody or antigen-binding fragment thereof labeled with a tracer marker; Optionally, the protein carrier includes bovine gamma globulin, bovine serum albumin, ovalbumin, keyhole limpet hemocyanin, human serum albumin, thyroglobulin, tetanus toxoid, artificially synthesized poly-lysine or purified protein derivatives.

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