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

Abstract

The application provides an anti-fluoxetine antibody or antigen binding fragment thereof and application thereof, and relates to the technical field of antibodies. The anti-fluoxetine antibody or antigen binding fragment thereof comprises a heavy chain variable region and a light chain variable region; the heavy chain variable region comprises VHCDR1 of the N-terminal 31-35th amino acid residues, VHCDR2 of the 50-66th amino acid residues and VHCDR3 of the 99-107th amino acid residues in the amino acid sequence shown in SEQ ID NO. 1; and the light chain variable region comprises VLCDR1 of the N-terminal 24-38th amino acid residues, VLCDR2 of the 54-60th amino acid residues and VLCDR3 of the 93-101th amino acid residues in the amino acid sequence shown in SEQ ID NO. 2. The antibody or antigen binding fragment thereof can be used for immunological detection, and has good specific binding capacity verified.

Description

TECHNICAL FIELD

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

[0002] Fluoxetine is a selective serotonin (5-hydroxytryptamine, 5-HT) reuptake inhibitor (SSRI) type of antidepressant, and its drug form is fluoxetine hydrochloride, with trade names of "Prozac" or "Prozact" (Prozac). Fluoxetine hydrochloride (FLX) is a selective neurotransmitter inhibitor (SSRI), namely (±) N-methyl-3-phenyl-3 (4-trifluoromethyl phenoxy) propylamine hydrochloride (C17H18F3NO·HCl), with a molecular formula of C17H19ClF3NO, a molecular weight of 345.787, an appearance of white to off-white crystalline solid, and a melting point of 158.4-158.9°C. It is easily soluble in methanol or ethanol, soluble in acetonitrile, acetone or chloroform, slightly soluble in ethyl acetate, dichloromethane or water (pH 1.2, 4.5, 7.0), and almost insoluble in cyclohexane, hexane or toluene, and its chemical structural formula is shown as formula (I).

[0003]

[0004] Fluoxetine hydrochloride selectively inhibits the absorption of 5-hydroxytryptamine, and is commonly used for the treatment of depression, and is also commonly used for the treatment of mental diseases such as obsessive-compulsive disorder, anxiety disorder and metabolic disorders. FLX is commonly used for the treatment of patients with moderate depression, and has small toxic and side effects. Common side effects include nausea and vomiting, and the frequency of side effects increases with the increase of the dose of the drug. The doctor can reduce the side effects by adjusting the dose according to the patient's condition. Fluoxetine hydrochloride is taken orally, and is well absorbed in the gastrointestinal tract. The bioavailability is not affected by the digestive substances in the gastrointestinal tract, and the peak blood concentration does not change when it is taken with food, and the peak time is delayed by 3-5 hours. The plasma protein binding rate is high (up to 95%), and the whole body is widely distributed. The elimination half-life of FLX is 1-4 days, and the metabolite fluoxetine is 7-15 days. 80% of FLX is excreted in urine, and 15% is excreted in feces. The pharmacokinetic characteristics of FLX are nonlinear, and patients with low liver function and weak metabolism (except for patients with kidney dysfunction, and there is no significant difference in the metabolism of FLX in patients with different degrees of kidney damage) should use FLX carefully under the advice of a doctor. Unlike other antidepressants, the metabolism of fluoxetine hydrochloride is not limited by age and gender, and compared with tricyclic antidepressants, FLX has better tolerance and smaller side effects, and is therefore particularly suitable for the treatment of elderly patients with depression.

[0005] Fluoxetine and its metabolite norfluoxetine are inhibitors of liver microsomal enzymes CYP2D6, CYP2C9, CYP2C19 and CYP3A4, respectively, and the blood concentration of fluoxetine at the same dose varies several times to several tens of times in different individuals. In view of the non-correlation between the plasma concentration of fluoxetine and the clinical response and the large difference in blood concentration between individuals, it is necessary to monitor the blood concentration of fluoxetine in clinical use.

[0006] At present, the detection of fluoxetine mainly relies on gas chromatography with electron capture detection or mass spectrometry, high performance liquid chromatography with ultraviolet or fluorescence detection. These methods usually have low sensitivity, use 0.5-2ml of plasma sample, and the minimum quantification limit is ≥15nmol / L. Moreover, they have the problems of expensive instruments, time-consuming detection and the need for professional technical personnel to operate, and cannot meet the requirements of modern detection for rapidness and accuracy. Therefore, it is necessary to establish a rapid, sensitive and accurate detection technology.

[0007] In order to solve the problem of large-scale application on site, immunological method is paid much attention due to its fast detection speed, low cost, high throughput, and some immunological detection methods (colloidal gold paper chromatography) also have the advantages of simple operation and no need for additional equipment. Therefore, developing an excellent antibody for the diagnosis of fluoxetine has become the key to determine the application prospect of immunological method.

[0008] In view of this, the present application is proposed. SUMMARY

[0009] The purpose of the present application is to provide an anti-fluoxetine antibody or antigen-binding fragment thereof, a monoclonal antibody is obtained by screening with fluoxetine-BSA as an antigen, and the CDR region sequence thereof is determined through cloning, identification and analysis of gene structure. Another purpose of the present application is to provide biological materials related to the antibody or antigen-binding fragment thereof and their applications.

[0010] To solve the above technical problems, the present application adopts the following technical solutions:

[0011] According to one aspect of the present application, an anti-fluoxetine antibody or antigen-binding fragment thereof is provided, which comprises a heavy chain variable region and a light chain variable region; the heavy chain variable region comprises VHCDR1 of N-terminal 31-35th amino acid residues, VHCDR2 of 50-66th amino acid residues and VHCDR3 of 99-107th amino acid residues of the amino acid sequence shown in SEQ ID NO. 1.

[0012] The light chain variable region comprises a VL CDR1 of amino acid residues 24-38, a VL CDR2 of amino acid residues 54-60, and a VL CDR3 of amino acid residues 93-101 in the N-terminal amino acid sequence of SEQ ID NO. 2.

[0013] According to an aspect of the present application, there is also provided a biological material selected from any one of (i) to (iii):

[0014] (i) a polynucleotide comprising a nucleotide sequence encoding the anti-fluoxetine antibody or antigen-binding fragment thereof according to any one of claims 1 to 4;

[0015] (ii) a vector carrying the polynucleotide of (i) above.

[0016] (iii) a cell carrying the polynucleotide of (i) above, or containing the vector of (ii) above, or expressing the anti-fluoxetine antibody or antigen-binding fragment thereof.

[0017] According to an aspect of the present application, there is also provided use of the anti-fluoxetine antibody or antigen-binding fragment thereof, or the biological material described above, in detecting fluoxetine for non-diagnostic and therapeutic purposes, or in preparing a product for detecting fluoxetine.

[0018] According to an aspect of the present application, there is also provided a reagent or kit for detecting fluoxetine, comprising the anti-fluoxetine antibody or antigen-binding fragment thereof described above.

[0019] Compared with the prior art, the present application has the following beneficial effects:

[0020] The present application uses fluoxetine-BSA to immunize Balb / c mice, fuses mouse spleen cells with myeloma cells, and obtains hybridoma cells with high specificity through specific high-throughput screening. A large amount of mouse ascites is obtained through culture and re-immunization, and high-purity, high-sensitivity and high-specificity anti-fluoxetine monoclonal antibody anti-Fluoxetine-mab1 is obtained through multi-step separation and purification, which provides the required raw material for developing an immunological test strip for detecting fluoxetine. The anti-fluoxetine monoclonal antibody anti-Fluoxetine-mab1 of the present application can be used for immunoblotting, immunofluorescence and other immunological detection, and the obtained antibody has been verified to have good specific binding capacity. BRIEF DESCRIPTION OF DRAWINGS

[0021] In order to illustrate the technical solutions in the embodiments of the present application or the prior art more clearly, hereinafter, the drawings required to be used in the description of the specific embodiments or the prior art will be briefly introduced. Obviously, the drawings described hereinafter are some embodiments of the present application, and for those skilled in the art, other drawings can also be obtained without any creative effort based on these drawings.

[0022] Figure 1 The detection results of the immunochromatography test paper coated with 73# antibody provided for Example 2 of the present application. DETAILED DESCRIPTION

[0023] The technical solutions of the present application will be described clearly and completely in combination with the embodiments hereinafter. Obviously, the described embodiments are some embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all the other embodiments obtained by those skilled in the art without any creative effort belong to the protection scope of the present application.

[0024] Generally, the nomenclature used in connection with, and the techniques of, cell and tissue culture, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are those well known and commonly used in the art. The methods and techniques of the present application are generally performed according to conventional methods in the art, unless otherwise indicated. Enzymatic reactions and purification techniques are performed according to manufacturer's specifications, as commonly accomplished in the art or as described herein. The nomenclature used in connection with, and the techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well known and commonly used in the art.

[0025] In the present application, "antibody or antigen-binding fragment thereof" refers to a protein that binds to a specific antigen, which generally refers to all proteins and protein fragments containing complementarity determining regions (CDR regions). In addition, "antibody or antigen-binding fragment thereof" also includes naturally occurring antibodies and non-naturally occurring antibodies. In the present application, "antigen-binding fragment" is a substance containing antibody CDRs, which lacks at least some amino acids present in the full-length chain but still can specifically bind to an antigen. Such fragments have biological activity because they bind to target antigens and can compete with other antigen-binding molecules (including intact antibodies) for binding to a given epitope. The antigen-binding fragment in the present application has the effect of specifically recognizing and binding to fluoxetine.

[0026] The "variable region" or "variable domain" of an antibody or antigen-binding fragment thereof refers to the domain of the antibody's heavy or light chain that recognizes and binds an antigen, the segment of the amino acid sequence that determines binding specificity. The heavy chain variable region can be referred to as "VH". The light chain variable region can be referred to as "VL". These domains are generally the most variable parts of an antibody and contain the antigen binding sites. Each of the variable regions of the heavy and light chains consists of three complementarity determining regions (CDRs) connected by four framework regions (FRs). The extent of the framework and CDR regions has been precisely defined, for example, in Kabat (see Sequences of Proteins of Immunological Interest, E. Kabat et al.) and Chothia, any CDR-determining method known in the art, including combinations of methods, can identify the CDRs of a variable domain. The CDRs in each chain are held together in close proximity by the FRs and with respect to each other, the variable regions of the heavy and light chains, VL / VH, can be obtained by arranging the numbered CDRs and FRs in the following combinations: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0027] The term "polynucleotide" refers to a polymeric form of nucleotides of any length, and includes both RNA and DNA that is single-stranded, double-stranded, or multi-stranded DNA or RNA, genomic DNA, cDNA, hybrid DNA-RNA, or a polymer containing purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases. A polynucleotide comprises a portion that encodes an antibody or antigen-binding fragment thereof as described above, optionally as a sense or antisense strand. A polynucleotide can be naturally occurring, synthetic, recombinant, or any combination thereof.

[0028] The term "vector" refers to a nucleic acid vehicle into which a polynucleotide can be inserted, for example, for expression of the polynucleotide encoded protein. A vector that is capable of directing the expression of a polynucleotide encoded protein is referred to as an expression vector. A vector can be introduced into a host cell by transformation, transduction or transfection, and the host cell into which the vector has been introduced can be used as a production cell to express the polynucleotide carried by the vector.

[0029] The vectors described herein are well-known to those skilled in the art and include, but are not limited to: plasmids; phage particles; Cos plasmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC); bacteriophages such as λ phage or M13 phage; and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retrotranscriptoviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses. In some embodiments, the vectors of this invention contain regulatory elements commonly used in genetic engineering, such as enhancers, promoters, internal ribosome entry sites (IRES), and other expression control elements (e.g., transcription termination signals, or polyadenylation signals and poly-U sequences, etc.).

[0030] The terms “cell,” “cell line,” and “cell culture” used herein are used interchangeably, and all such names include progeny. Progeny may not be identical to primary cells due to natural, accidental, or intentional mutations, for example, in morphological and / or genomic DNA differences. “Transformation” and “transformed cell” include primary test cells and cultures derived therefrom.

[0031] Unless otherwise expressly stated, throughout the specification and claims, the term "comprising" or its variations such as "including" or "comprising" shall be understood to include the stated elements or components without excluding other elements or other components.

[0032] According to one aspect of the present invention, an anti-fluoxetine antibody or an antigen-binding fragment thereof is provided, the antibody or the antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region; the heavy chain variable region comprises VHCDR1 (SYYIH) at amino acid residues 31-35 from the N-terminus of the amino acid sequence shown in SEQ ID NO.1, VHCDR2 (WIYPGNVNTKYNEKFKG) at amino acid residues 50-66, and VHCDR3 (LSYDYSFAY) at amino acid residues 99-107;

[0033] The light chain variable region contains VLCDR1 (RASQSVSTSTYTYMH) at amino acid residues 24-38 at the N-terminus of the amino acid sequence shown in SEQ ID NO.2, VLCDR2 (YASNLES) at amino acid residues 54-60, and VLCDR3 (QHSWEVPPT) at amino acid residues 93-101.

[0034] In an alternative embodiment, the heavy chain variable region has the structure VHFR1-VHCDR1-VHFR2-VHCDR2-VHFR3-VHCDR3-VHFR4; wherein the amino acid sequence of VHFR1 is set forth in SEQ ID NO. 3 (QVQLQQSGPELVKPGASVRISCKASGYTFT), and / or the amino acid sequence of VHFR2 is set forth in SEQ ID NO. 4 (WVKQRPGQGLEWIG), and / or the amino acid sequence of VHFR3 is set forth in SEQ ID NO. 5 (KATLTADKSSSTAYMQLSSLTSEDSAVYFCAS), and / or the amino acid sequence of VHFR4 is set forth in SEQ ID NO. 6 (WGQGTSVTVSS).

[0035] In an alternative embodiment, the light chain variable region has the structure VLFR1-VLCDR1-VLFR2-VLCDR2-VLFR3-VLCDR3-VLFR4; wherein the amino acid sequence of VLFR1 is set forth in SEQ ID NO. 7 (DIMLTQSPASLPVSLGQRATISC), and / or the amino acid sequence of VLFR2 is set forth in SEQ ID NO. 8 (WYQQKPGQPPKLLIK), and / or the amino acid sequence of VLFR3 is set forth in SEQ ID NO. 9 (GVPARFSGSGSGTDFTLNIHPVEEEDTATYYC), and / or the amino acid sequence of VLFR4 is set forth in SEQ ID NO. 10 (FGGGTKLEIK).

[0036] In an alternative embodiment, the heavy chain variable region has the amino acid sequence set forth in SEQ IN NO. 1.

[0037] In an alternative embodiment, the light chain variable region has the amino acid sequence set forth in SEQ IN NO. 2.

[0038] In an alternative embodiment, the antibody or antigen-binding fragment thereof further comprises a sequence of a portion or all of the constant region of any one of IgGl, IgG2, IgG3, IgG4, IgA, IgM, IgE, or IgD.

[0039] In an alternative embodiment, the antigen-binding fragment comprises one or more of F(ab')2, Fab', Fab, Fv, scFv, dsFv, a diabody, and a minimal recognition unit of an antibody.

[0040] In alternative embodiments, the antibody or antigen-binding fragment thereof, except for the CDR regions, the remaining sequence is derived from one or more of the following species: mouse, rat, guinea pig, hamster, rabbit, ferret, cat, dog, goat, sheep, cow, pig, horse, monkey, and human.

[0041] In alternative embodiments, the antibody further comprises a heavy chain constant region and a light chain constant region. In alternative embodiments, the heavy chain constant region and / or the light chain constant region is derived from mouse.

[0042] In alternative embodiments, the light chain of the antibody is a kappa chain.

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

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

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

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

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

[0048] According to another aspect of the present application, there is also provided use of the aforementioned fluoxetine antibody or antigen-binding fragment thereof, or the aforementioned biological material, for detecting fluoxetine for non-diagnostic and therapeutic purposes, or for preparing a product for detecting fluoxetine.

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

[0050] In alternative embodiments, a label is further included, the label comprising one or more of an enzyme, a fluorescent molecule label, a fluorescent microsphere, a colored microsphere, colloidal gold, biotin, or streptavidin.

[0051] In an alternative embodiment, the reagent or kit is used for immunoassay. In an alternative embodiment, the reagent or kit comprises an immunochromatographic assay reagent or kit, an ELISA assay reagent or kit, an immunomagnetic microparticle assay reagent or kit, an immunofluorescence assay reagent or kit, or an immunoblotting assay reagent or kit.

[0052] In an alternative embodiment, the kit comprises an immunochromatographic test strip, which is a competitive immunochromatographic test strip. The competitive immunochromatographic test strip is coated with the antibody or antigen-binding fragment thereof of any of the above embodiments for capturing fluoxetine in a sample to be tested.

[0053] In an alternative embodiment, the immunochromatographic test strip comprises a sample pad, a conjugate pad, and a detection pad along the sample flow direction, and the detection pad is provided with a test line and a quality control line; the conjugate pad is coated with the anti-fluoxetine antibody or antigen-binding fragment thereof labeled with a marker; the test line is coated with a fluoxetine hapten; and the quality control line is coated with an antibody that specifically binds to the anti-fluoxetine antibody or antigen-binding fragment thereof. When the sample contains fluoxetine, the anti-fluoxetine antibody or antigen-binding fragment thereof on the conjugate pad is competitively bound by the fluoxetine in the sample and the fluoxetine hapten coated on the test line. The higher the content of fluoxetine in the sample, the less anti-fluoxetine antibody or antigen-binding fragment thereof binds to the test line, and the lighter the test line. The marker-labeled anti-fluoxetine antibody or antigen-binding fragment thereof flows to the quality control line and is captured by the antibody coated on the quality control line to develop color, indicating that the immunochromatographic test strip is available.

[0054] In an alternative embodiment, the conjugate pad is coated with the anti-fluoxetine antibody or antigen-binding fragment thereof labeled with colloidal gold.

[0055] In an alternative embodiment, the immunochromatographic test strip is coated with a marker-labeled antibody that binds to fluoxetine, and the heavy chain variable region amino acid sequence of the antibody that binds to fluoxetine is shown in SEQ ID NO. 1, and the light chain variable region amino acid sequence is shown in SEQ ID NO. 2.

[0056] The application is further illustrated by the following specific examples, but it should be understood that these examples are merely intended to illustrate the application in more detail and should not be construed as limiting the application in any form.

[0057] Example 1 Preparation of Antibody

[0058] 1. Preparation of Fluoxetine-BSA Hapten

[0059] (1) Add 100 mg of fluoxetine to a dry 100 ml single-neck flask under nitrogen protection, then add 10 ml of pyridine to dissolve the raw material, stir until clear, then add succinic anhydride, and heat to reflux for 16 hours; the molar ratio of fluoxetine to succinic anhydride is 1:1.2-1.5.

[0060] (2) After the reaction, the solvent was concentrated and dried in a 55-degree water bath, the residue was dissolved in dichloromethane, and silica gel was added for mixing and column chromatography; the eluent ratio was DCM:MeOH=100:1, and 95 mg of product was obtained.

[0061] (3) 50 mg of product was dissolved in 1 ml of DMF to prepare a 50 mg / ml solution, and NHS and EDC were added for activation at room temperature overnight; the molar ratio of product, NHS, and EDC was 1:(1.2-1.5):(1.2-1.5).

[0062] (4) 100 mg of BSA was dissolved in 10 ml of PBS buffer to prepare a 10 mg / ml solution, and the activated small molecule was slowly added to the protein for 8 hours of reaction.

[0063] (5) The coupled antigen was loaded into a dialysis bag, and the solution was changed every two hours for more than six times, the sample was collected, and the concentration was measured by Lowry to obtain the synthetic antigen fluoxetine-BSA.

[0064] (II) Preparation of monoclonal antibody

[0065] 2.1 Select 6-8-week-old healthy female Balb / c mice, and perform immunization injection according to the following immunization scheme. As an immunogen, BALB / c mice are immunized, and the spleen lymphocytes of the successfully immunized mice are extracted, and the lymphocytes are fused with mouse myeloma cells SP2 / 0 through cell fusion technology, and after two rounds of subcloning screening, the hybridoma cell strain stably secreting anti-fluoxetine monoclonal antibody is obtained, thereby obtaining the anti-fluoxetine monoclonal antibody.

[0066] The fluoxetine hapten chemically synthesized in the above step is used for periodic immunization of experimental mice, and the specific steps include:

[0067] (1) Balb / c mice with consistent average body weight and age were randomly divided into two groups, an aluminum adjuvant (aluminum hydroxide adjuvant) group and a non-aluminum adjuvant group.

[0068] (2) Before the experiment, the pre-immune serum of each mouse was collected (the pre-immune serum was collected on the fifth day, and the blood was taken from the eyeball, and an appropriate amount of blood was taken to ensure the normal state of the mouse), and the collected serum was stored at -80°C.

[0069] (3) Aluminum adjuvant group: Before immunization, each antigen was diluted to the corresponding dose (75 μg per mouse) in 75 μL PBS and mixed with alum adjuvant (1 mg per mouse) according to the volume antigen: adjuvant = 3: 1 (i.e. 75 μl of immunogen diluent was added with 25 μl of adjuvant); before use, the adjuvant was shaken well, and the injection adjuvant (25 μl) was slowly added to the immunogen solution; after the adjuvant and the immunogen diluent were mixed well, they were mixed well for 30 minutes. The adjuvant effectively adsorbed the antigen; the subsequent operation was carried out according to the animal experiment operation.

[0070] (4) No aluminum adjuvant group: The antigen was diluted to the corresponding dose (75 μg per mouse) in 100 μL PBS according to the above table (100 μL of immunogen), and the subsequent operation was carried out according to the animal experiment operation.

[0071] (5) Subcutaneous injection at 2-week intervals: The experimental design was 4 times of immunization, but the supernatant of some mice was obtained by centrifugation after 4 days of each immunization, and the serum titer was detected first, and the maximum blood volume was taken by heart blood after 7 days of the last immunization, and the supernatant was obtained by centrifugation and stored at -80℃.

[0072] 2.2 Detection of serum titer, the results are shown in Table 1: The serum titer of immunized mice was detected by indirect ELISA using fluoxetine hapten as coating antigen, and the serum titer of immunized mice was detected by indirect competitive ELISA using fluoxetine hapten and fluoxetine small molecule.

[0073] The indirect ELISA method was used to add 50 μl of 1 μg / ml coating antigen diluted with coating solution to each well of the enzyme-labeled plate, and the plate was coated at 4℃ overnight, then washed with washing solution (PBST) 3 times (the same below), 200 μl of blocking solution (5% skim milk powder) was added to each well, and the plate was placed in a 37℃ incubator for 2h, then washed, 50 μl of diluted serum was added to each well, and the plate was reacted in a 37℃ incubator for 30 min. After washing, 50 μl of goat anti-mouse IgG-HRP solution was added, and the plate was reacted in a 37℃ incubator for 30 min. After washing, 100 μl of substrate solution was added, and the plate was colored in a 37℃ incubator for 10 min in the dark. Finally, 2 mol / L H2SO4, 50 μL was added to terminate the reaction, and the A450 value was read on the enzyme-labeled instrument. The orbital blood titer of 3 mice after the third immunization was >62500.

[0074] The process of indirect competitive ELISA is mostly the same as that of indirect ELISA, except that after blocking and washing the ELISA plate, 50 μl of diluted 200 ng / mL small molecule fluoxetine standard solution is added, and then 50 μl of diluted serum antibody is added to make the final concentration of small molecules reach 100 ng / mL, and the remaining steps are the same. The 100 ng / mL small molecule fluoxetine competition detection can reach more than 80% at 1:12500, and fusion is carried out.

[0075] Table 1 Serum titer detection data

[0076]

[0077]

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

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

[0080] The IgG antibody titer detection steps are as follows:

[0081] (1) Plate coating: dilute the used antigen to 3 μg / ml with coating diluent, add 100 μl of the prepared coating solution to each well, and place it in a 4°C refrigerator for 24 h.

[0082] (2) After 24 h, take it out from the refrigerator and place it at 37°C for 30 min, then discard the liquid in the well; wash the well with washing solution for 3 times, 3 min each time.

[0083] (3) Block the ELISA reaction well: add 200 μl of 5% calf serum to each well, and place it at 37°C for 90 min; after blocking, wash the well with washing solution for 3 times, 3 min each time.

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

[0085] (5) Add ELISA antibody: add the appropriate concentration of secondary antibody according to the instructions; 37°C, 90 min, 100 μl per well, and wash as before.

[0086] (6) Add substrate solution: 100 μl substrate solution per well, 37°C, dark, 15-30 min.

[0087] (7) Stop reaction: 50 μl stop solution per well, 20 min.

[0088] (4) Detection of the binding activity of the monoclonal antibody to fluoxetine:

[0089] 4.1 Cell fusion and cloning screening data:

[0090] The mouse numbers are A0, A1, A2, and A3, and four rounds of fusion were completed.

[0091] A total of 185 positive wells were selected from the A0 mouse fusion screening, and 2 cell strains were finally completed after subcloning. In the first subcloning screening, 153 positive clones with OD450 values > 2.2 were selected for detection of titer by doubling dilution. In the second subcloning screening, 47 positive cell strains were completed, and 22 cell strains were selected for the third subcloning, but the cells did not grow.

[0092] A total of 14 positive wells were selected from the A1 mouse fusion screening, and 43 cell strains were finally completed. In the first subcloning screening, 126 positive clones with OD450 values > 2.2 were selected. In the second subcloning screening, 28 cell strains were completed, and 36 cell strains were selected for the third subcloning, and 15 cell strains were completed.

[0093] The A2 and A3 mice failed to fuse, and there was no positive value.

[0094] A total of 90 complete cell strains were obtained after four cell fusions.

[0095] 4.2 Ascites preparation and detection data

[0096] Each complete cell strain was injected into one F1 mouse, and a total of 90 ascites were prepared. The titer detection data of some ascites are shown in Table 2.

[0097] Table 2 Ascites titer detection data

[0098]

[0099] 4.3 Antibody purification and detection data:

[0100] The above ascites were purified by 3.3% n-octanoic acid-thiamine precipitation method, and a total of 81 antibodies were obtained. 18#, 30#, 42#, 71#, 76#, 77#, 79#, 89#, and 90# had a small amount of ascites, and were not purified. The titer detection data of some antibodies are shown in Table 3:

[0101] Table 3 Purified antibody titer detection data

[0102]

[0103]

[0104] Ascites titer and antibody titer detection showed that the monoclonal antibodies of 81 cell strains had good specific binding capacity to fluoxetine antigen. The competition experiment with small molecule fluoxetine showed good competition, and therefore, the 81 antibodies were used for testing of fluoxetine colloidal gold products.

[0105] Example 2 Application of monoclonal antibodies in products.

[0106] The 81 antibodies of fluoxetine were verified by the immunocolloidal gold platform.

[0107] Specifically, the experimental group used 81 antibodies of fluoxetine 1# to 88# obtained by the above experiment to detect the fluoxetine standard product of Hangzhou Anxu Biological Technology Co., Ltd. The immunochromatography test strip includes a sample pad, a conjugate pad, and a detection pad, and the detection pad is provided with a detection line and a quality control line. The conjugate pad is coated with the screened anti-Fluoxetine-mab1 monoclonal antibody labeled with colloidal gold, the detection line is coated with the fluoxetine antigen, and the quality control line is coated with the goat anti-mouse IgG antibody.

[0108] The negative control is a urine sample. The above reagents are added, and then the POCT detection instrument ACG1000 (ID-A003) of Hangzhou Anxu Biological Technology Co., Ltd. is used to detect the results, and the 73# antibody is screened out as negative and with good gradient. The experimental data results are shown in the following table:

[0109] Table 4 Antibody preliminary evaluation results

[0110] Antibody Negative Fluoxetine 150 ng / ml Fluoxetine 450 ng / ml 73# G9 G4 G3.5 101# G9+ G7 G6 108# G8 G4 G3.5 148# G8 G4 G3.5 154# G9 G8 G7 172# G9 G4- G3.5 173# G9- G4 G4-

[0111] Note: In the above table, G3-G9 represents the color level of the test strip strip, and the higher the value, the darker the color, + / - represents slightly darker or lighter than the color of the level. The addition of the fluoxetine standard product with a lower value indicates that the small molecule has a competitive effect, and the antibody can be combined with the fluoxetine standard product.

[0112] The results show that the 73# antibody has a good gradient and can be used in the fluoxetine product, and the cut-off value is 300 ng / ml. Then the stability evaluation of the 73# antibody is performed, and three batches of small samples of the antibody are prepared, and the evaluation results are shown in the following table and Figure 1

[0113] Table 5 73# antibody 3 batch stability evaluation results ​

[0114] Antibody Lot Negative Fluoxetine 150 ng / ml Fluoxetine 450 ng / ml 73#-1 G9 / 9 / 9+ G5 / 5 G3.5 / 3.5 73#-2 G9+ / 9+ / 9+ G5 / 5 G3 / 3 73#-3 G9- / 9+ / 9+ G5- / 5+ G3.5 / 3.5

[0115] According to the evaluation results of the product, No. 73 antibody was named anti-Fluoxetine-mab1, which can be used for the detection of fluoxetine antigen detection kit.

[0116] Example 3 Sequence analysis of heavy chain V region (VH) and light chain V region (VL) of monoclonal antibody anti-Fluoxetine-mab1

[0117] (I) Analysis method:

[0118] (1) Design primers for amplifying heavy chain V region (VH) and light chain V region (VL) genes.

[0119] (2) Take the hybridoma cell strain of anti-Fluoxetine-mab1 in logarithmic growth phase (about 107 cells), extract total RNA from the cells according to the instructions of Trizol RNA extraction kit, and synthesize cDNA first strand using total RNA as template, and PCR amplify the VH / VL gene of the antibody using the above amplification product as template.

[0120] (3) Recover the heavy chain VH (about 360 bp) and light chain VL (about 300 bp) fragments of anti-Fluoxetine-mab1 and send them to the company for sequencing.

[0121] (4) Then analyze the VH / VL gene sequence

[0122] (II) Analysis results:

[0123] (1) Heavy chain variable region:

[0124] The amino acid sequence of the heavy chain variable region is:

[0125]

[0126] Among them, the underlined part represents VHCDR1-3 in turn.

[0127] The nucleotide sequence of the heavy chain variable region is:

[0128] (SEQ ID NO. 11, wherein the underlined part represents VHCDR1-3 in turn).

[0129] (2) Light chain variable region:

[0130] The amino acid sequence of the light chain variable region is:

[0131] wherein the underlined portions represent VL CDRs 1-3, respectively, in order.

[0132] The nucleotide sequence of the VL region is:

[0133] (SEQ ID NO. 12, wherein the underlined portions represent VL CDRs 1-3, respectively, in order).

[0134] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present application, and are not intended to limit the same; although the present application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that the technical solutions recorded in the foregoing embodiments can still be modified, or some or all of the technical features can be replaced by equivalents; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present application.

Claims

1. An anti-fluoxetine antibody or its antigen-binding fragment, characterized in that, It includes a heavy chain variable region and a light chain variable region; the heavy chain variable region contains VHCDR1 of amino acid residues 31-35 at the N-terminus of the amino acid sequence shown in SEQ ID NO.1, VHCDR2 of amino acid residues 50-66 and VHCDR3 of amino acid residues 99-107. The light chain variable region includes VLCDR1 of amino acid residues 24-38 at the N-terminus of the amino acid sequence shown in SEQ ID NO.2, VLCDR2 of amino acid residues 54-60, and VLCDR3 of amino acid residues 93-101.

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

10.

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

2.

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

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

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

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

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

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

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

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

11.

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

12.

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

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

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

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

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

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

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

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