CN117143245A

CN117143245A - Anti-fluoxetine antibodies or antigen binding fragments thereof and uses thereof

Info

Publication number: CN117143245A
Application number: CN202311091099.6A
Authority: CN
Inventors: 魏文涛; 陈媛; 成含锋; 汪燕燕; 魏昕洋
Original assignee: Sure Biotech (hangzhou) Ltd
Current assignee: Sure Biotech (hangzhou) Ltd
Priority date: 2023-08-28
Filing date: 2023-08-28
Publication date: 2023-12-01

Abstract

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

Description

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

Technical Field

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

Background

Fluoxetine (fluxetine) is an anti-depression drug of selective serotonin (5-hydroxytryptamine, 5-HT) reuptake inhibitor (SSRI), in the form of Fluoxetine hydrochloride (Fluoxetine hydrochloride), commercially known as "Bai you Jie" or "Bai you Jie" (Prozac). Fluoxetine hydrochloride (Fluoxetine Hydrochloride, FLX) is a selective neurotransmitter inhibitor (SSRI), namely (+ -) -N-methyl-3-phenyl-3 (4-trifluoromethylphenoxy) propylamine hydrochloride (C1H 18F 3 NO. HCl), and has a molecular formula of C17H19ClF3NO, a molecular weight of 345.787, a white to off-white crystalline solid with a melting point of 158.4-158.9 ℃. Is easily dissolved in methanol or ethanol, acetonitrile, acetone or chloroform, slightly dissolved in ethyl acetate, dichloromethane or water (pH l.2,4.5,7.0), and hardly dissolved in cyclohexane, hexane or toluene, and has a chemical structural formula shown in formula (I).

Fluoxetine hydrochloride selectively inhibits the absorption of 5-hydroxytryptamine, and is often used for treating depression, and also for treating mental diseases and metabolic disorders such as obsessive-compulsive disorder, anxiety disorder, and the like. FLX is mainly used for treating patients with moderate depression, has small toxic and side effects, has nausea and vomiting as common side effects, increases the frequency of side effect onset along with the increase of the dosage, and can reduce the side effects by regulating the dosage according to the illness state of the patients. The fluoxetine hydrochloride is taken orally, is well absorbed in the gastrointestinal tract, has bioavailability not influenced by digests in the gastrointestinal tract, has unchanged peak concentration of blood medicine when taken together with food, and has a delay of 3-5 hours in peak time. The binding rate of plasma protein is high (up to 95%), and the whole body is widely distributed. The elimination half-life of FLX is 1-4 days, the metabolite defluorinated statin is 7-15 days, 80% of FLX is excreted with urine, and 15% is excreted with feces. FLX has nonlinear pharmacokinetic characteristics, and patients with weak metabolic capacity such as liver hypofunction (except kidney dysfunction patients, and FLX metabolism of patients with different degrees of kidney injury has no obvious difference), and FLX should be carefully used under the advice of doctors. Unlike other antidepressants, fluoxetine hydrochloride is metabolized without age and sex limitation, and compared with tricyclic antidepressants, FLX has better tolerance and small side effects, so that the pharmaceutical composition is particularly suitable for the elderly depression patients.

Fluoxetine and its metabolites, norfluoxetine, are inhibitors of the liver microsomal enzymes CYP2D6, CYP2C9, CYP2C19 and CYP3A4, respectively, and the blood concentrations of fluoxetine in different individuals at the same dose differ by several times to tens of times. In view of the non-correlation of the plasma concentration and clinical response of fluoxetine and the large differences in blood concentration between individuals, it is necessary to monitor blood concentration when fluoxetine is used clinically.

Currently, fluoxetine detection mainly depends on gas chromatography using electron capture detection or mass spectrometry, high performance liquid chromatography using ultraviolet or fluorescence detection. These methods are generally less sensitive, using plasma samples of 0.5-2 ml, with a minimum limit of 15nmol/L or more. And all have the instrument expensive, detect time-consuming to need professional technician to operate, can not reach modern detection and to quick, accurate requirement. It is therefore necessary to establish a rapid, sensitive, accurate detection technique.

In order to solve the problem of large-scale field application, the immunological method has the advantages of high detection speed, low cost, high flux, simple operation, no need of additional equipment and the like due to the fact that part of immunological detection methods (colloidal gold paper chromatography) are important. Therefore, development of an excellent antibody for fluoxetine diagnosis becomes a key to determine the application prospect of an immunological method.

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

Disclosure of Invention

The invention aims to provide an anti-fluoxetine antibody or an antigen binding fragment thereof, a monoclonal antibody is obtained by taking fluoxetine-BSA as an antigen, the CDR region sequence is determined by cloning, identification and analysis of a gene structure, and the invention also aims to provide biological materials related to the antibody or the antigen binding fragment thereof and application thereof.

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

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

the light chain variable region comprises VLCDR1 of amino acid residues 24 to 38, VLCDR2 of amino acid residues 54 to 60 and VLCDR3 of amino acid residues 93 to 101 of the N-terminal amino acid sequence shown in SEQ ID NO. 2.

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

a polynucleotide comprising a nucleotide sequence encoding an anti-fluoxetine antibody or antigen binding fragment thereof according to any of claims 1 to 4;

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

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

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

According to one aspect of the present invention there is also provided a reagent or kit for the detection of fluoxetine, the reagent or kit comprising an anti-fluoxetine antibody or antigen binding fragment thereof as described above.

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

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

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the description of the embodiments or the prior art will be briefly described, and it is obvious that the drawings in the description below are some embodiments of the present invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.

FIG. 1 shows the detection results of the immunochromatographic test strip coated with the 73# antibody provided in example 2 of the present invention.

Detailed Description

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

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

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

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

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

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

Such vectors are well known to those skilled in the art and include, but are not limited to: a plasmid; phagemid; a cosmid; artificial chromosomes, such as Yeast Artificial Chromosome (YAC), bacterial Artificial Chromosome (BAC), or P1-derived artificial chromosome (PAC); phages such as lambda phage or M13 phage, animal viruses, etc. Animal viruses that may be used as vectors include, but are not limited to, retrovirus (including lentivirus), adenovirus, adeno-associated virus, herpes virus (e.g., herpes simplex virus), poxvirus, baculovirus, papilloma virus, and papilloma virus. In some embodiments, the vectors of the invention comprise regulatory elements commonly used in genetic engineering, such as enhancers, promoters, internal Ribosome Entry Sites (IRES) and other expression control elements (e.g., transcription termination signals, or polyadenylation signals, and poly U sequences, etc.).

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

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

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

the light chain variable region comprises VLCDR1 (RASQSVSTSTYTYMH) of amino acid residues 24 to 38, VLCDR2 (YASNLES) of amino acid residues 54 to 60 and VLCDR3 (QHSWEVPPT) of amino acid residues 93 to 101 of the N-terminal amino acid sequence shown in SEQ ID NO. 2.

In alternative embodiments, the heavy chain variable region is of the structure VHFR1-VHCDR1-VHFR2-VHCDR2-VHFR3-VHCDR3-VHFR4; wherein the amino acid sequence of VHFR1 is shown as SEQ ID NO.3 (QVQLQQSGPELVKPGASVRISCKASGYTFT), and/or the amino acid sequence of VHFR2 is shown as SEQ ID NO.4 (WVKQRPGQGLEWIG), and/or the amino acid sequence of VHFR3 is shown as SEQ ID NO.5 (KATLTADKSSSTAYMQLSSLTSEDSAVYFCAS), and/or the amino acid sequence of VHFR4 is shown as SEQ ID NO.6 (WGQGTSVTVSS).

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 shown as SEQ ID NO.7 (DIMLTQSPASLPVSLGQRATISC), and/or the amino acid sequence of VLFR2 is shown as SEQ ID NO.8 (WYQQKPGQPPKLLIK), and/or the amino acid sequence of VLFR3 is shown as SEQ ID NO.9 (GVPARFSGSGSGTDFTLNIHPVEEEDTATYYC), and/or the amino acid sequence of VLFR4 is shown as SEQ ID NO.10 (FGGGTKLEIK).

IN an alternative embodiment, the amino acid sequence of the heavy chain variable region is shown IN SEQ ID NO. 1.

IN an alternative embodiment, the amino acid sequence of the light chain variable region is shown IN SEQ ID NO. 2.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

In an alternative embodiment, the immunochromatographic test paper comprises a sample pad, a combination pad and a detection pad along the flowing direction of the sample, wherein the detection pad is provided with a detection line and a quality control line; the binding pad is coated with the anti-fluoxetine antibody or antigen binding fragment thereof labeled by a label; the detection line is coated with fluoxetine hapten; coating the quality control line with an antibody specifically combined with the anti-fluoxetine antibody or an antigen binding fragment thereof; when the sample contains fluoxetine, the anti-fluoxetine antibody or antigen binding fragment thereof on the binding pad is competitively combined by the fluoxetine in the sample and the fluoxetine hapten coated on the detection line. The higher the fluoxetine content in the sample, the less the anti-fluoxetine antibody or antigen binding sheet thereof binds to the detection line, and the shallower the detection line. And the anti-fluoxetine antibody or the antigen binding fragment thereof marked by the marker flows to the quality control line to be captured and developed by the antibody coated by the quality control line, so that the immunochromatographic test paper can be used.

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

In an alternative embodiment, the immunochromatography test paper is coated with a label-labeled antibody combined with fluoxetine, the amino acid sequence of a heavy chain variable region of the antibody combined with fluoxetine is shown as SEQ ID NO.1, and the amino acid sequence of a light chain variable region of the antibody combined with fluoxetine is shown as SEQ ID NO. 2.

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

Example 1 antibody preparation

1. Preparation of fluoxetine-BSA hapten

(1) Adding 100 mg of fluoxetine and nitrogen protection into a dried 100 ml single-port bottle, adding 10 ml of pyridine to dissolve raw materials, stirring until the raw materials are dissolved, adding succinic anhydride, and heating and refluxing for reaction for 16 hours; the molar ratio of fluoxetine to succinic anhydride is 1:1.2-1.5.

(2) Concentrating in 55 deg. water bath, air drying, dissolving the residue with dichloromethane, adding silica gel, stirring, and passing through column; the eluent ratio was DCM: meoh=100:1, yielding 95mg of product one.

(3) 50mg of the product I is dissolved in 1 ml of DMF to prepare 50mg/ml solution, NHS and EDC are added to activate the reaction at room temperature for overnight, and the mol ratio of the product I to the NHS and the EDC is 1 (1.2-1.5).

(4) 100 mg of BSA was weighed and dissolved in 10 ml of PBS buffer to prepare a 10mg/ml solution, and the activated small molecules were slowly added to the protein and reacted for 8 hours.

(5) And (3) filling the coupled antigen into a dialysis bag, changing the liquid once every two hours, changing the liquid more than six times, collecting samples, and measuring the concentration by Lowry to obtain the synthetic antigen fluoxetine-BSA.

(II) preparation of monoclonal antibodies

2.1 Balb/c healthy female mice of 6-8 weeks of age were selected and immunized as follows. As immunogen, immunizing BALB/c mouse, extracting spleen lymphocyte of the immunized mouse, fusing lymphocyte with myeloma cell SP2/0 of the mouse by cell fusion technology, and obtaining hybridoma cell strain for stably secreting monoclonal antibody of fluoxetine after two rounds of subcloning and screening, thereby obtaining the monoclonal antibody of fluoxetine.

Step-by-step immunization of experimental mice with the fluoxetine hapten chemically synthesized in the previous step, the specific steps include:

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

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

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

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

(5) Subcutaneous injections at 2 week intervals: the experiment is designed into a 4-time immunization mode, but partial mouse supernatant is obtained by taking blood from eyeballs after 4 days of each immunization injection respectively and a centrifugal method, serum titer is detected firstly, the heart takes blood to obtain the maximum blood volume after 7 days of the last immunization, the supernatant is obtained by centrifugation, and the supernatant is stored at-80 DEG C

2.2 serum titers were measured and the results are shown in Table 1: the serum titer of the immunized mice is detected by indirect ELISA using fluoxetine hapten as a coating antigen, and the serum titer of the immunized mice is detected by indirect competition ELISA using fluoxetine hapten and fluoxetine small molecules.

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

The indirect competition ELISA process is mostly the same as the indirect ELISA process, except that after the ELISA plate is blocked by a blocking solution and washed, 50 μl of diluted 200ng/mL small molecule fluoxetine standard solution is added, and 50 μl of diluted serum antibody is added, so that the final concentration of the small molecule reaches 100ng/mL, and the rest steps are the same. 100ng/ml small-molecule fluoxetine competition detection can reach more than 80% at the time of 1:12500, and fusion can be carried out.

TABLE 1 serum potency assay data

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

(III) ELISA detection of the binding Activity of the recognition of Fluoxetine

The IgG antibody titer detection procedure was as follows:

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

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

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

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

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

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

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

(IV) detecting the binding activity of the monoclonal antibody for recognizing fluoxetine:

4.1 cell fusion and clone screening data:

the serial numbers of the mice are A0, A1, A2 and A3 in sequence, and four rounds of fusion are completed.

A0 mice were screened for fusion and picked up 185 positive wells for subcloning, and finally 2 cell lines were completed. For the first subclone screening, 153 positive clones with OD450 value more than 2.2 are selected for multiple dilution detection titer. And then carrying out secondary subcloning screening, completely obtaining 47 positive cell strains, and selecting 22 cell strains for third subcloning, wherein the cells are not long.

A1 mice were screened for fusion to pick 14 positive wells, and finally 43 cell lines were completed. For the first subclone selection, 126 positive clones with OD450 values >2.2 or more were selected in total. And then carrying out secondary subcloning screening to obtain 28 cell strains completely, and selecting 36 cell strains for third subcloning to obtain 15 cell strains completely.

Mice A2 and A3 failed to fuse, with no positive value.

A total of 90 complete cell lines were obtained after four cell fusions.

4.2 ascites preparation and detection data

1F 1 mouse was used for each complete cell line, a total of 90 ascites were prepared, and the partial ascites detection titers were as shown in Table 2.

TABLE 2 ascites titer test data

4.3 antibody purification and detection data:

the ascites is purified by a 3.3% n-caprylic acid-thiamine precipitation method to obtain 81 antibodies, wherein the total amount of abdominal water of 9 cells, namely 18#, 30#, 42#, 71#, 76#, 77#, 79#, 89# and 90# is small, and the abdominal water is not purified. The potency detection data for a portion of the antibodies are shown in table 3:

TABLE 3 purification of antibody titer assay data

The monoclonal antibodies of 81 cell lines have good specific binding capacity to fluoxetine antigens through ascites titer and antibody titer detection, and the results of competition experiments are good, so that 81 antibodies are used for testing fluoxetine colloidal gold products.

Example 2 use of monoclonal antibodies in products.

The 81 antibodies to fluoxetine were validated by an immune colloidal gold platform.

Specifically, the experimental group is to use 81 antibodies of Fluoxetine antibodies 1# to 88# obtained by the above experiment to detect Fluoxetine standard substance of An Xu biotechnology limited, hangzhou. The immunochromatography test paper comprises a sample pad, a combination pad and a detection pad, wherein the detection pad is provided with a detection line and a quality control line. The binding pad is coated with a colloidal gold-labeled screened anti-Fluoxetine-mab1 monoclonal antibody, the detection line is coated with Fluoxetine antigen, and the quality control line is coated with a goat anti-mouse IgG antibody.

The negative control is a urine sample. The reagents are loaded, then, the results are detected by using POCT detection instrument ACG1000 (ID-A003) of Hangzhou An Xu biotechnology limited company, and 73# antibody negative and better gradient are screened, and experimental data are shown in the following table:

TABLE 4 initial evaluation results of antibodies

Antibodies to	Negative of	Fluoxetine 150ng/ml	Fluoxetine 450ng/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-

Note that: in the above table, G3-G9 represent the level of the strip color shade of the test strip, with higher values representing darker colors and +/-representing a slightly darker or lighter color than that level. The lower value of the fluoxetine standard substance is added to indicate that the small molecule has a competitive action, and the antibody can be combined with the fluoxetine standard substance.

The results show that the antibody # 73 has a better gradient, can be used in fluoxetine products, and has a cut-off value of 300ng/ml, and then stability evaluation of the antibody # 73 is carried out, 3 batches of antibody minisamples are prepared, and the evaluation results are shown in the following table and fig. 1:

table 5 # 73 antibody 3 batch stability evaluation results

Antibody lot number	Negative of	Fluoxetine 150ng/ml	Fluoxetine 450ng/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

The antibody No. 73 is named as anti-Fluoxetine-mab1 according to the evaluation result of the product, and can be used for detection of Fluoxetine antigen detection kit.

EXAMPLE 3 monoclonal antibody anti-Fluoxetine-mab1 heavy chain V region (VH) and light chain V region (VL) sequence analysis

The analysis method comprises the following steps:

(1) Primers were designed to amplify the heavy chain V region (VH) and light chain V region (VL) genes.

(2) Taking hybridoma cell lines (about 107 cells) in the logarithmic growth phase of anti-Fluoxetine-mab1, extracting total RNA of the cells according to the instruction of a Trizol RNA extraction kit, performing reverse transcription by taking the total RNA as a template to synthesize a first cDNA chain, and performing PCR (polymerase chain reaction) amplification on the VH/VL genes of the antibody by taking the amplified products as templates.

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

(4) Then analyzing the VH/VL gene sequence

(II) analysis results:

(1) Heavy chain variable region:

the heavy chain variable region amino acid sequence is:

wherein the underlined parts represent VHCDRs 1 to 3, respectively, in sequence).

The heavy chain variable region nucleotide sequence is:

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

(2) Light chain variable region:

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

wherein the underlined parts represent VLCDRs 1-3, respectively, in turn).

The light chain variable region nucleotide sequence is:

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

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

Claims

1. An anti-fluoxetine antibody, or an antigen binding fragment thereof, comprising a heavy chain variable region and a light chain variable region; the heavy chain variable region comprises VHCDR1 of 31 st to 35 th amino acid residues, VHCDR2 of 50 th to 66 th amino acid residues and VHCDR3 of 99 th to 107 th amino acid residues of the N-terminal of the amino acid sequence shown in SEQ ID NO. 1;

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

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

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

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

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

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

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

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

preferably, the antibody is an IgG antibody.

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

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

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

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

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

6. Use of an anti-fluoxetine antibody or antigen binding fragment thereof according to any of claims 1 to 4, or a biological material according to claim 5, for the detection of fluoxetine for non-diagnostic and therapeutic purposes, or for the preparation of a product for the detection of fluoxetine.

7. A reagent or kit for the detection of fluoxetine, comprising an anti-fluoxetine antibody or antigen binding fragment thereof according to any of claims 1 to 4.

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

9. The reagent or kit of claim 8, wherein the reagent or kit comprises an immunoassay reagent or kit;

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

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

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