A monoclonal antibody against carisoprodol and its application

Abstract

Some embodiments of the present specification provide an antibody or an antigen-binding fragment thereof that can bind to carisoprodol and its applications. The antibody or its antigen-binding fragment includes a heavy-chain variable region and a light-chain variable region, wherein: the heavy-chain variable region includes: the amino acid sequence of CDRH1 shown in SEQ ID NO: 1, the amino acid sequence RAN of CDRH2 shown in SEQ ID NO: 2, and the amino acid sequence of CDRH3 shown in SEQ ID NO: 3; the light-chain variable region includes: the amino acid sequence of CDRL1 shown in SEQ ID NO: 4, the amino acid sequence of CDRL2, and the amino acid sequence of CDRL3 shown in SEQ ID NO: 6.

Description

Technical Field

[0001] This specification relates to the field of bioengineering technology, and particularly relates to a monoclonal antibody against carisoprodol or its antigen-binding fragment and related applications. Background Art

[0002] Carisoprodol is a muscle relaxant drug, mainly used to treat symptoms such as chronic back pain, and also has sedative and anti-anxiety effects. Carisoprodol has been proven effective in treating back pain, but it is also considered a potentially abusable drug. Some patients taking carisoprodol have developed obvious drug dependence and even addiction tendencies. Investigations have found that many patients actually take more carisoprodol than the recommended dosage by doctors. In recent years, incidents of poisoning caused by overdose of carisoprodol have occurred frequently, which has worried the medical community. Therefore, there is a need to provide an antibody that can specifically bind to carisoprodol and a method for detecting the content of carisoprodol in a sample using this antibody. Summary of the Invention

[0003] Some embodiments of this specification provide an antibody or its antigen-binding fragment that can bind to carisoprodol, characterized in that the antibody or its antigen-binding fragment includes a heavy-chain variable region and a light-chain variable region, wherein: the heavy-chain variable region includes: the amino acid sequence of CDRH1 shown in SEQ ID NO:1, the amino acid sequence RAN of CDRH2 shown in SEQ ID NO:2, and the amino acid sequence of CDRH3 shown in SEQ ID NO:3; the light-chain variable region includes: the amino acid sequence of CDRL1 shown in SEQ ID NO:4, the amino acid sequence of CDRL2, and the amino acid sequence of CDRL3 shown in SEQ ID NO:6.

[0004] In some embodiments, the heavy-chain variable region consists of any one of the following: the amino acid sequence shown in SEQ ID NO:7; an amino acid sequence having at least 85%, 90% or 95% identity in the framework region compared to the amino acid sequence shown in SEQ ID NO:7; and an amino acid sequence having one or more amino acid additions, deletions and / or substitutions in the framework region compared to the amino acid sequence shown in SEQ ID NO:7.

[0005] In some embodiments, the light-chain variable region consists of any one of the following: the amino acid sequence shown in SEQ ID NO:8; an amino acid sequence having at least 85%, 90% or 95% identity in the framework region compared to the amino acid sequence shown in SEQ ID NO:8; and an amino acid sequence having one or more amino acid additions, deletions and / or substitutions in the framework region compared to the amino acid sequence shown in SEQ ID NO:8.

[0006] Some embodiments of the present specification also provide a nucleic acid molecule, characterized in that the nucleic acid molecule encodes the aforementioned antibody or its antigen-binding fragment.

[0007] In some embodiments, the nucleic acid molecule comprises a nucleotide sequence as shown in SEQ ID NO:9 encoding the heavy chain variable region, and a nucleotide sequence as shown in SEQ ID NO:10 encoding the light chain variable region.

[0008] Some embodiments of the present specification also provide an expression vector, characterized in that the expression vector comprises the aforementioned nucleic acid molecule.

[0009] Some embodiments of the present specification also provide a host cell, characterized in that the host cell comprises the aforementioned expression vector.

[0010] Some embodiments of the present specification also provide a kit for detecting carisoprodol, characterized in that the kit comprises the aforementioned antibody or its antigen-binding fragment.

[0011] In some embodiments, the kit is a kit for immunogold method.

[0012] In some embodiments, the minimum detection limit of the kit for detecting carisoprodol is 200 ng / mL.

[0013] Some embodiments of the present specification also provide the use of the aforementioned antibody or its antigen-binding fragment in the preparation of a kit for detecting carisoprodol.

[0014] Some embodiments of the present specification also provide a method for detecting carisoprodol, characterized in that it comprises using the aforementioned kit to detect the content of carisoprodol in a sample.

[0015] The present invention uses carisoprodol to immunize mice, fuses mouse spleen cells with myeloma cells, obtains highly specific hybridoma cells through specific high-throughput screening, obtains a large amount of mouse ascites through culture and re-immunization, and then obtains a monoclonal antibody against carisoprodol with high purity, high sensitivity and high specificity through multiple steps of separation and purification, providing the required raw materials for the development of a kit for detecting carisoprodol. The monoclonal antibody against carisoprodol provided by the present invention has been verified to have good specific binding ability and can be used in immunological detections such as immunoblotting and immunofluorescence. Detailed Description of the Invention

[0016] As used in this specification and the appended claims, unless the context clearly dictates otherwise, the words "a," "an," "the," and / or "said" are not intended to refer to the singular and may include the plural. In general, the terms "comprising" and "including" are used to indicate the inclusion of the specifically identified steps and elements, which do not form an exclusive listing, and a method or apparatus may also include other steps or elements.

[0017] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0018] The terms "about" and "around" may describe a range of values around a certain value, such as plus or minus 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of that value. For example, the term "about 10 mL" may include from 9 mL to 11 mL.

[0019] As used herein, the term "antibody" generally refers to a Y-shaped tetrameric protein comprising two heavy chains (H) and two light chains (L) polypeptide chains held together by covalent disulfide bonds and non-covalent interactions. For example, the light chains of an antibody can be divided into κ and λ light chains. The heavy chains can be divided into μ, δ, γ, α, and ε, which define the isotypes of the antibody as IgM, IgD, IgG, IgA, and IgE, respectively. In the light and heavy chains, the variable regions are joined to the constant regions by a "J" region of about 12 or more amino acids, and the heavy chain also contains a "D" region of about 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of 3 domains (CH1, CH2, and CH3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The VH and VL regions can be further divided into hypervariable regions (also known as complementarity-determining regions (CDRs)) separated by relatively conserved regions called framework regions (FRs). Typically, each VH and VL consists of 3 CDRs and 4 FRs in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from the N-terminus to the C-terminus. The variable regions (VH and VL) of each heavy chain / light chain pair form the antigen-binding site. Antibodies can have different types, such as IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtypes), IgA1, IgA2, IgD, IgE, or IgM antibodies.

[0020] The term "antigen-binding portion" or "antigen-binding fragment" refers to a polypeptide comprising a fragment of a full-length antibody that retains the ability to specifically bind an antigen that binds specifically to the full-length antibody, and / or that competes with the full-length antibody for binding to the same antigen. In some embodiments, antigen-binding fragments include Fab, Fab', F(ab')2, Fd, Fv, dAb, complementarity-determining region (CDR) fragments, single-chain antibodies (e.g., scFv), chimeric antibodies, diabodies, and polypeptides comprising at least a portion of an antibody sufficient to confer specific antigen-binding ability to the polypeptide. Antigen-binding fragments of an antibody can be obtained from a given antibody (e.g., the anti-carisoprodol monoclonal antibody provided in the present application) by conventional techniques known to those skilled in the art (e.g., recombinant DNA techniques or enzymatic or chemical cleavage methods), and can be screened for specificity in the same manner as intact antibodies.

[0021] The term "identity" refers to the relationship between the sequences of two or more polypeptide molecules or two or more nucleic acid molecules determined by alignment and comparison of the sequences. "Percent identity" refers to the percentage of identical residues between amino acids or nucleotides in the compared molecules and is calculated based on the size of the smallest molecule being compared.

[0022] The methods involved in the embodiments of the present specification will be described in detail below. It should be noted that the following embodiments are only used to explain the present specification and do not constitute a limitation on the present specification.

[0023] Some embodiments of the present specification provide an antibody or an antigen-binding fragment thereof that can bind to carisoprodol, the antibody or antigen-binding fragment thereof comprising a heavy-chain variable region and a light-chain variable region, wherein: the heavy-chain variable region comprises: CDRH1, CDRH2, and CDRH3; the light-chain variable region comprises: CDRL1, CDRL2, and CDRL3. Wherein, CDRH refers to the complementarity-determining region in the heavy-chain variable region; CDRL refers to the complementarity-determining region in the light-chain variable region.

[0024] In some embodiments, the amino acid sequence of CDRH1 is: GFTFSNYW (SEQ ID NO:1). The amino acid sequence of CDRH2 is: IRLKFNNYAT (SEQ ID NO:2). The amino acid sequence of CDRH3 is: TSLLWLRRDWYFDV (SEQ ID NO:3).

[0025] In some embodiments, the amino acid sequence of CDRL1 is: QDINGY (SEQ ID NO:4). The amino acid sequence of CDRL2 is: RAN. The amino acid sequence of CDRL3 is: LQSDEFPYT (SEQ ID NO:6).

[0026] In some embodiments, the heavy-chain variable region comprises the following sequence:

[0027] EVKLQQSGGGLVQPGGSMKLSCVASGFTFSNYWMNWVRQSPEKGLEWVAE

[0028] IRLKFNNYATHYSESVKGRFIISRDDSKSSVYLQMNNLRAEDTGIYYCTSLLWLRRDWYFDVWGAGTTVT (SEQ ID NO:7).

[0029] In some embodiments, the light chain variable region comprises the following sequence:

[0030] TQSPSSMYASLGERVTLTCKASQDINGYLSWFQQKPGKPPKTLIYRANRLVDGVPSRFSVSGSGQDYSLTISSLEYEDLGIYYCLQSDEFPYTFGGGTK (SEQ ID NO:8).

[0031] In some embodiments, the heavy chain variable region or the light chain variable region described above may contain no more than 2 amino acid or no more than 1 amino acid conservative substitutions. As used herein, the term "conservative substitution" refers to an amino acid substitution that does not adversely affect or alter the basic properties of the protein / polypeptide comprising the amino acid sequence. For example, conservative substitutions can be introduced by standard techniques known in the art (such as site-directed mutagenesis and PCR-mediated mutagenesis). Conservative amino acid substitutions include substitutions in which an amino acid residue is replaced with another amino acid residue having a similar side chain, such as substitution of a corresponding amino acid residue with a residue that is physically or functionally similar (e.g., having a similar size, shape, charge, chemical properties including the ability to form covalent or hydrogen bonds, etc.). Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids having basic side chains (e.g., lysine, arginine, and histidine), amino acids having acidic side chains (e.g., aspartic acid and glutamic acid), amino acids having uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), amino acids having nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), amino acids having β-branched side chains (e.g., threonine, valine, isoleucine), and amino acids having aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Accordingly, an amino acid residue is preferably replaced with another amino acid residue from the same side chain family.

[0032] In some embodiments, the heavy chain variable region comprises an amino acid sequence having at least 85%, 90%, or 95% identity in the framework region compared to the amino acid sequence shown in SEQ ID NO:7. In some embodiments, the heavy chain variable region comprises an amino acid sequence having one or more amino acid additions, deletions, and / or substitutions in the framework region compared to the amino acid sequence shown in SEQ ID NO:7.

[0033] In some embodiments, the light chain variable region comprises an amino acid sequence having at least 85%, 90%, or 95% identity in the framework region compared to the amino acid sequence shown in SEQ ID NO:8. In some embodiments, the light chain variable region comprises an amino acid sequence having one or more amino acid additions, deletions, and / or substitutions in the framework region compared to the amino acid sequence shown in SEQ ID NO:8.

[0034] In some embodiments, an antigen-binding fragment (or functional fragment) of the carisoprodol antibody can be obtained according to the sequences of the aforementioned heavy chain variable region, light chain variable region, and / or CDR region. For example, the antigen-binding fragment of the carisoprodol antibody includes Fab, Fab', F(ab')2, Fd, Fv, dAb, complementarity-determining region (CDR) fragments, single-chain antibodies (such as scFv), etc. Chimeric antibodies, bispecific antibodies, multispecific antibodies, etc. can also be designed using these antigen-binding fragments.

[0035] Some embodiments of the present specification also provide a nucleic acid molecule that encodes the aforementioned antibody or its antigen-binding fragment.

[0036] In some embodiments, the nucleic acid molecule may include the nucleotide sequence encoding CDRH1 in the heavy chain variable region: GGATTCACTTTCAGTAACTACTGG (SEQ ID NO:5). The nucleic acid molecule may include the nucleotide sequence encoding CDRH2 in the heavy chain variable region: ATTAGATTGAAATTTAATAATTATGCAACA (SEQ ID NO:11). The nucleic acid molecule may include the nucleotide sequence encoding CDRH3 in the heavy chain variable region: ACCAGTCTACTATGGTTACGACGGGACTGGTACTTCGATGTC (SEQ ID NO:12).

[0037] In some embodiments, the nucleic acid molecule may comprise a nucleotide sequence encoding CDRL1 in the light chain variable region: CAGGACATTAATGGCTAT (SEQ ID NO:13). The nucleic acid molecule may comprise a nucleotide sequence encoding CDRL2 in the light chain variable region: CGTGCAAAC. The nucleic acid molecule may comprise a nucleotide sequence encoding CDRL3 in the light chain variable region: CTACAGTCTGATGAGTTTCCGTACACG (SEQ ID NO:14).

[0038] In some embodiments, the nucleic acid molecule comprises the following nucleotide sequences encoding the heavy chain variable region:

[0039] GAGGTGAAACTGCAGCAGTCTGGAGGAGGCTTGGTGCAACCTGGAGGAT

[0040] CCATGAAACTCTCCTGTGTTGCCTCTGGATTCACTTTCAGTAACTACTGGA

[0041] TGAACTGGGTCCGCCAGTCTCCAGAGAAGGGACTTGAGTGGGTTGCTGAA

[0042] ATTAGATTGAAATTTAATAATTATGCAACACATTATTCGGAGTCTGTGAA

[0043] AGGGAGGTTCATCATCTCAAGAGATGATTCCAAAAGTAGTGTCTACCTGC

[0044] AAATGAACAACTTAAGAGCTGAAGACACTGGCATTTATTACTGTACCAGT

[0045] CTACTATGGTTACGACGGGACTGGTACTTCGATGTCTGGGGCGCAGGAACCACGGTCACC(SEQ IDNO:9).

[0046] In some embodiments, the nucleic acid molecule comprises the following nucleotide sequences encoding the light chain variable region:

[0047] ACCCAGTCTCCATCTTCCATGTATGCATCTCTAGGAGAGAGAGTCACTCTC

[0048] ACTTGCAAGGCGAGTCAGGACATTAATGGCTATTTAAGTTGGTTCCAGCA

[0049] GAAACCAGGGAAACCTCCTAAGACCCTGATTTATCGTGCAAACAGATTGG

[0050] TAGATGGGGTCCCATCAAGGTTCAGTGTCAGTGGATCTGGACAAGATTAT

[0051] TCTCTCACCATCAGCAGCCTGGAGTATGAAGATTTGGGAATTTATTATTGTCTACAGTCTGATGAGTTTCCGTACACGTTCGGAGGGGGGACCAAG(SEQ IDNO:10)。

[0052] Some embodiments of this specification also provide an expression vector, and the expression vector includes the foregoing nucleic acid molecule containing the nucleotide sequence shown in SEQ ID NO:9 and the nucleotide sequence shown in SEQ ID NO:10. In some embodiments, the foregoing nucleic acid molecule can be inserted into one or more expression vectors by using, for example, recombinant DNA techniques and gene transfection methods well known in the art, so that the gene is operably linked to transcription and translation regulatory sequences. In this context, the term "operably linked" is intended to mean that the antibody gene is linked into the vector such that the transcription and translation control sequences in the vector perform their intended functions of regulating the transcription and translation of the antibody gene, thereby expressing the anti-carisoprodol antibody provided by the present invention.

[0053] Some embodiments of this specification also provide a host cell, and the host cell includes the foregoing expression vector. The host cell includes, but is not limited to, microbial cells, animal cells, etc. For example, when an expression vector containing the nucleotide sequence encoding the anti-carisoprodol antibody or its antigen-binding fragment provided by the present invention is introduced into a mammalian host cell, the anti-carisoprodol antibody or its antigen-binding fragment can be obtained by culturing the host cell. By a similar method, the anti-carisoprodol antibody or its antigen-binding fragment provided by the present invention can be mass-produced.

[0054] Some embodiments of this specification also provide a kit for detecting carisoprodol, and the kit includes the foregoing antibody or its antigen-binding fragment.

[0055] In some embodiments, the kit can be a colloidal gold detection kit, an immunochromatographic detection kit, an enzyme immunoassay kit, a chemiluminescence kit, an immunoturbidimetry detection kit, etc. The present invention does not limit this.

[0056] As an example only, the kit may be a kit for immunogold assay. In some embodiments, the minimum detection limit of the kit for detecting carisoprodol is 200 ng / mL.

[0057] Some embodiments of the present specification also provide the use of the aforementioned antibody or its antigen-binding fragment in the preparation of a kit for detecting carisoprodol.

[0058] Some embodiments of the present specification also provide a method for detecting carisoprodol, including using the aforementioned kit to detect the content of carisoprodol in a sample. In some embodiments, the sample may be a body fluid sample from a subject, such as urine, blood, etc. The subject may be a human or other animal. By detecting the content of carisoprodol in the sample, it can be determined whether the subject has overdosed on carisoprodol. In some embodiments, the sample may be the waste liquid generated after purifying carisoprodol, and the method for detecting carisoprodol can be used to detect the content of carisoprodol in the waste liquid, so as to evaluate and improve the purification process, etc. In some embodiments, the method for detecting carisoprodol can also be used for scientific research purposes, such as research on the pharmacokinetics of carisoprodol, and the present invention is not limited thereto.

[0059] Examples

[0060] Unless otherwise specified, the experimental methods in the following examples are all conventional methods. Unless otherwise specified, the test materials used in the following examples are all purchased from regular biochemical reagent companies. For the quantitative tests in the following examples, unless otherwise specified, three repeated experiments are set, and the results are averaged. Example 1 Preparation of anti-carisoprodol monoclonal antibody

[0061] I. Antigen

[0062] Table 1 Information related to the antigen

[0063]

[0064]

[0065] The antigen used in this example is an artificial antigen synthesized with carisoprodol as the raw material, which is coupled with an immunogenic protein through a cross-linking arm on the amide N to obtain a complete antigen with reactivity and immunogenicity, and its structure is as follows:

[0066]

[0067] Where BSA is bovine serum albumin. For more descriptions of this antigen and its synthesis method, reference can be made to the Chinese patent application with the application number 202010736865.X.

[0068] II. Preparation of Monoclonal Antibodies

[0069] Healthy female Balb / c mice aged 6 - 8 weeks were selected and immunized by injection according to a pre - specified immunization protocol. The immunized BALB / c mice were used as immunogens, and spleen lymphocytes from successfully immunized mice were extracted. Lymphocytes were fused with mouse myeloma cells SP2 / 0 by cell fusion technology. After two rounds of sub - cloning and screening, a hybridoma cell line that stably secretes monoclonal antibodies against carisoprodol was obtained, thereby obtaining monoclonal antibodies against carisoprodol.

[0070] The synthetic antigen carisoprodol was used for animal immunization experiments.

[0071] The specific steps of the animal immunization experiment are as follows:

[0072] 1. Balb / c mice with consistent average body weight and age were randomly divided into two groups: the group with aluminum adjuvant (aluminum hydroxide adjuvant) and the group without aluminum adjuvant.

[0073] 2. Before the start of the experiment, pre - immune sera were collected from each mouse (the pre - immune sera were collected on the fifth day by eye - ball blood collection, taking an appropriate amount of blood to ensure the normal state of the mice), and the collected sera were stored at - 80 °C.

[0074] 3. Preparation method for the group with aluminum adjuvant (aluminum hydroxide adjuvant): Before immunization, each antigen was diluted to the corresponding dose (75 μg / mouse) in 75 μL of PBS and mixed with alum adjuvant (1 mg / mouse), according to the volume ratio of antigen:adjuvant = 3:1 (i.e., 25 μL of adjuvant was added to 75 μL of immunogen dilution); the adjuvant was shaken well before use, and the injection adjuvant (25 μL) was slowly added dropwise to the immunogen solution; after the adjuvant and the immunogen dilution were fully mixed, the two were mixed for 30 minutes to enable the adjuvant to effectively adsorb the antigen; subsequent operations were carried out according to the animal immunization experiment procedures.

[0075] 4. Group without aluminum adjuvant: The antigen was diluted to the corresponding dose in the above table (75 μg / mouse) in 100 μL of PBS, and 100 μL of immunogen was used. Subsequent operations were carried out according to the animal immunization experiment procedures.

[0076] 5. Subcutaneous injection at 2 - week intervals: The experiment was designed with a 3 - time immunization method, but eye - ball blood collection was performed 7 days after each immunization injection, and partial mouse supernatants were obtained by centrifugation. First, serum titer detection was carried out. 7 days after the last immunization, maximum blood volume was collected by cardiac puncture, and the supernatant was obtained by centrifugation and stored at - 80 °C.

[0077] 6. Detect the serum titer.

[0078] (1) Four mice were immunized, and their serial numbers were A0, A1, A2, and A3. After three immunizations, the serum titers were detected. The detection data are shown in Table 2 below.

[0079] Table 2 Serum Titer Detection Data

[0080]

[0081] In the human body, carisoprodol is converted into its active metabolite meprobamate (meprobamate) by CYP2C19 enzyme through N-dealkylation, and its metabolite is the small molecule carisoprodol. Antigen competition assays were performed using the carisoprodol prototype meprobamate and the small molecule metabolite carisoprodol respectively. Indirect ELISA and competitive ELISA were used to detect the serum titers of immunized mice with carisoprodol-BSA as the coated antigen. For the indirect ELISA method, 50 μl of the coated antigen diluted with coating buffer at 1 μg / ml was added to each well of the microtiter plate, and after coating overnight at 4°C, the plate was washed 3 times with washing buffer (PBST) (the same below). 200 μl of blocking solution (5% skim milk powder) was added to each well and incubated in a 37°C incubator for 2 h. After taking out and washing, 50 μl of diluted serum was added to each well, and then reacted in a 37°C incubator for 30 min. After washing, 50 μl of goat anti-mouse IgG-HRP solution was added and reacted in a 37°C incubator for 30 min. After washing, 100 μl of substrate solution was added and incubated in a 37°C incubator in the dark for 10 min. Finally, 50 μL of 2 mol / L H2SO4 was added to terminate the reaction, and the A450 value was read on an enzyme-linked immunosorbent assay reader. The titers of orbital blood of the four mice after the third immunization were all >62500.

[0082] The process of indirect competitive ELISA is mostly the same as that of indirect ELISA. The difference is that after blocking with the blocking solution and washing the microtiter plate, 50 μl of diluted meprobamate and carisoprodol standard solutions are added, and then 50 μl of diluted antibody is added, and the remaining steps are the same. For the competitive detection of 50 ng / ml of the small molecule metabolite carisoprodol, more than 50% can be achieved at 1:12500, and fusion can be arranged.

[0083] Immune spleen cells were fused with the 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 positive screening by ELISA and subcloning; for the selected positive monoclonal, ascites was taken, and the antibody was purified with a ProteinA / G antibody purification column. After detection, the purity of the purified antibody was >90%.

[0084] Example 2 Detection of the Binding Activity of Carisoprodol by Enzyme-Linked Reaction ELISA

[0085] 1. Detection Method for IgG Antibody Titer

[0086] (1) Coating of the bottom plate: Dilute the antigen used to 3 μg / ml with coating dilution buffer, add 100 μl of the prepared coating solution to each well, place it in a refrigerator at 4 °C for 24 h.

[0087] (2) After 24 h, take it out of the refrigerator and equilibrate at 37 °C for 30 min, then discard the liquid in the wells; wash the wells full with washing solution 3 times, 3 min for each time.

[0088] (3) Blocking 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 full with washing solution 3 times, 3 min for each time.

[0089] (4) Adding the sample to be detected: Dilute the sample according to the required ratio, add the diluted sample to the enzyme-labeled reaction wells, 100 μl for each well, place it at 37 °C for 90 min; wash the wells full with washing solution 3 times, 3 min for each time.

[0090] (5) Adding the enzyme-labeled antibody: Add the appropriate concentration of secondary antibody (goat anti-mouse secondary antibody labeled with HRP) according to the instruction manual; within 37 °C and 90 min, add 100 μl to each well and wash as before.

[0091] (6) Adding the substrate solution: Add 100 μl of the substrate to each well and place it at 37 °C in the dark for 15 - 30 min.

[0092] (7) Terminating the reaction: Add 50 μl of the termination solution to each well to terminate the reaction, and measure the experimental results within 20 min.

[0093] Use the purified antibody obtained in Example 1 to perform ELISA titer detection according to the above steps, and the result obtained is ELISA titer > 1:128,000.

[0094] Example 3 Detection of the Binding Activity of Monoclonal Antibody to Carisoprodol

[0095] (1). Cell Fusion and Clone Screening

[0096] The mouse numbers are successively A0, A1, A2, A3, and a total of four rounds of fusions were completed.

[0097] A total of 16 positive wells were selected for subcloning from the A0 mouse fusion screening, and finally 4 cell lines were obtained. After fusion screening, a total of 72 positive clones with OD450 values > 2.2 were selected for serial dilution to detect the titer, and then the second subcloning screening was carried out. Four cell lines were obtained and named A0-1 to A0-4 respectively.

[0098] A total of 4 positive wells were selected for subcloning from the A1 mouse fusion screening. After fusion screening, a total of 4 positive wells with OD450 values > 2.1 were selected for serial dilution to detect the titer, and then the second and third subcloning screenings were carried out. Finally, 1 cell line was obtained and named A1-1.

[0099] A total of 20 positive wells were selected for subcloning from the A2 mouse fusion screening, and then the second and third subcloning screenings were carried out. Finally, 4 cell lines were obtained and named A2-1 to A2-4 respectively.

[0100] A total of 8 positive wells were selected for subcloning from the A3 mouse fusion screening, and then the second and third subcloning screenings were carried out. Finally, 1 cell line was obtained and named A3-1.

[0101] A total of 10 cell lines were obtained after four cell fusions.

[0102] (2) Ascites preparation and test data

[0103] Three F1 mice were injected with each established cell line, and a total of 10 ascites were prepared. The titer test data of all ascites are shown in Table 3 below:

[0104] Table 3 Titer test of ascites

[0105]

[0106]

[0107] (3) Exploration of antibody purification conditions and test data

[0108] The above ascites were purified by 3.3% n-octanoic acid-thiamine precipitation method, and a total of 10 antibodies were obtained. The titer test data of all antibodies are shown in the following table:

[0109] Table 4 Titer test of antibodies

[0110]

[0111]

[0112] The above data show that the monoclonal antibodies of 10 cell lines have good specific binding ability to carisoprodol antigen. Since the carisoprodol colloidal gold product detects urine samples, a competition experiment was conducted using the small molecule metabolite carisoprodol. It was found that antibodies A0-1 and A3-1 have a competitive effect on the small molecule metabolite carisoprodol of carisoprodol. Therefore, antibodies A0-1 and A3-1 were used for the test of the carisoprodol colloidal gold product.

[0113] Example 4 Application of Monoclonal Antibody in Kit

[0114] In this example, taking the immunocolloidal gold detection method as an example, antibodies A0-1 and A3-1 against carisoprodol were verified through the immunocolloidal gold platform.

[0115] Specifically, the experimental group was to detect the carisoprodol standard product carisoprodol of Hangzhou Anxu Biotechnology Co., Ltd. using carisoprodol antibodies A0-1 and A3-1 obtained from the above experiment. The negative control was a urine sample. The above reagents were loaded, and then the POCT detection instrument ACG1000 (ID-A003) of Hangzhou Anxu Biotechnology Co., Ltd. was used to detect the results. The experimental data results are shown in Table 5.

[0116] Table 5 Preliminary Evaluation Results of Antibodies A0-1 / A3-1 in Immunocolloidal Gold Method

[0117]

[0118] Note: G4-G8 represent the levels of the strip color depth of the test strip. The higher the value, the darker the color. + / - indicates slightly darker or lighter than the color of this level. The decrease in the value of the added carisoprodol standard product carisoprodol indicates the competitive effect of the small molecule, and the antibody can bind to the small molecule metabolite carisoprodol.

[0119] The results show that the gradient of antibody A0-1 is good and can be used in carisoprodol products. The cut-off value is set at 200 ng / ml. Then, the stability evaluation of antibody A0-1 was carried out. Three batches of antibody samples were prepared, and the evaluation results are as follows:

[0120] Table 6 Stability Evaluation Results of Antibody A0-1

[0121]

[0122] According to the evaluation results of the product, antibody A0-1 was named anti-carisoprodol-mab1, and this antibody can be used for the detection of the carisoprodol antigen detection kit.

[0123] Example 5 Sequence Analysis of the Heavy Chain Variable Region (VH) and Light Chain Variable Region (VL) of Monoclonal Antibody anti-carisoprodol-mab1

[0124] The primers for amplifying the heavy chain V region (VH) and light chain V region (VL) genes are as follows:

[0125] Forward primer for the heavy chain variable region (VH-FOR):

[0126] GGGAATTCGAGGTGCAGCTGCAGGAGTCTGG (SEQ ID NO:15)

[0127] Reverse primer for the heavy chain variable region (VH-BACK):

[0128] GGAAGGTGTGCACACCGCTGGAC (SEQ ID NO:16)

[0129] Forward primer for the light chain variable region (VL-FOR): GATGGTGGGAAGATGGATACAGTT (SEQ ID NO:17)

[0130] Reverse primer for the light chain variable region (VL-BACK): ATGGAATCACAGRCYCWGGT (SEQ ID NO:18)

[0131] Take the hybridoma cell line in the logarithmic growth phase of anti-carisoprodol-mab1 (about 107 cells), extract the total RNA of the cells according to the instructions of the Trizol RNA extraction kit, reverse transcribe the first strand of cDNA using the total RNA as a template, and PCR amplify the VH / VL genes of the antibody using the above amplification products as templates.

[0132] Recover the heavy chain VH (about 360bp) and light chain VL (about 300bp) fragments of anti-carisoprodol-mab1 and send them to the company for sequencing.

[0133] Then analyze the VH / VL gene sequences:

[0134] The obtained sequences are as follows:

[0135] Variable region sequence of the heavy chain:

[0136] anti-carisoprodol-mab1-VH: 360bp

[0137] GAGGTGAAACTGCAGCAGTCTGGAGGAGGCTTGGTGCAACCTGGA GGATCCATGAAACTCTCCTGTGTTGCCTCTGGATTCACTTTCAGTAACTACTGGATGAACTGGGTCCGCCAGTCTCCAGAGAAGGGACTTGAGTGGGTTGCTGAAATTAGATTGAAATTTAATAATTATGCAACACATTATTCGGAGTCTGTGAAAGGGAGGTTCATCATCTCAAGAGATGATTCCAAAAGTAGTGTCTACCTGCAAATGAACAACTTAAGAGCTGAAGACACTGGCATTTATTACTGTACCAGTCTACTATGGTTACGACGGGACTGGTACTTCGATGTCTGGGGCGCAGGAACCACGGTCACC(SEQ ID NO:9)

[0138] anti-carisoprodol-mab1 protein: 120aa

[0139] EVKLQQSGGGLVQPGGSMKLSCVASGFTFSNYWMNWVRQSPEKGLE WVAEIRLKFNNYATHYSESVKGRFIISRDDSKSSVYLQMNNLRAEDTGIYYC TSLLWLRRDWYFDVWGAGTTVT(SEQ ID NO:7)

[0140] Variable region sequence of the light chain:

[0141] anti-carisoprodol-mab1LVκ: 297bp

[0142] ACCCAGTCTCCATCTTCCATGTATGCATCTCTAGGAGAGAGAGTCA CTCTCACTTGCAAGGCGAGTCAGGACATTAATGGCTATTTAAGTTGGTTCCAGCAGAAACCAGGGAAACCTCCTAAGACCCTGATTTATCGTGCAAACAGATTGGTAGATGGGGTCCCATCAAGGTTCAGTGTCAGTGGATCTGGACAAGATTATTCTCTCACCATCAGCAGCCTGGAGTATGAAGATTTGGGAATTTATTATTGTCTACAGTCTGATGAGTTTCCGTACACGTTCGGAGGGGGGACCAAG(SEQ ID NO:10)

[0143] anti-carisoprodol-mab1LVκ protein: 99aa

[0144] TQSPSSMYASLGERVTLTCKASQDINGYLSWFQQKPGKPPKTLIYRAN RLVDGVPSRFSVSGSGQDYSLTISSLEYEDLGIYYCLQSDEFPYTFGGGTK(SE Q ID NO:8)

[0145] A monoclonal antibody against carisoprodol or an antigen-binding fragment thereof and related applications disclosed by the present invention bring beneficial effects including but not limited to: (1) The novel monoclonal antibody against carisoprodol provided by the present invention has high titer and high specificity; (2) The monoclonal antibody against carisoprodol provided by the present invention can be used in immunological detections such as immunoblotting and immunofluorescence. For example, it can be used to detect the content of carisoprodol in a sample through a colloidal gold detection platform, with a minimum detection limit of about 200 ng / mL and high detection sensitivity. It should be noted that the beneficial effects that may be produced by different embodiments are different. In different embodiments, the beneficial effects that may be produced can be any one or several combinations of the above, or any other beneficial effects that may be obtained.

[0146] Those skilled in the art should understand that the above embodiments are only for illustrating the present invention and do not constitute a limitation to the present invention. Any modifications, equivalent replacements, and changes made within the spirit and principle of the present invention should be included within the protection scope of the present invention.

Claims

1. An antibody or antigen-binding fragment thereof that binds to carisoprodol, characterized in that, The antibody or its antigen-binding fragment comprises a heavy-chain variable region and a light-chain variable region, wherein: The heavy-chain variable region comprises: the amino acid sequence of CDRH1 as shown in SEQ ID NO: 1, the amino acid sequence of CDRH2 as shown in SEQ ID NO: 2, and the amino acid sequence of CDRH3 as shown in SEQ ID NO: 3; The light-chain variable region comprises: the amino acid sequence of CDRL1 as shown in SEQ ID NO: 4, the amino acid sequence of CDRL2 RAN, and the amino acid sequence of CDRL3 as shown in SEQ ID NO:

6.

2. The antibody or antigen-binding fragment thereof according to claim 1, wherein The heavy-chain variable region consists of any one of the following: The amino acid sequence as shown in SEQ ID NO: 7; An amino acid sequence having at least 85% identity in the framework region compared with the amino acid sequence as shown in SEQ ID NO: 7; and An amino acid sequence having one or more amino acid additions, deletions and / or substitutions in the framework region compared with the amino acid sequence as shown in SEQ ID NO:

7.

3. The antibody or antigen-binding fragment thereof according to claim 2, wherein The light-chain variable region consists of any one of the following Comprises: The amino acid sequence as shown in SEQ ID NO: 8; An amino acid sequence having at least 85% identity in the framework region compared with the amino acid sequence as shown in SEQ ID NO: 8; and An amino acid sequence having one or more amino acid additions, deletions and / or substitutions in the framework region compared with the amino acid sequence as shown in SEQ ID NO:

8.

4. The antibody or antigen-binding fragment thereof according to claim 3, wherein The ELISA titer of the binding ability of the antibody to the carisoprodol is greater than 1:128,000.

5. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the antibody or its antigen-binding fragment according to any one of claims 1-4.

6. The nucleic acid molecule according to claim 5, wherein The nucleic acid molecule comprises the nucleotide sequence as shown in SEQ ID NO: 9 encoding the heavy-chain variable region and the nucleotide sequence as shown in SEQ ID NO: 10 encoding the light-chain variable region.

7. An expression vector, characterized in that, The expression vector comprises the nucleic acid molecule according to claim 5.

8. A host cell, characterized in that, The host cell comprises the expression vector according to claim 7.

9. A kit for detecting carisoprodol, characterized in that, The kit comprises the antibody or its antigen-binding fragment according to any one of claims 1-4.

10. The kit according to claim 9, characterized in that, The kit is a kit for immunogold method.

11. The kit according to claim 10, wherein, The minimum detection limit of the kit for detecting carisoprodol is 200 ng / mL.

12. Use of the antibody or its antigen-binding fragment according to any one of claims 1-4 in the preparation of a kit for detecting carisoprodol.

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