Monoclonal antibody against trihexylamine, and application and product thereof

Abstract

The invention provides an anti-trihexylol monoclonal antibody as well as application and a product thereof, and relates to the technical field of biology. The method comprises the following steps: immunizing a Balb / c mouse by using trihexylamine, fusing splenocytes of the mouse with myeloma cells, carrying out specific high-throughput screening to obtain hybridoma cells with high specificity, and carrying out culture and re-immunization to obtain a large amount of mouse ascites. And the monoclonal antibody resisting the trihexylamine 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 the trihexylamine. Through verification, the monoclonal antibody for resisting the trihexylamine disclosed by the invention has good specific binding capacity.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to an anti-benzylhexol monoclonal antibody and its applications and products. Background Technology

[0002] Benzohexol is an anticholinergic drug with the chemical formula C64. 20 H 31 NO, the structural formula is as follows Figure 1 As shown, it is primarily used to treat Parkinson's disease and drug-induced extrapyramidal reactions. Due to its narrow therapeutic window, overdose can lead to serious adverse reactions (such as hallucinations, cognitive impairment, and arrhythmias), therefore precise monitoring of blood drug concentrations is necessary to optimize treatment regimens. Furthermore, benztropine also has important detection requirements in forensic toxicology and drug abuse screening.

[0003] Currently, the main methods for detecting benztropine include high-performance liquid chromatography (HPLC), gas chromatography-mass spectrometry (GC-MS), and liquid chromatography-tandem mass spectrometry (LC-MS / MS). While these methods are accurate, they suffer from drawbacks such as expensive equipment, complex operation, and long detection cycles, making it difficult to meet the needs of rapid clinical testing. Immunoassays (such as ELISA) offer advantages such as ease of operation, low cost, and high throughput; however, existing benztropine antibodies suffer from insufficient specificity and low sensitivity, limiting their application in clinical and forensic testing.

[0004] Therefore, developing a highly specific and sensitive anti-benzylhexol monoclonal antibody and establishing a rapid detection method based on this antibody (such as immunochromatographic test strips, ELISA, chemiluminescent immunoassay, etc.) has significant clinical application and market value.

[0005] In view of this, the present invention is hereby proposed. Summary of the Invention

[0006] The primary objective of this invention is to provide an anti-benzylhexol monoclonal antibody to solve the aforementioned technical problems.

[0007] A second objective of this invention is to provide biological materials.

[0008] A third objective of this invention is to provide a method for preparing the above-mentioned anti-benzylhexyl monoclonal antibody.

[0009] A fourth objective of this invention is to provide the application of the above-mentioned anti-benzhexol monoclonal antibody in the preparation of benzhexol detection products.

[0010] The fifth objective of this invention is to provide a benzylhexyl marker.

[0011] The sixth objective of this invention is to provide a kit for the detection of benztropine.

[0012] To achieve the above objectives, the following technical solution is adopted:

[0013] In a first aspect, the present invention provides an anti-benzylhexyl monoclonal antibody, wherein the variable region of the anti-benzylhexyl monoclonal antibody includes: a complementarity-determining region CDR1-VH of the amino acid sequence shown in SEQ ID NO.1, a complementarity-determining region CDR2-VH of the amino acid sequence shown in SEQ ID NO.2, a complementarity-determining region CDR3-VH of the amino acid sequence shown in SEQ ID NO.3, a complementarity-determining region CDR1-VL of the amino acid sequence shown in SEQ ID NO.4, a complementarity-determining region CDR2-VL of the amino acid sequence shown in SEQ ID NO.5, and a complementarity-determining region CDR3-VL of the amino acid sequence shown in SEQ ID NO.6.

[0014] As a further technical solution, the amino acid sequence of the heavy chain variable region VH of the anti-benzylhexyl monoclonal antibody is shown in SEQ ID NO.7;

[0015] The amino acid sequence of the light chain variable region VL of the anti-benzylhexyl monoclonal antibody is shown in SEQ ID NO.8;

[0016] As a further technical solution, the anti-benzylhexol monoclonal antibody is an IgG antibody.

[0017] Secondly, the present invention provides a biomaterial selected from any one of the following:

[0018] a. Nucleotides, said nucleotides comprising a nucleotide sequence encoding the anti-benzylhexol monoclonal antibody;

[0019] b. A carrier carrying the nucleotides in a;

[0020] c. A cell carrying the nucleotides in a, or containing the vector in b, or expressing the anti-benzylhexol monoclonal antibody.

[0021] Thirdly, the present invention provides a method for preparing the above-mentioned anti-benzylhexyl monoclonal antibody, which is obtained by cell fermentation.

[0022] Fourthly, the present invention provides the application of the above-mentioned anti-benzylhexyl monoclonal antibody in the preparation of benzylhexyl detection products.

[0023] Fifthly, the present invention provides a benztropine marker, comprising the aforementioned anti-benztropine monoclonal antibody and the marker;

[0024] The anti-benzylhexol monoclonal antibody and marker were conjugated.

[0025] As a further technical solution, the labeling material includes enzymes, fluorescent molecular labels, fluorescent microspheres, colored microspheres, colloidal gold, biotin, or streptavidin.

[0026] In a sixth aspect, the present invention provides a kit for the detection of benztropine, the kit comprising the aforementioned anti-benztropine monoclonal antibody or the aforementioned benztropine marker.

[0027] As a further technical solution, the kit includes an immunochromatographic detection kit, an ELISA detection kit, an immunomagnetic microparticle detection kit, an immunofluorescence detection kit, or an immunoblotting detection kit.

[0028] Compared with the prior art, the present invention has the following beneficial effects:

[0029] This invention utilizes trihexyphenidyl to immunize Balb / c mice, fusing mouse spleen cells with myeloma cells. Highly specific hybridoma cells are obtained through specific high-throughput screening. Large quantities of mouse ascites are obtained through culture and re-immunization. High-purity, high-sensitivity, and high-specificity anti-trihexyphenidyl monoclonal antibodies are then obtained through multi-step separation and purification, providing the necessary raw materials for developing immunoassay strips for the detection of trihexyphenidyl. The anti-trihexyphenidyl monoclonal antibody of this invention has been verified to have excellent specific binding ability. Attached Figure Description

[0030] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0031] Figure 1 The molecular structure of benzylhexyl;

[0032] Figure 2 The results are from the Hysso colloidal gold test strip. Detailed Implementation

[0033] The embodiments and examples of the present invention will be described in detail below. However, those skilled in the art will understand that the following embodiments and examples are for illustrative purposes only and should not be considered as limiting the scope of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention. Unless otherwise specified, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0034] Generally, the nomenclature and techniques used in cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization, together with those described herein, are those well-known and commonly used in the art. Unless otherwise stated, the methods and techniques of the present invention are generally carried out according to conventional methods well-known in the art and described in various general and more specific references, which are cited and discussed throughout this specification. Enzymatic reactions and purification techniques are carried out according to the manufacturer's instructions, as commonly practiced in the art, or as described herein. The nomenclature, laboratory procedures, and techniques used in analytical chemistry, synthetic organic chemistry, and medical and medicinal chemistry, together with those described herein, are those well-known and commonly used in the art.

[0035] The "variable region" or "variable domain" of an antibody refers to the domain at the amino terminus of the antibody's heavy or light chain that recognizes and binds to antigens. The composition and arrangement of the amino acids in this region determine the antibody's specificity in recognizing antigens. The heavy chain variable region can be called "VH," and the light chain variable region can be called "VL." These domains are usually the most variable parts of the antibody and contain antigen-binding sites. The variable regions of both the heavy and light chains consist of three complementarity-determining regions (CDRs) (also known as hypervariable regions) connected by four framework regions (FRs). The CDRs in each chain are held tightly together by the FRs to form the variable region. Typically, the VL / VH variable regions of the heavy and light chains are obtained by connecting the following numbered CDRs with FRs in the following combination: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0036] The term "vector" refers to a nucleic acid delivery vehicle into which nucleotides can be inserted. When a vector enables the expression of a protein encoded by the inserted polynucleotide, it is called an expression vector. Vectors can be introduced into host cells through transformation, transduction, or transfection, allowing the genetic material they carry to be expressed in the host cells.

[0037] 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.).

[0038] In a first aspect, the present invention provides an anti-benzylhexyl monoclonal antibody, wherein the variable region of the anti-benzylhexyl monoclonal antibody includes: a complementarity-determining region CDR1-VH of the amino acid sequence shown in SEQ ID NO.1, a complementarity-determining region CDR2-VH of the amino acid sequence shown in SEQ ID NO.2, a complementarity-determining region CDR3-VH of the amino acid sequence shown in SEQ ID NO.3, a complementarity-determining region CDR1-VL of the amino acid sequence shown in SEQ ID NO.4, a complementarity-determining region CDR2-VL of the amino acid sequence shown in SEQ ID NO.5, and a complementarity-determining region CDR3-VL of the amino acid sequence shown in SEQ ID NO.6.

[0039] The amino acid sequences of SEQ ID NO.1-SEQ ID NO.6 are shown in Table 1.

[0040] Table 1

[0041] High-variable area sequence serial number CDR1-VH SYCIV SEQ ID NO.1 CDR2-VH WIYPGNINTKYREKFKG SEQ ID NO.2 CDR3-VH SYIGASVD SEQ ID NO.3 CDR1-VL KVSQSLLYSYGKIILN SEQ ID NO.4 CDR2-VL QVSLLDS SEQ ID NO.5 CDR3-VL RQVSSIPLL SEQ ID NO.6

[0042] In some optional embodiments, the amino acid sequence of the heavy chain variable region VH of the anti-benzylhexyl monoclonal antibody is shown in SEQ ID NO.7:

[0043] EVQLQQSGPELVKPGLSVRISCKASYYTFTSYCIVWVRQRPGQGLE WIGWIYPGNINTKYREKFKGKATLTADKSSSTAYMQLSSLTYEDSAVYFC TRSYIGASVDWGQGTSVTVSS(SEQ ID NO.7);

[0044] The amino acid sequence of the light chain variable region VL of the anti-benzylhexyl monoclonal antibody is shown in SEQ ID NO. 8:

[0045] DIVLTQSPQTLSVTIGQAASISCKVSQSLLYSYGKIILNWLLQRPGQS PKRLIYQVSSLLDSGVPDKFAGSGSGTDFTLKISRMEAEDVATYYCRQVSS IPLLFGAGTKLELK (SEQ ID NO. 8).

[0046] In some optional embodiments, the nucleic acid sequence of the heavy chain variable region VH of the anti-benzylhexyl monoclonal antibody is shown in SEQ ID NO.9:

[0047] GAAGTGCAGCTGCAACAGAGTGGCCCAGAACTTGTAAAACCTGGCCTTAGTGTCAGGATATCATGTAAAGCCTCCTATTACAACCTTTACCAGTTACTGTATCGTTTGGGTTAGACAGCGCCCTGGCCAAGGACTGGAGTGGATCGGGTGGATCTACCCAGGCAACATCAACACCAAG TACAGAGAGAAGTTCAAAGGCAAGGCAACCCTTACAGCTGACAAATCAAGTCAACAGCTTACATGCAGCTGTCTTCTCTGACATACGAGGATAGCGCCGTGTACTTCTGTACCAGGTCCTACATCGGAGCCTCCGTTGATTGGGGACAGGGCACTAGCGTGACGGTGTCATCC(SEQ ID NO.9);

[0048] The nucleotide sequence of the light chain variable region VL of the anti-benzylhexyl monoclonal antibody is shown in SEQ ID NO.10:

[0049] GATATTGTTTTGACTCAATCCCCAAAACACTGAGCGTTACCATTGGGCAAGCCGCCAGCATCAGCTGTAAGGTATCTCAATCACTGCTCTATAGCTATGGGAAGATCATACTGAACTGGTTGCTGCAGCGGCCGGGGCAGTCCCCGAAAAGACTGATCTATCAGGTTTC CCTCCTCGACTCCGGTGTGCCTGACAAGTTCGCAGGCTCAGGCAGCGGAACTGACTTTACCCTCAAGATCTCCAGAATGGAAGCCGAAGATGTGGCCACATACTATTGTAGGCAGGTGTCTAGCATCCCTTTGCTCTTTTGGAGCCGGCACCAAACTCGAGCTCAAG(SEQ ID NO.10).

[0050] In some alternative embodiments, the anti-benzylhexol monoclonal antibody is an IgG antibody.

[0051] Secondly, the present invention provides a biomaterial selected from any one of the following:

[0052] a. Nucleotides, said nucleotides comprising a nucleotide sequence encoding the anti-benzylhexol monoclonal antibody;

[0053] b. A carrier carrying the nucleotides in a;

[0054] c. A cell carrying the nucleotides in a, or containing the vector in b, or expressing the anti-benzylhexol monoclonal antibody.

[0055] Thirdly, the present invention provides a method for preparing the above-mentioned anti-benzylhexyl monoclonal antibody, which is obtained by cell fermentation.

[0056] The preparation method is simple and efficient, and a large amount of anti-benzylhexol monoclonal antibody can be obtained through fermentation.

[0057] Fourthly, the present invention provides the application of the above-mentioned anti-benzylhexyl monoclonal antibody in the preparation of benzylhexyl detection products.

[0058] The anti-benzylhexol monoclonal antibody provided by this invention can specifically recognize benzylhexol, and therefore can be used for the detection of benzylhexol.

[0059] Fifthly, the present invention provides a benztropine marker, comprising the aforementioned anti-benztropine monoclonal antibody and the marker;

[0060] The anti-benzylhexol monoclonal antibody and marker were conjugated.

[0061] This marker can be used for the specific labeling of benztropine.

[0062] In some alternative embodiments, the labeling material includes, but is not limited to, enzymes, fluorescent molecular labels, fluorescent microspheres, colored microspheres, colloidal gold, biotin, or streptavidin.

[0063] In a sixth aspect, the present invention provides a kit for the detection of benztropine, the kit comprising the aforementioned anti-benztropine monoclonal antibody or the aforementioned benztropine marker.

[0064] This kit can be used for the detection of benztropine.

[0065] In some alternative implementations, the kit includes, but is not limited to, immunochromatographic assay kits, ELISA kits, immunomagnetic microparticle assay kits, immunofluorescence assay kits, or immunoblotting assay kits.

[0066] The present invention will be further illustrated below with specific embodiments. However, it should be understood that these embodiments are merely for the purpose of more detailed illustration and should not be construed as limiting the present invention in any way.

[0067] Example 1: Preparation of monoclonal antibody anti-Benzhexol-mab1

[0068] I. Preparation of Benzohexol-BGG Antigen

[0069] 1. Prepare a 6 mol / L hydrochloric acid solution by taking 100 mg of benztropine (structural formula as shown). Figure 1 (As shown) Dissolve in hydrochloric acid solution and react overnight in an oil bath at 50°C.

[0070] 2. After the reaction is complete, evaporate the solvent to dryness, add an appropriate amount of water and 4N sodium hydroxide solution to adjust the pH to neutral or weakly acidic.

[0071] 3. Add 20 ml of dichloromethane and extract three times. Collect the organic phase, add anhydrous sodium sulfate to dry, filter and evaporate the organic phase to obtain a light yellow viscous liquid—benzylhexyl hapten.

[0072] 4. Take 20 mg of benzylhexyl hapten, 8 mg of NHS (N-hydroxysuccinimide), and 13.4 mg of EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide) and dissolve them in DMF (N,N-dimethylformamide). Place the mixture on a turntable and react at room temperature overnight.

[0073] 5. Weigh 100 mg of BGG (bovine immunoglobulin) and dissolve it in 10 ml of PBS buffer. Stir well to obtain a protein solution with a concentration of 10 mg / ml.

[0074] 6. Under ice bath conditions, the benztropine hapten solution was slowly added dropwise to the protein solution. The resulting mixture was placed on a turntable at room temperature for 8 hours to obtain the artificial antigen mixture.

[0075] 7. Transfer the artificial antigen mixture into a dialysis bag and dialyze it 7 times with PBS buffer. After dialysis, centrifuge at 10000 r / min for 10 minutes, discard the precipitate, and take the supernatant to obtain the benztropine artificial antigen. Use the chemically synthesized benztropine antigen to perform phased immunization on experimental mice.

[0076] II. Preparation of monoclonal antibody anti-Benzhexol-mab1.

[0077] Generally, healthy female Balb / c mice aged 6-8 weeks are selected and immunized according to a pre-specified immunization protocol. These mice are then immunized with immunogens, and spleen lymphocytes are extracted from the successfully immunized mice. These lymphocytes are then fused with mouse myeloma cells SP2 / 0 using cell fusion technology. After two rounds of subclonal selection, a hybridoma cell line stably secreting anti-benzylhexol monoclonal antibody is obtained, thus yielding the anti-benzylhexol monoclonal antibody.

[0078] Chemically synthesized benztropine hapten was used to immunize experimental mice in stages.

[0079] The specific steps of animal immunization experiments include:

[0080] 1. Balb / c mice with the same weight and age were randomly divided into two groups: an aluminum adjuvant group (aluminum hydroxide adjuvant) and a non-aluminum adjuvant group.

[0081] 2. Before the experiment, pre-immunization serum was collected from each mouse and stored at -80℃.

[0082] 3. Preparation of aluminum adjuvant (aluminum hydroxide adjuvant) group: Before immunization, each antigen was diluted in 75 μL PBS to the corresponding dose (75 μg / mouse) and mixed with alum adjuvant (1 mg / mouse) at a volume ratio of antigen:adjuvant = 3:1 (i.e., 25 μl adjuvant was added to 75 μl of immunogen dilution). The adjuvant was shaken well before use, and the 25 μl adjuvant was slowly added dropwise to the immunogen solution. After thorough mixing of the adjuvant and immunogen dilution, the two solutions were allowed to mix thoroughly for 30 minutes to allow for effective adsorption of the antigen. Subsequent procedures were performed according to the animal immunization experiment procedures.

[0083] 4. Group without aluminum adjuvant: The antigen was diluted in 100 μL PBS to the corresponding dose (75 μg / mouse), and subsequent procedures were performed according to the animal immunization experiment procedure.

[0084] 5. Subcutaneous injection at 2-week intervals: The experiment was designed with 4 immunizations, but blood was collected from the eyeballs 4 days after each immunization injection. The supernatant of the mice was obtained by centrifugation and the serum titer was tested first. Seven days after the last immunization, the maximum blood volume was collected from the heart, and the supernatant was obtained by centrifugation and stored at -80℃.

[0085] 6. Detect serum titer.

[0086] (1) Two mice were immunized, numbered A0 and A1 respectively. After three immunizations, serum titers were measured. The data are shown in Table 2 below.

[0087] Table 2: Serum titer test data

[0088]

[0089] Antigen competition detection was performed using benztropine antigen (i.e., benztropine-BGG antigen) and benztropine small molecules, respectively. Indirect ELISA and competitive ELISA were performed using benztropine antigen as the coating antigen to detect the serum titer of immunized mice. For the indirect ELISA method, 50 μl of coating antigen diluted with coating buffer (1 μg / ml) was added to each well of the ELISA plate. After coating overnight at 4°C, the plate was washed 3 times with washing buffer (PBST). 200 μl of blocking buffer (5% skim milk powder) was added to each well, and the plate was incubated at 37°C for 2 h. After washing, 50 μl of diluted serum was added to each well, and the plate was incubated at 37°C for 30 min. After washing, 50 μl of goat anti-mouse IgG-HRP solution was added, and the plate was incubated at 37°C for 30 min. After washing, add 100 μl of substrate solution and incubate at 37 °C in the dark for 10 min to develop color. Finally, add 50 μL of 2 mol / L H2SO4 to terminate the reaction and read the A450 value using a microplate reader. The orbital blood titers of both mice after the third immunization were >312500.

[0090] The indirect competitive ELISA procedure is largely the same as the indirect ELISA procedure, except that blocking buffer is used. After washing the ELISA plate, 50 μl of diluted 2000 ng / mL trihexyphenidyl standard solution is added, followed by 50 μl of diluted serum antibody, to achieve a final concentration of 1000 ng / mL. The remaining steps are the same. At a dilution of 1:62500, the concentration of 1000 ng / mL trihexyphenidyl in competitive detection can reach over 80%, allowing for fusion assays.

[0091] Mouse immune spleen cells were fused with myeloma cell line SP2 / 0. Fusion cells were screened using HAT selective medium (containing hypoxanthine, aminopterin, and thymine), and ELISA positive screening and subcloning were performed on the fusion cells. Ascites fluid was collected from the selected positive monoclonal cells, and antibodies were purified using a Protein A / G antibody purification column. The purified antibody had an ELISA titer >1:128,000 and a purity >90%.

[0092] III. Enzyme-linked ELISA detection of the binding activity of benztropine.

[0093] 1. IgG antibody titer detection method

[0094] (1) Base plate coating: Dilute the antigen to 3 μg / ml with coating diluent, add 100 μl of the prepared coating solution to each well, and place in a 4℃ refrigerator for 24 h.

[0095] (2) After 24 hours, remove it from the refrigerator and place it at 37°C for 30 minutes to equilibrate. Then discard the liquid in the hole; wash the hole with washing solution 3 times, 3 minutes each time.

[0096] (3) Blocking enzyme-labeled reaction wells: Add 200 μl of 5% fetal bovine serum to each well and incubate at 37°C for 90 min. After blocking, wash the wells three times with washing buffer for 3 min each time.

[0097] (4) Add the sample to be tested: Dilute the sample according to the required ratio, add 100 μl of the diluted sample to each enzyme-labeled reaction well, place at 37℃ for 90 min; wash the well with washing solution 3 times, 3 min each time.

[0098] (5) Add enzyme-labeled antibody: Add the appropriate concentration of secondary antibody according to the instructions; wash 100 μl per well at 37℃ for 90 min as before.

[0099] (6) Add substrate solution: Add 100 μl of substrate per well and place at 37°C in the dark for 15 to 30 minutes.

[0100] (7) Termination of reaction: Add 50 μl of termination solution to each well to terminate the reaction, and measure the experimental results within 20 min.

[0101] IV. Detection of the binding activity of monoclonal antibodies to benztropine.

[0102] (1) Cell fusion and clone screening data

[0103] The mice were numbered A0 and A1, and a total of one round of fusion was completed.

[0104] A total of 23 complete cell lines were obtained after cell fusion.

[0105] (2) Supernatant preparation and detection data

[0106] Each complete cell line was separately expanded into a large-scale culture.

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

[0108] The cell supernatant was purified using a Protein G column, yielding a total of 23 antibodies. The antibody numbers are 1# to 23#. The titer data for some antibodies are shown in the table below:

[0109] Table 3: Data on purified antibody titer detection

[0110]

[0111]

[0112] Antibody titer testing showed that the monoclonal antibodies from 23 cell lines had good specific binding ability to the benztropine antigen. In a competition experiment using small molecule benztropine, 6 antibodies showed good competition. Therefore, the above antibodies were used for testing benztropine colloidal gold products.

[0113] V. Application of monoclonal antibody anti-Benzhexol-mab1 in products.

[0114] Six antibodies against benztropine were validated using an immunochromatographic colloidal gold platform.

[0115] Immunochromatographic test strips were used for detection. Specifically, in the experimental group, six antibodies against trihexyphenidyl obtained in the above experiment were labeled with gold and placed on the conjugation pad, while trihexyphenidyl antigen was coated on the test line. Trihexyphenidyl standards from Hangzhou Anxu Biotechnology Co., Ltd. were tested, and antibodies #1 and #7 showed good negative results and gradient. The experimental data results are shown in the table below:

[0116] Table 4: Preliminary Evaluation Results of Some Antibodies

[0117] Antibody negative 1 negative 2 Benzohexol 750ng / ml Benzohexol 250ng / ml 1# 9 9- 4- 3.5 7# 9 9 4 3.5

[0118] Note: G3-G10 represent the color intensity levels of the test strips; higher values ​​indicate a darker color, and + / - indicates a slightly darker or lighter color than that level. A decrease in the value after adding benztropine standard indicates that the small molecule has a competitive effect, meaning the antibody can bind to the benztropine standard.

[0119] The results showed that antibody #7 had a good gradient (test strip results as shown). Figure 2 (As shown), it can be used in benztropine detection products, with a cut-off value of 500 ng / ml. Antibody stability was then evaluated, and three batches of antibody samples were prepared. The evaluation results are shown in the table below:

[0120] Table 5: Stability assessment results of three batches of antibodies #9 and #25

[0121] antibody batch number Negative Benzohexol 750ng / ml Benzohexol 250ng / ml 7#-1 9 4 3.5+ 7#-2 9 4 3.5+ 7#-3 9 4 3.5+

[0122] The results showed that the three batches were consistent, indicating that the antibody can be stably used in the benztropine antigen detection kit.

[0123] VI. Sequence analysis of the V region (VH) of the heavy chain and the V region (VL) of the monoclonal antibody anti-Benzhexol-mab1.

[0124] Design primers to amplify the heavy chain V region (VH) and light chain V region (VL) genes.

[0125] Take approximately 10 hybridoma cell lines in the logarithmic growth phase of anti-Benzhexol-mab1. 7 Total RNA was extracted from cells according to the instructions of the Trizol RNA Extraction Kit. The total RNA was used as a template to reverse transcribe and synthesize the first strand of cDNA. The VH / VL gene of the antibody was amplified by PCR using the amplification product as a template.

[0126] The heavy chain VH (approximately 360 bp) and light chain VL (approximately 300 bp) fragments of anti-Benzhexol-mab1 were recovered and sent to the company for sequencing.

[0127] Then, the VH / VL gene sequences were analyzed, and the resulting sequences are as follows:

[0128] Variable region sequence of heavy chain:

[0129] anti-Benzhexol-mab1-VH:351bp.

[0130] GAAGTGCAGCTGCAACAGAGTGGCCCAGAACTTGTAAAACCTGGCCTTAGTGTCAGGATATCATGTAAAGCCTCCTATTACACCTTTACCAGTTACTGTATCGTTTGGGTTAGACAGCGCCCTGGCCAAGGACTGGAGTGGATCGGGTGGATCTACCCAGGCAACATCAACACCAAGTACAGAGAGAAGTTCAAAGGCAAGGCAACCCTTACAGCTGACAAATCAAGCTCAACAGCTTACATGCAGCTGTCTTCTCTGACATACGAGGATAGCGCCGTGTACTTCTGTACCAGGTCCTACATCGGAGCCTCCGTTGATTGGGGACAGGGCACTAGCGTGACGGTGTCATCC(SEQ ID NO.9).

[0131] anti-Benzhexol-mab1 protein: 117 aa.

[0132] EVQLQQSGPELVKPGLSVRISCKASYYTFTSYCIVWVRQRPGQGLE WIGWIYPGNINTKYREKFKGKATLTADKSSSTAYMQLSSLTYEDSAVYFC TRSYIGASVDWGQGTSVTVSS(SEQ ID NO.7).

[0133] Variable region sequence of the light chain:

[0134] anti-Benzhexol-mab1LVκ: 336 bp.

[0135] GATATTGTTTTGACTCAATCCCCAAAACACTGAGCGTTACCATTGGGCAAGCCGCCAGCATCAGCTGTAAGGTATCTCAATCACTGCTCTATAGCTATGGGAAGATCATACTGAACTGGTTGCTGCAGCGGCCGGGGCAGTCCCCGAAAAGACTGATCTATCAGGTTTC CCTCCTCGACTCCGGTGTGCCTGACAAGTTCGCAGGCTCAGGCAGCGGAACTGACTTTACCCTCAAGATCTCCAGAATGGAAGCCGAAGATGTGGCCACATACTATTGTAGGCAGGTGTCTAGCATCCCTTTGCTCTTTTGGAGCCGGCACCAAACTCGAGCTCAAG(SEQ ID NO.10).

[0136] anti-Benzhexol-mab1LVκprotein:112aa.

[0137] DIVLTQSPQTLSVTIGQAASISCKVSQSLLYSYGKIILNWLLQRPGQS PKRLIYQVSSLLDSGVPDKFAGSGSGTDFTLKISRMEAEDVATYYCRQVSS IPLLFGAGTKLELK (SEQ ID NO. 8).

[0138] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A monoclonal antibody against benztropine, characterized in that, The variable region of the anti-benzylhexol monoclonal antibody includes: the complementarity-determining region CDR1-VH of the amino acid sequence shown in SEQ ID NO.1, the complementarity-determining region CDR2-VH of the amino acid sequence shown in SEQ ID NO.2, the complementarity-determining region CDR3-VH of the amino acid sequence shown in SEQ ID NO.3, the complementarity-determining region CDR1-VL of the amino acid sequence shown in SEQ ID NO.4, the complementarity-determining region CDR2-VL of the amino acid sequence shown in SEQ ID NO.5, and the complementarity-determining region CDR3-VL of the amino acid sequence shown in SEQ ID NO.

6.

2. The anti-benzylhexol monoclonal antibody according to claim 1, characterized in that, The amino acid sequence of the heavy chain variable region VH of the anti-benzylhexyl monoclonal antibody is shown in SEQ ID NO.7; The amino acid sequence of the light chain variable region VL of the anti-benzylhexyl monoclonal antibody is shown in SEQ ID NO.

8.

3. The anti-benzylhexol monoclonal antibody according to claim 1, characterized in that, The anti-benzylhexol monoclonal antibody is an IgG antibody.

4. A biomaterial, characterized in that, The biomaterial is selected from any one of ac: a. A nucleotide, wherein the nucleotide comprises a nucleotide sequence encoding the anti-benzylhexol monoclonal antibody according to any one of claims 1-3; b. A carrier carrying the nucleotides in a; c. A cell carrying the nucleotides of a, or containing the vector of b, or expressing the anti-benzylhexol monoclonal antibody according to any one of claims 1-3.

5. The method for preparing the anti-benzylhexol monoclonal antibody according to any one of claims 1-3, characterized in that, It is obtained by cell fermentation as described in claim 4.

6. The use of the anti-benzylhexol monoclonal antibody according to any one of claims 1-3 in the preparation of benzylhexol detection products.

7. A benzylhexyl marker, characterized in that, Includes the anti-benzylhexol monoclonal antibody and marker as described in any one of claims 1-3; The anti-benzylhexol monoclonal antibody and marker were conjugated.

8. The benzylhexyl marker according to claim 7, characterized in that, The labeling agents include enzymes, fluorescent molecular labels, fluorescent microspheres, colored microspheres, colloidal gold, biotin, or streptavidin.

9. A kit for the detection of benztropine, characterized in that, The kit comprises the anti-benzylhexyl monoclonal antibody as described in any one of claims 1-3 or the benzylhexyl marker as described in claim 7 or 8.

10. The reagent kit according to claim 9, characterized in that, The kits include immunochromatographic assay kits, ELISA kits, immunomagnetic microparticle assay kits, immunofluorescence assay kits, or immunoblotting assay kits.

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