Monoclonal antibodies against hyoscine and their use and products

Abstract

The application provides an anti-hyoscine monoclonal antibody and application and product thereof, and relates to the technical field of biology.The application immunizes Balb / c mice with hyoscine, fuses mouse spleen cells with myeloma cells, obtains hybridoma cells with high specificity through specific high-throughput screening, obtains a large amount of mouse ascites through culture and re-immunization, and further obtains high-purity, high-sensitivity and high-specificity anti-hyoscine monoclonal antibodies through multi-step separation and purification, thereby providing raw materials required for developing an immunological test strip for detecting hyoscine.The anti-hyoscine monoclonal antibody anti-hyoscine-mab1 of the application can be used for immunological detection such as immunoblotting and immunofluorescence, and the obtained antibody has good specific binding capacity.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to a monoclonal antibody against hyoscine and application and product thereof. BACKGROUND

[0002] Hyoscine is a tropane alkaloid, mainly existing in Solanaceae plants, and is the main alkaloid in datura stramonium, anisodus tangutorum and stramonium. In clinical practice, hyoscine is mainly used for analgesia, anesthesia, anti-motion sickness, Parkinson's disease, improvement of microcirculation, drug withdrawal, pesticide poisoning and the like, and has a huge market demand.

[0003] In recent years, the application range of hyoscine in clinical treatment has become increasingly wide, and remarkable effects have been achieved. For example, it has important uses in the treatment of respiratory diseases, infectious shock, cardiovascular diseases, digestive system diseases, nervous system diseases and the like. Its adverse reactions generally include dry mouth, thickened secretions, flushed face, restlessness, agitation, hallucinations, delirium, urinary retention, blurred vision, abdominal distension, elevated body temperature and the like; and occasionally, arrhythmia, convulsions, coma and the like. Small doses of hyoscine show sedative effects, and large doses produce hypnotic effects. Therefore, it is necessary to establish a rapid, sensitive and accurate method for detecting hyoscine, which is helpful to determine the abuse of hyoscine.

[0004] At present, the detection of hyoscine mainly relies on methods such as gas chromatography-mass spectrometry (GC-MS), liquid chromatography-quadrupole time-of-flight mass spectrometry (Q-TOFLC / MS), nuclear magnetic resonance hydrogen spectrum (HNMR), nuclear magnetic resonance carbon spectrum (13CNMR), nuclear magnetic resonance fluorine spectrum (19FNMR) and infrared spectrum (IR), but all have the shortcomings of expensive instruments, time-consuming detection and the need for professional technical personnel to operate, and therefore cannot meet the requirements of modern detection for simplicity, rapidness and accuracy.

[0005] Therefore, the present application is proposed. SUMMARY

[0006] The first object of the present application is to provide a monoclonal antibody against hyoscine to solve the problem of detection of hyoscine.

[0007] The second object of the present application is to provide a biological material.

[0008] The third object of the present application is to provide a preparation method of the monoclonal antibody against hyoscine.

[0009] The fourth object of the present application is to provide application of the monoclonal antibody against hyoscine in the preparation of a detection product of hyoscine.

[0010] A fifth object of the present application is to provide a marker for scopolamine.

[0011] A sixth object of the present application is to provide a kit for detection of scopolamine.

[0012] To achieve the above objects, the following technical solutions are adopted:

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

[0014] As a further technical solution, the variable region comprises a heavy chain variable region VH having an amino acid sequence as shown in SEQ ID NO. 6.

[0015] The variable region comprises a light chain variable region VL having an amino acid sequence as shown in SEQ ID NO. 7.

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

[0017] In a second aspect, the present application provides a biological material selected from any one of a-c:

[0018] a. a nucleic acid comprising a nucleic acid sequence encoding the anti-scopolamine monoclonal antibody;

[0019] b. a vector carrying the nucleic acid in a;

[0020] c. a cell carrying the nucleic acid in a, or containing the vector in b, or expressing the anti-scopolamine monoclonal antibody.

[0021] In a third aspect, the present application provides a preparation method of the above anti-scopolamine monoclonal antibody, which is obtained by fermentation of the cell.

[0022] In a fourth aspect, the present application provides a use of the above anti-scopolamine monoclonal antibody in the preparation of a detection product for scopolamine.

[0023] In a fifth aspect, the present application provides a marker for scopolamine, comprising the anti-scopolamine monoclonal antibody and a marker.

[0024] The anti-scopolamine monoclonal antibody and the marker are coupled.

[0025] As a further technical solution, the marker comprises an enzyme, a fluorescent molecule marker, fluorescent microspheres, colored microspheres, colloidal gold, biotin or streptavidin.

[0026] In a sixth aspect, the present application provides a kit for detecting scopolamine, comprising the anti-scopolamine monoclonal antibody or the marker for scopolamine.

[0027] As a further technical solution, the kit comprises an immunochromatographic detection kit, an ELISA detection kit, an immunomagnetic micro-particle detection kit, an immunofluorescence detection kit or an immunoblotting detection kit.

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

[0029] The present application uses scopolamine to immunize Balb / c mice, fuses mouse spleen cells with myeloma cells, and obtains hybridoma cells with high specificity through specific high-throughput screening. A large amount of mouse ascites is obtained through culture and re-immunization, and high-purity, high-sensitivity and high-specificity anti-scopolamine monoclonal antibody anti-hyoscine-mab1 is obtained through multi-step separation and purification, which provides the required raw material for developing an immunological test strip for detecting scopolamine. The anti-scopolamine monoclonal antibody anti-hyoscine-mab1 of the present application can be used for immunoblotting, immunofluorescence and other immunological detection, and the obtained antibody has been verified to have good specific binding capacity. DETAILED DESCRIPTION

[0030] The embodiments of the present application will be described in detail below with reference to the embodiments and examples, but those skilled in the art will understand that the following embodiments and examples are only used to illustrate the present application and should not be regarded as limiting the scope of the present application. Based on the examples in the present application, all other examples obtained by those of ordinary skill in the art without making creative efforts fall within the scope of the present application. If the specific conditions are not specified, the conventional conditions or the conditions recommended by the manufacturer are used. If the manufacturers of the reagents or instruments are not specified, they are all conventional products that can be purchased on the market.

[0031] Generally, the nomenclature used in connection with, and the techniques of, cell and tissue culture, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are those well-known and commonly used in the art. The methods and techniques of the present application are generally performed according to conventional methods well-known in the art and as described in various general and more specific references that are cited throughout the present specification unless otherwise indicated. Enzymatic reactions and purification techniques are performed according to the manufacturer's specifications, as commonly accomplished in the art or as described herein. The nomenclature used in connection with, and the techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well-known and commonly used in the art.

[0032] The "variable region" or "variable domain" of an antibody refers to the domain of the heavy or light chain of the antibody that recognizes and binds an antigen, the segment of the amino acid sequence that determines binding specificity. The heavy chain variable region can be referred to as "VH". The light chain variable region can be referred to as "VL". These domains are generally the most variable parts of an antibody and contain the antigen binding site. Each of the variable regions of the heavy chain and light chain is composed of three complementarity determining regions (CDRs) connected by four framework regions (FRs), also known as hypervariable regions. The CDRs in each chain are held together in close proximity by the FRs and with the CDRs from the other chain to form the variable region, typically, the variable regions of the heavy and light chains, VL / VH, can be arranged in the following order: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0033] The term "vector" refers to a nucleic acid vehicle into which a nucleotide can be inserted. When the vector is capable of directing the expression of the inserted polynucleotide, the vector is called an expression vector. The vector can be introduced into a host cell by transformation, transduction or transfection, so that the host cell assumes the genetic material carried by the vector and expresses it.

[0034] The vectors are well known to those skilled in the art and include, but are not limited to, plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1 -derived artificial chromosomes (PAC); bacteriophages such as lambda phage or M13 phage, and animal viruses, etc. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex viruses), poxviruses, baculoviruses, papillomaviruses, papova viruses. In some embodiments, the vectors described in the present application comprise regulatory elements commonly used in genetic engineering, such as enhancers, promoters, internal ribosome entry sites (IRES), and other expression control elements (such as transcription termination signals, or polyadenylation signals and poly U sequences, etc.).

[0035] In a first aspect, the present application provides an anti-hyoscyamine monoclonal antibody, wherein the variable region of the anti-hyoscyamine monoclonal antibody comprises: a complementarity determining region CDR1-VH having an amino acid sequence as shown in SEQ ID NO. 1, a complementarity determining region CDR2-VH having an amino acid sequence as shown in SEQ ID NO. 2, a complementarity determining region CDR3-VH having an amino acid sequence as shown in SEQ ID NO. 3, a complementarity determining region CDR1-VL having an amino acid sequence as shown in SEQ ID NO. 4, a complementarity determining region CDR2-VL having an amino acid sequence of KVS, and a complementarity determining region CDR3-VL having an amino acid sequence as shown in SEQ ID NO. 5.

[0036] The amino acid sequences of SEQ ID NO. 1-SEQ ID NO. 5 are shown in Table 1.

[0037] Table 1

[0038]

[0039] In some alternative embodiments, the variable region comprises a heavy chain variable region VH having an amino acid sequence as shown in SEQ ID NO. 6.

[0040] VKLQQSGGGLVQPGGSLKVSCATSGFTFSDYYMYWVRQTPEKRLEWVAYISNGGGSTYYPDTVKGRFTISRDNAKNTLYLQMSRLKSEDTAMYYCARHTLYAMDYWGQGTSVTCLL (SEQ ID NO. 6).

[0041] The variable region comprises a light chain variable region VL having an amino acid sequence as shown in SEQ ID NO. 7.

[0042] DIQMTQTPASLSASVGETVTITCRTSGNIHNYLAWYQQKRGRSPQLLVYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVTLTFGAGTKLELK (SEQ ID NO. 7).

[0043] In some alternative embodiments, the anti-scopolamine monoclonal antibody is an IgG antibody.

[0044] In a second aspect, the present application provides a biological material selected from any one of a-c:

[0045] a. a nucleic acid comprising a nucleic acid sequence encoding the anti-scopolamine monoclonal antibody;

[0046] b. a vector carrying the nucleic acid of a;

[0047] c. a cell carrying the nucleic acid of a, or containing the vector of b, or expressing the anti-scopolamine monoclonal antibody.

[0048] In a third aspect, the present application provides a preparation method of the anti-scopolamine monoclonal antibody described above, which is obtained by fermentation of the cell.

[0049] The preparation method is simple and efficient, and a large amount of anti-scopolamine monoclonal antibody can be obtained by fermentation.

[0050] In a fourth aspect, the present application provides a use of the anti-scopolamine monoclonal antibody described above in the preparation of a detection product of scopolamine.

[0051] The anti-scopolamine monoclonal antibody provided by the present application can specifically recognize anti-scopolamine, and thus can be used for detection of scopolamine.

[0052] In a fifth aspect, the present application provides a marker of scopolamine, comprising the anti-scopolamine monoclonal antibody and a marker.

[0053] The anti-scopolamine monoclonal antibody and the marker are coupled.

[0054] The marker can be used for specific labeling of anti-scopolamine.

[0055] In some alternative embodiments, the marker includes but is not limited to an enzyme, a fluorescent molecule marker, a fluorescent microsphere, a colored microsphere, colloidal gold, biotin or streptavidin.

[0056] In a sixth aspect, the present application provides a kit for detecting scopolamine, which comprises the anti-scopolamine monoclonal antibody or the scopolamine label.

[0057] In some alternative embodiments, the kit comprises, but is not limited to, an immunochromatographic detection kit, an ELISA detection kit, an immunomagnetic micro-particle detection kit, an immunofluorescence detection kit or an immunoblotting detection kit.

[0058] The present application is further illustrated by the following specific examples, but it should be understood that these examples are only used to illustrate in more detail and should not be understood as limiting the present application in any form.

[0059] Example 1, discovery of the anti-hyoscine-mab1 monoclonal antibody

[0060] I. Preparation of scopolamine-BSA antigen

[0061] The antigen information is shown in the following table.

[0062] Table 2

[0063]

[0064] The scopolamine and BSA were reacted to synthesize the scopolamine hapten hyoscine-BSA, and the hapten was used to immunize the experimental mice in stages.

[0065] II. Preparation of the anti-hyoscine-mab1 monoclonal antibody

[0066] Generally, 6-8 week old healthy female Balb / c mice are selected and immunized according to the pre-specified immunization scheme. The prepared hapten is used as an immunogen to immunize BALB / c mice, and the spleen lymphocytes of the successfully immunized mice are extracted. The lymphocytes are fused with mouse myeloma cells SP2 / 0 through cell fusion technology, and after two rounds of subcloning screening, a hybridoma cell strain stably secreting anti-scopolamine monoclonal antibody is obtained, thereby obtaining the anti-scopolamine monoclonal antibody.

[0067] The chemically synthesized scopolamine hapten hyoscine-BSA is used to immunize the experimental mice in stages.

[0068] The specific steps of the animal immunization experiment include:

[0069] 1. The Balb / c mice with consistent average body weight and age were randomly divided into two groups, an aluminum adjuvant (aluminum hydroxide adjuvant) group and a non-aluminum adjuvant group.

[0070] 2. Before the experiment, pre-immune serum was collected from each mouse (pre-immune serum was collected on the fifth day by blood collection through the eyeball, and an appropriate amount of blood was collected to ensure the normal condition of the mice). The collected serum was stored at -80℃.

[0071] 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 mixture was allowed to sit for 30 minutes to allow for effective adsorption of the antigen. Subsequent procedures were performed according to the animal immunization experiment procedures.

[0072] 4. Group without aluminum adjuvant: The antigen was diluted in 100 μL PBS to the corresponding dose in the table above (75 μg / mouse), and 100 μL of immunogen was added. Subsequent procedures were carried out according to the animal immunization experiment procedures.

[0073] 5. Subcutaneous injection at 2-week intervals: The experiment was designed as a 3-immunization method, but blood was collected from the eyeballs 7 days after each immunization injection. Part of the mouse supernatant was obtained by centrifugation and the serum titer was first tested. 7 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℃.

[0074] 6. Detect serum titer.

[0075] (1) Three mice were immunized, numbered A0, A1, and A2 respectively. After three immunizations, scopolamine standard was used for antigen competition detection, and scopolamine hapten hyoscine-BSA was used as the coating antigen for indirect ELISA and competitive ELISA to detect the serum titer of the immunized mice. The results of the indirect ELISA were read by an enzyme-linked immunosorbent assay (ELISA) reader; the results of the competitive ELISA are shown in Table 3.

[0076] Indirect ELISA method: 1 μg / ml of coated antigen diluted with coating solution was added to each well of the enzyme-labeled plate, and the plate was coated at 4°C overnight. After washing the plate 3 times with washing solution (PBST), 200 μl of blocking solution (5% skim milk powder) was added to each well, and the plate was placed in a 37°C incubator for 2 hours. After removing and washing, 50 μl of diluted serum was added to each well, and the plate was incubated at 37°C for 30 minutes. After washing, 50 μl of goat anti-mouse IgG-HRP solution was added, and the plate was incubated at 37°C for 30 minutes. After washing, 100 μl of substrate solution was added, and the plate was incubated at 37°C for 10 minutes in the dark. Finally, 50 μl of 2 mol / L H2SO4 was added to terminate the reaction, and the A450 value was read on an enzyme-labeled instrument. The orbital blood titers of three mice after three immunizations were all >62500.

[0077] Indirect competitive ELISA method: The process of indirect competitive ELISA is mostly the same as that of indirect ELISA, except that after blocking and washing the enzyme-labeled plate, 50 μl of diluted 100 ng / ml small molecule scopolamine standard solution is added, and then 50 μl of diluted serum antibody is added. The remaining steps are the same. The 100 ng / ml small molecule scopolamine competition detection was used, and the competition rate of three mice reached more than 50% at 1:12500, so fusion can be arranged.

[0078] Unless otherwise specified, the indirect ELISA method and indirect competitive ELISA method used in the following experiments are in accordance with the indirect ELISA method and indirect competitive ELISA method of this experiment.

[0079] Table 3 Serum titer detection data

[0080]

[0081] Note: The hyoscine-BSA serum titer detection column in the table is the detection result of indirect ELISA detection, and the scopolamine standard 100 ng / ml column is the competitive ELISA detection result of competition with 100 ng / ml scopolamine standard (unless otherwise specified, the meanings of the hyoscine-BSA serum titer detection column and the scopolamine standard 100 ng / ml column in the following tables are the same as in this table).

[0082] The 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 containing hypoxanthine, aminopterin and thymine), and the fused cells were subjected to ELISA positive screening and subcloning; the positive monoclonal selected was taken from ascites, and the antibody was purified by Protein A / G antibody purification column. The ELISA titer of the purified antibody was >1:128,000, and the purity was >90%.

[0083] III. ELISA assay to detect the binding activity of scopolamine recognized by the monoclonal antibody

[0084] 1. IgG antibody titer detection method

[0085] (1) Coating of the bottom plate: dilute the used antigen with coating diluent to 3 μg / ml, add 100 μl of the prepared coating solution to each well, and place it in a refrigerator at 4°C for 24 h.

[0086] (2) After 24 h, take it out of the refrigerator and place it at 37°C for 30 min, and then discard the liquid in the well; wash the well with washing solution for 3 times, 3 min each time.

[0087] (3) Seal the enzyme-labeled reaction well: add 200 μl of 5% calf serum to each well, and place it at 37°C for 90 min. After sealing, wash the well with washing solution for 3 times, 3 min each time.

[0088] (4) Add the sample to be detected: dilute the sample according to the required ratio, add the diluted sample to the enzyme-labeled reaction well, 100 μl per well, and place it at 37°C for 90 min; wash the well with washing solution for 3 times, 3 min each time.

[0089] (5) Add enzyme-labeled antibody: add the secondary antibody with the appropriate concentration according to the instructions; 37°C, 90 min, 100 μl per well, and wash as before.

[0090] (6) Add substrate solution: add 100 μl of substrate to each well, and place it at 37°C in the dark for 15-30 min.

[0091] (7) Stop the reaction: add 50 μl of stop solution to each well to stop the reaction, and measure the experimental results within 20 min.

[0092] IV. Detection of the binding activity of scopolamine recognized by the monoclonal antibody

[0093] (1) Cell fusion and cloning screening data

[0094] The mouse numbers are A0, A1, A2, and a total of four rounds of fusion were completed.

[0095] A0 mouse fusion screening selected 1 positive well, subcloned, and finally completed 1 cell strain. After fusion screening, 1 positive clone with OD450 value > 2.2 was selected for detection of titer by doubling dilution, and then subcloned for the second time. One cell strain was obtained, named A0-1.

[0096] A1 and A2 mice had poor fusion effect, with no fusion positive value.

[0097] Through 3 rounds of fusion, a complete cell strain was obtained.

[0098] (2) Ascites preparation and detection data

[0099] A0-1 cell strain was injected into 3 F1 mice, ascites was prepared, and the titer data of the mixed ascites of the 3 mice was as follows:

[0100] Table 4

[0101]

[0102] (3) Antibody purification condition exploration and detection data

[0103] The above ascites was purified by 3.3% n-octanoic acid-thiamine precipitation to obtain A0-1 antibody, and the titer detection data of the antibody are shown in the following table:

[0104] Table 5

[0105]

[0106] The above data show that the monoclonal antibody of A0-1 cell strain has good specific binding ability to hyoscine antigen. The hyoscine colloidal gold product detects urine samples, so the standard hyoscine is used for competition experiment. The results show that the A0-1 antibody has competition effect on small molecule hyoscine, therefore, the A0-1 antibody is selected for testing of the hyoscine colloidal gold product.

[0107] Five, application of monoclonal antibody anti-hyoscine-mab1 in product.

[0108] Verify the hyoscine antibody A0-1 antibody through the immune colloidal gold platform.

[0109] The A0-1 antibody of hyoscine is labeled with colloidal gold through the immune colloidal gold platform, and then a colloidal gold chromatography test paper is prepared. The immune chromatography test paper comprises a sample pad, a binding pad and a detection pad, and the detection pad is provided with a detection line and a quality control line. The binding pad is coated with the screened monoclonal antibody labeled with colloidal gold, the detection line is coated with the antigen of hyoscine, and the quality control line is coated with a goat anti-mouse IgG antibody. The negative buffer solution sample and the small molecule standard of different concentrations of hyoscine are detected in parallel. When detecting the negative sample, the small molecule of hyoscine does not compete with the antigen of hyoscine to bind the monoclonal antibody labeled with colloidal gold, so the antigen of hyoscine coated on the detection line binds the monoclonal antibody labeled with colloidal gold, and the T line develops color.

[0110] Specifically, the experimental group is to use the scopolamine antibody A0-1 obtained by the above experiment to detect the scopolamine standard product of Hangzhou Anxu Biological Technology Co., Ltd. The negative control is a urine sample. The above reagents are added, and then the POCT detection instrument ACG1000 (ID-A003) of Hangzhou Anxu Biological Technology Co., Ltd. is used to detect the results, and the experimental data results are shown in Table 6.

[0111] Table 6

[0112]

[0113] Note: G4-G8 represents the level of strip color, the higher the value, the darker the color, + / - indicates slightly darker or lighter than the color of the level. The addition of scopolamine standard product with lower value indicates that the small molecule has a competitive effect, and the antibody can be combined with the scopolamine standard product.

[0114] The results show that the A0-1 antibody gradient is good and can be used in the scopolamine product, and the cut-off value is 100 ng / ml. Then the stability evaluation of A0-1 antibody is carried out, and three batches of antibody samples are prepared, and the evaluation results are as follows:

[0115] Table 7

[0116]

[0117] According to the evaluation results of the product, the A0-1 antibody is named anti-hyoscine-mab1, which can be used for detection of scopolamine antigen detection kit.

[0118] Six, sequence analysis of the heavy chain V region (VH) and light chain V region (VL) of the monoclonal antibody anti-hyoscine-mab1.

[0119] The primers for amplifying the heavy chain V region (VH) and light chain V region (VL) genes are designed.

[0120] The primers are as follows:

[0121] Heavy chain variable region forward primer (VH-FOR):

[0122] GGGAATTCGAGGTGCAGCTGCAGGAGTCTGG (SEQ ID NO. 10);

[0123] Heavy chain variable region reverse primer (VH-BACK):

[0124] GGAAGGTGTGCACACCGCTGGAC (SEQ ID NO. 11);

[0125] Light chain variable region forward primer (VL-FOR):

[0126] CACGCTAGGGGCGGCCACTGTGGATCCGGATACAGTTGGTGCAGCATC (SEQ ID NO. 12);

[0127] Light chain variable region reverse primer (VL-BACK):

[0128] GGCTGAGCGGGGCTAGATGCCTCGAGGATATTGTGATAACCCAG (SEQ ID NO. 13).

[0129] Take anti-hyoscine-mab1 hybridoma cell strain in logarithmic growth phase (about 10 7 cells), according to the instructions of Trizol RNA extraction kit to extract total RNA of cells, and synthesize cDNA first strand with total RNA as template, and PCR amplify VH / VL gene of antibody with the above amplification product as template.

[0130] Recycle heavy chain VH (about 360 bp) and light chain VL (about 300 bp) fragments of anti-hyoscine-mab1, and send to company for sequencing.

[0131] Then analyze VH / VL gene sequence:

[0132] The obtained sequence is as follows:

[0133] The sequence of variable region of heavy chain is as follows:

[0134] The sequence of variable region of heavy chain is as follows:

[0134] anti-hyoscine-mab1 VH: 348 bp.

[0135] GTGAAACTGCAGCAGTCAGGGGGAGGCTTAGTGCAGCCTGGAGGGTCCCTGAAAGTCTCCTGTGCAACCTCTGGATTCACTTTCAGTGACTATTACATGTATTGGGTTCGCCAGACTCCAGAGAAGAGGCTGGAGTGGGTCGCATACATTAGTAATGGTGGTGGTAGCACCTATTATCCAGACACTGTAAAGGGCCGATTCACCATCTCCAGAGACAATGCCAAGAACACCCTGTACCTGCAAATGAGCCGTCTGAAGTCTGAGGACACAGCCATGTATTACTGTGCAAGACATACCCTCTATGCTATGGACTACTGGGGTCAAGGAACCTCAGTCACCTGTCTCCTC (SEQ ID NO. 8).

[0136] anti-hyoscine-mab1 protein: 116 aa.

[0137] VKLQQSGGGLVQPGGSLKVSCATSGFTFSDYYMYWVRQTPEKRLEWVAYISNGGGSTYYPDTVKGRFTISRDNAKNTLYLQMSRLKSEDTAMYYCARHTLYAMDYWGQGTSVTCLL (SEQ ID NO. 6).

[0138] Variable region sequence of the light chain:

[0139] anti-hyoscine-mab1 LVK: 321 bp.

[0140] GATATCCAGATGACACAGACTCCAGCCTCCCTATCTGCATCTGTGGGAGAAACTGTCACCATCACATGTCGAACAAGTGGGAATATTCACAATTATTTAGCATGGTATCAGCAGAAACGGGGAAGATCTCCTCAGCTCCTGGTCTATAAAGTTTCCAACCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAGCAGAGTGGAGGCTGAGGATCTGGGAGTTTATTTCTGCTCTCAAAGTACACATGTTACGCTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAA (SEQ ID NO. 9).

[0141] anti-hyoscine-mab1 LVκ protein: 107 aa.

[0142] DIQMTQTPASLSASVGETVTITCRTSGNIHNYLAWYQQKRGRSPQLLVYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVTLTFGAGTKLELK (SEQ ID NO. 7).

[0143] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present application, and are not limited thereto; although the present application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that they can still modify the technical solutions recorded in the foregoing embodiments, or make equivalent replacement for part or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present application.

Claims

1. A monoclonal antibody against scopolamine, characterized in that, The variable region of the monoclonal antibody against scopolamine 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 KVS, and the complementarity-determining region CDR3-VL of the amino acid sequence shown in SEQ ID NO.

5.

2. The monoclonal antibody against scopolamine according to claim 1, characterized in that, The amino acid sequence of the heavy chain variable region VH is shown in SEQ ID NO. 6; The amino acid sequence of the light chain variable region VL is shown in SEQ ID NO.

7.

3. The monoclonal antibody against scopolamine according to claim 1, characterized in that, The monoclonal antibody against scopolamine is an IgG antibody.

4. A biomaterial, characterized in that, The biomaterial is selected from any one of ac: a. Nucleic acid, said nucleic acid comprising a nucleic acid sequence encoding a monoclonal antibody against scopolamine as claimed in any one of claims 1-3; b. A vector carrying the nucleic acid from a; c. A cell carrying the nucleic acid of a, or containing the vector of b, or expressing a monoclonal antibody against scopolamine as described in any one of claims 1-3.

5. The method for preparing the monoclonal antibody against scopolamine 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-scopolamine monoclonal antibody according to any one of claims 1-3 in the preparation of a scopolamine detection product.

7. A kit for the detection of scopolamine, characterized in that, The kit comprises a monoclonal antibody against scopolamine as described in any one of claims 1-3.

8. The reagent kit according to claim 7, 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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