Anti-tolnate monoclonal antibody and application thereof

Abstract

The present application utilizes toluene thiazide-BSA conjugate to immunize mice, and then according to the hybridoma technology, a high-purity, high-sensitivity and high-specificity anti-toluene thiazide monoclonal antibody is obtained, which can be used for developing an immune test product for detecting toluene thiazide. The anti-toluene thiazide monoclonal antibody can provide reliable laboratory diagnosis for relevant inspection and quarantine departments at the fastest speed, is suitable for screening in drug rehabilitation centers, hospitals, high-risk population census, special industries and physical examination for recruitment, and supervision of toluene thiazide drug abuse and food inspection by health and epidemic prevention departments.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of genetic engineering, and relates to an anti-xylazine monoclonal antibody and application thereof. BACKGROUND

[0002] Xylazine (C12H16N2S), also known as methallylthiazide and Celazine, is a widely used veterinary sedative and analgesic with anesthetic effect. The compound has a local anesthetic and muscle relaxant effect by acting on alpha2 receptors and peripheral nerves. Xylazine is an alpha2-adrenergic receptor agonist. After systemic application, it can produce certain anesthetic effect, has a short anesthetic induction time, has good sedative and muscle relaxant effect, but only has weak analgesic effect, and has inhibitory effect on the respiratory and cardiac conduction system, and stimulates the increased secretion of salivary glands and tracheal glands. After intraperitoneal injection of xylazine, it can penetrate the blood-brain barrier into the brain, activate the peptide neurons of the hypothalamus, increase the secretion and synthesis of enkephalins and endorphins in the pituitary gland, and enter the blood circulation to regulate the functions of blood vessels and respiratory system, causing blood pressure drop, respiratory depression, etc.

[0003] Xylazine has a rapid anesthetic effect on humans. Intramuscular injection for 15-20 min or intravenous injection for 1-2 min produces an effect, and the drug effect can last for more than 4 hours. Xylazine is rapidly absorbed, metabolized and decomposed in the body, and only about 8% of the original body is excreted from urine. After oral administration, it is almost completely excreted after 10-15 hours. The original prototype in the blood is less, and it is difficult to extract and purify, and is easy to be missed. Therefore, it is very important to establish a rapid, simple and accurate test method for xylazine in blood for the test of related cases.

[0004] At present, the commonly used preliminary screening methods for xylazine include radioimmunoassay, enzyme-linked immunoassay, immunochromatographic technology, etc. The confirmation method mainly uses physical and chemical detection methods such as high performance liquid chromatography (HPLC) and gas chromatography-mass spectrometry (GC / MS). The above methods all need precise instruments and specific test conditions, and there are great limitations in actual application. In order to solve the problem of large-scale application on site, immunological methods are paid much attention due to their fast detection speed, low cost, high throughput, and some immunological detection methods (colloidal gold paper chromatography) also have the advantages of simple operation and no need for additional equipment. Therefore, developing excellent xylazine diagnostic antigen-antibody has become the key to determine the application prospect of immunological methods. SUMMARY

[0005] In view of the problems in the prior art, the application provides a tolonazine antibody, which can effectively improve the detection rate of enzyme-labeled products and gold-labeled products for detecting tolonazine and reduce the false positive rate.

[0006] The application provides an anti-tolonazine monoclonal antibody, which comprises a heavy chain variable region and a light chain variable region, wherein the CDR1, CDR2 and CDR3 amino acid sequences of the heavy chain variable region are SEQ ID NO: 3-5, and the CDR1, CDR2 and CDR3 amino acid sequences of the light chain variable region are SEQ ID NO: 11-13.

[0007] Further, the amino acid sequence of the heavy chain variable region is SEQ ID NO: 2, and the amino acid sequence of the light chain variable region is SEQ ID NO: 10.

[0008] The application further provides an anti-tolonazine monoclonal antibody, which comprises a heavy chain variable region and a light chain variable region, wherein the nucleotide sequences encoding the CDR1, CDR2 and CDR3 of the heavy chain variable region are SEQ ID NO: 6-8, and the nucleotide sequences of the CDR1, CDR2 and CDR3 of the light chain variable region are SEQ ID NO: 14-16.

[0009] Further, the nucleotide sequence encoding the heavy chain variable region is SEQ ID NO: 1, and the nucleotide sequence of the light chain variable region is SEQ ID NO: 9.

[0010] Further, the anti-tolonazine monoclonal antibody is a complete antibody or a fragment having tolonazine binding activity after treatment.

[0011] The application further provides a kit for detecting tolonazine in a sample, wherein the kit comprises the anti-tolonazine monoclonal antibody.

[0012] Further, the sample is urine.

[0013] The application provides an application of the anti-tolonazine monoclonal antibody in tolonazine immune detection.

[0014] Beneficial effects: the present application applies xylazine-BSA conjugate to immunize Balb / c mice, fuses mouse spleen cells with myeloma cells, and 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-xylazine monoclonal antibody anti-xylazine-mab1 through multi-step separation and purification, which provides the required raw material for developing xylazine detection immunological test products; the anti-xylazine monoclonal antibody anti-xylazine-mab1 of the present application can be used for immunological detection such as immunoblotting and immunofluorescence, and the obtained antibody has been verified to have good specific binding capacity. DETAILED DESCRIPTION

[0015] Example 1: Preparation of antigen xylazine-BSA conjugate

[0016] 1. Dissolve 100 mg of xylazine in 5 ml of pyridine at room temperature, add 55 mg of succinic anhydride under vigorous stirring, and react at room temperature overnight. Post-treatment: evaporate the pyridine at 45°C using a rotary evaporator, then add 5 ml of dichloromethane and 3 ml of water, extract the succinic anhydride in the organic phase, extract the organic phase with 3 ml of saturated sodium chloride again, and dry the organic phase with anhydrous sodium sulfate. Concentrate the combined organic phase to obtain 150 mg of crude product. Purify through a column to obtain 120 mg of product xylazine.

[0017] 2. Dissolve 120 mg of xylazine in 2 ml of N,N-dimethylformamide, then add 53.4 mg of N-hydroxysuccinimide and 88.5 mg of N-ethyl-N'-(3-dimethylaminopropyl) carbodiimide, and react on a rotating disc at a speed of 15 revolutions per minute for 24 hours. Obtain activated xylazine.

[0018] 3. Prepare a 24 ml solution of 10 mg / ml BSA-PBS (BSA content: 10 mg / ml, pH: 7.4), then slowly add the activated xylazine solution to the BSA-PBS solution under the condition of ice water bath, and couple for 24 hours on a rotating disc at a speed of 15 revolutions per minute.

[0019] 4. After the coupling is completed, dialyze with 1xPBS, and change the PBS every 2 hours. After changing the PBS for 7-9 times, collect the sample to obtain 8.5 mg / ml of xylazine antigen, a total of 26 ml.

[0020] Among them, the synthesis process of xylazine antigen is as follows:

[0021]

[0022] Example 2: ELISA detection of antibody titer in samples

[0023] (1) Coating of the bottom plate: The toluene thiazide antigen prepared in Example 1 was diluted to 3 μg / ml with coating diluent (carbonate buffer at pH 9.6, CB), 100 μl of the prepared coating solution (carbonate buffer at pH 9.6, CB) was added to each well, and the plate was placed in a refrigerator at 4°C for 24 hours.

[0024] (2) After 24 hours, the plate was taken out of the refrigerator and equilibrated at 37°C for 30 minutes, and then the liquid in the wells was discarded; the wells were washed with washing solution (phosphate buffer at pH 7.4, PBS) for 3 times, 3 minutes each time.

[0025] (3) Blocking of the enzyme-labeled reaction wells: 200 μl of 5% calf serum was added to each well, and the plate was blocked at 37°C for 90 minutes; after the blocking was completed, the wells were washed with washing solution (phosphate buffer at pH 7.4, PBS) for 3 times, 3 minutes each time.

[0026] (4) Addition of the sample to be detected: The sample to be detected (such as serum, ascites, supernatant of hybridoma cell in vitro culture, or purified antibody solution) was diluted according to the required ratio, and the diluted sample was added to the enzyme-labeled reaction wells, 100 μl per well, and the plate was placed at 37°C for 90 minutes; the wells were washed with washing solution (phosphate buffer at pH 7.4, PBS) for 3 times, 3 minutes each time.

[0027] (5) Addition of enzyme-labeled antibody: HRP-labeled goat anti-mouse secondary antibody was added at a ratio of 1:4000; 100 μl of the same washing solution was added to each well between 37°C and 90 minutes.

[0028] (6) Addition of substrate solution (A solution: TMB (3,3',5,5'-tetramethylbenzidine) at pH 5.0, TMB concentration 2.5 mg / ml, and B solution: H2O2 concentration 0.03% (V / V)): 100 μl of substrate solution was added to each well, and the plate was placed at 37°C in the dark for 15-30 minutes.

[0029] (7) Termination of the reaction: 50 μl of 10.87% sulfuric acid was added to each well to terminate the reaction, and the experimental results were determined within 20 minutes.

[0030] Example 3: Preparation of anti-toluene thiazide monoclonal antibody

[0031] Generally, 6-8 week old healthy female Balb / c mice were used, and the mice were immunized according to the following animal immunization steps, and the toluene thiazide-BSA conjugate synthesized by Hangzhou Anxu Biological Technology Co., Ltd. in Example 1 was used for animal immunization experiments. The specific steps of the animal immunization experiment are as follows:

[0032] 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.

[0033] 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℃.

[0034] 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.

[0035] 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.

[0036] 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. Serum was obtained from the blood of some mice by centrifugation. The serum antibody titer was first tested according to the method in Example 2. Seven days after the last immunization, blood was collected from the heart, centrifuged to obtain serum from the heart blood, and stored at -80℃.

[0037] 6. Serum titer detection: Four mice were immunized, numbered A0, A1, A2, and A3 respectively. After three immunizations, their ocular serum titers were detected according to the method in Example 2.

[0038] The test data are shown in Table 1 below.

[0039] Table 1. Serum titer test data

[0040]

[0041] Conclusion: After three immunizations, the ocular blood titers of the four mice were all >312500. Using competitive detection with 25 ng / ml and 100 ng / ml toluenethiazide, the serum of the four mice achieved an inhibitory effect of more than 50% at a dilution of 1:62500, which can be used for fusion.

[0042] The spleen lymphocytes of the immunized mice were extracted, and the lymphocytes were fused with mouse myeloma cells SP2 / 0 by cell fusion technology. The fused cells were screened by HAT selection medium (HAT selection medium contains hypoxanthine, aminopterin and thymine), and the fused cells were subjected to ELISA positive screening and subcloning. The positive monoclonal obtained was subjected to ascites preparation, and the antibody was purified by Protein A / G antibody purification column. The titer of the purified antibody was detected according to the method in Example 2, and the titer of the antibody was >1:128000, and the purity was >90%. After two rounds of subcloning screening, a hybridoma cell strain stably secreting the monoclonal antibody against toluene thiazide was obtained, thereby obtaining the monoclonal antibody against toluene thiazide.

[0043] Example 4: Detection of the binding activity of the monoclonal antibody recognizing toluene thiazide

[0044] (1) Cell fusion and cloning screening data

[0045] The mouse numbers are A0, A1, A2, A3 in turn, and four rounds of fusion are completed.

[0046] A total of 16 positive wells were selected from the A0 mouse fusion screening, subcloned, and finally four cell strains were completed. After fusion screening, a total of 56 positive clones with OD450 values >2.2 were selected. The titer was detected by multiple dilution according to the method in Example 2, and the second subcloning screening was carried out. Four cell strains were obtained, which were named A0-1 to A0-4.

[0047] A total of 12 positive wells were selected from the A1 mouse fusion screening, subcloned, and after fusion screening, a total of 4 positive wells with OD450 values >2.1 were selected. The titer was detected by multiple dilution according to the method in Example 2, and the second and third subcloning screening was carried out. Finally, two cell strains were obtained, which were named A1-1 and A1-2.

[0048] A total of 8 positive wells were selected from the A2 mouse fusion screening, subcloned, and the second and third subcloning screening was carried out. Finally, one cell strain was obtained, which was named A2-1.

[0049] A total of 26 positive wells were selected from the A3 mouse fusion screening, subcloned, and the second and third subcloning screening was carried out. Finally, three cell strains were obtained, which were named A3-1 to A3-3.

[0050] A total of 10 cell strains were obtained after four cell fusions.

[0051] (2) Ascites preparation and detection data

[0052] Each complete cell strain was injected into 3 F1 mice, and 10 ascites were prepared. The ascites antibody titer was detected according to the method in Example 2, and the data are shown in Table 2.

[0053] Table 2. Ascites antibody titer

[0054]

[0055] (3) Antibody purification condition exploration and detection data

[0056] The above 10 ascites were purified by 3.3% n-octanoic acid-thiamine precipitation method, and 10 purified antibody solutions were obtained. The antibody titer was detected according to the method in Example 2, and 100 ng / ml of the standard tolonazoline was added as a sample into the tolonazoline colloidal gold rapid diagnosis product (the purified antibody solution was used as the capture antibody and was coupled with colloidal gold, and was coated on the labeled pad) for competitive detection. The detection results are shown in Table 3.

[0057] Table 3. Antibody solution titer after purification and competitive detection results

[0058]

[0059]

[0060] The above data show that the monoclonal antibodies of the 10 cell strains have good specific binding capacity to tolonazoline. The tolonazoline colloidal gold rapid diagnosis product detects urine samples, so 100 ng / ml of the standard tolonazoline is used for competitive experiment. The results show that the A1-1 and A1-2 antibodies have competitive effect on small molecule tolonazoline, and therefore, the A1-1 and A1-2 antibodies are used for testing of the tolonazoline colloidal gold rapid diagnosis product.

[0061] Example 5: Application of anti-tolonazoline monoclonal antibody in product

[0062] The anti-tolonazoline antibodies A1-1 and A1-2 were verified. Specifically, the anti-tolonazoline antibodies A1-1 and A1-2 obtained in Example 4 were used to detect the tolonazoline standard product of Hangzhou Anxu Biological Technology Co., Ltd. The negative control was a urine sample without tolonazoline. The above A1-1 and A1-2 antibodies were added, and then the POCT detection instrument ACG1000 (ID-A003) of Hangzhou Anxu Biological Technology Co., Ltd. was used to detect the results. The experimental data results are shown in Table 4.

[0063] Table 4. Initial evaluation results of two antibodies

[0064] Antibody Negative Tolcapone 100 ng / ml Tolcapone 300 ng / ml A1-1 G8 G5+ G4 A1-2 G8 G5+ G4

[0065] Note: G4-G8 represents the level of strip color of test strip, the higher the value, the darker the color, + / - indicates slightly darker or lighter than the color of the level. The value decreases after adding the standard of xylazine indicates that the small molecule has a competitive effect, and the antibody can bind to the xylazine standard.

[0066] The results show that the gradient of A1-1 and A1-2 antibodies is good, which can be used in the xylazine product, and the cut-off value reaches 200 ng / ml. Then the stability of A1-1 antibody is evaluated, and three batches of antibody samples are prepared, and the evaluation results are shown in Table 5:

[0067] Table 5. Evaluation results of three batches of stability A1-1 samples

[0068]

[0069] According to the evaluation results of the product, the A1-1 antibody is named anti-xylazine-mab1, which can be used for detection of xylazine antigen detection kit.

[0070] Example 6: Analysis of V region (VH) and light chain V region (VL) sequences of anti-xylazine-mab1 heavy chain

[0071] (1) Design amplification primers for amplifying the V region (VH) and light chain V region (VL) genes of anti-xylazine-mab1.

[0072] (2) Take the hybridoma cell strain of anti-xylazine-mab1 in logarithmic growth phase (about 107 cells), extract the total RNA of the hybridoma cell strain according to the instructions of Trizol RNA extraction kit (TAKARA company), and synthesize the first strand of cDNA with total RNA as template. Reverse transcription, and the VH / VL gene of anti-xylazine-mab1 is amplified by PCR with the amplification product designed in (1) as template.

[0073] (3) Recover the heavy chain VH and light chain VL fragments of anti-xylazine-mab1, perform gene sequencing, obtain the VH / VL gene sequence, and according to the correspondence between codon and amino acid, obtain the amino acid sequence encoded by the VH / VL gene sequence of anti-xylazine-mab1.

[0074] (4) Analysis of VH / VL gene sequence and amino acid sequence.

[0075] The obtained VH / VL gene sequence and amino acid sequence are shown as follows, wherein the underlined region is the CDR region (CDR1, CDR2 and CDR3).

[0076] Anti-Xylazine-mab1 heavy chain VH variable region gene sequence.

[0077] Anti-Xylazine-mab1-VH: 342 bp

[0078] GTGCAGCTGCAGCAGTCAGGAGCTGAGCTGGTAAGGCCTGGGACTTCAGT

[0079] GAAGATATCCTGCAAGGCTTCT GGCTACACCTTCACTAACTACTTA CTAGGT

[0080] TGGGTAAAGCAGAGGCCTGGACATGGACTTGAGTGGATTGGAGAT ATTTAC

[0081] CCTGGAGGTGCTTATACT AACTACAATGAGAAATTCAAGGGCAGGGCCACA

[0082] CTGACTGCAGACACATCCTCCAGCACTGCCTACATGCAGCTCAGTAGCCTG

[0083] ACATCTGAGGACTCTGCTGTCTATTTCTGT GCAAAAAACGACCTTTTTCCTTAC TGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA (SEQ ID NO: 1)

[0084] Amino acid sequence corresponding to the anti-xylazine-mab1 heavy chain VH variable region gene sequence:

[0085] Anti-Xylazine-mab1 protein: 114 aa

[0086] VQLQQSGAELVRPGTSVKISCKAS GYTFTNYL LGWVKQRPGHGLEWIGDIYP

[0087] GGAYTNYNEKFKGRATLTADTSSSTAYMQLSSLTSEDSAVYFC AKNDLFPY W GQGTLVTVSA (SEQ ID NO: 2)

[0088] Therefore, it is known that the amino acid sequences of CDR1, CDR2 and CDR3 of the heavy chain VH variable region of the antibody anti-xylazine-mab1 are GYTFTNYL (SEQ ID NO: 3), IYPGGAYT (SEQ ID NO: 4) and AKNDLFPY (SEQ ID NO: 5), respectively, and the nucleotide sequences encoding CDR1, CDR2 and CDR3 of the heavy chain VH variable region are GGCTACACCTTCACTAACTACTTA (SEQ ID NO: 6), ATTTACCCTGGAGGTGCTTATACT (SEQ ID NO: 7) and GCAAAAAACGACCTTTTTCCTTAC (SEQ ID NO: 8), respectively.

[0089] The nucleotide sequence of the variable region gene of the light chain VL of the antibody anti-xylazine-mab1.

[0090] The nucleotide sequence of the variable region gene of the light chain VL of the antibody anti-xylazine-mab1.

[0091] GACATCCAGCTGACTCAGTCTCCATCCTCCTTATCTGCCTCTCTGGGAGAAA

[0092] GAGTCAGTCTCACTTGTCGGGCAAGT CAGGATATTGGTAGTAGG TTAAACT

[0093] GGCTTCAACAGGAACCAGATGGAACTATTAAACGCCTGATCTAC GCCACAT

[0094] CC AGTTTAGATTCTGGTGTCCCCAAAAGGTTCAGTGGCAGTAGGTCTGGGT

[0095] CAGATTATTCTCTCACCATCAGCAGCCTTGAGTCTGAAGATTTTGTAGACTA

[0096] TTACTGT CTACAATATGCTAGTTCTCCGTACACG TTCGGAGGGGGGACCAAGCTGGAAATAAAA (SEQ ID NO: 9)

[0097] The amino acid sequence corresponding to the nucleotide sequence of the variable region gene of the light chain VL of the antibody anti-xylazine-mab1:

[0098] anti-xylazine-mab1 LV kappa protein: 107 aa

[0099] DIQLTQSPSSLSASLGERVSLTCRASQDIGSRLNWLQQEPDGTIKRLIYATSSLD SGVPKRFSGSRSGSDYSLTISSLESEDFVDYYC LQYASSPYT FGGGTKLEIK (SEQ ID NO: 10)

[0100] Therefore, it is known that the amino acid sequences of CDR1, CDR2 and CDR3 of the VL variable region of the light chain of the antibody anti-xylazine-mab1 are QDIGSR (SEQ ID NO: 11), ATS (SEQ ID NO: 12) and LQYASSPYT (SEQ ID NO: 13), respectively, and the nucleotide sequences encoding CDR1, CDR2 and CDR3 of the VL variable region of the light chain are CAGGATATTGGTAGTAGG (SEQ ID NO: 14), GCCACATCC (SEQ ID NO: 15) and CTACAATATGCTAGTTCTCCGTACACG (SEQ ID NO: 16), respectively.

Claims

1. An anti-toluidine monoclonal antibody, said anti-toluidine monoclonal antibody comprising a heavy chain variable region and a light chain variable region, characterized in that, The amino acid sequences of CDR1, CDR2 and CDR3 of the heavy chain variable region are SEQ ID NO:3, 4 and 5, respectively; the amino acid sequences of CDR1, CDR2 and CDR3 of the light chain variable region are SEQ ID NO:11, 12 and 13, respectively, wherein the amino acid sequence of SEQ ID NO:12 is ATS.

2. The anti-toluidine monoclonal antibody as described in claim 1, characterized in that, The amino acid sequence of the heavy chain variable region is SEQ ID NO:2, and the amino acid sequence of the light chain variable region is SEQ ID NO:

10.

3. A gene encoding an anti-toluidine monoclonal antibody, said anti-toluidine monoclonal antibody comprising a heavy chain variable region and a light chain variable region, characterized in that, The nucleotide sequences encoding the heavy chain variable regions CDR1, CDR2, and CDR3 are SEQ ID NO: 6, 7, and 8, respectively; the nucleotide sequences encoding the light chain variable regions CDR1, CDR2, and CDR3 are SEQ ID NO: 14, 15, and 16, respectively; wherein, the nucleotide sequence of SEQ ID NO: 15 is GCCACATCC.

4. The gene encoding an anti-toluidine monoclonal antibody as described in claim 3, characterized in that, The nucleotide sequence encoding the heavy chain variable region is SEQ ID NO:1, and the nucleotide sequence encoding the light chain variable region is SEQ ID NO:

9.

5. A kit for detecting toluenethiazide in a sample, characterized in that, The kit includes the anti-toluidine monoclonal antibody as described in claim 1 or 2.

6. The reagent kit as described in claim 5, characterized in that, The sample was urine.

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