Apigenine hapten, hybridoma cell strain, antibody and application

Abstract

The invention relates to an apigenine hapten, a hybridoma cell strain, an antibody and application, and belongs to the technical field of immunochemistry. The hybridoma cell strain IS0 is preserved in China General Microbiological Culture Collection Center (CGMCC) in Institute of Microbiology, Chinese Academy of Sciences, No.3, No.1 Yard, Beichen West Road, Chaoyang District, Beijing, on April 18, 2024, and has a preservation number of CGMCC No.45918. The hybridoma cell strain IS0 can be used for preparing a drug for preventing and treating tumors. The monoclonal antibody secreted by the cell strain provided by the invention has better detection sensitivity (the IC50 value is 9.69 mu g / mL) and specificity to the apigenine, can realize rapid detection of the apigenine, provides a raw material for immunodetection of apigenine in apigenin and cosmetics, and has a practical application value.

Description

Technical Field

[0001] The present invention relates to the technical field of immunochemistry, and in particular to a coniine hapten, a hybridoma cell line, an antibody and applications thereof. Background Art

[0002] Coniine (CON) is an alkaloid that represents the main toxic effect of poison hemlock. It exists in plants such as poison hemlock in the form of salt. CON is a colorless alkaline liquid with a rat-like odor and is highly toxic! Ingestion can cause fatigue, dizziness, vomiting, lethargy, paralysis, suffocation, and death in severe cases. The lethal dose is 60mg to 120mg. Since poison hemlock is similar in appearance to edible plants such as celery and fennel, it often causes coniine poisoning due to accidental ingestion. In 399 BC, Socrates was poisoned and died after drinking a mixture containing coniine. Coniine is also a banned raw material for cosmetics.

[0003] There are relatively few methods for detecting coniine at present, and the detection methods currently under research are mainly instrumental analysis methods, including thin layer chromatography (TLC) and high performance liquid chromatography-mass spectrometry (HPLC-MS). Instrumental analysis methods have good sensitivity, high accuracy, low detection limit, low false positive rate, and strong specificity, but the sample pretreatment is complicated and requires professional personnel. In addition, the instruments and equipment are expensive and difficult to meet the requirements of rapid on-site detection. Compared with instrumental analysis, immunological detection methods have simple pretreatment and have many advantages such as convenience, speed, and simple operation. They can also improve detection sensitivity and reduce detection costs, thus having unique advantages and great development potential in the initial screening of a large number of samples and rapid on-site detection.

[0004] Immunological detection is a method that uses antigen-antibody specific binding reactions to detect various substances. Currently, there are almost no monoclonal antibodies prepared for coniine. Summary of the invention

[0005] In order to solve the above technical problems, the present invention provides a coniine hapten, a hybridoma cell line, an antibody and applications, which have good specificity and detection sensitivity for CON and can be used to detect CON residues in coniine and cosmetics.

[0006] The present invention is achieved through the following technical solutions:

[0007] The first object of the present invention is to provide a coniine hapten, the structural formula of the coniine hapten is:

[0008]

[0009] The second object of the present invention is to provide a method for preparing the coniine hapten, comprising the following steps:

[0010] S1: dissolving 2-piperidineacetic acid in an organic solvent, adding EDC and NHS to react;

[0011] S2: adding ethyl 4-aminobutyrate to the reaction product obtained in step S1 to react at room temperature;

[0012] S3: alkaline hydrolyze the reaction product obtained in step S2 to obtain the coniine hapten.

[0013] The reaction route is:

[0014]

[0015] In one embodiment of the present invention, in step S1, the reaction temperature is room temperature and the reaction time is 0.5 h.

[0016] In one embodiment of the present invention, in step S2, the reaction temperature is room temperature and the reaction time is 4 hours.

[0017] In one embodiment of the present invention, in step S3, the alkaline hydrolysis temperature is 80° C. and the time is 1 hour.

[0018] In one embodiment of the present invention, the organic solvent is DMF.

[0019] The third object of the present invention is to provide a complete coniine antigen, which is prepared from the coniine hapten and a carrier protein.

[0020] In one embodiment of the present invention, the carrier protein is one or more of bovine serum albumin, keyhole limpet hemocyanin and ovalbumin.

[0021] In one embodiment of the present invention, the coniine immunogen is prepared from coniine hapten and bovine serum albumin or keyhole limpet hemocyanin; the coniine coating agent is prepared from coniine hapten and ovalbumin.

[0022] When the carrier protein is bovine serum albumin, the structural formula of the obtained coniine immunogen is:

[0023]

[0024] When the carrier protein is ovalbumin, the structural formula of the obtained coniine coating source is:

[0025]

[0026] The fourth object of the present invention is to provide a hybridoma cell line that secretes coniine monoclonal antibodies, wherein the hybridoma cell line is obtained by immunizing mice with the coniine hapten by synthesizing the coniine complete antigen; on April 18, 2024, it was deposited in the General Microbiology Center of China National Microbiological Culture Collection Administration, with the deposit address at No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No.45918.

[0027] The fifth object of the present invention is to provide a monoclonal antibody against coniine, which is secreted and produced by the hybridoma cell line.

[0028] The sixth object of the present invention is to provide a composition comprising the coniine monoclonal antibody.

[0029] The seventh object of the present invention is to provide a test strip for detecting coniine, comprising the coniine monoclonal antibody or the composition.

[0030] The eighth object of the present invention is to provide a kit for detecting coniine, comprising the coniine monoclonal antibody, the composition or the test strip.

[0031] In one embodiment of the present invention, the kit is selected from an enzyme-linked immunosorbent assay kit, a fluorescent immunoassay kit or a chemiluminescent immunoassay kit.

[0032] The ninth objective of the present invention is to provide the use of the composition, the test strip or the kit in detecting coniine.

[0033] The present invention also provides a method for preparing the above CON monoclonal antibody hybridoma cell line, comprising the following steps:

[0034] (1) Extending the carbon chain of the carboxyl group of 2-piperidinylacetic acid as a hapten;

[0035] (2) coupling the hapten with a carrier protein to obtain an immunogen;

[0036] (3) mixing the immunogen with Freund's adjuvant and emulsifying it, subcutaneously immunizing mice, and screening mice with good immune effects;

[0037] (4) fusing the spleen cells of the mice screened in step (3) with myeloma cells to obtain the CON monoclonal antibody hybridoma cell line.

[0038] The above technical solution of the present invention has the following advantages compared with the prior art:

[0039] The present invention provides a coniine hapten, a hybridoma cell line, an antibody and an application thereof. The monoclonal antibody secreted by the cell line provided by the present invention has a good detection sensitivity (IC) for CON.50 The value was 9.69 μg / mL) and specificity, which can realize the detection of CON, especially the detection of CON residues in poison hemlock and cosmetics, and has practical application value.

[0040] Deposit of biological material samples: A coniine monoclonal antibody hybridoma cell line IS0 has been deposited in the General Microbiology Center of China Microbiological Culture Collection Committee CGMCC, located at the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. It is classified and named as monoclonal cell line. The deposit date is April 18, 2024, and the deposit number is CGMCC No.45918. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] In order to make the content of the present invention more clearly understood, the present invention is further described in detail below according to specific embodiments of the present invention in conjunction with the accompanying drawings, wherein:

[0042] Figure 1 It is the inhibition standard curve of the CON monoclonal antibody of the present invention. DETAILED DESCRIPTION

[0043] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.

[0044] The invention immunizes mice with CON complete antigen, performs cell fusion, culture with HAT selective medium, and screens cell supernatant with ic-ELISA, thereby finally obtaining a hybridoma cell strain with high secretion specific antibody against CON.

[0045] In the following examples, the solution is configured as follows:

[0046] Carbonate buffer (CBS): Weigh 1.59 g of Na2CO3 and 2.93 g of NaHCO3, dissolve them in a small amount of double distilled water respectively and mix them, add double distilled water to about 800 mL and mix well, adjust the pH to 9.6, add double distilled water to make up to 1000 mL, and store at 4°C for later use.

[0047] Phosphate buffered saline (PBS): 8.00 g NaCl, 0.2 g KCl, 0.2 g KH2PO4, 2.9 g Na2HPO4·12H2O, dissolved in 800 mL pure water, adjusted to pH 7.2-7.4 with NaOH or HCl, and made up to 1000 mL;

[0048] PBST: PBS containing 0.05% Tween 20;

[0049] TMB colorimetric solution: Solution A: Na2HPO 4.12H2O 18.43g, citric acid 9.33g, pure water to 1000mL; Solution B: 60mg TMB dissolved in 100mL ethylene glycol. Mix solution A and solution B in a ratio of 5:1 to obtain TMB colorimetric solution, which should be mixed before use.

[0050] The culture medium involved in the following examples is as follows:

[0051] RPMI-1640 medium (mg / L): L-arginine 290, L-asparagine 50, L-aspartic acid 20, L-cystine dihydrochloride 65.15, L-glutamic acid 20, glycine 10, L-histidine 15, L-hydroxyproline 20, L-isoleucine 50, L-leucine 50, L-lysine hydrochloride 40, L-methionine 15, L-phenylalanine 15, L-proline 20, L-serine 30, L-threonine 20, L-tryptophan 5, L-tyrosine 23. 19. L-valine 20. p-Aminobenzoic acid 1. Calcium nitrate 100. Anhydrous magnesium sulfate 48.84. Anhydrous sodium dihydrogen phosphate 676.13. Potassium chloride 400. Sodium chloride 6000. Glucose 2000. Reduced glutathione 1. Phenol red 5. L-glutamine 300. Biotin 0.2. D-calcium pantothenate 0.25. Folic acid 1. i-inositol 35. Nicotinamide 1. Choline chloride 3. Pyridoxine hydrochloride 1. Riboflavin 0.2. Thiamine hydrochloride 1. Vitamin B12 0.005. Sodium bicarbonate 2000.

[0052] 8-10 week old BALB / c mice were purchased from SPIEF (Suzhou) Biotechnology Co., Ltd.

[0053] Example 1 Preparation of hybridoma cell lines.

[0054] (1) Derivatization of hapten: Extend the carbon chain of the carboxyl group of 2-piperidinylacetic acid to form hapten S1, the steps are as follows:

[0055] Weigh 10 mg of 2-piperidineacetic acid and dissolve it in 300 mL of DMF, add 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC, 20 mg) and N-hydroxysuccinimide (NHS, 12 mg), react at room temperature for 0.5 h, add ethyl 4-aminobutyrate (13 mg) and react at room temperature for 4 h, use 2 mL of 1M NaOH-methanol solution to alkaline hydrolyze at 80 ° C for 1 h, blow dry with nitrogen, dissolve with 2 mL of double distilled water, adjust the pH to 3.5 with hydrochloric acid, and extract with 3 mL of ethyl acetate three times, discard the aqueous phase, add anhydrous magnesium sulfate to dry, blow dry with nitrogen to obtain the product hapten S1 (HaptenS1)

[0056] (2) Preparation of immunogen HaptenS1-BSA: The hapten S1 was coupled to the carrier protein by the EDC method to obtain the CON artificial antigen. The steps are as follows:

[0057] Weigh 0.58 mg haptenS1 (haptenS1 to bovine serum albumin (BSA) molar ratio is 40:1), dissolve in 100 μL N,N-dimethylformamide (DMF), add 0.83 mg (N-hydroxysuccinimide) NHS first, react at room temperature for 10 min, then add 1.37 mg EDC (1-ethyl-carbodiimide hydrochloride), and continue to react at room temperature for 4 h (called A solution). Take 4 mg BSA, dissolve it in 2 mL 0.01 M carbonate buffer solution (CB, pH = 9.0) (called B solution), then slowly add A solution to B solution drop by drop, and couple overnight; then dialyze with 0.01 M PBS solution to remove unreacted small molecule haptens to obtain the complete antigen haptenS1-BSA.

[0058] (3) Preparation of coated Hapten S1-OVA: The hapten S1 is coupled to the carrier protein by the EDC method to obtain the CON artificial antigen, and the steps are as follows:

[0059] Weigh 1.08 mg haptenS1 (haptenS1 to ovalbumin (OVA) molar ratio is 60:1), dissolve in 100 μL N,N-dimethylformamide (DMF), add 1.32 mg (N-hydroxysuccinimide) NHS first, react at room temperature for 10 min, then add 2.56 mg EDC (1-ethyl-carbodiimide hydrochloride), and continue to react at room temperature for 4 h (called A solution). Take 4 mg OVA, dissolve it in 2 mL 0.01 M carbonate buffer solution (CB, pH = 9.0) (called B solution), then slowly add A solution to B solution drop by drop, and couple overnight; then dialyze with 0.01 M PBS solution to remove unreacted small molecule haptens to obtain the original haptenS1-OVA coating.

[0060] (4) Animal immunization: The complete antigen haptenS1-BSA was mixed with an equal amount of Freund's adjuvant and emulsified, and then BALB / c mice were immunized by subcutaneous injection at multiple points on the back of the neck (except for sprint immunization).

[0061] Complete Freund's adjuvant was used for the first immunization, with a dose of 100 μg / mouse; incomplete Freund's adjuvant was used for multiple booster immunizations and the dose was halved to 50 μg / mouse; no adjuvant was used for sprint immunizations, and it was directly diluted with normal saline and injected intraperitoneally, and the dose was halved again to 25 μg / mouse. There was a one-month interval between the first immunization and the second booster immunization, a 21-day interval between multiple booster immunizations, and an 18-21-day interval between the sprint immunization and the last booster immunization. The immune effect of mice was observed by indirect competitive enzyme-linked immunosorbent assay (ic-Elisa), that is, the titer and inhibition of mouse serum were detected.

[0062] (5) Cell fusion: Three days after the sprint immunization, cell fusion was performed according to the conventional PEG (polyethylene glycol, molecular weight 4000) method. The specific steps are as follows:

[0063] a. Eyeballs of mice were removed for blood collection. After killing the mice by cervical dislocation, the mice were immediately placed in 75% alcohol for disinfection and soaked for about 5 minutes. The spleen of the mice was removed by aseptic operation. The spleen was moderately ground with a syringe rubber tip and passed through a 200-mesh cell sieve to obtain a spleen cell suspension. The suspension was collected and centrifuged (1200 rpm, 8 minutes). The spleen cells were washed three times with RPMI-1640 medium. After the last centrifugation, the spleen cells were diluted to a certain volume, counted, and set aside.

[0064] b. Collect SP 2 / 0 Cells: 7-10 days before fusion, place SP 2 / 0 Tumor cells were cultured in RPMI-1640 medium containing 10% FBS (fetal bovine serum) in a 5% CO2 incubator. 2 / 0 The number of tumor cells reaches 1-4×10 7 , ensure the pre-fusion SP 2 / 0 Tumor cells are in the logarithmic growth phase. When fused, tumor cells are collected, suspended in RPMI-1640 basal culture medium, and cell counts are performed;

[0065] c. Fusion process 7min: In the first minute, 1mL of PEG 4000 was added to the cells from slow to fast; in the second minute, it was left to stand. In the third and fourth minutes, 1mL of RPMI-1640 medium was added within 1min; in the fifth and sixth minutes, 2mL of RPMI-1640 medium was added within 1min; in the seventh minute, 1mL of RPMI-1640 medium was added every 10s. Then, it was incubated at 37℃ for 5min. Centrifuged (800rpm, 10min), the supernatant was discarded, the cells were gently knocked apart, and RPMI-1640 selective medium (HAT medium) containing 20% ​​fetal bovine serum and 2% 50×HAT was added thereto, and added to the 96-well cell plate at 200μL / well, and cultured in a 37℃, 5% CO2 incubator.

[0066] (6) Cell screening and cell line establishment: On the third day after cell fusion, half of the fused cells were replaced with HAT medium; on the fifth day, the medium was fully replaced with RPMI-1640 transition medium (HT medium) containing 20% ​​fetal bovine serum and 1% 100×HT; on the seventh day, the cell supernatant was taken for screening. The screening was divided into two steps: the first step was to screen out the positive cell wells by ic-ELISA, and the second step was to select CON as the standard and use ic-ELISA to measure the inhibitory effect of positive cells. The cell wells with better inhibition of the CON standard were selected, and subcloned by the limiting dilution method. After seven days, the same method was used for detection. Three subclonings were performed according to the above method, and finally the CON monoclonal antibody cell line IS0 was obtained.

[0067] Example 2 Preparation and identification of monoclonal antibodies.

[0068] 8-10 week old BALB / c mice were taken and each mouse was intraperitoneally injected with 1 mL of sterile paraffin oil. Seven days later, each mouse was intraperitoneally injected with 1×10 6 CON hybridoma cells, ascites was collected from the seventh day, and the ascites was purified by the caprylic acid-saturated ammonium sulfate method. Under slightly acidic conditions, caprylic acid can precipitate other miscellaneous proteins in the ascites except IgG immunoglobulin, and then centrifuge and discard the precipitate; then use an equal amount of saturated ammonium sulfate solution to precipitate IgG type monoclonal antibodies, centrifuge, discard the supernatant, dissolve with 0.01M PBS solution (pH7.4), dialyze and desalt, and finally obtain the purified monoclonal antibody and store it at -20℃.

[0069] 2.1 Coating: Dilute the original haptenS1-OVA to 0.3 μg / mL with CBS, coat the ELISA plate, 100 μL / well, react at 37℃ for 2 hours;

[0070] 2.2 Washing: Pour off the solution in the plate and wash with PBST 3 times, 3 min each time, 200 μL / well, and spin dry;

[0071] 2.3 Blocking: Add 200 μL / well blocking solution and react at 37°C for 2 h;

[0072] 2.4 Washing: Same as 2.2;

[0073] 2.5 Sample addition: First add 50 μL of serially diluted CON standard working solution (concentrations are 0.4 μg / mL, 1.0 μg / mL, 3.0 μg / mL, 10 μg / mL, 30 μg / mL, 90 μg / mL, 270 μg / mL) to each well, dilute the antibody to 0.3 μg / mL with PBS, add 50 μL / well, and react at 37°C for 30 min;

[0074] 2.6 Washing: Same as 2.2;

[0075] 2.7 Add 1:3000 diluted HRP-goat anti-mouse IgG, 100 μL / well, and react at 37°C for 30 min.

[0076] 2.8 Washing: Same as 2.2;

[0077] 2.9 Color development: Add 100 μL of TMB color development solution to each well and react at 37°C in the dark for 15 minutes.

[0078] 2.10 Termination and determination: Add 50 μL of stop solution to each well to terminate the reaction, and then use a microplate reader to determine the OD450 value of each well.

[0079] The inhibition standard curve of CON monoclonal antibody is as follows Figure 1 IC of monoclonal antibody CON was determined by ic-ELISA 50 The value was 9.69 μg / mL, indicating good sensitivity to CON and can be used for CON immunoassay detection.

[0080] The cross-reaction results of CON monoclonal antibody to nicotine, arecoline and theophylline are shown in Table 1:

[0081] Table 1

[0082]

[0083] As can be seen from Table 1, the CON monoclonal antibody only cross-reacts well with CON and has no cross-reactions with other analogs, indicating that the antibody has a strong specificity and will not recognize other alkaloids, thus avoiding false positive results when testing samples.

[0084] Obviously, the above embodiments are merely examples for clear explanation and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived from these are still within the protection scope of the invention.

Claims

1. A coniine hapten, characterized in that The structural formula of the coniine hapten is:

2. The method for preparing the coniine hapten according to claim 1, characterized in that: The following steps are involved: S1: dissolving 2-piperidineacetic acid in an organic solvent, adding EDC and NHS to react; S2: adding ethyl 4-aminobutyrate to the reaction product obtained in step S1 to react at room temperature; S3: alkaline hydrolyze the reaction product obtained in step S2 to obtain the coniine hapten.

3. A complete coniine antigen, characterized in that: The coniine hapten according to claim 1 is prepared with a carrier protein.

4. The complete coniine antigen according to claim 3, characterized in that The carrier protein is one or more of bovine serum albumin, keyhole limpet hemocyanin and ovalbumin.

5. A hybridoma cell line secreting a monoclonal antibody against coniine, characterized in that: The hybridoma cell line is obtained by immunizing mice with the coniine hapten described in claim 1 by synthesizing the coniine complete antigen; it was deposited in the General Microbiology Center of China National Microbiological Culture Collection Committee on April 18, 2024, with the deposit address at No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 45918.

6. A coniine monoclonal antibody, characterized in that: The hybridoma cell line according to claim 5 is secreted and produced.

7. A composition, characterized in that The invention comprises the coniine monoclonal antibody as claimed in claim 6.

8. A test strip for detecting coniine, characterized in that: The invention comprises the coniine monoclonal antibody according to claim 6, or the composition according to claim 4.

9. A kit for detecting coniine, characterized in that: The invention comprises the coniine monoclonal antibody according to claim 6, the composition according to claim 7 or the test strip according to claim 8.

10. Use of the composition according to claim 7, the test strip according to claim 8 or the kit according to claim 9 in detecting coniine.

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