A cicutine hapten, hybridoma cell line, antibody and its application

Abstract

This invention relates to a cicutine hapten, a hybridoma cell line, an antibody, and their applications, belonging to the field of immunochemistry. The hybridoma cell line, ISO 400, is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, on April 18, 2024, with accession number CGMCC No. 45918. The monoclonal antibody secreted by the cell line provided by this invention exhibits good detection sensitivity (IC50) for cicutine. 50 With a value of 9.69 μg / mL and high specificity, it enables rapid detection of coniine, providing raw materials for the immunoassay of coniine in hemlock and cosmetics, and has practical application value.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of immunochemistry, in particular to a coniine hapten, hybridoma cell strain, antibody and application. BACKGROUND

[0002] Coniine (CON) is a representative alkaloid of the main toxic effect of poison hemlock, which exists in the form of salt in plants such as poison hemlock. CON is a colorless alkaline liquid with a mouse odor, and is highly toxic! Mistakenly eating can cause fatigue, dizziness, vomiting, coma, paralysis, suffocation, and even death in severe cases, with a lethal dose of 60mg-120mg. Poison hemlock often causes coniine poisoning due to mistaken eating because of its similar appearance to edible plants such as celery and fennel. In 399 BC, Socrates died of poisoning after drinking a mixture containing coniine. And coniine is a banned cosmetic raw material.

[0003] At present, there are few methods for detecting coniine. The current detection methods mainly include instrument analysis methods, such as thin layer chromatography (TLC) and high performance liquid chromatography-mass spectrometry (HPLC-MS). Instrument analysis methods have good sensitivity, high accuracy, low detection limit, low false positive rate and strong specificity, but the sample pretreatment is complex, requires professional personnel, and the instrument equipment is expensive, which is difficult to meet the requirements of on-site rapid detection. Compared with instrument analysis, immunological detection method has the advantages of simple pretreatment, convenience, rapidness, simple operation, improved detection sensitivity and reduced detection cost, thereby having unique advantages and great development potential in large sample preliminary screening detection and on-site rapid detection.

[0004] Immunological detection method is a method for detecting various substances by using specific antigen-antibody binding reaction. At present, there is almost no preparation of monoclonal antibody for coniine. SUMMARY

[0005] To solve the above technical problems, the present application provides a coniine hapten, hybridoma cell strain, antibody and application, which has good specificity and detection sensitivity for CON, and can be used for detecting the CON residual amount in poison hemlock and cosmetics.

[0006] The present application is realized by the following technical solutions:

[0007] The first object of the present application is to provide a coniine hapten, the structure of which is as follows:

[0008]

[0009] The second object of the present application is to provide a preparation method of the coniine hapten, which comprises the following steps:

[0010] S1: 2-piperidine acetic acid is dissolved in an organic solvent, EDC and NHS are added to react;

[0011] S2: 4-amino butyric acid ethyl ester is added to the reaction product obtained in step S1 to react at room temperature;

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

[0013] The reaction route is as follows:

[0014]

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

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

[0017] In one embodiment of the present application, in step S3, the alkaline hydrolysis temperature is 80℃, and the reaction time is 1 h.

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

[0019] A third object of the present application is to provide a coniine complete antigen prepared from the coniine hapten and a carrier protein.

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

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

[0022] When the carrier protein is bovine serum albumin, the structure of the obtained coniine immunogen is as follows:

[0023]

[0024] When the carrier protein is ovalbumin, the structure of the obtained coniine coating antigen is as follows:

[0025]

[0026] The fourth object of the present application is to provide a hybridoma cell strain secreting monoclonal antibody of coniine, which is obtained by immunizing mice with the complete antigen of coniine hapten synthesized from the coniine hapten; and has been preserved in the China General Microbiological Culture Collection Center on April 18, 2024, at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with a preservation number of CGMCC No. 45918.

[0027] The fifth object of the present application is to provide a monoclonal antibody of coniine, which is secreted by the hybridoma cell strain.

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

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

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

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

[0032] The ninth object of the present application is to provide the composition, the test strip, or the kit for detecting coniine.

[0033] The present application also provides a preparation method of the above-mentioned CON monoclonal antibody hybridoma cell strain, comprising the following steps,

[0034] (1) extending the carbon chain of the carboxyl group of 2-piperidine acetic acid as a hapten;

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

[0036] (3) mixing and emulsifying the immunogen with Freund's adjuvant to immunize mice subcutaneously, and screening mice with good immunization effect;

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

[0038] The above technical solutions of the present application have the following advantages compared with the prior art:

[0039] The present application provides a coniine hapten, a hybridoma cell strain, an antibody, and applications. The monoclonal antibody secreted by the cell strain provided by the present application has good detection sensitivity (IC50 The value is 9.69 μg / mL) and specificity, the detection of CON can be realized, especially the detection of CON residues in poison parsnip and cosmetics, which has practical application value.

[0040] Biological material sample preservation: a strain of coniin monoclonal antibody hybridoma cell line ISO has been preserved in the China General Microbiological Culture Collection Center, located at No. 1, Beichen West Road, Haidian District, Beijing, China Institute of Microbiology, classified as a monoclonal cell line, preserved on April 18, 2024, and the preservation number is CGMCC No. 45918. BRIEF DESCRIPTION OF DRAWINGS

[0041] In order to make the content of the application more easily understood, the application will be further described in detail below according to specific embodiments of the application and in conjunction with the drawings, wherein,

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

[0043] The application will be further described below in conjunction with the drawings and specific embodiments, so that those skilled in the art can better understand the application and implement it, but the embodiments are not limiting the application.

[0044] The application finally obtained a hybridoma cell strain with high secretion specific antibody against CON by immunizing mice with CON complete antigen, cell fusion, HAT selective medium culture, and ic-ELISA screening of cell supernatant.

[0045] In the following examples, the solutions are configured as follows:

[0046] Carbonate buffer solution (CBS): weigh 1.59 g of Na2CO3 and 2.93 g of NaHCO3, mix after dissolving in a small amount of double distilled water, add double distilled water to about 800 mL, mix well, adjust the pH value to 9.6, add double distilled water to 1000 mL, store at 4℃ for standby.

[0047] Phosphate buffer solution (PBS): 8.00 g of NaCl, 0.2 g of KCl, 0.2 g of KH2PO4, 2.9 g of Na2HPO4·12H2O, dissolved in 800 mL of pure water, adjust the pH to 7.2-7.4 with NaOH or HCl, and dilute to 1000 mL;

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

[0049] TMB developing solution: A solution: Na2HPO 4.12H2O 18.43g, citric acid 9.33g, pure water to 1000mL; B solution: 60mg TMB dissolved in 100mL ethylene glycol. A, B solution mixed in 5:1 as TMB chromogenic solution, now mixed.

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

[0051] RPMI-1640 culture 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 phosphate dibasic 676.13, potassium chloride 400, sodium chloride 6000, glucose 2000, reduced glutathione 1, phenol red 5, L-glutamine 300, biotin 0.2, calcium D-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 Sibeifu (Suzhou) Biotechnology Co., Ltd.

[0053] Example 1 Preparation of hybridoma cell strain.

[0054] (1) Derivation of hapten: 2-piperidine acetic acid carboxyl group is extended carbon chain as hapten S1, the steps are as follows:

[0055] Take 2-piperidine acetic acid 10mg dissolved in 300mL DMF, add 1-ethyl-(3- dimethylaminopropyl) carbodiimide (EDC, 20mg) and N-hydroxysuccinimide (NHS, 12mg), react at room temperature for 0.5h, add 4-aminobutyric acid ethyl ester (13mg), react at room temperature for 4h, alkali hydrolysis with 2mL 1M NaOH-methanol solution at 80℃ for 1h, dry with nitrogen, dissolve with 2mL double distilled water, adjust PH to 3.5 with hydrochloric acid, extract with 3mL ethyl acetate for 3 times, discard the water phase, dry with anhydrous magnesium sulfate, dry with nitrogen, and the product hapten S1 (Hapten S1) is obtained.

[0056] (2) Preparation of immunogen Hapten S1-BSA: coupling hapten S1 with carrier protein by EDC method, and the CON artificial antigen is obtained, the steps are as follows:

[0057] Take 0.58 mg hapten S1 (hapten S1 and bovine serum albumin (BSA) molar ratio of 40:1), dissolved in 100 μL N,N-dimethylformamide (DMF), first add 0.83 mg (N-hydroxysuccinimide) NHS, react for 10 min at room temperature, then add 1.37 mg EDC (1-ethyl-carbodiimide hydrochloride), continue to react for 4 h at room temperature (referred to as A liquid). Take 4 mg BSA, dissolved with 2 mL 0.01M carbonate buffer solution (CB, pH = 9.0) (referred to as B liquid), then slowly add A liquid drop by drop to B liquid, couple overnight; then dialyze with 0.01M PBS solution to remove unreacted small molecule hapten, to obtain the complete antigen hapten S1-BSA.

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

[0059] Take 1.08 mg hapten S1 (hapten S1 and ovalbumin (OVA) molar ratio of 60:1), dissolved in 100 μL N,N-dimethylformamide (DMF), first add 1.32 mg (N-hydroxysuccinimide) NHS, react for 10 min at room temperature, then add 2.56 mg EDC (1-ethyl-carbodiimide hydrochloride), continue to react for 4 h at room temperature (referred to as A liquid). Take 4 mg OVA, dissolved with 2 mL 0.01M carbonate buffer solution (CB, pH = 9.0) (referred to as B liquid), then slowly add A liquid drop by drop to B liquid, couple overnight; then dialyze with 0.01M PBS solution to remove unreacted small molecule hapten, to obtain the coated hapten S1-OVA.

[0060] (4) Animal immunization: the complete antigen hapten S1-BSA is mixed and emulsified with an equal amount of Freund's adjuvant, and then subcutaneously injected into the neck and back of BALB / c mice (except for the boost immunization).

[0061] The first immunization uses complete Freund's adjuvant, with a dose of 100 μg per mouse; the multiple booster immunization uses incomplete Freund's adjuvant, with a dose of 50 μg per mouse; the boost immunization does not use adjuvant, but is diluted with normal saline, with a dose of 25 μg per mouse. The interval between the first immunization and the second booster immunization is one month, the interval between the multiple booster immunizations is 21 days, and the interval between the boost immunization and the last booster immunization is 18-21 days. The immune effect of the mice is observed by indirect competitive enzyme-linked immunosorbent assay (ic-Elisa), that is, the titer and inhibition of the mouse serum are detected.

[0062] (5) Cell fusion: three days after the sprint immunization, the cell fusion was carried out according to the conventional PEG (polyethylene glycol, molecular weight 4000) method, and the specific steps were as follows:

[0063] a. Mice were enucleated and bled, and immediately after the mice were executed by cervical dislocation, they were disinfected in 75% alcohol for about 5 min. The spleen of the mouse was taken out by sterile operation, and the spleen cell suspension was obtained by moderately grinding with a syringe rubber head and passing through a 200 mesh cell screen. After collection and centrifugation (1200 rpm, 8 min), the spleen cells were washed three times with RPMI-1640 medium, and after the last centrifugation, the spleen cells were diluted to a certain volume, counted, and reserved;

[0064] b. Collect SP 2 / 0 Cells: 7-10 days before fusion, collect SP 2 / 0 tumor cells in logarithmic growth phase. At the time of fusion, collect the tumor cells, suspend them in RPMI-1640 base culture solution, and count the cells; 2 / 0 tumor cells to reach 1-4×10 7 , to ensure that the SP 2 / 0 tumor cells are in logarithmic growth phase before fusion. At the time of fusion, collect the tumor cells, suspend them in RPMI-1640 base culture solution, and count the cells;

[0065] c. 7 min of fusion process: 1 mL of PEG 4000 was added to the cells from slow to fast dropwise at the first minute; at the second minute, stand still. At the third and fourth minutes, 1 mL of RPMI-1640 medium was added within 1 min; at the fifth and sixth minutes, 2 mL of RPMI-1640 medium was added within 1 min; at the seventh minute, 1 mL of RPMI-1640 medium was added every 10 seconds. Then 37°C warm bath for 5 min. Centrifugation (800 rpm, 10 min), discard the supernatant, gently knock the cells apart, and add 20% fetal bovine serum and 2% 50×HAT RPMI-1640 selective medium (HAT medium) to the inside, add 200 μL / well to a 96-well cell plate, and place it in a 37°C, 5% CO2 incubator for culture.

[0066] (6) Cell screening and cell strain establishment: on the third day after cell fusion, the fused cells were semi-replaced with HAT medium; on the fifth day, the cells were fully replaced with RPMI-1640 transition medium containing 20% fetal bovine serum and 1% 100xHT (HT medium); on the seventh day, the cell supernatant was taken for screening. The screening was divided into two steps: first, positive cell wells were screened by ic-ELISA method; second, the positive cells were determined for inhibition effect by ic-ELISA method using CON as a standard. The cell wells with better inhibition to the CON standard were selected, and subcloning was performed by limiting dilution method. After seven days, the same method was used for detection. The above method was performed three times for subcloning, and finally the CON monoclonal antibody cell strain ISO was obtained.

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

[0068] Eight to ten-week-old BALB / c mice were taken, and each mouse was injected with 1 mL of sterile paraffin oil in the abdominal cavity; after 7 days, each mouse was injected with 1x10 6 CON hybridoma cells, starting from the seventh day, the ascites were collected, and the antibodies were purified by octanoic acid-saturated ammonium sulfate method. Under slightly acidic conditions, n-octanoic acid can precipitate other impurities in the ascites except IgG immunoglobulin, and then centrifugation is performed to discard the precipitate; then, the IgG type monoclonal antibody is precipitated by an equal amount of saturated ammonium sulfate solution, centrifugation is performed, the supernatant is discarded, and then the solution is dissolved in 0.01M PBS solution (pH 7.4), dialyzed and desalted, and finally the purified monoclonal antibody is obtained and stored at -20°C.

[0069] 2.1 Coating: coat the enzyme-labeled plate with the coating hapten S1-OVA diluted with CBS to 0.3 μg / mL, 100 μL / well, and react at 37°C for 2 h;

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

[0071] 2.3 Blocking: add 200 μL / well of 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 gradient-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, and 270 μg / mL) to each well, dilute the antibody with PBS to 0.3 μg / mL, 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, 37°C for 30 min.

[0076] 2.8 Wash: same as 2.2;

[0077] 2.9 Color development: add 100 μL TMB color developing solution to each well, 37°C for 15 min in dark.

[0078] 2.10 Stop and determination: add 50 μL stop solution to each well to stop the reaction, then determine the OD450 value of each well by microplate reader.

[0079] The inhibition standard curve of CON monoclonal antibody is shown in Figure 1. Figure 1 The IC50 value of CON monoclonal antibody determined by ic-ELISA is 9.69 μg / mL, which indicates that the antibody has good sensitivity to CON and can be used for the detection of CON by immunoassay. 50

[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 shown in Table 1, the CON monoclonal antibody has good cross-reaction to CON only and has no cross-reaction to other similar substances, which indicates that the antibody has strong specificity and cannot recognize other alkaloids, thus avoiding false positive results in the detection of samples.

[0084] Obviously, the above examples are only examples for clear illustration and are not limitation to the embodiments. Other different forms of changes or variations can be made by those skilled in the art based on the above description. Here, all the embodiments are not required to be exhausted and the obvious changes or variations derived therefrom are still within the protection scope of the present application.​

Claims

1. A complete antigen of coniine, characterized in that, is prepared from a coniine hapten and a carrier protein; a structural formula of the coniine hapten is: 。 2. The complete antigen of coniine according to claim 1, characterized in that, the carrier protein is one or more of bovine serum albumin, keyhole limpet hemocyanin and ovalbumin.

3. A hybridoma cell line secreting monoclonal antibodies to coniine, characterized in that, The hybridoma cell strain is obtained by synthesizing a complete coniine antigen from the coniine hapten of claim 1, immunizing a mouse, and has been preserved in the China General Microbiological Culture Collection Center on April 18, 2024, at an address of No. 1, Beichen West Road, Haidian District, Beijing, with a preservation number of CGMCC No. 45918.

4. A monoclonal antibody to coniine, characterized in that, is secreted by the hybridoma cell strain of claim 3.

5. A composition characterized in that, includes the coniine monoclonal antibody of claim 4.

6. A test strip for detecting coniine, characterized in that includes the coniine monoclonal antibody of claim 4 or the composition of claim 5.

7. A kit for detecting coniine, characterized by includes the coniine monoclonal antibody of claim 4, the composition of claim 5 or the test strip of claim 6.

8. Use of the composition of claim 5, the test strip of claim 6 or the kit of claim 7 in detecting coniine; the use is not related to diagnosis and treatment of diseases.

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