A amygdalin hapten, hybridoma cell strain, antibody and application

Abstract

The present application relates to a kind of amygdalin hapten, hybridoma cell strain, antibody and application, belong to the field of immunochemistry.The hybridoma cell strain DMN has been preserved in China Microbial Culture Collection Center CGMCC, address Beijing Chaoyang District Beichen West Road No. 1 Yard No. 3, Institute of Microbiology, Chinese Academy of Sciences, preservation date is April 18, 2024, and the preservation number is CGMCC No.45923.The monoclonal antibody secreted by the cell strain provided in the present application has good detection sensitivity (IC 50 Values are 8.65 ng / mL) and specificity, which can realize the rapid detection of amygdalin, provide raw materials for the immunodetection of amygdalin in amygdalin and products, and have practical application value.

Description

Technical Field

[0001] This invention relates to the field of immunochemical technology, and in particular to an amygdalin hapten, a hybridoma cell line, an antibody, and their applications. Background Technology

[0002] Amygdalin (Amy), also known as amygdalin, is a toxic cyanogenic glycoside found in many plants. It is widely present in the seeds of various Rosaceae plants, including apricots, bitter almonds, apples, peaches, black cherries, loquats, and plums, with the highest content (approximately 2-3%) in bitter almonds. Amygdalin consists of one benzaldehyde unit, one hydrocyanic acid unit, and two glucose units. It is the active ingredient in the traditional Chinese medicine bitter almond, possessing expectorant, antitussive, laxative, and adjuvant anticancer effects. While amygdalin itself is non-toxic, it is hydrolyzed by amygdalinase, producing highly toxic hydrocyanic acid. Therefore, establishing methods for detecting amygdalin content is crucial.

[0003] Currently, there are relatively few methods for detecting amygdalin, including indirect and direct methods. Indirect methods require pretreatment and determine the amygdalin content by measuring the hydrocyanic acid content, making the detection process relatively complex. Direct methods mainly involve instrumental analysis, including thin-layer chromatography (TLC) and high-performance liquid chromatography-mass spectrometry (HPLC-MS). Instrumental analysis methods offer high sensitivity, accuracy, low detection limits, low false positive rates, and high specificity; however, sample pretreatment is complex, requiring specialized personnel, and the equipment is expensive, making them unsuitable for rapid on-site testing. Compared to instrumental analysis, immunological detection methods offer simpler pretreatment, convenience, speed, and ease of operation, while also improving detection sensitivity and reducing costs. Therefore, they possess unique advantages and significant development potential for large-scale sample screening and rapid on-site detection.

[0004] Immunological detection is a method that uses the specific binding reaction of antigen and antibody to detect various substances. Currently, there is very little preparation of monoclonal antibodies against amygdalin. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides an amygdalin hapten, a hybridoma cell line, an antibody, and its applications. The amygdalin monoclonal antibody obtained by this invention exhibits good specificity and detection sensitivity for amygdalin and can be used to detect amygdalin residues in bitter almonds and their products.

[0006] This invention is achieved through the following technical solution:

[0007] The first objective of this invention is to provide an amygdalin hapten, the structural formula of which is:

[0008]

[0009] A second objective of this invention is to provide a method for preparing the amygdalin hapten, comprising the following steps:

[0010] Amygdalin was dissolved in an organic solvent, and then succinic anhydride and DMAP were added to react and obtain amygdalin hapten.

[0011] In one embodiment of the present invention, the reaction temperature is 60°C to 80°C and the reaction time is 8h to 12h.

[0012] The third objective of this invention is to provide a complete amygdalin antigen, prepared from the amygdalin hapten and a carrier protein.

[0013] In one embodiment of the present invention, the carrier protein is one or more of bovine serum albumin, keyhole hemocyanin, and ovalbumin.

[0014] The fourth objective of this invention is to provide a hybridoma cell line containing amygdalin monoclonal antibody, characterized in that the hybridoma cell line is obtained by synthesizing amygdalin complete antigen from amygdalin hapten and then immunizing mice; it was deposited on April 18, 2024, at the China General Microbiological Culture Collection Center, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 45923.

[0015] The fifth objective of this invention is to provide an amygdalin monoclonal antibody secreted by the hybridoma cell line described above.

[0016] A sixth object of the present invention is to provide a composition characterized by comprising the aforementioned amygdalin monoclonal antibody.

[0017] A seventh object of the present invention is to provide a test strip for detecting amygdalin, comprising the amygdalin monoclonal antibody or the composition thereof.

[0018] An eighth object of the present invention is to provide a kit for detecting amygdalin, comprising the amygdalin monoclonal antibody, the composition, or the test strip.

[0019] A ninth object of the present invention is to provide the use of the said composition, the said test strip, or the said kit in the detection of amygdalin.

[0020] This invention also provides a method for preparing the above-mentioned amygdalin monoclonal antibody hybridoma cell line, comprising the following steps:

[0021] S1: The carbon chain of amygdalin hydroxyl group is extended by succinic anhydride to serve as amygdalin hapten.

[0022] S2: The hapten is coupled to the carrier protein to obtain the amygdalin complete antigen;

[0023] S3: Mix and emulsify the amygdalin complete antigen with Freund's adjuvant, and subcutaneously immunize mice to screen for mice with good immunization effects;

[0024] S4: The spleen cells and myeloma cells of the mice selected in step S3 are fused to obtain the amygdalin monoclonal antibody hybridoma cell line.

[0025] The technical solution of the present invention has the following advantages compared with the prior art:

[0026] This invention provides an amygdalin hapten, a hybridoma cell line, an antibody, and its applications. The monoclonal antibody secreted by this hybridoma cell line exhibits good detection sensitivity (IC50) for amygdalin. 50 With a value of 8.65 ng / mL and high specificity, it can be used to detect amygdalin, especially the content of amygdalin in bitter almonds, and has practical application value.

[0027] Biological material sample deposit: A hybridoma cell line DMN with amygdalin monoclonal antibody has been 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. It is classified as a monoclonal cell line, deposited on April 18, 2024, with accession number CGMCC No. 45923. Attached Figure Description

[0028] To make the content of this invention easier to understand, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings, wherein...

[0029] Figure 1 This is the inhibition standard curve of the amygdalin (Amy) monoclonal antibody of this invention. Detailed Implementation

[0030] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0031] This invention obtains a hybridoma cell line with high secretion specific antibodies against Amy by immunizing mice with the Amy complete antigen, fusion cells, culturing them in HAT selective medium, and screening the cell supernatant by ic-ELISA.

[0032] In the following examples, the solutions are prepared as follows:

[0033] Carbonate buffer (CBS): Weigh 1.59 g of Na2CO3 and 2.93 g of NaHCO3, dissolve them separately in a small amount of double-distilled water and mix them together. Add double-distilled water to about 800 mL and mix well. Adjust the pH to 9.6 and add double-distilled water to a final volume of 1000 mL. Store at 4°C for later use.

[0034] Phosphate-buffered saline (PBS): 8.00g NaCl, 0.2g KCl, 0.2g KH2PO4, 2.9g Na2HPO4·12H2O, dissolved in 800mL pure water, pH adjusted to 7.2-7.4 with NaOH or HCl, and then brought to a final volume of 1000mL.

[0035] PBST: PBS containing 0.05% Tween 20;

[0036] TMB colorimetric solution: Solution A: Na₂HPO₄ 4. 12H₂O 18.43g, citric acid 9.33g, diluted to 1000mL with pure water; Solution B: 60mg TMB dissolved in 100mL ethylene glycol. Mix solutions A and B in a 5:1 ratio to obtain the TMB colorimetric solution, mix fresh before use.

[0037] The culture media involved in the following examples are as follows:

[0038] RPMI-1640 medium (mg / L): L-arginine 290, L-asparagine 50, L-aspartic acid 20, L-cysteine ​​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. Para-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.

[0039] 8-10 week old BALB / c mice were purchased from Spiford (Suzhou) Biotechnology Co., Ltd.

[0040] Example 1: Preparation of hybridoma cell lines.

[0041] (1) Derivatization of the hapten: 50 mg of amygdalin was dissolved in 400 mL of pyridine, 13 mg of succinic anhydride and 16 mg of DMAP were added, and the mixture was reacted at 60 °C for 12 h. After the reaction was completed, the mixture was dried under nitrogen, dissolved in 2 mL of double-distilled water, and the pH was adjusted to 2-4 with hydrochloric acid. The mixture was then extracted five times with 5 mL of ethyl acetate. The aqueous phase was discarded, and the mixture was dried under anhydrous magnesium sulfate and nitrogen to obtain the product hapten S1 (HaptenS1), whose structural formula is:

[0042]

[0043] The reaction route is as follows:

[0044]

[0045] (2) Preparation of immunogen HaptenS1-BSA: The hapten S1 was coupled to the carrier protein BSA using the EDC method to obtain the amygdalin immunogen. The steps are as follows:

[0046] Weigh 2 mg of haptenS1 (haptenS1 to bovine serum albumin (BSA) molar ratio 60:1), dissolve in 100 μL of N,N-dimethylformamide (DMF), add 1.3 mg of N-hydroxysuccinimide (NHS), react at room temperature for 10 min, then add 2.1 mg of EDC (1-ethyl-carbodiimide hydrochloride), and continue the reaction at room temperature for 4 h (referred to as solution A). Take 4 mg of BSA, dissolve in 2 mL of 0.01 M carbonate buffer (CB, pH = 9.0) (referred to as solution B), then slowly add solution A dropwise to solution B, and couple overnight; then dialyze with 0.01 M PBS solution to remove unreacted small molecule haptens, obtaining the complete antigen haptenS1-BSA, whose structural formula is:

[0047]

[0048] (3) Preparation of the coating antigen haptenS1-OVA: The hapten S1 is coupled to the carrier protein OVA using the EDC method to obtain the amygdalin coating antigen. The steps are as follows:

[0049] Weigh 2.5 mg of haptenS1 (haptenS1 to ovalbumin (OVA) molar ratio 60:1), dissolve in 100 μL of N,N-dimethylformamide (DMF), add 1.6 mg of N-hydroxysuccinimide (NHS), react at room temperature for 10 min, then add 2.6 mg of EDC (1-ethyl-carbodiimide hydrochloride), and continue the reaction at room temperature for 4 h (referred to as solution A). Take 4 mg of OVA, dissolve in 2 mL of 0.01 M carbonate buffer (CB, pH = 9.0) (referred to as solution B), then slowly add solution A dropwise to solution B, and couple overnight; then dialyze with 0.01 M PBS solution to remove unreacted small molecule haptens, obtaining the complete antigen haptenS1-OVA, whose structural formula is:

[0050]

[0051] (3) Animal immunization: After mixing and emulsifying the haptenS1-BSA complete antigen with an equal amount of Freund's adjuvant, BALB / c mice were immunized by multiple subcutaneous injections at the nape of the neck (except for sprint immunization).

[0052] The initial immunization used complete Freund's adjuvant at a dose of 100 μg / mouse. Multiple booster immunizations used incomplete Freund's adjuvant at half the dose (50 μg / mouse). The final sprint immunization did not use adjuvant; it was directly diluted with physiological saline and injected intraperitoneally at a dose halved again (25 μg / mouse). The interval between the initial and second booster immunizations was one month, between multiple booster immunizations was 21 days, and between the final sprint immunization and the last booster immunization was 18-21 days. The immune efficacy in mice was observed using an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA), specifically by detecting serum titers and inhibition.

[0053] (4) Cell fusion: Three days after the sprint immunization, cell fusion was performed using the conventional PEG (polyethylene glycol, molecular weight 4000) method. The specific steps are as follows:

[0054] a. Blood was collected from the eyeballs of mice. After euthanizing the mice by cervical dislocation, the mice were immediately disinfected in 75% alcohol and soaked for about 5 minutes. The spleen of the mice was removed under aseptic conditions. The spleen cells were moderately ground with the tip of a syringe and passed through a 200-mesh cell sieve to obtain a spleen cell suspension. The suspension was collected, centrifuged (1200 rpm, 8 min), and washed three times with RPMI-1640 medium. After the last centrifugation, the spleen cells were diluted to a certain volume, counted, and set aside for later use.

[0055] b. Collect SP 2 / 0 Cells: 7-10 days before fusion, SP 2 / 0 Tumor cells were cultured in RPMI-1640 medium containing 10% FBS (fetal bovine serum) in a 5% CO2 incubator. SP cells were required before fusion.2 / 0 The number of tumor cells reached 1-4 × 10 7 Ensure SP before fusion 2 / 0 Tumor cells are in the logarithmic growth phase. Upon fusion, tumor cells are collected, suspended in RPMI-1640 basal culture medium, and cell counting is performed.

[0056] c. Fusion process (7 min): At min 1, add 1 mL of PEG 4000 dropwise to the cells, gradually increasing the speed. At min 2, allow to stand. At min 3 and min 4, add 1 mL of RPMI-1640 medium dropwise over 1 min. At min 5 and min 6, add 2 mL of RPMI-1640 medium dropwise over 1 min. At min 7, add 1 mL of RPMI-1640 medium dropwise every 10 s. Then incubate at 37°C for 5 min. Centrifuge (800 rpm, 10 min), discard the supernatant, gently break up the cells, and add RPMI-1640 selective medium (HAT medium) containing 20% ​​fetal bovine serum and 2% 50×HAT to the cells. Add 200 μL / well to a 96-well cell plate and incubate at 37°C in a 5% CO2 incubator.

[0057] (5) Cell screening and cell line establishment: On day 3 after cell fusion, the fused cells were partially replaced with HAT medium; on day 5, the medium was completely replaced with RPMI-1640 transition medium (HT medium) containing 20% ​​fetal bovine serum and 1% 100×HT; on day 7, the cell supernatant was collected for screening. Screening was performed in two steps: first, positive cell wells were selected using ic-ELISA; second, amygdalin was used as a standard, and the inhibitory effect on positive cells was determined using ic-ELISA. Cell wells that showed good inhibition of the amygdalin standard were selected, and subcloning was performed using the limiting dilution method. The same method was used for detection seven days later. Subcloning was performed three times according to the above method to finally obtain the amygdalin monoclonal antibody cell line DMN.

[0058] Example 2: Preparation and identification of monoclonal antibodies.

[0059] 8-10 week old BALB / c mice were injected intraperitoneally with 1 mL of sterile paraffin oil; 7 days later, each mouse was injected intraperitoneally with 1×10 6 Ascites fluid was collected from amygdalin hybridoma cells starting on day 7, and the ascites fluid was purified for antibody using the caprylic acid-saturated ammonium sulfate method. Under slightly acidic conditions, caprylic acid precipitates other proteins in the ascites fluid besides IgG immunoglobulins. The precipitate was then discarded by centrifugation. IgG-type monoclonal antibodies were then precipitated with an equal volume of saturated ammonium sulfate solution, centrifuged, and the supernatant was discarded. The precipitate was dissolved in 0.01M PBS solution (pH 7.4), dialyzed to desalt, and finally the purified monoclonal antibodies were stored at -20°C.

[0060] 2.1 Coating: Dilute the original haptenS1-OVA with CBS to 0.3 μg / mL, coat the microplate with 100 μL / well, and incubate at 37℃ for 2 h;

[0061] 2.2 Washing: Discard the solution in the plate and wash 3 times with PBST for 3 min each time, 200 μL / well, and spin dry;

[0062] 2.3 Blocking: Add 200 μL / well blocking solution and react at 37℃ for 2 h;

[0063] 2.4 Washing: Same as 5.2;

[0064] 2.5 Sample addition: Add 50 μL of serially diluted Amy standard working solution (concentrations of 0, 0.5 ng / mL, 2.0 ng / mL, 6.5 ng / mL, 20 ng / mL, 60 ng / mL, 180 ng / mL, and 540 ng / mL) to each well. Dilute the antibody with PBS to 0.3 μg / mL. Add 50 μL to each well and incubate at 37°C for 30 min.

[0065] 2.6 Washing: Same as 2.2;

[0066] 2.7 Add 100 μL of HRP-goat anti-mouse IgG diluted 1:3000 to each well and react at 37°C for 30 min.

[0067] 2.8 Washing: Same as 2.2;

[0068] 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 min.

[0069] 2.10 Termination and Measurement: Add 50 μL of stop solution to each well to terminate the reaction, and then measure the OD450 value of each well using a microplate reader.

[0070] The IC50 of the monoclonal antibody Amy was determined by ic-ELISA. 50 The value was 8.65 ng / mL, indicating good sensitivity to Amy and suitable for the immunoassay of amygdalin.

[0071] The cross-reactivity results of amygdalin monoclonal antibodies against safflower glycoside, safflower glycoside, linalool, and linalool are shown in Table 1.

[0072] Table 1

[0073]

[0074]

[0075] The amygdalin monoclonal antibody shows good cross-reactivity only with Amy and almost no cross-reactivity with other analogues, indicating that the antibody has strong specificity and will not recognize other cyanogenic glycosides, thus avoiding false positive results when testing samples.

[0076] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A monoclonal antibody hybridoma cell line for amygdalin, characterized in that, The hybridoma cell strain is obtained by immunizing a mouse with a synthesized complete antigen of amygdalin after an amygdalin hapten; the hybridoma cell strain was deposited on April 18, 2024, in the China General Microbiological Culture Collection Center, located at No. 1, Huayuan Road, Beijing, China, and has a preservation number of CGMCC No. 45923.

2. The monoclonal antibody hybridoma cell line of claim 1, wherein the monoclonal antibody is an IgG antibody. The structural formula of the amygdalin hapten is: 。 3. The monoclonal antibody hybridoma cell strain of amygdalin according to claim 1 or 2, characterized in that, The preparation method of the amygdalin hapten comprises the following steps: The amygdalin is dissolved in an organic solvent, succinic anhydride and DMAP are added for reaction to obtain the amygdalin hapten.

4. A monoclonal antibody to amygdalin, characterized in that, Secretion is produced by the hybridoma cell strain of claim 1.

5. A composition for detecting amygdalin, characterized by, The amygdalin monoclonal antibody of claim 4 is included.

6. A test strip for detecting amygdalin, characterized by The amygdalin monoclonal antibody of claim 4 or the composition of claim 5 is included.

7. A kit for detecting amygdalin, characterized by, The amygdalin monoclonal antibody of claim 4, the composition of claim 5 or the test strip of claim 6 is included.

8. Use of the composition of claim 5, the test strip of claim 6 or the kit of claim 7 in detecting amygdalin.

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