Amygdalin hapten, hybridoma cell strain, antibody and application

Abstract

The invention relates to an amygdalin hapten, a hybridoma cell strain, an antibody and application, and belongs to the technical field of immunochemistry. The hybridoma cell strain DMN is preserved in the China General Microbiological Culture Collection Center (CGMCC), the address is Institute of Microbiology, Chinese Academy of Sciences, No.3, No.1 Yard, Beichen West Road, Chaoyang District, Beijing, the preservation date is April 18, 2024, and the preservation number is CGMCC No.45923. The monoclonal antibody secreted by the cell strain provided by the invention has good detection sensitivity (IC50 value is 8.65 ng / mL) and specificity on amygdalin, can realize rapid detection of amygdalin, provides a raw material for immunodetection of amygdalin in bitter apricot kernels and products thereof, and has practical application value.

Description

Technical Field

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

[0002] Amygdalin (Amy), also known as amygdalin, is a toxic cyanogenic glycoside found in many plants. It is widely found in the seeds of various Rosaceae fruits such as apricot, bitter almond, apple, peach, black cherry, loquat and plum, especially in bitter almond, with the highest content of about 2-3%. Amygdalin is composed of one unit of benzaldehyde, one unit of hydrocyanic acid and two units of glucose. It is an effective ingredient in the traditional Chinese medicine bitter almond, which has the effects of expectoration and cough relief, laxative and auxiliary anti-cancer. Amygdalin itself is non-toxic, but after being hydrolyzed by the amygdalin enzyme contained in itself, it produces highly toxic hydrocyanic acid. Therefore, it is important to establish a method to detect the content of amygdalin.

[0003] At present, there are relatively few methods for detecting amygdalin, including indirect and direct methods. The indirect method requires pretreatment, and the content of amygdalin is determined by measuring the content of hydrocyanic acid, and the detection steps are relatively complicated. The direct method is mainly an instrumental analysis method, including thin layer chromatography (TLC) and high performance liquid chromatography-mass spectrometry (HPLC-MS method). The instrumental analysis method has 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, the immunological detection method has simple pretreatment, and has many advantages such as convenience, rapidity, and simple operation. It can also improve the detection sensitivity and reduce the detection cost, 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 the specific binding reaction of antigen and antibody to detect various substances. Currently, there are few monoclonal antibodies prepared for amygdalin. Summary of the invention

[0005] To solve the above technical problems, the present invention provides amygdalin hapten, hybridoma cell line, antibody and application. The amygdalin monoclonal antibody obtained by the present invention has good specificity and detection sensitivity for amygdalin and can be used to detect the residual amount of amygdalin in bitter almonds and their products.

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

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

[0008]

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

[0010] Amygdalin is dissolved in an organic solvent, and succinic anhydride and DMAP are added to react to 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 8 h to 12 h.

[0012] The third object of the present invention is to provide a complete amygdalin antigen, which is 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 limpet hemocyanin and ovalbumin.

[0014] The fourth object of the present invention is to provide a hybridoma cell line of amygdalin monoclonal antibody, characterized in that the hybridoma cell line is obtained by synthesizing the amygdalin complete antigen from the amygdalin hapten described in claim 1 and then immunizing mice; it has been 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.45923.

[0015] The fifth object of the present invention is to provide a monoclonal antibody against amygdalin, which is secreted and produced by the hybridoma cell line.

[0016] The sixth object of the present invention is to provide a composition, characterized in that it comprises the amygdalin monoclonal antibody.

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

[0018] The 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] The ninth objective of the present invention is to provide the use of the composition, the test strip or the kit in detecting amygdalin.

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

[0021] S1: Extend the carbon chain of amygdalin hydroxyl group through succinic anhydride to make amygdalin hapten;

[0022] S2: coupling the hapten with a carrier protein to obtain amygdalin complete antigen;

[0023] S3: mixing the complete amygdalin antigen with Freund's adjuvant to emulsify the mixture, subcutaneously immunizing mice, and screening mice with good immune effects;

[0024] S4: fusing the spleen cells of the mice screened in step S3 with myeloma cells to obtain the amygdalin monoclonal antibody hybridoma cell line.

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

[0026] The present invention provides amygdalin hapten, hybridoma cell line, antibody and application. The monoclonal antibody secreted by the hybridoma cell line has good detection sensitivity (IC 50 The value is 8.65ng / mL) and specificity, which can realize the detection of amygdalin, especially the detection of the amygdalin content in bitter almonds, and has practical application value.

[0027] Deposit of biological material samples: A amygdalin monoclonal antibody hybridoma cell line DMN has been deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration 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, with a deposit date of April 18, 2024 and a deposit number of CGMCC No.45923. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] 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:

[0029] Figure 1 It is the inhibition standard curve of the amygdalin (Amy) monoclonal antibody of the present invention. DETAILED DESCRIPTION

[0030] 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.

[0031] The invention immunizes mice with Amy 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 Amy.

[0032] In the following examples, the solution is configured as follows:

[0033] Carbonate buffer (CBS): weigh Na 2 CO 3 1.59 g, NaHCO 3 2.93 g, respectively dissolved in a small amount of double distilled water and mixed, added double distilled water to about 800 mL and mixed evenly, adjusted the pH value to 9.6, added double distilled water to 1000 mL, and stored at 4 °C for later use.

[0034] Phosphate buffered saline (PBS): 8.00 g NaCl, 0.2 g KCl, 0.2 g KH 2 PO 4 , 2.9gNa 2 HPO 4 12H 2 O, dissolved in 800 mL pure water, adjusted pH to 7.2-7.4 with NaOH or HCl, and made up to 1000 mL;

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

[0036] TMB colorimetric solution: Solution A: Na 2 HPO 4. 12H 2 O 18.43g, citric acid 9.33g, pure water to 1000mL; Solution B: 60mg TMB dissolved in 100mL ethylene glycol. Mix A and B in a ratio of 5:1 to make TMB colorimetric solution, mix before use.

[0037] The culture medium involved in the following examples is as follows:

[0038] 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.

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

[0040] Example 1 Preparation of hybridoma cell lines.

[0041] (1) Derivatization of hapten: Weigh 50 mg of amygdalin and dissolve it in 400 mL of pyridine. Add succinic anhydride (13 mg) and DMAP (16 mg) and react at 60°C for 12 h. After the reaction, blow dry with nitrogen. Dissolve it in 2 mL of double distilled water. Adjust the pH to 2-4 with hydrochloric acid. Extract it with 5 mL of ethyl acetate for 5 times. Discard the aqueous phase, dry it with anhydrous magnesium sulfate, and blow dry it with nitrogen to obtain the product hapten S1 (Hapten S1). Its structural formula is:

[0042]

[0043] The reaction route is:

[0044]

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

[0046] Weigh 2 mg haptenS1 (haptenS1 to bovine serum albumin (BSA) molar ratio is 60:1), dissolve in 100 μL N, N-dimethylformamide (DMF), add 1.3 mg (N-hydroxysuccinimide) NHS first, react at room temperature for 10 min, then add 2.1 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, whose structural formula is:

[0047]

[0048] (3) Preparation of coating source haptenS1-OVA: The hapten S1 is coupled with the carrier protein OVA by the EDC method to obtain the amygdalin coating source, and the steps are as follows:

[0049] Weigh 2.5 mg haptenS1 (haptenS1 to ovalbumin (OVA) molar ratio is 60:1), dissolve in 100 μL N, N-dimethylformamide (DMF), add 1.6 mg (N-hydroxysuccinimide) NHS first, react at room temperature for 10 min, then add 2.6 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 complete antigen haptenS1-OVA, whose structural formula is:

[0050]

[0051] (3) Animal immunization: The haptenS1-BSA complete antigen 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).

[0052] 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.

[0053] (4) 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:

[0054] 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.

[0055] 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 an atmosphere of 5% CO. 2Culture in an incubator. SP is required before fusion 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;

[0056] c. Fusion process 7min: In the first minute, 1mL of PEG 4000 was added to the cells from slow to fast speed; in the second minute, it was allowed 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 200μL / well was added to a 96-well cell plate and placed at 37℃, 5% CO 2 Culture in an incubator.

[0057] (5) 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 completely 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 the positive cell wells by ic-ELISA, and the second step was to select amygdalin as the standard and use ic-ELISA to measure the inhibitory effect of positive cells. The cell wells with better inhibition of the amygdalin standard were selected, and subcloned by limiting dilution method, and tested by the same method seven days later. Three subclonings were performed according to the above method, and finally the amygdalin monoclonal antibody cell line DMN was obtained.

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

[0059] 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 6Amygdalin 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 except IgG immunoglobulin in the ascites, 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℃.

[0060] 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;

[0061] 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;

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

[0063] 2.4 Washing: Same as 5.2;

[0064] 2.5 Sample addition: First, add 50 μL of the serially diluted Amy standard working solution (concentrations are 0, 0.5 ng / mL, 2.0 ng / mL, 6.5 ng / mL, 20 ng / mL, 60 ng / mL, 180 ng / mL, 540 ng / 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;

[0065] 2.6 Washing: Same as 2.2;

[0066] 2.7 Add 1:3000 diluted HRP-goat anti-mouse IgG, 100 μL / 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 minutes.

[0069] 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.

[0070] Determination of IC of monoclonal antibody Amy by ic-ELISA 50 The value was 8.65 ng / mL, indicating good sensitivity to Amy and can be used for immunoassay detection of amygdalin.

[0071] The cross-reaction results of the amygdalin monoclonal antibody to safflower glycosides, linamarin, linamarin and radix sutchurensis are shown in Table 1:

[0072] Table 1

[0073]

[0074]

[0075] The monoclonal antibody against amygdalin only cross-reacts well with Amy and has almost no cross-reaction with other analogs, 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 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 laetrile hapten, characterized in that: The structural formula of the amygdalin hapten is:

2. The method for preparing the amygdalin hapten according to claim 1, characterized in that: The following steps are involved: Amygdalin is dissolved in an organic solvent, and succinic anhydride and DMAP are added to react to obtain amygdalin hapten.

3. A complete amygdalin antigen, characterized in that: The method is prepared from the amygdalin hapten described in claim 1 and a carrier protein.

4. The complete amygdalin 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 of amygdalin monoclonal antibody, characterized in that: The hybridoma cell line is obtained by synthesizing the complete amygdalin antigen from the amygdalin hapten described in claim 1 and then immunizing mice; it was deposited in the General Microbiology Center of the China Culture Collection Administration 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. 45923.

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

7. A composition, characterized in that Including the amygdalin monoclonal antibody according to claim 6.

8. A test strip for detecting amygdalin, characterized in that: Comprising the amygdalin monoclonal antibody according to claim 6, or the composition according to claim 7.

9. A kit for detecting amygdalin, characterized in that: It comprises the amygdalin 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 amygdalin.

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