Hybridoma cell strain secreting alpha-tocopherol monoclonal antibody and application of hybridoma cell strain

Abstract

The invention discloses a hybridoma cell strain capable of secreting an alpha-tocopherol monoclonal antibody and application of the hybridoma cell strain, and belongs to the field of immunodetection. The hybridoma cell strain secreting the alpha-tocopherol monoclonal antibody has been preserved in the China General Microbiological Culture Collection Center on April 18, 2024, and the preservation number is CGMCC No.45925. The alpha-tocopherol monoclonal antibody secreted by the hybridoma cell strain can be used for immunoassay and detection, and can be used for detecting the alpha-tocopherol monoclonal antibody. According to the present invention, the alpha-tocopherol immunodetection kit has characteristics of good alpha-tocopherol detection sensitivity and good alpha-tocopherol detection specificity, IC50 value of 0.2 [mu] g / mL, and alpha-tocopherol analogue crossing rate less than 1%, and can be used for preparing the alpha-tocopherol immunodetection product so as to provide the efficient alpha-tocopherol detection method and the efficient alpha-tocopherol detection method;

Description

Technical Field

[0001] The invention belongs to the technical field of immune detection, and particularly relates to a hybridoma cell strain secreting alpha-tocopherol monoclonal antibody and application thereof. Background Art

[0002] Vitamin E (VE), also known as tocopherol, was first discovered in 1992. VE has eight forms, including α, β, γ, and δ tocotrienols, all of which have VE biological activity. Among them, α-tocopherol has the greatest biological activity. α-Tocopherol is insoluble in water, heat-resistant, and is destroyed by oxygen, ultraviolet rays, and alkali. In infants aged 1-11 months, the average α-tocopherol intake for girls is 2.9-4.9 mg / day, and for boys is 3.2-5.4 mg / day. Wheat germ oil and sunflower seeds are the best sources of VE. Meat, eggs, dairy products, fish, seafood, grains, green leafy vegetables, and nuts are good sources of VE. VE is an intracellular antioxidant that delays the rancidity of fatty acids in the digestive tract of plants and animals and protects cells from damage by toxic unsaturated fatty acid peroxides. In addition to its antioxidant physiological effects, VE can also maintain the integrity of red blood cells and protect the male reproductive system. VE is also related to nucleic acid metabolism, coenzyme Q synthesis, hormone synthesis, and vitamin C synthesis. VE deficiency can cause nerve and muscle damage, leading to loss of sensation in the arms and legs, loss of control over body movements, muscle weakness, vision degeneration, and a weakened immune system. Therefore, it is necessary to invent a technology to measure VE in food.

[0003] At present, the main methods for determining vitamin E are chromatographic analysis, microbiological analysis, sensor analysis, and immunoassay. Among them, chromatographic analysis has the characteristics of high sample throughput, high analysis efficiency, high sensitivity and good selectivity, and can quantify the target by special monitoring of multiple fragment ions. However, chromatographic analysis requires expensive instruments and has certain requirements on the operator's skills. Microbiological analysis can achieve high-throughput detection. However, the cultivation time is long, which wastes time. Sensors have made great progress in the detection of vitamins, but still face the problems of high instrument cost, susceptibility to food matrix, and difficulty in rapid on-site detection. Based on the specific reaction between antigens and antibodies, immunoassay has become a potential alternative method for determining vitamins, and its core is specific antibodies. Therefore, the present invention is proposed. Summary of the invention

[0004] To solve the above technical problems, the present invention provides a hybridoma cell line that secretes α-tocopherol monoclonal antibodies. The α-tocopherol monoclonal antibodies secreted by the hybridoma cell line have good specificity and detection sensitivity for α-tocopherol, and can be used to establish an immunological detection method for α-tocopherol and detect the vitamin content in food, health products, etc.

[0005] The first object of the present invention is to provide a hybridoma cell line that secretes α-tocopherol monoclonal antibodies. The hybridoma cell line is named monoclonal cell line SLLB, which was deposited in the General Microbiology Center of China National Microbiological Culture Collection Committee on April 18, 2024, with a deposit number of CGMCC No.45925.

[0006] Further, the hybridoma cell line is obtained by immunizing an animal with a complete antigen prepared with the α-tocopherol hapten shown in the following structure:

[0007] The preparation of the hybridoma cell line specifically comprises the following steps:

[0008] S1. preparing α-tocopherol hapten into a complete antigen, and using the complete antigen to immunize animals;

[0009] S2. Collect blood from the immunized animals and screen the serum immune titer and immunosuppressive ability of the immunized animals;

[0010] S3. Fusing and culturing the screened spleen cells of the immune animals with myeloma cells to obtain the hybridoma cell line secreting the α-tocopherol monoclonal antibody.

[0011] Furthermore, in step S1, the animal immunization process includes primary immunization, booster immunization and sprint immunization. The primary immunization uses complete antigen and complete Freund's adjuvant, the booster immunization uses complete antigen and incomplete Freund's adjuvant, and the sprint immunization uses complete antigen.

[0012] Furthermore, the animal is a mouse.

[0013] Furthermore, the complete antigen is obtained by coupling α-tocopherol hapten with a carrier protein.

[0014] Furthermore, the carrier protein includes but is not limited to bovine serum albumin BSA, ovalbumin OVA and the like.

[0015] Furthermore, the above-mentioned α-tocopherol complete antigen is coupled to the α-tocopherol hapten and the carrier protein by the carbodiimide method. The specific preparation method comprises the following steps:

[0016] (1) activating the α-tocopherol hapten to obtain an activation solution;

[0017] (2) adding the activation solution obtained in the above step (1) to the carrier protein solution to react and obtain the complete α-tocopherol antigen.

[0018] Furthermore, in step (1), the activation is performed by dissolving the α-tocopherol hapten and adding ethylene glycol and sodium borohydride to react.

[0019] Furthermore, α-tocopherol hapten was dissolved using N,N-dimethylformamide.

[0020] Furthermore, in step (2), the solution after the reaction is dialyzed and separated to obtain the complete α-tocopherol antigen.

[0021] Furthermore, in step (3), the carrier protein solution is obtained by dissolving the carrier protein in a carbonate buffer solution.

[0022] Furthermore, the carbonate buffer solution has a concentration of 0.01-0.5 mol / L (preferably 0.05 mol / L) and a pH of 8.0-10.0 (preferably 9.6).

[0023] Furthermore, in step S2, the serum immune titer and immunosuppressive ability are detected by indirect competitive enzyme-linked immunosorbent assay (ic-ELISA).

[0024] The second object of the present invention is to provide the use of the hybridoma cell line in secreting α-tocopherol monoclonal antibody.

[0025] The third object of the present invention is to provide an α-tocopherol monoclonal antibody, wherein the α-tocopherol monoclonal antibody is secreted by the above hybridoma cell line.

[0026] The fourth object of the present invention is to provide the use of the hybridoma cell line or α-tocopherol monoclonal antibody in vitamin detection, especially α-tocopherol detection.

[0027] The fifth object of the present invention is to provide an α-tocopherol detection product, wherein the α-tocopherol detection product comprises the above-mentioned α-tocopherol monoclonal antibody. Of course, the detection product can be prepared into any form such as a kit, a reagent, a test strip, etc.

[0028] Furthermore, the α-tocopherol detection product also includes α-tocopherol coating source.

[0029] Furthermore, the α-tocopherol coating is obtained by activating the α-tocopherol hapten and coupling it with a carrier protein (such as ovalbumin).

[0030] The sixth object of the present invention is to provide the application of the detection product in vitamin detection or α-tocopherol detection.

[0031] The above technical solution of the present invention has the following advantages compared with the prior art:

[0032] The present invention provides a monoclonal antibody hybridoma cell line with high affinity, good specificity and detection sensitivity to α-tocopherol, and the IC value of the α-tocopherol monoclonal antibody to α-tocopherol is50 The concentration of the purified ELISA kit is 0.2 μg / mL, and the cross-over rate to α-tocopherol analogs is less than 1%. It can be used to establish an enzyme-linked immunosorbent assay for α-tocopherol, laying a foundation for the research, development and promotion of detection products such as indirect competitive ELISA kits, and is helpful to realize the detection of α-tocopherol. It provides an immunoassay method and raw materials for the immunoassay of α-tocopherol, and has practical application value.

[0033] Biomaterial Deposit

[0034] The monoclonal cell line SLLB was deposited in the General Microbiology Center of China Culture Collection Administration on April 18, 2024, with the deposit number CGMCC No.45925, and the deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] 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

[0036] Figure 1 This is the standard inhibition curve of the α-tocopherol monoclonal antibody of the present invention against α-tocopherol. DETAILED DESCRIPTION

[0037] 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.

[0038] The scheme of the present invention is as follows:

[0039] The present invention uses α-tocopherol analogs as haptens, uses the EDC method to couple the haptens with carrier proteins, and uses an ultraviolet spectrophotometer to determine whether the coupling is successful. During the first immunization, the complete antigen of α-tocopherol is mixed and emulsified with an equal amount of complete Freund's adjuvant at an amount of 100 μg / mouse for injection immunization, and BALB / c mice are immunized by multiple subcutaneous injections on the back of the neck; multiple booster immunizations (50 μg / mouse) are performed with incomplete Freund's adjuvant; the last sprint immunization is performed with α-tocopherol complete antigen (25 μg / mouse, the complete antigen is diluted with physiological saline and does not contain adjuvant), and the mice are injected intraperitoneally. Select high titer, low IC 50 Mouse spleen cells were fused with SP2 / 0 myeloma cells by PEG 4000, and the hybrid cells after the three cell fusions were screened by selective culture medium; the cells were screened by indirect competitive enzyme-linked immunosorbent assay and subcloned four times, and finally a monoclonal antibody hybridoma cell line was obtained. The monoclonal antibody secreted by this cell line has good specificity and detection sensitivity for α-tocopherol (IC 50value is 0.2μg / mL), which can realize the detection of dietary foods for special medical purposes and provide raw materials for the immunological detection of α-tocopherol in food, and has practical application value.

[0040] Example 1 Preparation of complete antigen

[0041] The α-tocopherol hapten is coupled to the carrier protein by the EDC method to obtain the α-tocopherol artificial antigen. Among them, the α-tocopherol hapten prepared in this embodiment is coupled with bovine serum albumin BSA or ovalbumin OVA to obtain the complete antigen VE-EDC-BSA / OVA. Among them, VE-EDC-BSA is used as an immunogen to immunize mice, and VE-EDC-OVA is used as a coating source for cell determination of mouse serum and subclones after fusion.

[0042] The structure of the α-tocopherol hapten is shown below:

[0043]

[0044] The preparation method of the complete antigen VE-EDC-BSA is as follows:

[0045] a. Weigh 2.46 mg of hapten VE, 1.45 mg of N-hydroxysuccinimide, and 1.23 mg of 1-ethylcarbodiimide hydrochloride, dissolve them in 400 μL of N,N-dimethylformamide (the reaction solution is called A solution), and react at room temperature for 4-6 hours. Weigh 6.0 mg of BSA, add 2 mL of carbonate buffer solution (BSA protein is dissolved and called B solution), add A solution dropwise to B solution at room temperature, adjust the pH of the mixture to 8-9 with 1M NaOH solution, and react at room temperature overnight to obtain the conjugate VE-EDC-BSA. The method for coupling VE-EDC-OVA is similar to the above method.

[0046] b. Dialysis: Cut an 8 cm dialysis bag, boil it in boiling water for 3 min and cool it, store it in deionized water at 4°C for later use; put the conjugate VE-EDC-BSA / OVA conjugate solution into the dialysis bag and dialyze it in 0.01 mol / L PBS, changing it every 8 hours. Dialyze for 3 days to obtain the complete antigen VE-EDC-BSA / OVA, take it out and store it at -20°C.

[0047] Example 2 Immunization of mice

[0048] For the first immunization, the complete antigen of α-tocopherol (VE-EDC-BSA) was mixed with an equal amount of complete Freund's adjuvant and emulsified at a dose of 100 μg / mouse for injection, and BALB / c mice were immunized by multiple subcutaneous injections on the back of the neck; after a lapse of 4 weeks, a booster immunization was performed, and the dose of the complete antigen was halved (50 μg / mouse), and the complete antigen was mixed and emulsified with incomplete Freund's adjuvant, and the interval between subsequent booster immunizations was 3 weeks; for the sprint immunization, the dose was halved again (25 μg / mouse), and the complete antigen was diluted with saline and injected intraperitoneally into the mice. After the third immunization, the mice can be tail-cut and blood can be collected for testing, and the titer and IC of the mouse serum can be tested by indirect competitive enzyme-linked immunosorbent assay (ic-ELISA). 50 , select high titer, IC 50 Low mice were fused;

[0049] Example 3 Cell fusion and screening

[0050] (1) Three days after the sprint immunization, cell fusion was performed according to the conventional PEG 4000 (polyethylene glycol) method. The specific steps are as follows:

[0051] a. Collect SP2 / 0 tumor cells: 7-10 days before fusion, culture SP2 / 0 tumor cells in RPMI-1640 medium containing 10% FBS (fetal bovine serum) in a 5% CO2 incubator. Before fusion, the number of SP2 / 0 tumor cells should reach 1-4*10 7 , to ensure that SP2 / 0 tumor cells are in the logarithmic growth phase before fusion. When fusion occurs, tumor cells are collected, suspended in RPMI-1640 basal culture medium, and cell counts are performed;

[0052] b. After killing the mouse by dislocating the neck, immediately sterilize it in 75% alcohol, soak it for about 5 minutes, remove the mouse spleen by aseptic operation, grind it moderately with a syringe rubber tip and pass it through a 200-mesh cell sieve to obtain a spleen cell suspension. Collect it in a 50mL sterile centrifuge tube and centrifuge it at 1200r / min for 8 minutes. Wash the spleen cells with RPMI-1640 medium, pick out the larger tissue impurities, and repeat the operation three times. After the last centrifugation, dilute the spleen cells to a certain volume, count them, and set aside;

[0053] 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, the tube was left to stand and held tightly with both hands. In the third and fourth minutes, 1mL of RPMI-1640 medium was added within 1min; in the fifth and sixth minutes, 1mL of RPMI-1640 medium was added every 30s; in the seventh minute, 1mL of RPMI-1640 medium was added every 10s. Then, the cells were incubated at 37°C for 5min. Centrifuged at 800r / min for 10min, the supernatant was discarded, the cells in the centrifuge tube 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°C, 5% CO2 incubator.

[0054] (2) 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 out the positive cell wells by ic-ELISA, and the second step was to select α-tocopherol standard and measure the inhibitory effect of positive cells by ic-ELISA. The cell wells with better inhibition of α-tocopherol standard were selected, subcloned by limiting dilution method, and tested by the same method seven days later. Four subclonings were performed according to the above method, and finally the α-tocopherol monoclonal antibody cell line SLLB was obtained.

[0055] Example 4 Preparation and identification of monoclonal antibodies

[0056] 1. Sensitivity test

[0057] 8-10 week old BALB / c mice were intraperitoneally injected with 1 mL of sterile paraffin oil. Seven days later, 2 × 10 6 α-tocopherol 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 (pH 7.4), dialyze and desalt, and finally obtain the purified monoclonal antibody and store it at -20℃.

[0058] (1) Coating: The coating source (VE-EDC-OVA) was diluted 3-fold from 1 μg / mL in 0.05 M (pH 9.6) carbonate buffer, 100 μL / well, and reacted at 37°C for 2 h.

[0059] (2) Washing: Pour off the solution in the plate and wash with washing solution three times, 3 minutes each time.

[0060] (3) Blocking: After patting dry, add 200 μL / well blocking solution and react at 37°C for 2 h. Wash and dry for later use.

[0061] (4) Sample addition: dilute the antiserum (blood was collected from the mouse tail and diluted with antibody diluent to the corresponding multiples) starting from 1:1000 and add it to the coated wells of each dilution, 100 μL / well, and react at 37°C for 30 min. After thorough washing, add 1:3000 diluted HRP-goat anti-mouse IgG, 100 μL / well, and react at 37°C for 30 min.

[0062] (5) Color development: Take out the ELISA plate, wash it thoroughly, add 100 μL of TMB color development solution to each well, and react at 37°C in the dark for 15 min.

[0063] (6) 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 an ELISA reader.

[0064] Determination of IC of monoclonal antibody α-tocopherol by IC-ELISA 50 The sensitivity of the sample to α-tocopherol is 0.2 μg / mL, which indicates that the sample has good sensitivity to α-tocopherol and can be used for α-tocopherol immunoassay detection.

[0065] Solution configuration:

[0066] Carbonate buffer (CBS): Weigh 1.59 g Na2CO3 and 2.93 g 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 1000 mL, and store at 4°C for later use.

[0067] Phosphate buffered saline (PBS): 8.0 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;

[0068] PBST: PBS containing 0.05% Tween 20;

[0069] 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.

[0070] 2. Specificity experiment

[0071] The IC of monoclonal antibodies against α-tocopherol was determined using an indirect competitive ELISA method. 50 The IC values ​​of vitamin A, vitamin K1, vitamin K2, vitamin K3, vitamin K4, etc. were verified. 50 And the cross-reaction rate, the cross-reaction value is calculated as follows:

[0072] (IC of α-tocopherol 50 / IC of other compounds 50 )×100%, as shown in Table 1.

[0073] Table 1 IC of α-tocopherol monoclonal antibody against α-tocopherol and cross- 50 and cross-reaction rate

[0074]

[0075]

[0076] The cross-reaction rate of the monoclonal antibody to α-tocopherol is 100%, and the cross-reaction rate to vitamin A, vitamin K1, vitamin K2, vitamin K3, and vitamin K4 is less than 1%, indicating that the monoclonal antibody obtained by the present invention has high sensitivity to α-tocopherol and high specificity (the cross-reaction rate with analogs is less than 1%).

[0077] 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 hybridoma cell line, characterized in that: The deposit number of the hybridoma cell line is CGMCC No.45925.

2. Use of the hybridoma cell line according to claim 1 in the preparation of α-tocopherol monoclonal antibodies.

3. A monoclonal antibody, characterized in that: The monoclonal antibody is secreted by the hybridoma cell line according to claim 1.

4. Use of the hybridoma cell line according to claim 1 or the monoclonal antibody according to claim 3 in vitamin detection, characterized in that: The vitamins include alpha-tocopherol.

5. A detection product, characterized in that: The detection product contains the monoclonal antibody according to claim 3.

6. The detection product according to claim 5, characterized in that: The detection product also includes a coating source.

7. The detection product according to claim 6, characterized in that: The coating source is obtained by coupling with a carrier protein after hapten activation.

8. The detection product according to claim 7, characterized in that: The structure of the hapten is shown below:

9. The detection product according to claim 7, characterized in that: The carrier protein includes ovalbumin or bovine serum albumin.

10. Use of the detection product according to any one of claims 5 to 9 in vitamin detection, characterized in that: The vitamins include alpha-tocopherol.

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