Hybridoma cell strain secreting alpha-tocopherol monoclonal antibody and its application

Abstract

The application discloses a hybridoma cell strain secreting alpha-tocopherol monoclonal antibody and an application thereof, and belongs to the field of immune detection. The hybridoma cell strain secreting alpha-tocopherol monoclonal antibody is preserved in the China General Microbiological Culture Collection Center on April 18, 2024, and has a preservation number of CGMCC No.45925. The alpha-tocopherol monoclonal antibody secreted from the hybridoma cell strain can be used for immune analysis and detection, has good detection sensitivity and specificity for alpha-tocopherol, has an IC 50 value of 0.2 mu g / mL, has a cross rate of less than 1% for alpha-tocopherol analogs, can be used for preparing an immune detection product of alpha-tocopherol, and provides an efficient detection method and means for detection of alpha-tocopherol.

Description

Technical Field

[0001] This invention belongs to the field of immunoassay technology, and in particular relates to a hybridoma cell line that secretes α-tocopherol monoclonal antibody and its application. Background Technology

[0002] Vitamin E (VE), also known as tocopherol, was first discovered in 1992. VE exists in eight forms, including α, β, γ, and δ-tocotrienols, all of which possess biological activity. α-Tocopherol exhibits the greatest biological activity. α-Tocopherol is insoluble in water, heat-resistant, but destroyed by oxygen, ultraviolet light, and alkalis. In infants aged 1-11 months, the average α-tocopherol intake for girls is 2.9-4.9 mg / day, and for boys, it 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, leafy green vegetables, and nuts are also good sources. Vitamin E is an intracellular antioxidant that delays the rancidity of fatty acids in the digestive tract of plants and animals, protecting cells from damage caused by toxic unsaturated fatty acid peroxides. In addition to its antioxidant physiological effects, vitamin E also maintains the integrity of red blood cells and protects the male reproductive system. Vitamin E is also involved in nucleic acid metabolism, coenzyme Q synthesis, hormone synthesis, and vitamin C synthesis. Vitamin E deficiency can lead to nerve and muscle damage, resulting in loss of sensation in the arms and legs, loss of motor control, muscle weakness, vision deterioration, and a weakened immune system. Therefore, a technology is needed to determine the vitamin E content in food.

[0003] Currently, the main methods for vitamin E determination include chromatographic analysis, microbiological analysis, sensor analysis, and immunoassay. Chromatographic analysis boasts high sample throughput, high analytical efficiency, high sensitivity, and good selectivity, and can quantify target analytes through specific monitoring of multiple fragment ions. However, chromatographic analysis requires expensive instruments and demands a certain level of operator skill. Microbiological analysis can achieve high-throughput detection, but the long culture time is time-consuming. Sensor analysis has made significant progress in vitamin detection, but still faces challenges such as high instrument costs, susceptibility to food matrices, and difficulty in rapid on-site detection. Based on the specific reaction between antigens and antibodies, immunoassay has emerged as a promising alternative method for vitamin determination, with specific antibodies at its core. This invention is proposed based on this understanding. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention provides a hybridoma cell line that secretes α-tocopherol monoclonal antibodies. The α-tocopherol monoclonal antibodies secreted by this 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 to detect the vitamin content in food, health products, etc.

[0005] The first objective of this invention is to provide a hybridoma cell line that secretes an α-tocopherol monoclonal antibody, the hybridoma cell line being named monoclonal cell line SLLB, which was deposited on April 18, 2024, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 45925.

[0006] Furthermore, hybridoma cell lines were obtained by immunizing animals with a complete antigen prepared from the α-tocopherol hapten, as shown in the following structure:

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

[0008] S1. Prepare a complete antigen from the α-tocopherol hapten, and use the complete antigen to immunize animals.

[0009] S2. Blood samples are collected from immunized animals to screen for serum immunogenicity and immunosuppressive capacity.

[0010] S3. The spleen cells and myeloma cells of the selected immunized animals are fused and cultured to obtain the hybridoma cell line that secretes α-tocopherol monoclonal antibody.

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

[0012] Furthermore, the animal in question is a mouse.

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

[0014] Furthermore, the carrier proteins include, but are not limited to, bovine serum albumin (BSA) and ovalbumin (OVA).

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

[0016] (1) The above α-tocopherol hapten was activated to obtain an activated solution;

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

[0018] Further, in step (1), the activation is to dissolve the α-tocopherol hapten and add ethylene glycol and sodium borohydride to react.

[0019] Furthermore, the 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 α-tocopherol complete antigen.

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

[0022] Further, 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, serum immunotiter and immunosuppressive capacity are detected by indirect competitive enzyme-linked immunosorbent assay (ic-ELISA).

[0024] A second objective of this invention is to provide the application of the above-mentioned hybridoma cell line in the secretion of α-tocopherol monoclonal antibodies.

[0025] A third objective of this invention is to provide an α-tocopherol monoclonal antibody, which is secreted by the aforementioned hybridoma cell line.

[0026] A fourth objective of this invention is to provide the application of the above-mentioned hybridoma cell lines or α-tocopherol monoclonal antibodies in vitamin detection, especially in the detection of α-tocopherol.

[0027] A fifth objective of this invention is to provide an α-tocopherol detection product comprising the aforementioned α-tocopherol monoclonal antibody. Of course, the detection product can be prepared in any form, such as a kit, reagent, or test strip.

[0028] Furthermore, the α-tocopherol detection product also includes α-tocopherol coated antigen.

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

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

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

[0032] This invention provides a monoclonal antibody hybridoma cell line with high affinity for α-tocopherol, good specificity, and high detection sensitivity. The IC50 of the α-tocopherol monoclonal antibody against α-tocopherol is...50 With a concentration of 0.2 μg / mL and a cross-conversion rate of less than 1% with α-tocopherol analogs, it can be used to establish an enzyme-linked immunosorbent assay (ELISA) method for α-tocopherol. This lays the foundation for the research and development and promotion of detection products such as indirect competitive ELISA kits, helps to realize the detection of α-tocopherol, and provides an immunoassay method and raw materials for the immunoassay of α-tocopherol, thus having practical application value.

[0033] Preservation of biological materials

[0034] The monoclonal cell line SLLB was deposited on April 18, 2024, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 45925, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. Attached Figure Description

[0035] 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...

[0036] Figure 1 This is the standard inhibition curve of α-tocopherol by the α-tocopherol monoclonal antibody of the present invention. Detailed Implementation

[0037] 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.

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

[0039] This invention uses an α-tocopherol analogue as a hapten, and conjugates the hapten to a carrier protein using the EDC method. The success of the conjugation is determined using a UV spectrophotometer. For the initial immunization, BALB / c mice are immunized with a mixture of 100 μg of complete α-tocopherol antigen and an equal volume of complete Freund's adjuvant via subcutaneous injection at multiple sites on the neck and back. Multiple booster immunizations (50 μg / mouse) are administered using incomplete Freund's adjuvant. The final immunization is performed via intraperitoneal injection using 25 μg of complete α-tocopherol antigen (diluted with physiological saline and without adjuvant). High titer and low IC50 are selected. 50 Mouse spleen cells were fused with SP2 / 0 myeloma cells using the PEG 4000 method. Hybrid cells from the three cell lines were selected using selective culture medium. Cells were then screened using an indirect competitive enzyme-linked immunosorbent assay (ELISA) and subcloned four times to obtain a monoclonal antibody hybridoma cell line. The monoclonal antibody secreted by this cell line exhibited good specificity and detection sensitivity (IC50) for α-tocopherol. 50With a value of 0.2 μg / mL, it can be used to detect dietary foods for special medical purposes, providing raw materials for the immunoassay of α-tocopherol in food, and has practical application value.

[0040] Example 1: Preparation of complete antigen

[0041] The α-tocopherol hapten is conjugated to a carrier protein using the EDC method to obtain the α-tocopherol artificial antigen. In this example, the α-tocopherol hapten prepared is conjugated with bovine serum albumin (BSA) or ovalbumin (OVA) to obtain the complete antigen VE-EDC-BSA / OVA. VE-EDC-BSA is used as an immunogen to immunize mice, and VE-EDC-OVA is used as a coating agent for assays in mouse serum and fusion subclones.

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

[0043]

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

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

[0046] b. Dialysis: Cut an 8cm dialysis bag, boil it in boiling water for 3 minutes and cool it, then store it in deionized water at 4℃ for later use; put the VE-EDC-BSA / OVA conjugate into the dialysis bag and dialyze it in 0.01mol / L PBS, changing it every 8 hours, and dialyze for 3 days to obtain the complete antigen VE-EDC-BSA / OVA, which should be taken out and stored at -20℃.

[0047] Example 2: Immunization of mice

[0048] For the initial immunization, BALB / c mice were immunized with a mixture of 100 μg of the complete α-tocopherol antigen (VE-EDC-BSA) and an equal volume of complete Freund's adjuvant, emulsified, and injected subcutaneously at multiple sites on the neck and back. Four weeks later, a booster immunization was performed with half the dose of the complete antigen (50 μg / mouse), emulsified with incomplete Freund's adjuvant. Subsequent booster immunizations were administered at 3-week intervals. For the final sprint immunization, the dose was again halved (25 μg / mouse), and the complete antigen was diluted with physiological saline and injected intraperitoneally. After the third immunization, tail-disconnected blood samples were collected for testing. Serum titers and IC50 were determined using an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA). 50 Choose high-performance ICs 50 Low-grade 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. Collection of 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. The required number of SP2 / 0 tumor cells before fusion should be 1-4 * 102. 7 To ensure that SP2 / 0 tumor cells are in the logarithmic growth phase before fusion. During fusion, tumor cells are collected, suspended in RPMI-1640 basal culture medium, and cell counting is performed.

[0052] b. After euthanizing mice by cervical dislocation, immediately sterilize them in 75% alcohol for about 5 minutes. Aseptically remove the spleen, gently grind it using a syringe tip, and pass it through a 200-mesh cell sieve to obtain a spleen cell suspension. Collect the suspension in 50 mL sterile centrifuge tubes, centrifuge at 1200 rpm for 8 minutes, wash the spleen cells with RPMI-1640 medium, remove any large tissue impurities, and repeat the process three times. After the final centrifugation, dilute the spleen cells to a specific volume, count them, and set aside for later use.

[0053] 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 the centrifuge tube to stand and hold it firmly with both hands. At min 3 and min 4, add 1 mL of RPMI-1640 medium dropwise every 1 min. At min 5 and min 6, add 1 mL of RPMI-1640 medium dropwise every 30 s. At min 7, add 1 mL of RPMI-1640 medium dropwise every 10 s. Then incubate at 37°C for 5 min. Centrifuge at 800 rpm for 10 min, discard the supernatant, gently break up the cells in the centrifuge tube, and add RPMI-1640 selective medium (HAT medium) containing 20% ​​fetal bovine serum and 2% 50×HAT at a rate of 200 μL / well to a 96-well cell plate. Incubate at 37°C in a 5% CO2 incubator.

[0054] (2) 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, α-tocopherol standards were used, and the inhibitory effect on positive cells was determined using ic-ELISA. Cell wells showing good inhibition of α-tocopherol standards were selected, and subcloning was performed using the limiting dilution method. Seven days later, the same method was used for detection. Subcloning was performed four times according to the above method to finally obtain the α-tocopherol monoclonal antibody cell line SLLB.

[0055] Example 4: Preparation and Identification of Monoclonal Antibodies

[0056] 1. Sensitivity Experiment

[0057] 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 2 × 10⁻⁶ g of paraffin oil. 6 Ascites fluid was collected from α-tocopherol hybridoma cells starting on day 7. The ascites fluid was then purified 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 after centrifugation. Next, an equal volume of saturated ammonium sulfate solution was used to precipitate IgG-type monoclonal antibodies. After centrifugation and discarding the supernatant, the antibodies were dissolved in 0.01M PBS solution (pH 7.4), dialyzed to desalt, and finally the purified monoclonal antibodies were stored at -20°C.

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

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

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

[0061] (4) Sample addition: The antiserum (antiserum obtained by diluting the blood from the tail of mice with antibody diluent) was serially diluted from 1:1000 and added to each well of the coated sample at 100 μL / well. The mixture was reacted at 37°C for 30 min. After thorough washing, HRP-goat anti-mouse IgG diluted at 1:3000 was added at 100 μL / well. The mixture was reacted at 37°C for 30 min.

[0062] (5) Color development: Remove the microplate, 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] The IC50 of monoclonal antibody α-tocopherol was determined by ic-ELISA. 50 The value was 0.2 μg / mL, indicating that it has good sensitivity to α-tocopherol and can be used for the immunoassay detection of α-tocopherol.

[0065] Solution preparation:

[0066] 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.

[0067] Phosphate-buffered saline (PBS): 8.0g 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.

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

[0069] 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.

[0070] 2. Specificity test

[0071] The IC50 of monoclonal antibodies against α-tocopherol was determined using an indirect competitive ELISA method. 50 The concentration was 0.2 μg / mL, and its IC50 for compounds such as vitamin A, vitamin K1, vitamin K2, vitamin K3, and vitamin K4 was verified. 50 The cross-reactivity rate and cross-reactivity value are calculated as follows:

[0072] (IC50 of α-tocopherol) 50 IC of other compounds 50 )×100%, as shown in Table 1.

[0073] Table 1 IC50 of α-tocopherol monoclonal antibody against α-tocopherol and cross-conjugates 50 and cross-reactivity

[0074]

[0075]

[0076] The monoclonal antibody showed 100% cross-reactivity with α-tocopherol, while the cross-reactivity with vitamins A, K1, K2, K3, and K4 was less than 1%. This indicates that the monoclonal antibody obtained in this invention has high sensitivity to α-tocopherol and also high specificity (cross-reactivity rate with analogs less than 1%).

[0077] 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 hybridoma cell line, characterized in that: The hybridoma cell line secretes α-tocopherol monoclonal antibody, with accession number CGMCC No. 45925.

2. The use of the hybridoma cell line according to claim 1 in the preparation of α-tocopherol monoclonal antibody.

3. An alpha-tocopherol monoclonal antibody, characterized by: The α-tocopherol monoclonal antibody is secreted by the hybridoma cell line described in claim 1.

4. The application of the hybridoma cell line of claim 1 or the α-tocopherol monoclonal antibody of claim 3 in the detection of α-tocopherol.

5. A test product characterized by: The test product contains the α-tocopherol monoclonal antibody as described in claim 3.

6. The test product of claim 5, wherein: The tested products also include coating agents.

7. The test product of claim 6, wherein: The coating is obtained by coupling the activated hapten with a carrier protein.

8. The detection product of claim 7, wherein: The structure of the hapten is shown in the following formula: 。 9. The detection product of claim 7, wherein, The carrier protein includes ovalbumin or bovine serum albumin.

10. The application of the detection product according to any one of claims 5-9 in the detection of α-tocopherol.

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