Hybridoma cell strain secreting plant menadione monoclonal antibody and application of hybridoma cell strain

Abstract

The invention relates to a hybridoma cell strain capable of secreting a plant menadione monoclonal antibody and application of the hybridoma cell strain, and belongs to the field of food safety immunodetection. The menadione hapten is obtained through multiple times of screening and experiments, a menadione complete antigen is prepared from the hapten, an animal is immunized with the complete antigen to obtain a hybridoma cell strain, a menadione monoclonal antibody secreted by the cell strain has excellent affinity and sensitivity to menadione, the IC50 of the menadione monoclonal antibody to plant menadione reaches 0.2 microgram / mL, and the menadione monoclonal antibody secreted by the cell strain has high sensitivity to menadione. The method can be used for preparing an immunodetection product of menadione, and an efficient detection method and means are provided for residual detection of menadione in food.

Description

Technical Field

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

[0002] VK is a class of compounds with a common chemical structure of 2-methyl-1,4-naphthoquinone. VK has four forms. Vitamin K1 (Vitamin K1, VK1) and vitamin K2 (Vitamin K2, VK2) are naturally occurring fat-soluble vitamins, while vitamin K3 (Vitamin K3, VK3) and vitamin K4 (Vitamin K4, VK4) are synthetic vitamins. It is easily soluble in organic solvents, insoluble in water, and sensitive to acid, alkali, light, heat, oxygen, and ultraviolet light. VK is a cofactor for blood coagulation and promotes blood coagulation. VK is related to osteocalcin-containing proteins in the bone matrix and participates in bone metabolism. It can also help the body use insulin, regulate blood sugar and type II diabetes; VK promotes sphingolipid synthesis and can protect developing oligodendrocytes and neurons from intracellular oxidative stress. VK deficiency reduces bone mineralization, leading to osteoporosis, prolonged coagulation time, and bleeding in severe cases. In addition, VK deficiency can cause neonatal hemorrhagic disease. Conversely, consuming more than the recommended daily intake of VK can have adverse health effects, including loss of appetite, pale complexion, muscle stiffness, or difficulty breathing.

[0003] At present, the main methods for determining plant menadiones are chromatography, microbial analysis, sensor analysis, and immunoassay. Among them, chromatography has the characteristics of high analytical efficiency, high sensitivity, and good selectivity. However, chromatography requires expensive instruments and a certain data processing capability. The microbial culture time of microbial analysis is long, which wastes time and cannot achieve rapid detection. Sensor analysis has made great progress in the detection of vitamins, but it still faces problems such as high cost of detection instruments, 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. Compared with instrumental methods, immunoassay methods are low in cost, fast in speed, high in accuracy, and high in sensitivity, and have been rapidly developed in the detection of hormones, antibiotics, heavy metals, biological toxins, environmental pollutants, and clinical diagnosis. Summary of the invention

[0004] In order to solve the above technical problems, the present invention provides a hybridoma cell line secreting a monoclonal antibody to phytomenadione and its application. The product prepared by the present invention is used for the study of phytomenadione immunoassay method, providing necessary artificial antigens for future research. The antibody prepared by the cell line has good specificity and detection sensitivity to phytomenadione, and can be used to establish an immunological detection method for phytomenadione.

[0005] The present invention is achieved through the following technical solutions:

[0006] The first object of the present invention is to provide a hybridoma cell line that secretes phytomenadione monoclonal antibodies. The hybridoma cell line was deposited in the General Microbiology Center (CGMCC) of the China Culture Collection Administration on October 31, 2024. The deposit address is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The deposit number is CGMCC No. 46221, and the classification name is a monoclonal cell line.

[0007] The second object of the present invention is to provide a phytomenadione monoclonal antibody secreted and produced by the hybridoma cell line.

[0008] The third object of the present invention is to provide a phytomenadione hapten for preparing the hybridoma cell line, wherein the phytomenadione hapten has the structural formula:

[0009]

[0010] The fourth object of the present invention is to provide a phytomenadione complete antigen, which is prepared from the phytomenadione hapten and an immunogenic carrier.

[0011] In one embodiment of the present invention, the immunogenic carrier is selected from at least one of chicken ovalbumin, bovine serum albumin, keyhole limpet hemocyanin, human serum albumin, lactoferrin, horseradish peroxidase, thyroglobulin and immunoglobulin.

[0012] The fifth object of the present invention is to provide a composition, which contains the hybridoma cell line and / or the phytomenadione monoclonal antibody.

[0013] The sixth object of the present invention is to provide a kit for detecting phytomenadione, wherein the kit contains at least one of the hybridoma cell line, the phytomenadione monoclonal antibody and the composition.

[0014] In one embodiment of the present invention, the kit is selected from an enzyme-linked immunosorbent assay kit, a fluorescent immunoassay kit or a chemiluminescent immunoassay kit.

[0015] The seventh object of the present invention is to provide a test strip for detecting phytomenadione, wherein the test strip contains at least one of the hybridoma cell line, the phytomenadione monoclonal antibody and the composition.

[0016] In one embodiment of the present invention, the test strip comprises a sample pad, a conjugation pad, a nitrocellulose membrane and a water absorbent pad, the nitrocellulose membrane is provided with a detection line and a quality control line in sequence, and the conjugation pad is coated with the phytomenadione monoclonal antibody according to claim 2.

[0017] The eighth object of the present invention is to provide a chip for detecting phytomenadione, wherein the chip contains at least one of the hybridoma cell line, the phytomenadione monoclonal antibody and the composition.

[0018] The ninth objective of the present invention is to provide the use of the hybridoma cell line, the phytomenadione monoclonal antibody, the composition, the kit, the test strip or the chip in detecting phytomenadione.

[0019] The present invention also provides a method for preparing a hybridoma cell line secreting a phytomenadione monoclonal antibody, comprising the following steps:

[0020] S1, preparing the phytomenadione hapten into a complete antigen, and using the complete antigen to immunize animals;

[0021] S2. Collect blood from the immunized animals and screen the serum immune titer and immunosuppressive ability of the immunized animals;

[0022] S3. Fusing and culturing the screened spleen cells of the immune animals with myeloma cells to obtain the hybridoma cell line secreting the phytomenadione monoclonal antibody.

[0023] In one embodiment of the present invention, 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.

[0024] In one embodiment of the present invention, the animal is a mouse.

[0025] In one embodiment of the present invention, in step S1, the structure of the phytomenaquinone hapten is as follows:

[0026]

[0027] In one embodiment of the present invention, in step S1, the phytomenadione complete antigen is obtained by coupling the phytomenadione hapten mentioned above with a carrier protein.

[0028] In one embodiment of the present invention, the carrier protein includes bovine serum albumin BSA, ovalbumin OVA and the like.

[0029] In one embodiment of the present invention, the phytomenadione complete antigen is coupled to the phytomenadione hapten and the carrier protein by the sodium periodate method, and the specific preparation method comprises the following steps:

[0030] (1) activating the phytomenadione hapten to obtain an activation solution;

[0031] (2) Add the activation solution obtained in the above step (1) to the carrier protein solution to react and obtain the complete phytomenadione antigen.

[0032] In one embodiment of the present invention, in step (1), the activation is to dissolve the phytomenadione hapten, add ethylene glycol and sodium borohydride to react.

[0033] In one embodiment of the present invention, N,N-dimethylformamide is used to dissolve the phytomenadione hapten.

[0034] In one embodiment of the present invention, in step (2), the solution after the reaction is dialyzed and separated to obtain the complete phytomenadione antigen.

[0035] In one embodiment of the present invention, in step (3), the carrier protein solution is obtained by dissolving the carrier protein in a carbonate buffer solution.

[0036] In one embodiment of the present invention, the carbonate buffer solution is 0.01 mol / L to 0.5 mol / L, preferably 0.05 mol / L, and the pH is 8.0 to 10.0, preferably 9.6.

[0037] In one embodiment of the present invention, in step S2, the serum immune titer and immunosuppressive ability are detected by indirect competitive enzyme-linked immunosorbent assay (ic-ELISA).

[0038] In one embodiment of the present invention, the phytonadione detection product further comprises a coating source, and the coating source is obtained by coupling the phytonadione hapten with ovalbumin.

[0039] In one embodiment of the present invention, the above-mentioned phytonadione detection kit is used for the residual analysis and detection of phytonadione in food safety detection of dietary food for special medical purposes.

[0040] The above technical solution of the present invention has the following advantages compared with the prior art:

[0041] The present invention provides a hybridoma cell line secreting a monoclonal antibody against phytomenadione and its application. The synthesis steps of the phytomenadione hapten are simple and effective, and can be completely used in immunoassays, providing a convenient approach for future research; the complete antigen prepared based on the hapten, the hybridoma cell line obtained by immunizing mice with the complete antigen, and the monoclonal antibody secreted by the hapten have good specificity and detection sensitivity to phytomenadione (ICP of the monoclonal antibody against phytomenadione is 0.04). 50 0.2 μg / mL), which can realize the detection of plant menadione residues, and provide an immunoassay method and raw materials for the immunoassay of plant menadione residues in food, which has practical application value.

[0042] Biomaterial Deposit

[0043] A hybridoma cell line GAPHD that secretes phytomenadione monoclonal antibodies has been deposited in the General Microbiology Center (CGMCC) of the China Microbiological Culture Collection Administration. The deposit address is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. It is classified and named as a monoclonal cell line. The deposit date is October 31, 2024, and the deposit number is CGMCC No.46221. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] 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:

[0045] Figure 1 This is a standard inhibition curve of the phytonadione monoclonal antibody prepared in Example. DETAILED DESCRIPTION

[0046] 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.

[0047] The present invention uses phytonadione analogs as haptens, uses the sodium periodate method to couple the haptens to the carrier protein, and uses an ultraviolet spectrophotometer to determine whether the coupling is successful. During the first immunization, the complete phytonadione antigen 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 phytonadione 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 50Mouse 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 (IC 50 value 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 plant menadione residues in food, and has practical application value.

[0048] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods, and the materials, reagents, etc. used are all available from commercial sources unless otherwise specified.

[0049] The solution used in the embodiment of the present invention is configured as follows:

[0050] 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.

[0051] 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;

[0052] PBST: PBS containing 0.05% Tween 20;

[0053] 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.

[0054] The phytomenadione hapten used in the examples of the present invention is CAS 132080-60-5, manufactured by Kaiwei Chemical; product number: JZ00116A.

[0055] Example 1 Preparation of complete antigen

[0056] The phytomenadione hapten was coupled to the carrier protein by the sodium periodate method to obtain the phytomenadione artificial antigen. The phytomenadione hapten prepared in Example 1 was coupled with bovine serum albumin BSA to obtain the complete antigen VK1-CHO-BSA; or coupled with chicken ovalbumin OVA to obtain the complete antigen VK1-CHO-OVA.

[0057] The preparation method of complete antigen VK1-CHO-BSA is as follows:

[0058] a. Weigh 4.26 mg of hapten VK1-CHO, dissolve it with 300 μL N,N-dimethylformamide, add 30 μL ethylene glycol, and react for 30 minutes (the reaction solution is called A solution). 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, then add 1.0 mg of sodium borohydride, and stir and react at 4°C for 2 hours. The conjugate VK1-CHO-BSA is obtained; the conjugation method of VK1-CHO-OVA is similar to the above method.

[0059] 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 VK1-CHO-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 VK1-CHO-BSA / OVA, take it out and store it at -20°C.

[0060] Example 2 Immunization of mice

[0061] For the first immunization, the complete antigen of phytonadione 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 it was mixed and emulsified with incomplete Freund's adjuvant, and the intervals between subsequent booster immunizations were 3 weeks; for the sprint immunization, the dose was halved again (25 μg / mouse), and the complete antigen was diluted with physiological saline and injected intraperitoneally into the mice. After the third immunization, the mice could be tail-cut and blood was collected for testing, and the titer and IC of the mouse serum were tested by indirect competitive enzyme-linked immunosorbent assay (ic-ELISA). 50 , select high titer, IC 50 Low mice were fused;

[0062] Example 3 Cell fusion and screening

[0063] (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:

[0064] 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;

[0065] 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;

[0066] 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.

[0067] (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 the positive cell wells by ic-ELISA, and the second step was to select the plant menadione standard and measure the inhibitory effect of the positive cells by ic-ELISA. The cell wells with better inhibition of the plant menadione 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 plant menadione monoclonal antibody cell line GAPHD was obtained.

[0068] Example 4 Preparation and identification of monoclonal antibodies

[0069] 8-10 week old BALB / c mice were intraperitoneally injected with 1 mL of sterile paraffin oil. Seven days later, 2 × 10 6 Phytomenadione hybridoma cells, ascites was collected from the seventh day, and the ascites was purified by the caprylic acid-saturated ammonium sulfate method. Under 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℃.

[0070] (1) Coating: The original VK1-CHO-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.

[0071] (2) Washing: Pour off the solution in the plate and wash with washing solution three times, each time for 3 minutes.

[0072] (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.

[0073] (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.

[0074] (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.

[0075] (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.

[0076] Determination of IC of monoclonal antibody phytonadione by IC-ELISA 50 The value is: 0.2μg / mL, which indicates that it has good sensitivity to phytomenadione and can be used for phytomenadione immunoassay detection.

[0077] Example 5 Specificity Experiment

[0078] Determination of IC of monoclonal antibodies against phytonadione using indirect competitive ELISA 50The IC values ​​of vitamin A, vitamin K2, vitamin K3, vitamin K4, vitamin E and other compounds were verified. 50 And the cross-reaction rate, the cross-reaction value is calculated as follows:

[0079] (IC of phytonadione 50 / IC of other compounds 50 )×100%, as shown in Table 1.

[0080] Table 1 IC of phytomenadione monoclonal antibody against phytomenadione and its cross-compounds 50 and cross-reaction rate

[0081] <![CDATA[IC 50 (μg / mL)]]> Cross-reaction rate Phytonadione 0.2 100％ Vitamin A ＞50 ＜1％ Vitamin K2 ＞50 ＜1％ Vitamin K3 ＞50 ＜1％ Vitamin K4 ＞50 ＜1％ Vitamin E ＞50 ＜1％

[0082] As can be seen from Table 1, the cross-reactivity of the monoclonal antibody to phytomenadione is 100%, and the cross-reactivity to vitamin A, vitamin K2, vitamin K3, vitamin K4, and vitamin E is less than 1%. This indicates that the monoclonal antibody obtained by the present invention has a high sensitivity to phytomenadione (IC 50 value is 0.2 μg / mL) and has high specificity (the cross-reaction rate with analogues is less than 1%).

[0083] 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 secreting a monoclonal antibody against phytomenadione, characterized in that: The hybridoma cell line was deposited in the General Microbiology Center (CGMCC) of the China Microbiological Culture Collection Administration on October 31, 2024. The deposit address is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Building 1, Beichen West Road, Chaoyang District, Beijing. The deposit number is CGMCC No. 46221, and the classification name is monoclonal cell line.

2. A phytomenadione monoclonal antibody, characterized in that: The hybridoma cell line according to claim 1 is secreted and produced.

3. A phytomenadione hapten for preparing the hybridoma cell line according to claim 1, characterized in that: The structural formula of the phytonadione hapten is:

4. A composition, characterized in that The composition contains the hybridoma cell line according to claim 1 and / or the phytomenadione monoclonal antibody according to claim 2.

5. A kit for detecting phytonadione, characterized in that: The kit contains at least one of the hybridoma cell line according to claim 1, the phytomenadione monoclonal antibody according to claim 2, and the composition according to claim 4.

6. The kit according to claim 5, characterized in that The kit is selected from an enzyme-linked immunosorbent assay kit, a fluorescent immunoassay kit or a chemiluminescent immunoassay kit.

7. A test strip for detecting phytonadione, characterized in that: The test strip contains at least one of the hybridoma cell line according to claim 1, the phytomenadione monoclonal antibody according to claim 2, and the composition according to claim 4.

8. The test strip according to claim 7, characterized in that: The test strip comprises a sample pad, a conjugation pad, a nitrocellulose membrane and a water absorbent pad, the nitrocellulose membrane is provided with a detection line and a quality control line in sequence, and the conjugation pad is coated with the phytomenadione monoclonal antibody according to claim 2.

9. A chip for detecting phytonadione, characterized in that: The chip contains at least one of the hybridoma cell line according to claim 1 , the phytomenadione monoclonal antibody according to claim 2 , and the composition according to claim 4 .

10. Use of the hybridoma cell line according to claim 1, the phytomenadione monoclonal antibody according to claim 2, the composition according to claim 4, the kit according to claims 5-6, the test strip according to claims 6-7 or the chip according to claim 9 in detecting phytomenadione.

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