Hybridoma cell strain secreting vitamin B12 monoclonal antibody and application of hybridoma cell strain

Abstract

The invention relates to a hybridoma cell strain secreting a vitamin B12 monoclonal antibody and application of the hybridoma cell strain. A monoclonal antibody with high sensitivity and specificity to the vitamin B12 is screened out, and the time-resolved fluorescence immunoassay kit for detecting the vitamin B12 is prepared by combining a time-resolved fluorescence analysis technology. The time-resolved fluorescence immunoassay kit disclosed by the invention has high sensitivity to vitamin B12, can detect 0.05 mu g / kg of vitamin B12 in milk powder, and is far lower than the detection limit of 4 mu g / kg in the prior art. And the time-resolved fluorescence immunoassay kit disclosed by the invention has very high specificity on the vitamin B12, and has no inhibition effect on VB3, VB9, VB5 and VB6. Therefore, the time-resolved fluorescence immunoassay kit disclosed by the invention can be used for accurately detecting the vitamin B12, and is particularly widely applied to the detection of special medical foods.

Description

Technical Field

[0001] The present invention relates to the technical field of immune detection, in particular to a hybridoma cell line secreting vitamin B12 monoclonal antibody and application thereof. Background Art

[0002] Vitamin B12 (VB12), also known as cobalamin, is an essential water-soluble vitamin found in foods such as meat, eggs and dairy products. Supplementing VB12 can prevent diseases such as pernicious anemia, Alzheimer's disease, and depression. Therefore, VB12 plays an important role in maintaining human life and health. However, excessive VB12 can also produce toxic side effects, such as folic acid deficiency. Therefore, the detection of VB12 is very important.

[0003] The methods commonly used to detect VB12 include instrumental analysis, immunoassay and microbiological methods. Among the instrumental analysis methods, the more common ones are high performance liquid chromatography (HPLC) and LC-MS / MS methods, which have high sensitivity and accurate qualitative analysis, but the equipment is relatively expensive and requires professional operators to perform sample analysis. The microbiological method requires VB12 by virtue of the growth of microorganisms, but the growth time of microorganisms is long and cannot meet the needs of rapid on-site detection. Immunoassay methods based on antigen-antibody reactions, such as colloidal gold test strips, time-resolved fluorescence test strips, etc., have the advantages of simple operation and high detection flux, which can meet the needs of rapid on-site detection. However, the existing immunoassay methods have low sensitivity and low specificity for VB12 detection, especially in special medical foods (special medical foods are specially processed and prepared to meet the special needs of people with limited food intake, digestion and absorption disorders, metabolic disorders or specific disease states for nutrients or diets), and it is particularly important to accurately detect vitamin B12. Summary of the invention

[0004] Therefore, the technical problem to be solved by the present invention is to overcome the problem of low sensitivity and low specificity of the immunoassay method in the prior art for VB12 detection.

[0005] In order to solve the above technical problems, the present invention provides a hybridoma cell line secreting a vitamin B12 monoclonal antibody and its application. The present invention prepares a time-resolved fluorescence immunoassay kit capable of detecting vitamin B12 by screening a monoclonal antibody with high sensitivity and high specificity to vitamin B12 and combining it with a time-resolved fluorescence analysis technique. The test strip of the present invention has high sensitivity to vitamin B12, can detect 0.05 μg / kg of vitamin B12 in milk powder, and has no inhibitory effect on VB3, VB9, VB5 and VB6, and has good specificity.

[0006] The first object of the present invention is to provide a hybridoma cell line, wherein the hybridoma cell line SDHI was deposited in the General Microbiology Center of China Culture Collection Administration on April 18, 2024, with the deposit number CGMCC No. 45920, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

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

[0008] The third object of the present invention is to provide a use of the above monoclonal antibody in the preparation of vitamin B12 detection products.

[0009] The fourth object of the present invention is to provide a detection product containing the above monoclonal antibody.

[0010] Furthermore, the detection product includes a time-resolved fluorescence immunoassay kit.

[0011] Furthermore, the time-resolved fluorescent immunoassay kit includes a base plate, a sample pad, a conjugate pad, a detection pad and a water absorbent pad;

[0012] The detection pad is formed in the middle of the bottom plate, and a quality control line and a detection line are provided on the detection pad, the quality control line contains a vitamin B12 secondary antibody, and the detection line contains a vitamin B12-carrier protein complex;

[0013] The sample pad is formed on one side of the bottom plate close to the detection line of the detection pad, the conjugate pad is overlapped and arranged between the sample pad and the detection pad, and the conjugate pad contains the monoclonal antibody labeled with time-resolved fluorescent microspheres;

[0014] The water absorbent pad is formed on one side of the bottom plate close to the quality control line of the detection pad, and the water absorbent pad partially overlaps with the detection pad;

[0015] Wherein, the marker of the time-resolved fluorescent microspheres includes rare earth elements; the rare earth elements are lanthanide elements.

[0016] Furthermore, the vitamin B12 secondary antibody includes goat anti-mouse secondary antibody.

[0017] Furthermore, the detection pad is made of nitrocellulose membrane.

[0018] Furthermore, the fluorescent microspheres are modified with active groups that are connected to the monoclonal antibodies.

[0019] Furthermore, the movement direction of the sample to be tested is perpendicular to the detection line and the quality control line.

[0020] Furthermore, the kit also includes an extracting solution and a diluent.

[0021] Furthermore, the extract comprises acetic acid with a mass concentration of 4%-5%.

[0022] Furthermore, the diluent includes PBS solution.

[0023] Furthermore, when preparing the monoclonal antibody labeled with time-resolved fluorescent microspheres used in the conjugate pad, a solution containing time-resolved fluorescent microspheres is co-incubated with the monoclonal antibody, and the concentration of the solution containing time-resolved fluorescent microspheres is 20-80 μg / mL.

[0024] The fifth object of the present invention is to provide an application of the above-mentioned detection product in the preparation of vitamin B12 detection products.

[0025] Beneficial effects of the present invention:

[0026] The present invention screens out a monoclonal antibody with high sensitivity and specificity for vitamin B12, and combines the time-resolved fluorescence analysis technology to prepare a time-resolved fluorescence test strip for detecting vitamin B12. The time-resolved fluorescence test strip of the present invention has high sensitivity to vitamin B12, and can detect 0.05 μg / kg of vitamin B12 in milk powder, which is much lower than the detection limit of 4 μg / kg in the prior art. And the time-resolved fluorescence test strip of the present invention has high specificity for vitamin B12, and has no inhibitory effect on VB3, VB9, VB5 and VB6. Therefore, the time-resolved fluorescence test strip of the present invention can accurately detect vitamin B12, and is widely used, especially in the detection of special medical foods.

[0027] Biomaterial Deposit

[0028] The hybridoma cell line SDHI was deposited in the General Microbiology Center of China Culture Collection Administration on April 18, 2024, with the deposit number CGMCC No. 45920, and the deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] 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

[0030] Figure 1 This is the schematic diagram of the time-resolved fluorescence test strip;

[0031] Figure 2 This is the optimization diagram of the dilution multiple of the fluorescent microsphere dilution solution;

[0032] Figure 3 It is the establishment of the standard curve of the test strips;

[0033] Figure 4 It is the standard curve of the time-resolved fluorescence test strip. DETAILED DESCRIPTION

[0034] 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.

[0035] In the description of the present invention, it should be noted that the terms "front", "rear", "center", "up", "down", "left", "right", "vertical", "horizontal", "inside", "outside" and the like indicate directions or positional relationships based on the directions or positional relationships shown in the drawings, and are only for the convenience of describing the present invention and simplifying the description, and do not indicate or imply that the devices or elements referred to must have directions or positional relationships.

[0036] The invention has a specific orientation, is constructed and operated in a specific orientation, and therefore cannot be understood as limiting the invention. In addition, the terms "first", "second", and "third" are only used for descriptive purposes and cannot be understood as indicating or implying relative importance.

[0037] In the description of the present invention, it should be noted that, unless otherwise clearly specified and limited, the terms "installed", "connected", and "connected" should be understood in a broad sense, for example, it can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection or an electrical connection; it can be a direct connection, or it can be indirectly connected through an intermediate medium, it can also be the internal connection of two components, it can be a wireless connection, or it can be a wired connection. For ordinary technicians in this field, the specific meanings of the above terms in the present invention can be understood according to specific circumstances.

[0038] The technical features involved in the different embodiments of the present invention described above can be combined with each other as long as they do not conflict with each other.

[0039] In addition, unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or prepared by existing methods.

[0040] Example 1: Preparation of monoclonal antibodies

[0041] 1. Obtaining the complete antigen: Add 2M hydrochloric acid to 25 mg of the VB12 hapten shown in Formula I, stir overnight for 12 hours, blow dry the liquid in the bottle with nitrogen, add 0.01M PBS to dissolve, adjust the pH value of the reconstituted solution to 4.0, and then couple it with KLH by EDC method to prepare the VB12 complete antigen.

[0042]

[0043] 2. Animal immunization: Mix VB12 complete antigen with an equal amount of complete Freund's adjuvant to obtain complete Freund's adjuvant containing antigen, mix VB12 complete antigen with an equal amount of incomplete Freund's adjuvant to obtain incomplete Freund's adjuvant containing antigen, and perform multiple subcutaneous injections of BALB / c mice on the back of the neck for immunization (except for sprint immunization); the first immunization uses complete Freund's adjuvant containing antigen, and the dose is 100ug / mouse; multiple booster immunizations use incomplete Freund's adjuvant containing antigen and the dose is halved to 50ug / mouse; sprint immunizations do not use adjuvants, and the complete antigen diluted with physiological saline is directly injected intraperitoneally, and the dose is further halved to 25ug / mouse; the first immunization and the second booster immunization are separated by one month, the multiple booster immunizations are separated by 21 days, and the sprint immunization and the last booster immunization are separated by 18-21 days; the immune effect of mice is observed by indirect competitive enzyme-linked immunosorbent assay (ic-ELISA), that is, the titer and inhibition of mouse serum are detected; at the same time, the cross-reactivity of mouse serum to other vitamins (VB3, VB9, VB5, VB6) is detected. 0.5 ng / mL VB12, 0.5 ng / mL VB9, 0.5 ng / mL VB3, 0.5 ng / mL VB5, and 0.5 ng / mL VB6 were added to the experimental groups, and the inhibition rate of mouse serum was calculated using the following formula (results are shown in Table 1):

[0044] Inhibition rate (%) = 1-(serum OD value of mice in the experimental group / serum OD value of mice in the control group) × 100%

[0045] Table 1 Mouse serum screening results

[0046]

[0047] Among them, the antibody produced by 4# mouse had an inhibition rate of 91% on VB12, was highly sensitive to VB12 and had no obvious cross-reaction with other B vitamins, so this mouse was selected for cell fusion.

[0048] 3. 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:

[0049] a. Cut off the tail to collect blood. After killing the mice by cervical dislocation, immediately put them in 75% alcohol for disinfection and soak for about 5 minutes. Take out the spleen of the mice aseptically, grind them moderately with the rubber tip of the syringe and pass them through a 200-mesh cell sieve to obtain a spleen cell suspension, collect them, centrifuge them (1200rpm, 8min), wash the spleen cells three times with RPMI-1640 medium, dilute the spleen cells to a certain volume after the last centrifugation, count them, and set aside;

[0050] b. Collect SP2 / 0 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 per ml of culture medium should reach (1-4)×10 7 To ensure that SP2 / 0 tumor cells are in the logarithmic growth phase before fusion, tumor cells are collected and suspended in RPMI-1640 basal culture medium during fusion, and cell counting is performed;

[0051] c. Fusion process 7min: In the first minute, 1mL of PEG 1500 was added to the cells from slow to fast speed; in the second minute, the cells were 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 the cells were incubated at 37°C for 5min; centrifuged (800rpm, 8min), the supernatant was discarded, and the cells were resuspended in RPMI-1640 screening culture medium containing 20% ​​fetal bovine serum and 2% 50×HAT, and added to a 96-well cell plate at 200μL / well, and cultured in a 37°C, 5% CO2 incubator;

[0052] 4. Cell screening and cell line establishment: On the third day of cell fusion, the fused cells were half-changed with RPMI-1640 screening medium. On the fifth day, the medium was fully replaced with RPMI-1640 transition medium containing 20% ​​fetal bovine serum and 1% 100×HT. On the seventh day, the cell supernatant was collected for screening.

[0053] The screening was divided into two steps: the first step was to screen the positive cell wells by ic-ELISA method, and the second step was to use pantothenic acid as the standard and measure the inhibitory effect of positive cells by ic-ELISA method;

[0054] The cell wells with good inhibition of VB12 standard were selected and subcloned by limiting dilution method, and tested by the same method 7 days later;

[0055] Subcloning was performed three times according to the above method, and finally a hybridoma cell line secreting VB12 monoclonal antibody was obtained.

[0056] The preserved hybridoma cell line was expanded and cultured, and injected into mice to induce ascites; 1 mL of ascites was taken, and 1 mL of sodium acetate buffer of equal volume was added, 33 μL of caprylic acid was added dropwise under stirring at room temperature, and the mixture was shaken for 30 minutes, and centrifuged at 8000 r / min for 5 minutes to obtain a supernatant; 2 mL of saturated ammonium sulfate was added, and the mixture was allowed to stand at 4°C for 2 hours; centrifuged at 8000 r / min for 5 minutes, the supernatant was discarded, and the precipitate was dissolved in PBS solution and dialyzed for 2-3 days to obtain a monoclonal antibody against VB12.

[0057] Example 2: Preparation of fluorescent microsphere conjugate pad

[0058] Using the carboxyl groups on the surface of TRFM, fluorescent microsphere-labeled antibodies were prepared by the EDC-mediated active ester method. 100 μL of TRFMs (2 mg / mL) and 500 μL of 0.04 mol / L borate buffer were mixed and shaken in a 2 mL centrifuge tube. Then, 15 μL of EDC (20 mg / mL) was added and reacted under magnetic stirring at room temperature for 20 minutes to activate the carboxyl groups on the surface of TRFM. Centrifuge at 12000 × g for 20 minutes at 4 ° C, discard the supernatant, add 600 μL of resuspended TRFM labeled with fluorescent microspheres, and place it in an ultrasonic device for 1 minute. Subsequently, 30 μg of the monoclonal antibody of Example 1 was added to the resuspension for labeling and magnetically stirred at room temperature for 2 hours. After labeling, 100 μL of blocking solution (BSA (1%, w / v)) was slowly added and reacted at room temperature for 2 hours to block the remaining sites on the surface of the microspheres.

[0059] The final solution was centrifuged at 12,000 × g for 10 min and the supernatant was discarded. The pellet was dissolved in 500 μL of fluorescent microsphere marker resuspension buffer and stored at 4°C for future use. The monoclonal antibody-fluorescent microsphere marker was then sprayed onto the glass fiber membrane.

[0060] Example 3: Preparation of a time-resolved fluorescence immunoassay kit for quantitative detection of VB12

[0061] The structure of the time-resolved fluorescent immunoassay kit of this embodiment includes a bottom plate, a sample pad, a conjugate pad, a detection pad and a water absorbent pad (see Figure 1 ). Among them, the material of the bottom plate is polyvinyl chloride (PVC), the material of the detection pad is nitrocellulose membrane, the fluorescent microsphere pad is prepared by spraying VB12 monoclonal antibody labeled with fluorescent microspheres on the glass fiber membrane, and the detection pad is provided with a quality control line and a detection line. The quality control line is sprayed with goat anti-mouse secondary antibody, and the detection line is sprayed with VB12-BSA (bovine serum albumin) solution. Then the sample pad, fluorescent microsphere pad, detection pad and water absorbent pad are sequentially pasted on the bottom plate.

[0062] In this embodiment, the specification of the time-resolved fluorescent test strip is 8 cm long × 4 mm wide. The sample pad, fluorescent microsphere pad, detection pad and water absorbent pad are sequentially pasted on the PVC board from top to bottom, wherein the overlap length between the sample pad and the fluorescent microsphere pad is 15 mm, the overlap length between the fluorescent microsphere pad and the nitrocellulose membrane is 3 mm, the overlap length between the detection pad and the water absorbent pad is 15 mm, and the sample pad and the water absorbent pad are aligned with the upper and lower edges of the bottom plate.

[0063] Example 4: Detection of time-resolved fluorescence immunoassay kit

[0064] 1. Prepare the sample to be tested

[0065] (1) Test strip optimization experimental samples: Take milk powder samples without VB12 and add VB12 to the concentrations of 0 and 0.5 μg / kg respectively.

[0066] (2) Test strip standard curve samples: Take milk powder samples without VB12 and add VB12 to concentrations of 0, 50, 100, 200, 500, 1000 and 5000 μg / kg respectively.

[0067] (3) Samples to be tested (comparison of recovery rate and detection method): Take three positive samples containing VB12.

[0068] 2. Quantitative immunochromatographic detection of VB12 in food

[0069] (1) Sample detection optimization

[0070] Weigh 1 g of the milk powder sample to be tested prepared in step 1 (1) into a 15 mL centrifuge tube, add 3 mL of water (heated in a 55°C water bath), shake well for 3 min, sonicate in a 60°C water bath for 30 min, cool, add 1 mL of 4% acetic acid, shake well for 3 min, centrifuge at 12000 rpm for 15 min, filter the clarified liquid with a 0.22 m filter membrane, take 100 μL of the filtrate, add 200 μL of the diluent, mix well, add to the sample well (marked with S) of the test card that has been preheated to 37°C, cover the incubator lid, and time for 8 min; after the timer ends, take out the test card and read the result within 20 s (see Table 2).

[0071] According to the ratio of the T line to the C line of the test strip, the dilution multiple with the largest difference in T / C values ​​when the VB12 addition amount was 0 μg / kg and 0.5 μg / kg was selected as the optimal value of the dilution multiple of the diluent, and finally a dilution multiple of 10 was selected.

[0072] Table 2 Optimization of dilution multiples of fluorescent microsphere diluent

[0073]

[0074] (2) Establishing a standard curve

[0075] Take 1 g of the milk powder standard curve sample prepared in step 1 (2) in a 15 mL centrifuge tube, add 3 mL of water (heated in a 55°C water bath), shake well for 3 min, sonicate in a 60°C water bath for 30 min, cool, add 1 mL of 4% acetic acid, shake well for 3 min, centrifuge at 12000 rpm for 15 min, filter the clarified liquid with a 0.22 m filter membrane, take 100 μL of the filtrate, add 200 μL of the diluent, mix well, add to the sample well (marked with S) of the test card that has been preheated to 37°C, cover the incubator lid, and time for 8 min; after the timer ends, take out the test card, read the result within 20 s, and obtain the standard curve ( Figure 4 ), the detection limit was calculated to be 0.05 μg / kg using the formula LOD=yblank+3×SDblank (where yblank and SDblank are the average T / C value and standard deviation (n=20) of the blank control).

[0076] (3) Recovery rate test

[0077] Take 1g of the milk powder sample prepared in step 1 (3) in a 15mL centrifuge tube, add 3mL of water (heated in a 55℃ water bath), shake well for 3min, ultrasonicate in a 60℃ water bath for 30min, add 1mL of 4% acetic acid after cooling, shake well for 3min, centrifuge at 12000rpm for 15min, take the clarified liquid and filter it with a 0.22m filter membrane, take 100L of the filtrate, add 200L of the diluent, mix well and add it to the sample well (marked with S) of the test card that has been preheated to 37℃, cover the incubator lid, and count for 8min; after the count ends, take out the test card and read the result within 20s. The recovery rate of VB12 is shown in Table 3:

[0078] Table 3 Recovery rate of VB12 in three milk powder samples

[0079] sample Detection concentration (μg / kg) Recovery rate (%) 1 68.1 86 2 64.0 160 3 72.2 91

[0080] It can be seen from the above table that the test strips provided by the present invention have good results in the three milk powder samples and meet the recovery rate requirements of the analytical method.

[0081] 4. Comparison between rapid immune test method and traditional method (microbiological method)

[0082] The microbiological detection of VB12 in special medical foods was determined by Wuxi Food Safety Inspection and Testing Center, and the same samples were tested using the method of the present invention. The results are shown in Table 4.

[0083] As shown in Table 4, compared with the microbiological method, the immunochromatographic method provided by the present invention not only has similar accuracy and repeatability, but also has a safer detection process, low cost and short time.

[0084] Table 4 Comparison of different detection methods for VB12 in special medical foods

[0085] Detection Methods Recovery rate Microbiological method 80-120 Time-resolved fluorescence test strip method 80-120

[0086] 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 hybridoma cell line was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on April 18, 2024, with the deposit number CGMCC No.45920, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

2. A monoclonal antibody secreted by the hybridoma cell line according to claim 1.

3. Use of the monoclonal antibody according to claim 2 in the preparation of a vitamin B12 detection product.

4. A detection product, characterized in that: Containing the monoclonal antibody according to claim 2.

5. The detection product according to claim 4, characterized in that: The detection product includes a time-resolved fluorescent immunoassay kit.

6. The detection product according to claim 5, characterized in that: The time-resolved fluorescent immunoassay kit comprises a base plate, a sample pad, a conjugate pad, a detection pad and a water absorbent pad; The detection pad is formed in the middle of the bottom plate, and a quality control line and a detection line are provided on the detection pad, the quality control line contains a vitamin B12 secondary antibody, and the detection line contains a vitamin B12-carrier protein complex; The sample pad is formed on one side of the bottom plate close to the detection line of the detection pad, the conjugate pad is overlapped and arranged between the sample pad and the detection pad, and the conjugate pad contains the monoclonal antibody according to claim 2 labeled with time-resolved fluorescent microspheres; The water absorption pad is formed on one side of the bottom plate close to the quality control line of the detection pad, and the water absorption pad partially overlaps with the detection pad.

7. The detection product according to claim 6, characterized in that: The vitamin B12 secondary antibody includes goat anti-mouse secondary antibody.

8. The detection product according to claim 6, characterized in that: The marker of the time-resolved fluorescent microspheres includes rare earth elements; the rare earth elements are lanthanide elements.

9. The detection product according to claim 6, characterized in that: When preparing the monoclonal antibody labeled with time-resolved fluorescent microspheres used in the conjugate pad, a solution containing time-resolved fluorescent microspheres is co-incubated with the monoclonal antibody, wherein the concentration of the solution containing time-resolved fluorescent microspheres is 20-80 μg / mL.

10. Use of the detection product according to any one of claims 4 to 9 in the preparation of a vitamin B12 detection product.

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