Hybridoma cell strain secreting vitamin b3 monoclonal antibody and application thereof

Abstract

The present application relates to a kind of hybridoma cell strains secreting vitamin B3 monoclonal antibody and its application, belong to immune detection technical field.The present application has obtained a kind of hybridoma cell strains capable of secreting vitamin B3 monoclonal antibody after screening many times, the vitamin B3 monoclonal antibody secreted by the hybridoma cell strains of the present application has excellent affinity and sensitivity to vitamin B3, the IC 50 It reaches 14.36 ng / mL, and for the structural analog of vitamin B3, such as vitamin A, vitamin K2, vitamin K3, vitamin K4, vitamin E, there is no cross reaction.Therefore the monoclonal antibody of the present application can be used for preparing the immune detection product of vitamin B3, provides efficient detection method and means for the residual detection of vitamin B3.

Description

Technical Field

[0001] This invention relates to the field of immunoassay technology, and in particular to a hybridoma cell line that secretes a vitamin B3 monoclonal antibody and its application. Background Technology

[0002] Vitamin B3 (also known as niacin) is a class of compounds sharing the common chemical structure of pyridine-3-carboxylic acid. Vitamin B3 exists primarily in three forms: vitamin B3 (nicotinic acid), nicotinamide, and related derivatives. Vitamin B3 and nicotinamide are naturally occurring water-soluble vitamins (vitamin B3), while vitamin B3 derivatives (such as vitamin B3 inositol esters) are mostly synthetically produced. Vitamin B3 is readily soluble in water and ethanol, slightly soluble in organic solvents, and relatively stable to light, heat, and oxygen. It can undergo hydrolysis in acidic and alkaline solutions. Vitamin B3 is an important component of coenzymes NAD (nicotinamide adenine dinucleotide) and NADP (nicotinamide adenine dinucleotide phosphate), participating in energy metabolism, redox reactions, and DNA repair. Simultaneously, vitamin B3 also regulates blood lipids, lowering LDL cholesterol and triglycerides while raising HDL cholesterol; it participates in glucose metabolism, helping to maintain stable blood sugar levels; and it contributes to the health of the skin, digestive system, and nervous system. Vitamin B3 deficiency can cause pellagra, with typical symptoms including dermatitis, diarrhea, and dementia. Severe deficiency may also be accompanied by angular cheilitis, glossitis, and peripheral neuropathy. Conversely, long-term intake of vitamin B3 exceeding the tolerable upper intake level may cause adverse reactions, commonly including skin flushing, itching, headache, abnormal liver function, and gastrointestinal discomfort. In severe cases, it may lead to hyperuricemia and glucose intolerance.

[0003] Currently, the main methods for determining vitamin B3 include chromatographic analysis, microbiological analysis, sensor analysis, and immunoassay. Chromatographic analysis is characterized by high efficiency, high sensitivity, and good selectivity. However, it requires expensive instruments and significant data processing capabilities. Microbiological analysis involves lengthy microbial culture times, which is time-consuming and hinders rapid detection. Sensor analysis has made significant progress in vitamin detection, but it still faces challenges such as high instrument costs, susceptibility to food matrices, and difficulty in rapid on-site detection. Enzyme-linked immunosorbent assay (ELISA) is an extremely efficient, sensitive, and rapid detection method suitable for rapid on-site detection of large numbers of samples, providing a new approach for vitamin B3 detection. ELISA requires a monoclonal antibody against vitamin B3, but existing vitamin B3 monoclonal antibodies have limited IC50... 50 The lowest concentration was 603.41 ng / mL, making it impossible to detect low concentrations of vitamin B3. Summary of the Invention

[0004] Therefore, the technical problem to be solved by the present invention is to overcome the lack of a monoclonal antibody for detecting low concentrations of vitamin B3 in the prior art.

[0005] To address the aforementioned technical problems, this invention provides a hybridoma cell line that secretes vitamin B3 monoclonal antibodies and its applications. Through multiple screening processes, this invention has obtained a hybridoma cell line capable of secreting vitamin B3 monoclonal antibodies. The vitamin B3 monoclonal antibodies secreted by this hybridoma cell line exhibit excellent affinity and sensitivity to vitamin B3, with an IC50 value of [missing information - likely referring to an IC50 value]. 50 The concentration reached 14.36 ng / mL, and there was no cross-reactivity with structural analogs of vitamin B3, such as vitamin A, vitamin K2, vitamin K3, vitamin K4, and vitamin E. Therefore, the monoclonal antibody of this invention can be used to prepare immunoassay products for vitamin B3, providing an efficient detection method and means for the detection of vitamin B3 residues.

[0006] The first objective of this invention is to provide a hybridoma cell line, which is proposed to be taxonomically named a monoclonal cell line and has the accession number CGMCC NO.46738.

[0007] Furthermore, the hybridoma cell line is obtained by immunizing animals with a complete antigen prepared from a hapten, wherein the structural formula of the hapten is shown in Formula I:

[0008] .

[0009] Furthermore, the complete antigen is obtained from the hapten coupled with a carrier protein.

[0010] Furthermore, the carrier protein includes bovine serum albumin.

[0011] A second objective of this invention is to provide an application of the above-mentioned hybridoma cell line in the detection of vitamin B3.

[0012] A third objective of this invention is to provide a monoclonal antibody secreted by the aforementioned hybridoma cell line.

[0013] A fourth objective of this invention is to provide an application of the above-mentioned monoclonal antibody in the detection of vitamin B3.

[0014] A fifth objective of this invention is to provide a detection product for vitamin B3, the detection product comprising the aforementioned monoclonal antibody.

[0015] Furthermore, the test product also includes a coating agent.

[0016] Furthermore, the coating is prepared from a hapten-conjugated carrier protein; the carrier protein includes chicken oocyte albumin.

[0017] Compared with the prior art, the above-described technical solution of the present invention has the following advantages:

[0018] The vitamin B3 monoclonal antibody secreted by the hybridoma cell line of the present invention exhibits excellent affinity and sensitivity to vitamin B3, with an IC50 value of [missing information - likely related to an IC50 value]. 50 The concentration reached 14.36 ng / mL, and there was no cross-reactivity with structural analogs of vitamin B3, such as vitamin A, vitamin K2, vitamin K3, vitamin K4, and vitamin E. Therefore, the monoclonal antibody of this invention can be used to prepare immunoassay products for vitamin B3, providing an efficient detection method and means for the detection of vitamin B3 residues.

[0019] Preservation of biological materials

[0020] The cell line SLM is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences. It is classified as a monoclonal cell line, deposited on November 12, 2025, with accession number CGMCC No. 46738. Attached Figure Description

[0021] 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.

[0022] Figure 1 This is the standard inhibition curve of the vitamin B3 (niacin) monoclonal antibody of the present invention. Detailed Implementation

[0023] 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.

[0024] The culture media involved in the following examples are as follows:

[0025] RPMI-1640 medium (mg / L): L-arginine 290, L-asparagine 50, L-aspartic acid 20, L-cysteine ​​dihydrochloride 65.15, L-glutamic acid 20, glycine 10, L-histidine 15, L-hydroxyproline 20, L-isoleucine 50, L-leucine 50, L-lysine hydrochloride 40, L-methionine 15, L-phenylalanine 15, L-proline 20, L-serine 30, L-threonine 20, L-tryptophan 5. L-Tyrosine 23.19, L-Valine 20, Para-aminobenzoic acid 1, Calcium nitrate 100, Anhydrous magnesium sulfate 48.84, Anhydrous sodium dihydrogen phosphate 676.13, Potassium chloride 400, Sodium chloride 6000, Glucose 2000, Reduced glutathione 1, Phenol red 5, L-Glutamine 300, Biotin 0.2, D-Calcium pantothenate 0.25, Folic acid 1, I-Inositol 35, Nicotinamide 1, Choline chloride 3, Pyridoxine hydrochloride 1, Riboflavin 0.2, Thiamine hydrochloride 1, Vitamin B12 0.005, Sodium bicarbonate 2000.

[0026] The solutions used in these embodiments of the invention are prepared as follows:

[0027] 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.

[0028] 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, pH adjusted to 7.2-7.4 with NaOH or HCl, and then brought to a final volume of 1000 mL.

[0029] PBST: PBS containing 0.05% Tween 20;

[0030] TMB colorimetric solution: Solution A: 18.43 g Na2HPO4·12H2O, 9.33 g citric acid, diluted to 1000 mL with pure water; Solution B: 60 mg TMB dissolved in 100 mL ethylene glycol. Mix solutions A and B in a 5:1 ratio to obtain the TMB colorimetric solution. Mix fresh before use.

[0031] The detection methods involved in the following embodiments are as follows:

[0032] Vitamin B3 inhibition rate detection method: The optimal antigen and antibody concentrations for ic-ELISA were selected using a checkerboard assay. The antigen was diluted to 0.1 μg / mL with carbonate buffer (CBS), and the antibody was diluted to 0.03 μg / mL with antibody dilution buffer. After selecting the optimal operating point, the vitamin B3 standard was diluted to eight concentrations (0, 2, 5, 10, 20, 50, 100, 200 ng / mL). Following the ic-ELISA procedure, the results were plotted using OriginPro 8.5 (see results below). Figure 1 (As shown), obtain the standard inhibition curve of vitamin B3 and calculate IC50. 50 .

[0033] Example 1: Preparation of Immunogen

[0034] The vitamin B3 hapten is coupled to a carrier protein using the carbodiimide method to obtain the vitamin B3 coated antigen. The vitamin B3 hapten is then coupled to bovine serum albumin (BSA) to obtain the immunogen Hapten-BSA.

[0035] The method for preparing the immunogen Hapten-BSA is as follows:

[0036] a. Weigh 1.26 mg of the hapten Hapten, dissolve it in 300 μL of methanol, add 1.34 mg of NHS, and react for 0.5 hours. Then add 1.84 mg of EDC and continue the activation reaction for 4-6 hours. Weigh 5.0 mg of BSA and add 2 mL of carbonate buffer solution (the dissolved BSA protein is called solution B). Add the activation solution to the protein solution. The conjugate Hapten-EDC-BSA is then obtained.

[0037] b. Dialysis: Cut an 8 cm dialysis bag, boil it in boiling water for 3 min and cool it, then store it in deionized water at 4℃ for later use; put the Hapten-EDC-BSA conjugate into the dialysis bag and dialyze it in 0.01 mol / L PBS, changing it every 8 h, and dialyze for 3 days to obtain the complete antigen Hapten-EDC-BSA, which should be taken out and stored at -20℃.

[0038] Example 2: Preparation of the coating agent

[0039] The vitamin B3 hapten is coupled to chicken ovalbumin using the carbodiimide method to obtain the vitamin B3 coated antigen. The vitamin B3 hapten is then coupled to chicken ovalbumin (OVA) to obtain the coated antigen Hapten-OVA.

[0040] The method for preparing the immunogen Hapten-OVA is as follows:

[0041] a. Weigh 1.26 mg of the hapten Hapten, dissolve it in 300 μL of methanol, add 1.34 mg of NHS, and react for 0.5 hours. Then add 1.84 mg of EDC and continue the activation reaction for 4-6 hours. Weigh 5.0 mg of OVA, add it to 2 mL of carbonate buffer (the OVA protein is dissolved and called solution B), and add the activation solution to the protein solution to obtain the conjugate Hapten-EDC-OVA.

[0042] b. Dialysis: Cut an 8 cm dialysis bag, boil it in boiling water for 3 min and cool it, then store it in deionized water at 4℃ for later use; put the Hapten-EDC-OVA conjugate into the dialysis bag and dialyze it in 0.01 mol / L PBS, changing it every 8 h, and dialyze for 3 days to obtain the immunogen Hapten-EDC-OVA, which should be taken out and stored at -20℃.

[0043] Example 3: Preparation of hybridoma cell lines

[0044] 1. Immunity in mice

[0045] For the initial immunization, BALB / c mice were immunized with a mixture of 100 μg of vitamin B3 immunogen 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 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;

[0046] 2. Cell fusion and screening

[0047] (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:

[0048] 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.

[0049] b. After euthanizing mice by cervical dislocation, immediately sterilize them in 75% alcohol for about 5 minutes. Aseptically remove the spleen, gently grind it with a syringe tip, and pass it through a 200-mesh cell sieve to obtain a spleen cell suspension. Collect 50 mL of the suspension in a sterile centrifuge tube, centrifuge at 1200 r / min 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.

[0050] 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 r / min 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 to the medium. Add 200 μL / well to a 96-well cell plate and incubate at 37°C in a 5% CO2 incubator.

[0051] (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. The screening was carried out in two steps: first, positive cell wells were screened using ic-ELISA; second, vitamin B3 standard was selected, and the inhibitory effect on positive cells was determined using ic-ELISA. Cell wells that showed good inhibition of vitamin B3 standard were selected, and subcloning was performed using the limiting dilution method. The same method was used for detection seven days later. Subcloning was performed four times according to the above method to finally obtain the vitamin B3 monoclonal antibody cell line SLM.

[0052] Example 4: Preparation and Identification of Monoclonal Antibodies

[0053] 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. 6Vitamin B3 hybridoma cells were used. Ascites fluid was collected starting on day 7, and the ascites fluid was purified for antibody purification 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 by centrifugation. IgG-type monoclonal antibodies were then precipitated with an equal volume of saturated ammonium sulfate solution, centrifuged, and the supernatant was discarded. The precipitate was dissolved in 0.01 M PBS solution (pH 7.4), dialyzed to desalt, and finally the purified monoclonal antibodies were stored at -20°C.

[0054] (1) Coating: The original Hapten-EDC-OVA was diluted 3-fold from 1 µg / mL with 0.05M (pH 9.6) carbonate buffer, 100 μL / well, and reacted at 37℃ for 2 h.

[0055] (2) Washing: Pour out the solution in the plate and wash with washing solution 3 times, 3 min each time.

[0056] (3) Sealing: After patting dry, add 200 μL / well sealing solution and react at 37℃ for 2 h. Wash and dry for later use.

[0057] (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 coating at 100 μL / well. The reaction was carried out at 37℃ for 30 min. After thorough washing, HRP-goat anti-mouse IgG diluted at 1:3000 was added at 100 μL / well. The reaction was carried out at 37℃ for 30 min.

[0058] (5) Color development: Take out 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.

[0059] (6) Termination and measurement: Add 50 μL of stop solution to each well to terminate the reaction, and then measure the OD of each well using a microplate reader. 450 value.

[0060] The IC50 of monoclonal antibody against vitamin B3 was determined using ic-ELISA. 50 The value was 14.36 ng / mL, indicating good sensitivity to vitamin B3, and it can be used for the immunoassay of vitamin B3.

[0061] The IC50 of monoclonal antibodies against vitamin B3 was determined using an indirect competitive ELISA method. 50 The concentration was 14.36 ng / mL, and its IC50 for compounds such as vitamin A, vitamin K2, vitamin K3, vitamin K4, and vitamin E was verified. 50 And the cross-reactivity rate, the cross-reactivity value is calculated as follows: (IC50 of vitamin B3) 50IC of other compounds 50 () × 100%, as shown in Table 1.

[0062] Table 1. IC50 of vitamin B3 monoclonal antibodies against vitamin B3 and cross-links 50 and cross-reactivity

[0063]

[0064] As shown in Table 1, the monoclonal antibody exhibits a 100% cross-link with vitamin B3, while the cross-links with vitamins A, K2, K3, K4, and E are all less than 1%. This indicates that the monoclonal antibody obtained in this invention has high sensitivity (IC50) to vitamin B3. 50 The value is 14.36 ng / mL, and it also has high specificity (cross-reactivity with analogs is less than 1%).

[0065] 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 proposed taxonomic name for the hybridoma cell line is monoclonal cell line, and its accession number is CGMCC NO.46738.

2. The hybridoma cell line according to claim 1, characterized in that, The hybridoma cell line was obtained by immunizing animals with a complete antigen prepared from a hapten, wherein the structural formula of the hapten is shown in Formula I: 。 3. The hybridoma cell line according to claim 1, characterized in that, The complete antigen is obtained by conjugating the hapten to a carrier protein.

4. The hybridoma cell line according to claim 1, characterized in that, The carrier protein includes bovine serum albumin.

5. The use of the hybridoma cell line according to any one of claims 1-4 in the detection of vitamin B3.

6. A monoclonal antibody secreted by a hybridoma cell line according to any one of claims 1-4.

7. The use of the monoclonal antibody according to claim 6 in the detection of vitamin B3.

8. A vitamin B3 detection product, characterized in that, The detection product includes the monoclonal antibody as described in claim 6.

9. The testing product according to claim 8, characterized in that, The tested products also include coating agents.

10. The testing product according to claim 9, characterized in that, The coating is prepared by conjugating a hapten with a carrier protein; the carrier protein includes chicken oocyte albumin.

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