Hybridoma cell strain capable of secreting pantothenic acid monoclonal antibody and application of hybridoma cell strain

Abstract

The invention relates to a hybridoma cell strain capable of secreting pantothenic acid monoclonal antibodies and application of the hybridoma cell strain, and belongs to the technical field of immunochemistry. A pantothenic acid complete antigen is prepared by synthesizing a pantothenic acid hapten, a hybridoma cell strain is obtained through cell fusion and screening of an indirect competitive enzyme-linked immunosorbent assay, a pantothenic acid monoclonal antibody secreted by the cell strain has good specificity and detection sensitivity on pantothenic acid, and the IC50 value is 25.67 ng / mL; the time-resolved fluorescent test strip for quantitative detection of pantothenic acid is prepared from the pantothenic acid monoclonal antibody, the detection sensitivity of the test strip is further improved by optimizing the addition amount of fluorescent microspheres of the test strip, the lowest detection limit of the obtained test strip to pantothenic acid is 5.08 mu g / kg, and the linear detection range is 3.79-40.15 mu g / kg. According to the present invention, the results can be used for rapid quantitative detection of pantothenic acid in food, and the problem of poor specificity and poor detection sensitivity in the existing pantothenic acid immunodetection method is solved.

Description

Technical Field

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

[0002] Pantothenic acid (VB5) is a water-soluble B vitamin that can be converted into coenzyme A or acyl carrier protein in the body and participate in sugar, fat, protein and energy metabolism. It is an essential nutrient for nerves and brain, and is also an essential substance for fatty acid synthesis of steroids. Pantothenic acid helps the secretion of steroids in the body and maintains the health of skin and hair. At the same time, pantothenic acid can also participate in the synthesis of steroid lipids, melatonin and heme. Pantothenic acid can also help the formation of cells and maintain the normal development of the central nervous system. Pantothenic acid has the function of antibody production, which can help the body resist infectious diseases, alleviate the side effects and toxicity of various antibiotics, and help alleviate allergic symptoms. When pantothenic acid is deficient, it can cause symptoms such as hypoglycemia, skin abnormalities, fatigue and insomnia, loss of appetite and indigestion. Postoperative patients, malnourished patients, and pregnant and lactating women all need to supplement pantothenic acid. Pantothenic acid is widely present in many foods and is also an additive to many foods. Commercial vitamin supplements and vitamin-fortified foods have become an effective means to solve this defect. Therefore, rapid, accurate and reliable detection of pantothenic acid content in food and vitamin supplements is necessary for quality control. Therefore, it is necessary to establish a rapid and accurate method for detecting pantothenic acid.

[0003] High performance liquid chromatography, gas chromatography, gas or liquid chromatography-mass spectrometry have the characteristics of high sensitivity and accurate qualitative analysis, but the equipment is relatively expensive and requires professional operators to perform sample analysis. The immunoassay method based on antigen-antibody reaction has the advantages of simple operation and high detection throughput, which can meet the needs of rapid on-site detection. However, the pantothenic acid immunoassay method that has been reported so far has poor specificity and detection sensitivity and cannot meet the needs of rapid and accurate detection of pantothenic acid. Therefore, it is very important to develop a pantothenic acid monoclonal antibody with high specificity and detection sensitivity, and to develop a method for efficient detection of pantothenic acid using this monoclonal antibody to achieve rapid and accurate detection of pantothenic acid. Summary of the invention

[0004] In order to solve the above technical problems, the present invention provides a hybridoma cell line secreting pantothenic acid monoclonal antibody and its application. The pantothenic acid monoclonal antibody secreted by the hybridoma cell line provided by the present invention has good specificity and detection sensitivity for pantothenic acid, IC 50The value is 25.67ng / mL; the present invention uses pantothenic acid monoclonal antibody to prepare a time-resolved fluorescent test strip for quantitative detection of pantothenic acid, combined with the optimization of the amount of fluorescent microspheres added, the minimum detection limit of the test strip for pantothenic acid is 5.08μg / kg, and the linear detection range is 3.79-40.15μg / kg. The results of the present invention can be used for the rapid quantitative detection of pantothenic acid in food, solving the problem of poor specificity and detection sensitivity in the current pantothenic acid immunoassay method.

[0005] The first object of the present invention is to provide a hybridoma cell line that secretes pantothenic acid monoclonal antibodies, which has been deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration CGMCC, located at the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and is classified and named as a monoclonal cell line. The deposit date is April 18, 2024, and the deposit number is CGMCC No.45919.

[0006] The second object of the present invention is to provide a method for preparing a hybridoma cell line that secretes pantothenic acid monoclonal antibodies. The molecular structure of the pantothenic acid hapten used in the preparation process is:

[0007]

[0008] Furthermore, the method for preparing the pantothenic acid hapten comprises the following steps:

[0009] Weigh 4-(bromomethyl)-phenylacetic acid phenacyl ester and dissolve it in 20 mL of N,N-dimethylformamide (DMF) solution, weigh pantothenic acid and add it to the solution, add potassium fluoride to the solution, and stir at room temperature for 2 hours. Combine the organic phases, wash with water, extract with ethyl acetate, dry over Na2SO4, and concentrate to obtain a yellow oily compound. The compound is dissolved in a CH3COOH solution, zinc powder is added thereto, and stirred at room temperature overnight. Concentrate and combine the organic phases, and purify by a preparative column to obtain a yellow oily compound, i.e., pantothenic acid hapten.

[0010] Furthermore, the method for preparing the complete pantothenic acid antigen comprises the following steps:

[0011] Weigh the pantothenic acid hapten and dissolve it in N,N-dimethylformamide (DMF), then add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS), and stir the mixture at room temperature for 6 hours to obtain solution A; weigh bovine serum albumin BSA, dissolve it in carbonate buffer (CBS) and dilute it to 3 mg / mL to obtain solution B; then, add solution A dropwise into solution B, react at room temperature for 8 hours to obtain a reaction solution, dialyze the reaction solution with PBS solution to remove unreacted small molecule hapten, and obtain the pantothenic acid complete antigen VB5-BSA.

[0012] Furthermore, the method for preparing the pantothenic acid-coated antigen comprises the following steps:

[0013] Weigh pantothenic acid hapten and dissolve it in N,N-dimethylformamide (DMF), then add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS), and stir the mixture at room temperature for 6 hours to obtain solution C; weigh chicken ovalbumin (OVA), and dissolve it in carbonate buffer (CBS) to obtain solution D; then, add solution C dropwise into solution D, react at room temperature for 8 hours to obtain a reaction solution, dialyze the reaction solution with PBS solution to remove unreacted small molecule hapten, and obtain pantothenic acid coated antigen VB5-OVA.

[0014] Furthermore, the method for preparing a hybridoma cell line secreting a pantothenic acid monoclonal antibody comprises the following steps:

[0015] Step S1, preparing pantothenic acid hapten, using the pantothenic acid hapten to prepare pantothenic acid complete antigen, mixing the obtained pantothenic acid complete antigen with complete Freund's adjuvant to obtain antigen-containing complete Freund's adjuvant, and mixing the obtained pantothenic acid complete antigen with incomplete Freund's adjuvant to obtain antigen-containing incomplete Freund's adjuvant;

[0016] Step S2, subcutaneously immunizing the immunized animal with the antigen-containing complete Freund's adjuvant obtained in step S1;

[0017] Step S3, the immunized animals immunized in step S2 are boosted with the incomplete Freund's adjuvant containing the antigen obtained in step S1, and are sprinted with the complete pantothenic acid antigen;

[0018] Step S4: taking spleen cells from the immunized animal subjected to sprint immunization in step S3 and performing cell fusion with myeloma cells.

[0019] Step S5, using the indirect ELISA method to screen out positive cell wells, selecting pantothenic acid as a standard, using the indirect ELISA method to measure the inhibitory effect of positive cells, using the limiting dilution method to perform subcloning, and finally screening out the hybridoma cell line that secretes the pantothenic acid monoclonal antibody.

[0020] The third object of the present invention is to provide a pantothenic acid monoclonal antibody secreted by the above hybridoma cell line.

[0021] Furthermore, the method for preparing the pantothenic acid monoclonal antibody comprises the following steps:

[0022] 8-10 week old BALB / c mice were injected intraperitoneally with 1 mL of sterile paraffin oil. Seven days later, 1×10 6For pantothenic acid hybridoma cells, ascites was collected from the 7th day, and antibodies were purified from the ascites by the caprylic acid-saturated ammonium sulfate method. The purified monoclonal antibodies were stored at -20°C.

[0023] The fourth object of the present invention is to provide a test strip comprising the above-mentioned pantothenic acid monoclonal antibody.

[0024] Furthermore, the test strip includes a backing and a sample pad, a conjugation pad, a coating membrane and a water-absorbing pad arranged on the backing (the sample pad, the conjugation pad, the coating membrane and the water-absorbing pad are sequentially pasted on the bottom plate from top to bottom, and each part overlaps by a certain length);

[0025] A binding pad, which is disposed between the sample pad and the coating membrane and is modified with a labeled pantothenic acid monoclonal antibody;

[0026] A coating membrane, wherein a detection line and a quality control line are provided on the coating membrane; wherein the detection line is provided at one end close to the binding pad and fixed with a pantothenic acid antigen, and the quality control line is provided at one end away from the binding pad and fixed with a pantothenic acid secondary antibody;

[0027] The water absorbent pad is arranged at one end of the coating membrane away from the binding pad.

[0028] Furthermore, the test strip comprises a time-resolved fluorescence test strip.

[0029] Furthermore, the label comprises time-resolved fluorescent microspheres.

[0030] Furthermore, the concentration of the time-resolved fluorescent microspheres used to modify the conjugate pad is 20-80 μg / mL.

[0031] A fifth object of the present invention is to provide a test kit, which contains the above-mentioned test strip.

[0032] The sixth object of the present invention is to provide the use of the above-mentioned pantothenic acid monoclonal antibody, the above-mentioned test strip or the above-mentioned kit in detecting pantothenic acid.

[0033] The above technical solution of the present invention has the following advantages compared with the prior art:

[0034] The pantothenic acid monoclonal antibody secreted by the hybridoma cell line of the present invention has better specificity and detection sensitivity for pantothenic acid (IC50 value is 25.67 ng / mL); the time-resolved fluorescent test strip for quantitative detection of pantothenic acid prepared with the pantothenic acid monoclonal antibody has a minimum detection limit of 5.08 μg / kg for pantothenic acid and a linear detection range of 3.79-40.15 μg / kg. It has better specificity and detection sensitivity in pantothenic acid detection and can be used for rapid and accurate quantitative detection of pantothenic acid in food.

[0035] Biomaterial Deposit

[0036] The hybridoma cell line secreting pantothenic acid monoclonal antibody is deposited in the General Microbiology Center of China Culture Collection Administration, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, and is classified and named as monoclonal cell line. The deposit date is April 18, 2024, and the deposit number is CGMCC No.45919. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] 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

[0038] Figure 1 This is the standard curve of the inhibition of pantothenic acid by the pantothenic acid monoclonal antibody of the present invention.

[0039] Figure 2 It is a structural diagram of a time-resolved fluorescent test strip for quantitatively detecting pantothenic acid according to the present invention.

[0040] Figure 3 It is the standard curve for detecting pantothenic acid by the time-resolved fluorescent test strip of the present invention. DETAILED DESCRIPTION

[0041] 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.

[0042] The culture medium involved in the embodiment is as follows:

[0043] RPMI-1640 medium (mg / L): L-arginine 290, L-asparagine 50, L-aspartic acid 20, L-cystine 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. p-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.

[0044] The reagents involved in the following examples are as follows:

[0045] 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.

[0046] Phosphate buffered saline (PBS): 8.00 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;

[0047] Borate buffer solution: weigh 6.18 g of H3BO3, dissolve it in pure water and make up to 500 ml to obtain a boric acid solution; weigh 5.02 g of Na2B4O7·10H2O, dissolve it in pure water and make up to 500 ml to obtain a borax solution; prepare boric acid and borax solution in a volume ratio of 7:3 to obtain a borate buffer solution.

[0048] PBST: PBS containing 0.05% Tween 20;

[0049] Antibody diluent: PBS with 0.1% gelatin.

[0050] TMB colorimetric solution: Solution A: 18.43g Na2HPO4.12H2O, 9.33g citric acid, dilute to 1000mL with pure water; Solution B: 60mg TMB dissolved in 100mL ethylene glycol. Mix solution A and solution B in a ratio of 5:1 to obtain the TMB colorimetric solution, which can be mixed before use.

[0051] The detection methods involved in the following embodiments are as follows:

[0052] Pantothenic acid inhibition rate detection method: The most suitable antigen and antibody concentrations for ic-ELISA were selected by chessboard test. The antigen was diluted to 0.3, 1, 3 and 10 μg / mL with carbonate buffer (CBS), and the antibody was diluted to 0.3, 1, 3 and 10 μg / mL with antibody diluent. After selecting the best working point, the pantothenic acid standard was diluted to concentrations of 0, 3, 10, 30, 90, 270 and 810 ng / mL, and the ic-ELISA operation steps were followed. Finally, OriginPro 8.5 was used to draw the graph (the results are shown in Figure 2). Figure 1 As shown), obtain the pantothenic acid standard inhibition curve and calculate the IC 50 .

[0053] Example 1: Synthesis of pantothenic acid hapten

[0054] The molecular structure of the pantothenic acid hapten used in the preparation process is:

[0055]

[0056] The synthetic route of pantothenic acid hapten is as follows:

[0057]

[0058] Preparation of pantothenic acid hapten: Weigh 1000 mg (2.89 mmol) of 4-(bromomethyl)-phenylacetic acid phenacyl ester and dissolve it in 20 mL of N,N-dimethylformamide (DMF) solution, weigh 632 mg (2.89 mmol) of pantothenic acid and add it to the solution, add potassium fluoride (670 mg, 11.56 mmol) to the solution, and stir at room temperature for 2 h. Combine the organic phases, wash with 40 mL of water, extract with ethyl acetate, dry with Na2SO4, and concentrate to obtain a yellow oily compound. The compound (1000 mg, 2.06 mmol) is dissolved in 10 mL of CH3COOH solution, zinc powder is added thereto, and stirred at room temperature overnight. Concentrate and combine the organic phases, and purify by preparative column to obtain a yellow oily compound (250 mg), i.e., pantothenic acid hapten.

[0059] Example 2: Synthesis of complete pantothenic acid antigen

[0060] Weigh 1.64 mg of pantothenic acid hapten and dissolve it in 600 μL N,N-dimethylformamide.

[0061] (DMF), then add 2.58 mg 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and 1.55 mg N-hydroxysuccinimide (NHS), and the mixture is stirred at room temperature for 6 hours to obtain solution A; weigh 5 mg bovine serum albumin (BSA), dilute it to 3 mg / mL with 0.01 M carbonate buffer (CBS) to obtain solution B; then, add solution A dropwise into solution B, react at room temperature for 8 hours to obtain a reaction solution, dialyze the reaction solution with PBS solution to remove unreacted small molecule haptens, and obtain the pantothenic acid complete antigen VB5-BSA.

[0062] Example 3: Synthesis of pantothenic acid-coated antigen

[0063] 1.64 mg of pantothenic acid hapten was weighed and dissolved in 600 μL N,N-dimethylformamide (DMF), and then 2.58 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and 1.55 mg of N-hydroxysuccinimide (NHS) were added, and the mixture was stirred at room temperature for 6 hours to obtain solution C; 6 mg of chicken ovalbumin (OVA) was weighed and dissolved in 1 ml of 0.01 M carbonate buffer (CBS) to obtain solution D; then, solution C was added dropwise to solution D, and the reaction was carried out at room temperature for 8 hours to obtain a reaction solution, and the reaction solution was dialyzed with PBS solution to remove unreacted small molecule hapten to obtain the pantothenic acid coated antigen VB5-OVA.

[0064] Example 4: Preparation of hybridoma cell lines secreting pantothenic acid monoclonal antibodies

[0065] 1. Animal immunization: Mix pantothenic acid complete antigen with an equal amount of complete Freund's adjuvant to obtain antigen-containing complete Freund's adjuvant, and mix pantothenic acid complete antigen with an equal amount of incomplete Freund's adjuvant to obtain antigen-containing incomplete Freund's adjuvant, and perform multiple subcutaneous injections on the back of the neck of BALB / c mice 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 directly use complete antigen diluted with physiological saline for intraperitoneal injection, and the dose is further halved to 25ug / mouse; the interval between the first immunization and the second booster immunization is one month, the interval between multiple booster immunizations is 21 days, and the interval between the sprint immunization and the last booster immunization is 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;

[0066] 2. 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:

[0067] 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;

[0068] 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;

[0069] 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;

[0070] 3. Cell screening and cell line establishment: On the third day of cell fusion, the fused cells were half-changed with RPMI-1640 screening medium, and 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 taken for screening.

[0071] 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;

[0072] The cell wells with good inhibition of pantothenic acid standard were selected and subcloned by limiting dilution method, and tested by the same method 7 days later;

[0073] Subcloning was performed three times according to the above method, and finally a hybridoma cell line secreting pantothenic acid monoclonal antibody was obtained.

[0074] Example 5: Preparation and identification of pantothenic acid monoclonal antibodies

[0075] 8-10 week old BALB / c mice were taken and each mouse was intraperitoneally injected with 1 mL of sterile paraffin oil. Seven days later, each mouse was intraperitoneally injected with 1×10 6 For pantothenic acid hybridoma cells, ascites was collected from day 7 and the antibodies were purified by the caprylic acid-saturated ammonium sulfate method.

[0076] Under slightly acidic conditions, octanoic acid can precipitate other impurities in the ascites except IgG immunoglobulin, 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°C.

[0077] Pantothenic acid was detected by indirect competitive ELISA using a monoclonal antibody against pantothenic acid, and the IC 50 The value was 25.67ng / mL, indicating that the detection sensitivity of pantothenic acid was very good; at the same time, the pantothenic acid monoclonal antibody was tested against other analogs (vitamin B3, vitamin B6, vitamin B7, vitamin B9, vitamin B12), and the cross-rate was less than 5% (IC 50 >600ng / mL), indicating that the monoclonal antibody has good specificity, as shown in Table 1, and can therefore be used for pantothenic acid immunoassay detection. (Crossover rate = (IC 50 / Analog IC 50 )×100%).

[0078] Table 1 Cross-reaction rate of pantothenic acid monoclonal antibody

[0079] Vitamins <![CDATA[IC 50 Value (ng / ml)]]> Cross-reaction rate Pantothenic acid 25.67 100％ Vitamin B3 >600 ＜5％ Vitamin B6 >600 ＜5％ Vitamin B7 >600 ＜5％ Vitamin B9 >600 ＜5％ Vitamin B12 >600 ＜5％

[0080] Example 6: Preparation of a time-resolved fluorescence test strip for quantitative detection of pantothenic acid

[0081] (1) Preparation of fluorescent microsphere pads: Using the carboxyl groups on the surface of time-resolved fluorescent microspheres (TRFM), the time-resolved fluorescent microsphere-labeled antibodies were prepared by the EDC-mediated active ester method. Briefly, 100 μL TRFM (2 mg / mL) and 500 μL 0.04 mol / L borate buffer were mixed and shaken in a 2 mL centrifuge tube. Then, 15 μL EDC (20 mg / mL) was added and reacted with magnetic stirring at room temperature for 20 min to activate the carboxyl groups on the TRFM surface. Centrifuge at 12000 × g for 20 min at 4 °C, discard the supernatant, add 600 μL PBS solution and place it in an ultrasonic device for 1 min. Subsequently, 30 μg of pantothenic acid monoclonal antibody was added to the resuspension for labeling and magnetic stirring was performed at room temperature for 2 h. After labeling, 100 μL blocking solution (BSA (1%, w / v)) was slowly added and reacted at room temperature for 2 h to block the remaining sites on the microsphere surface. The final solution was centrifuged at 12000×g for 10 min, and the supernatant was discarded. The precipitate was dissolved in 500 μL PBS solution, and placed in an ultrasonic device for 1 min and stored at 4°C for future use. Subsequently, the pantothenic acid monoclonal antibody-time-resolved fluorescent microsphere marker was sprayed on the glass cellulose membrane at 7 μL / cm to prepare a fluorescent microsphere pad.

[0082] (2) Spraying the quality control line and the detection line on the nitrocellulose membrane: Spray 0.1 mg / mL goat anti-mouse secondary antibody and 0.2 mg / mL VB5-BSA solution on the nitrocellulose membrane at a spraying amount of 7 μL / cm to form two lines, which serve as the quality control line and the detection line, respectively, and then dry them in an environment of 40°C for 48 h.

[0083] (3) Assembling and cutting the test strip: assemble the PVC plate, sample pad, fluorescent microsphere pad, nitrocellulose membrane and absorbent pad in sequence. After assembly, cut into strips to obtain the time-resolved fluorescent test strip for quantitative detection of pantothenic acid. The specification of the time-resolved fluorescent test strip is 8 cm long × 4 mm wide. Figure 2 .

[0084] Example 7: Application of time-resolved fluorescence test strips for quantitative detection of pantothenic acid

[0085] 1. Prepare the sample to be tested

[0086] (1) Test strip optimization experimental samples: Take milk powder samples without pantothenic acid and add pantothenic acid to concentrations of 0 and 100 μg / kg respectively.

[0087] (2) Test strip standard curve samples: Take milk powder samples without pantothenic acid and add pantothenic acid to concentrations of 0, 5, 10, 20, 50, 100, 200 and 500 μg / kg.

[0088] (3) Samples to be tested (comparison of recovery rate and detection method): Take three positive samples containing pantothenic acid.

[0089] 2. Quantitative detection of pantothenic acid in food

[0090] (1) Preparation of the test card: Fix the time-resolved fluorescence test strip in a plastic card shell and press it to obtain the test card.

[0091] (2) Optimization of fluorescent microsphere concentration

[0092] Take 1 g of the milk powder sample to be tested prepared in step 1 (1) and place it in a 15 mL centrifuge tube. Add 3 mL of water (heated in a 55°C water bath), shake well for 3 min, and sonicate in a 60°C water bath for 30 min. After cooling, add 1 mL of extract (3% v / v acetic acid aqueous solution), shake well for 3 min, and centrifuge at 12000 rpm for 15 min. Take the clarified liquid and filter it with a 0.22 μm filter membrane. Take 100 μL of the filtrate, add 200 μL of diluent (0.01 M PBS (1% v / v ON 870) solution), mix well, and add it to the sample well of the test card that has been preheated to 37°C. Cover the incubator lid and time it for 8 min. After the timer ends, take out the test card and read the result within 20 s.

[0093] According to the ratio of the test strip T line (test line) to the C line (quality control line), when the amount of pantothenic acid added was 0 μg / kg, the T / C value was between 1.5 and 4.0; when the amount of pantothenic acid added was 0 μg / kg and 100 μg / kg, the maximum difference in T / C value was 0.65, so the concentration of time-resolved fluorescent microspheres was selected to be 40 μg / mL, as shown in Table 2.

[0094] Table 2 Optimization of time-resolved fluorescent microsphere concentration

[0095]

[0096] (3) Establishing a standard curve

[0097] Take 1g of the standard curve sample of the milk powder to be tested prepared in step 1 (2) in a 15mL centrifuge tube, add 3mL of water (heated in a 55℃ water bath), shake well for 3min, sonicate in a 60℃ water bath for 30min, cool and add 1mL of extract (3% v / v acetic acid aqueous solution), shake well for 3min, centrifuge at 12000rpm for 15min, take the clarified liquid and filter it with a 0.22μm filter membrane, take 100μL of the filtrate, add 200μL of diluent (0.01M PBS (1% v / v ON 870) solution), mix well and add it to the sample well of the test card that has been preheated to 37℃, cover the incubator lid, and count for 8min; after the timing ends, take out the test card and read the result within 20s. The standard curve of the time-resolved fluorescence test strip for detecting pantothenic acid was obtained, with a minimum detection limit of 5.08μg / kg and a linear detection range of 3.79-40.15μg / kg. Figure 3 .

[0098] (4) Recovery rate test

[0099] 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, sonicate in a 60℃ water bath for 30min, cool, add 1mL of extract (3% v / v acetic acid aqueous solution), shake well for 3min, centrifuge at 12000rpm for 15min, filter the clarified liquid with a 0.22μm filter membrane, take 100μL of the filtrate, add 200μL of diluent (0.01M PBS (1% v / v ON 870) solution), mix well, add to the sample well 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 pantothenic acid is obtained, see Table 3.

[0100] Table 3 Recovery of pantothenic acid in three milk powder samples

[0101] sample Detection concentration (μg / kg) Recovery rate (%) 1 261 86 2 475 106 3 275 91

[0102] 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.

[0103] Obviously, the above embodiments are merely examples for clear explanation and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived from these are still within the protection scope of the invention.

Claims

1. A hybridoma cell line, characterized in that The deposit number of the hybridoma cell line is CGMCC No.45919.

2. The method for preparing a hybridoma cell line according to claim 1, characterized in that: The molecular structure of the pantothenic acid hapten used in the preparation process is:

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

4. A test strip, characterized in that: The test strip comprises the pantothenic acid monoclonal antibody according to claim 3.

5. The test strip according to claim 4, characterized in that: The test strip comprises a base liner and a sample pad, a conjugation pad, a coating film and a water-absorbing pad arranged on the base liner; A binding pad, which is disposed between the sample pad and the coating membrane and is modified with a labeled pantothenic acid monoclonal antibody; A coating membrane, wherein a detection line and a quality control line are provided on the coating membrane; wherein the detection line is provided at one end close to the binding pad and fixed with a pantothenic acid antigen, and the quality control line is provided at one end away from the binding pad and fixed with a pantothenic acid secondary antibody; The water absorbent pad is arranged at one end of the coating membrane away from the binding pad.

6. The test strip according to claim 5, characterized in that: The test strips include time-resolved fluorescence test strips.

7. The test strip according to claim 6, characterized in that: The labels include time-resolved fluorescent microspheres.

8. The test strip according to claim 5, characterized in that: The concentration of time-resolved fluorescent microspheres used to modify the conjugate pad was 20-80 μg / mL.

9. A kit, characterized in that: The kit contains the test strip according to any one of claims 4 to 8.

10. Use of the pantothenic acid monoclonal antibody according to claim 3, the test strip according to any one of claims 4 to 8, or the kit according to claim 9 in detecting pantothenic acid.

Images