Hybridoma cell strain secreting pantothenic acid monoclonal antibody and application thereof

Abstract

The present application relates to a kind of pantothenic acid secreting monoclonal antibody hybridoma cell strain and its application, belong to the field of immunochemistry technology.The present application is prepared by synthesizing pantothenic acid hapten, pantothenic acid complete antigen, after cell fusion and the screening of indirect competition enzyme-linked immunoassay, hybridoma cell strain is obtained, and the pantothenic acid monoclonal antibody secreted by cell strain has good specificity and detection sensitivity to pantothenic acid, IC 50 Value is 25.67ng / mL;With pantothenic acid monoclonal antibody preparation obtained quantitative detection pantothenic acid time resolution fluorescent test strip, by optimizing the amount of fluorescent microspheres of test strip, further improve the detection sensitivity of test strip, the minimum detection limit of obtained test strip to pantothenic acid is 5.08 μg / kg, and the linear detection range is 3.79-40.15 μg / kg.The present application can be used for the rapid quantitative detection of pantothenic acid in food, and solves the problem of poor specificity and detection sensitivity in the current pantothenic acid immunodetection method.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of immunochemistry, in particular to a hybridoma cell strain secreting pantothenic acid monoclonal antibody and application thereof. BACKGROUND

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

[0003] High performance liquid chromatography, gas chromatography, gas or liquid chromatography-mass spectrometry have high sensitivity and accurate qualitative analysis characteristics, but the equipment is relatively expensive and requires professional operators to analyze samples. The immunological analysis method based on antigen-antibody reaction has the advantages of simple operation and high detection throughput, which can meet the needs of on-site rapid detection. However, the specificity and detection sensitivity of the pantothenic acid immunoassay methods reported so far are poor, which 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 efficiently detecting pantothenic acid with the monoclonal antibody. SUMMARY

[0004] To solve the above technical problems, the present application provides a hybridoma cell strain secreting pantothenic acid monoclonal antibody and application thereof. The pantothenic acid monoclonal antibody secreted by the hybridoma cell strain provided by the present application has good specificity and detection sensitivity for pantothenic acid, and the IC 50The value is 25.67 ng / mL; the time-resolved fluorescence test strip for quantitatively detecting pantothenic acid is prepared from the pantothenic acid monoclonal antibody, and the optimal amount of the fluorescence microspheres is added, so that the minimum detection limit of the test strip for pantothenic acid is 5.08 mu g / kg, and the linear detection range is 3.79-40.15 mu g / kg. The achievement can be used for rapid quantitative detection of pantothenic acid in food, and solves the problems of poor specificity and detection sensitivity in the current pantothenic acid immunodetection method.

[0005] The first object of the present application is to provide a hybridoma cell strain secreting pantothenic acid monoclonal antibody, which has been preserved in the China General Microbiological Culture Collection Center, No. 1, Xibahe Road, Haidian District, Beijing, China, and classified as a monoclonal cell strain, with a preservation date of April 18, 2024, and a preservation number of CGMCC No. 45919.

[0006] The second object of the present application is to provide a preparation method of a hybridoma cell strain secreting pantothenic acid monoclonal antibody, and the molecular structure formula of the pantothenic acid hapten used in the preparation process is as follows:

[0007]

[0008] Further, the preparation method of the pantothenic acid hapten comprises the following steps:

[0009] Weigh 4-(bromomethyl)-phenylacetic acid benzoylmethyl ester, 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 h. Combine the organic phase, wash with water, extract with ethyl acetate, and dry over Na2SO4, concentrate to obtain a yellow oil compound. Dissolve the compound in CH3COOH solution, add zinc powder to it, stir overnight at room temperature. Concentrate the combined organic phase, and purify by preparative column to obtain a yellow oil compound, i.e. pantothenic acid hapten.

[0010] Further, the preparation method of the pantothenic acid hapten 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), N-hydroxysuccinimide (NHS), stir the mixture at room temperature for 6 h to obtain solution A; weigh bovine serum albumin BSA, dissolve it in carbonate buffer (CBS) and dilute to 3 mg / mL to obtain solution B; then, add solution A dropwise to solution B, react at room temperature for 8 h to obtain a reaction solution, dialyze the reaction solution against a PBS solution to remove unreacted small molecule hapten, and obtain pantothenic acid complete antigen VB5-BSA.

[0012] Further, the preparation method of the pantothenic acid-coated antigen comprises the following steps:

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

[0014] Further, the preparation method of the pantothenic acid monoclonal antibody secreting hybridoma cell strain comprises the following steps:

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

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

[0017] Step S3, boosting the immunized animal in step S2 by using the antigen-containing incomplete Freund's adjuvant obtained in step S1, and priming the animal by using the pantothenic acid complete antigen;

[0018] Step S4, taking the spleen cells and myeloma cells of the primed animal in step S3 to perform cell fusion.

[0019] Step S5, screening positive cell wells by using an indirect ELISA method, selecting pantothenic acid as a standard, determining the inhibition effect of the positive cells by using an indirect ELISA method, performing subcloning by using a limited dilution method, and finally screening the pantothenic acid monoclonal antibody secreting hybridoma cell strain.

[0020] A third object of the present application is to provide a pantothenic acid monoclonal antibody secreted by the hybridoma cell strain.

[0021] Further, the preparation method of the pantothenic acid monoclonal antibody comprises the following steps:

[0022] Taking 8-10-week-old BALB / c mice, injecting 1 mL of sterile paraffin oil into the abdominal cavity of each mouse, injecting 1×10 6The ascites of the pantothenic acid hybridoma cells are collected from the 7th day, and the antibodies are purified by the caprylic acid-saturated ammonium sulfate method, and the purified monoclonal antibodies are stored at -20 DEG C.

[0023] A fourth object of the present application is to provide a test strip comprising the pantothenic acid monoclonal antibody described above.

[0024] Further, the test strip comprises a base plate, and a sample pad, a conjugate pad, a coated membrane and an absorbent pad arranged on the base plate (the sample pad, the conjugate pad, the coated membrane and the absorbent pad are sequentially adhered to the base plate in the order from top to bottom, and each part has a certain length of overlap);

[0025] The conjugate pad is arranged between the sample pad and the coated membrane, and the conjugate pad is modified with the labeled pantothenic acid monoclonal antibody;

[0026] The coated membrane is provided with a detection line and a quality control line; wherein the detection line is arranged near one end of the conjugate pad and fixed with the pantothenic acid antigen, and the quality control line is arranged away from the conjugate pad and fixed with the pantothenic acid secondary antibody;

[0027] The absorbent pad is arranged away from the conjugate pad.

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

[0029] Further, the label comprises a time-resolved fluorescence microsphere.

[0030] Further, the concentration of the time-resolved fluorescence microsphere used to modify the conjugate pad is 20-80 μg / mL.

[0031] A fifth object of the present application is to provide a kit comprising the test strip described above.

[0032] A sixth object of the present application is to provide the use of the pantothenic acid monoclonal antibody described above, the test strip described above or the kit described above in the detection of pantothenic acid.

[0033] The above technical solutions of the present application have the following advantages compared with the prior art:

[0034] The pantothenic acid monoclonal antibody secreted by the hybridoma cell strain has better specificity and detection sensitivity (IC50 value is 25.67 ng / mL) for pantothenic acid; the time-resolved fluorescence test strip prepared from the pantothenic acid monoclonal antibody for quantitative detection of pantothenic acid 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, and has better specificity and detection sensitivity in the detection of pantothenic acid, and can be used for rapid and accurate quantitative detection of pantothenic acid in food.

[0035] Biological material preservation

[0036] The hybridoma cell strain secreting the pantothenic acid monoclonal antibody is preserved in the China General Microbiological Culture Collection Center, located at No. 1, Beichen West Road, No. 3, Chaoyang District, Beijing, Institute of Microbiology of Chinese Academy of Sciences, and is classified and named as a monoclonal cell strain, and the preservation date is April 18, 2024, and the preservation number is CGMCC No. 45919. BRIEF DESCRIPTION OF DRAWINGS

[0037] In order to make the content of the present application more easily understood, the present application will be further described in detail below according to specific embodiments of the present application and in conjunction with the drawings, in which

[0038] Figure 1 is the inhibition standard curve of the pantothenic acid monoclonal antibody of the present application to pantothenic acid.

[0039] Figure 2 is the structure diagram of the time-resolved fluorescence test strip for quantitatively detecting pantothenic acid of the present application.

[0040] Figure 3 is the standard curve of the time-resolved fluorescence test strip for detecting pantothenic acid of the present application. DETAILED DESCRIPTION

[0041] The present application will be further described below in conjunction with the drawings and specific embodiments, so that those skilled in the art can better understand the present application and implement it, but the embodiments are not limiting to the present application.

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

[0043] RPMI-1640 culture 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 phosphate 676.13, potassium chloride 400, sodium chloride 6000, glucose 2000, reduced glutathione 1, phenol red 5, L-glutamine 300, biotin 0.2, D-pantothenic acid calcium 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 of Na2CO3 and 2.93 g of NaHCO3, respectively, dissolve them in a small amount of double distilled water, mix, add double distilled water to about 800 mL, mix well, adjust the pH to 9.6, add double distilled water to 1000 mL, and store at 4°C for standby use.

[0046] Phosphate buffer (PBS): 8.00 g of NaCl, 0.2 g of KCl, 0.2 g of KH2PO4, and 2.9 g of Na2HPO4·12H2O are dissolved in 800 mL of pure water, the pH is adjusted to 7.2-7.4 with NaOH or HCl, and the volume is made up to 1000 mL;

[0047] Borate buffer: 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; mix the boric acid and borax solutions in a volume ratio of 7:3 to obtain the borate buffer.

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

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

[0050] TMB developing solution: A solution: 18.43 g of Na2HPO4·12H2O and 9.33 g of citric acid are dissolved in pure water to make up to 1000 mL; B solution: 60 mg of TMB is dissolved in 100 mL of ethylene glycol. Mix A and B solutions in a ratio of 5:1 to obtain TMB developing solution, which is mixed fresh.

[0051] The detection method involved in the following examples is as follows:

[0052] Pantothenic acid inhibition rate detection method: The most suitable antigen and antibody concentrations in ic-ELISA are selected by chessboard test. Dilute the antigen to 0.3, 1, 3, and 10 μg / mL with carbonate buffer (CBS), and dilute the antibody to 0.3, 1, 3, and 10 μg / mL with antibody diluent. After selecting the optimal working point, dilute the pantothenic acid standard to concentrations of 0, 3, 10, 30, 90, 270, and 810 ng / mL, follow the ic-ELISA operation steps, and finally plot the results using OriginPro 8.5 (as shown in Figure 1 50 .

[0053] Example 1: Synthesis of pantothenic acid hapten

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

[0055]

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

[0057]

[0058] Preparation of the pantothenic acid hapten: 1000 mg of 4-(bromomethyl)-phenylacetic acid benzoylmethyl ester (2.89 mmol) was weighed and dissolved in 20 mL of N,N-dimethylformamide (DMF) solution, 632 mg of pantothenic acid (2.89 mmol) was added to the solution, and potassium fluoride (670 mg, 11.56 mmol) was added to the solution, and stirred at room temperature for 2 h. The organic phase was combined and washed with 40 mL of water, extracted with ethyl acetate, and dried over Na2SO4, concentrated to obtain a yellow oil compound. The compound (1000 mg, 2.06 mmol) was dissolved in 10 mL of CH3COOH solution, zinc powder was added, and stirred at room temperature overnight. The combined organic phase was concentrated and purified by a preparative column to obtain a yellow oil compound (250 mg), which was the pantothenic acid hapten.

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

[0060] 1.64 mg of the pantothenic acid hapten was weighed and dissolved in 600 μL of N,N-dimethylformamide (DMF), then 2.58 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), 1.55 mg of N-hydroxysuccinimide (NHS) was added, and the mixture was stirred at room temperature for 6 h to obtain solution A; 5 mg of bovine serum albumin (BSA) was weighed and diluted to 3 mg / mL with 0.01 M carbonate buffer (CBS) to obtain solution B; then, solution A was added dropwise to solution B, and reacted at room temperature for 8 h to obtain a reaction solution, which was dialyzed against a PBS solution to remove unreacted small molecule haptens, and a pantothenic acid complete antigen VB5-BSA was obtained.

[0061] Example 2: Synthesis of pantothenic acid complete antigen

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

[0063] Take 1.64 mg of pantothenic acid hapten and dissolve it in 600 μL of N,N- dimethylformamide (DMF), then add 2.58 mg of 1-(3-dimethylaminopropyl)-3- ethylcarbodiimide hydrochloride (EDC), 1.55 mg of N-hydroxysuccinimide (NHS), stir the mixture at room temperature for 6 h to obtain solution C; take 6 mg of chicken ovalbumin (OVA) and dissolve it in 1 ml of carbonate buffer solution (CBS) with a concentration of 0.01 M to obtain solution D; then, add solution C dropwise to solution D, react at room temperature for 8 h to obtain a reaction solution, dialyze the reaction solution with a PBS solution to remove unreacted small molecule haptens, and obtain a pantothenic acid-coated antigen VB5-OVA.

[0064] Example 4: Preparation of hybridoma cell strain secreting pantothenic acid monoclonal antibody

[0065] 1. Obtaining of animal immunization: mix the pantothenic acid complete antigen with an equal amount of complete Freund's adjuvant to obtain an antigen-containing complete Freund's adjuvant, mix the pantothenic acid complete antigen with an equal amount of incomplete Freund's adjuvant to obtain an antigen-containing incomplete Freund's adjuvant, and perform subcutaneous multi-point injection immunization (except for the boost immunization) on the neck of BALB / c mice; the first immunization uses the antigen-containing complete Freund's adjuvant with a dose of 100 ug per mouse; the multiple booster immunizations use the antigen-containing incomplete Freund's adjuvant with a dose of 50 ug per mouse; the boost immunization does not use an adjuvant, but directly uses the complete antigen diluted with normal saline for intraperitoneal injection with a dose of 25 ug per mouse; the interval between the first immunization and the second booster immunization is one month, the interval between the multiple booster immunizations is 21 days, and the interval between the boost immunization and the last booster immunization is 18-21 days; observe the immunization effect of the mice by indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) to detect the titer and inhibition of the mouse serum;

[0066] 2. Cell fusion: three days after the boost immunization, perform cell fusion according to the conventional PEG (polyethylene glycol, molecular weight 4000) method, and the specific steps are as follows:

[0067] a. Tail bleeding, immediately after the mice are sacrificed by cervical dislocation, disinfect them in 75% alcohol for about 5 min, perform aseptic operation to take out the spleen of the mice, gently grind it with the rubber head of a syringe, and pass it through a 200-mesh cell sieve to obtain a spleen cell suspension, collect it, centrifuge (1200 rpm, 8 min), wash the spleen cells with RPMI-1640 culture medium three times, and after the last centrifugation, dilute the spleen cells to a certain volume, count them, and reserve them for use;

[0068] b, collection of SP2 / 0 cells: 7-10 days before fusion, SP2 / 0 tumor cells are cultured in 10% FBS (fetal bovine serum) RPMI-1640 medium in a 5% CO2 incubator, and the number of SP2 / 0 tumor cells per milliliter of medium is required to reach (1-4) x 10 7 To ensure that the SP2 / 0 tumor cells are in the logarithmic growth phase before fusion, collect the tumor cells and suspend them in RPMI-1640 base medium for cell counting before fusion;

[0069] c, fusion process 7min: 1mL of PEG 1500 is added to the cells from slow to fast in the first minute; 2min, stand; 3min and 4min, add 1mL of RPMI-1640 medium within 1min; 5min and 6min, add 2mL of RPMI-1640 medium within 1min; 7min, add 1mL of RPMI-1640 medium every 10s; then 37℃ incubation for 5min; centrifugation (800rpm, 8min), discard the supernatant, resuspend in RPMI-1640 screening medium containing 20% fetal bovine serum, 2% 50x HAT, add 200μL / well to a 96-well cell plate, and incubate in a 37℃, 5% CO2 incubator;

[0070] 3, cell screening and cell strain establishment: on the 3rd day of cell fusion, the fusion cells are subjected to RPMI-1640 screening medium semi-replacement, on the 5th day, the cells are subjected to full replacement with RPMI-1640 transition medium containing 20% fetal bovine serum, 1% 100x HT, and on the 7th day, the cell supernatant is screened.

[0071] The screening is divided into two steps: first, positive cell wells are screened by ic-ELISA method, and second, the positive cells are subjected to inhibition effect determination by ic-ELISA method using pantothenic acid as a standard;

[0072] The cell wells with good inhibition to the pantothenic acid standard are selected, and subcloned by limited dilution method, and detected by the same method after 7 days;

[0073] According to the above method, subcloning is carried out three times, and finally the hybridoma cell strain secreting pantothenic acid monoclonal antibody is obtained.

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

[0075] Take 8-10 week old BALB / c mice, inject 1mL of sterile paraffin oil into the abdominal cavity of each mouse; 7 days later, inject 1x10 6 Pantothenic acid hybridoma cells, starting from the 7th day, collect ascites, and purify the antibodies from the ascites by caprylic acid-saturated ammonium sulfate method.

[0076] Under the condition of partial acid, n-octanoic acid can precipitate other impurities except IgG immunoglobulin in ascites, then centrifugal, discard the precipitate; then use equal amount of saturated ammonium sulfate solution to precipitate IgG type monoclonal antibody, centrifugal, discard the supernatant, then dissolve with 0.01M PBS solution (pH 7.4), dialysis and desalination, finally obtain the purified monoclonal antibody and store at -20℃.

[0077] Using indirect competitive ELISA, pantothenic acid monoclonal antibody was used to detect pantothenic acid, and the IC 50 value of pantothenic acid was 25.67 ng / mL, indicating that it had good detection sensitivity for pantothenic acid; at the same time, pantothenic acid monoclonal antibody was used to detect other similar substances (vitamin B3, vitamin B6, vitamin B7, vitamin B9, vitamin B12), and the cross rate was less than 5% (IC 50 >600 ng / mL), indicating that the monoclonal antibody had good specificity, and see Table 1, so it can be used for the immunological analysis and detection of pantothenic acid. (Cross rate = (IC 50 of pantothenic acid / IC 50 of similar substance) x 100%).

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

[0079] Vitamin IC 50 values (ng / ml) Cross-reactivity 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 time-resolved fluorescence test strip for quantitative detection of pantothenic acid

[0081] (1) Preparation of fluorescent microsphere pad: using the carboxyl group on the surface of time-resolved fluorescent microspheres (TRFM), EDC-mediated active ester method was used to prepare time-resolved fluorescent microsphere-labeled antibody. Briefly, 100 μL of TRFM (2 mg / mL) and 500 μL of 0.04 mol / L borate buffer were mixed in a 2 mL centrifuge tube and shaken. Then, 15 μL of EDC (20 mg / mL) was added, and the reaction was magnetically stirred at room temperature for 20 min to activate the carboxyl group on the surface of TRFM. Centrifugation was performed at 12000 x g for 20 min at 4°C, the supernatant was discarded, 600 μL of PBS solution was added and ultrasonicated in an ultrasonic device for 1 min. Subsequently, 30 μg of pantothenic acid monoclonal antibody was added to the resuspension for labeling, and magnetically stirred at room temperature for 2 h. After labeling, 100 μL of blocking solution (BSA (1%, w / v)) was slowly added and reacted at room temperature for 2 h to block the remaining sites on the surface of the microspheres. The final solution was centrifuged at 12000 x g for 10 min, and the supernatant was discarded. The precipitate was dissolved in 500 μL of PBS solution and ultrasonicated in an ultrasonic device for 1 min, and stored at 4°C for standby. The pantothenic acid monoclonal antibody-time-resolved fluorescent microsphere label was then sprayed on the glass fiber membrane at a rate of 7 μL / cm to prepare the fluorescent microsphere pad.

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

[0083] (3) Assembly and segmentation of test strip: PVC plate, sample pad, fluorescent microsphere pad, nitrocellulose membrane and water absorption pad were sequentially assembled. After assembly, the strip was cut to obtain the time-resolved fluorescent test strip for quantitative detection of pantothenic acid, and the size of the time-resolved fluorescent test strip was 8 cm in length and 4 mm in width. Figure 2 .

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

[0085] 1. Preparation of sample to be tested

[0086] (1) Test strip optimization experiment sample: Take the pantothenic acid-free milk powder sample, and add pantothenic acid to a concentration of 0 and 100 μg / kg, respectively.

[0087] (2) Test strip standard curve sample: Take the pantothenic acid-free milk powder sample, and add pantothenic acid to a concentration of 0, 5, 10, 20, 50, 100, 200 and 500 μg / kg, respectively.

[0088] (3) Test sample (recovery rate and detection method comparison): Take three positive samples containing pantothenic acid.

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

[0090] (1) Preparation of detection card: Fix the time-resolved fluorescent test strip in the plastic card shell, and press tightly to obtain the detection card.

[0091] (2) Optimization of fluorescent microsphere concentration

[0092] Take 1 g of the test milk powder sample prepared in step 1(1) in a 15 mL centrifuge tube, add 3 mL of water (55°C water bath heating), shake and shake for 3 min, ultrasonic at 60°C water bath for 30 min, after cooling, add 1 mL of extraction solution (3% v / v acetic acid aqueous solution), shake and shake for 3 min, centrifuge at 12000 rpm for 15 min, take the clear liquid and filter with 0.22 μm filter membrane, take 100 μL of the filtrate, add 200 μL of diluent (0.01M PBS (1% v / v ON 870) solution), mix well and add to the sample well of the detection card which has been preheated to 37°C, cover the incubator cover, and count for 8 min; after the timing is over, take out the detection card and read the results within 20s.

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

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

[0095]

[0096] (3) Establishment of standard curve

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

[0098] (4) Recovery detection

[0099] Take the sample 1 g prepared in step 1 (3) in a 15 mL centrifuge tube, add 3 mL of water (55 ℃ water bath heating), shake and shake for 3 min, 60 ℃ water bath ultrasonic for 30 min, after cooling, add 1 mL of extraction liquid (3% v / v acetic acid aqueous solution), shake and shake for 3 min, centrifuge at 12000 rpm for 15 min, take the clear liquid and filter with 0.22 μm filter membrane, take 100 μL of filtrate, add 200 μL of diluent (0.01M PBS (1% v / v ON 870) solution), mix well and add to the sample well of the detection card which has been preheated to 37 ℃, cover the incubator cover, time for 8 min; after timing, take out the detection card and read the result within 20 s. The recovery rate of pantothenic acid is obtained, as shown in Table 3.

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

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

[0102] From the above table, it can be seen that the detection test strip provided by the present application has good results in three milk powder samples, which meets the recovery rate requirement of the analysis method.

[0103] Obviously, the above examples are only examples for clearly illustrating, and are not limited to the embodiments. For those skilled in the art, other different forms of changes or variations can be made on the basis of the above description. Here, all the embodiments are not required to be exhausted, and the obvious changes or variations derived therefrom are still within the protection scope of the present application.

Claims

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

2. A monoclonal antibody to pantothenic acid, characterized in that, The pantothenic acid monoclonal antibody is secreted by the hybridoma cell strain of claim 1.

3. A test strip, characterized in that The test strip comprises the pantothenic acid monoclonal antibody of claim 2.

4. The test strip of claim 3, wherein, The test strip comprises a base support and a sample pad, a conjugate pad, a coated membrane and an absorbent pad arranged on the base support. The conjugate pad is arranged between the sample pad and the coated membrane, and the conjugate pad is modified with the labeled pantothenic acid monoclonal antibody. The coated membrane is provided with a detection line and a quality control line; wherein the detection line is arranged near one end of the conjugate pad and fixed with the pantothenic acid antigen, and the quality control line is arranged away from the conjugate pad and fixed with the goat anti-mouse secondary antibody. The absorbent pad is arranged away from the conjugate pad of the coated membrane.

5. The test strip of claim 4, wherein, The test strip comprises a time-resolved fluorescence test strip.

6. The test strip of claim 5, wherein, The label comprises time-resolved fluorescence microspheres.

7. The test strip of claim 6, wherein, The concentration of the time-resolved fluorescence microspheres used for modifying the conjugate pad is 20-80 μg / mL.

8. A kit characterized in that, The kit contains the test strip of any one of claims 3-7.

9. Use of the pantothenic acid monoclonal antibody of claim 2, the test strip of any one of claims 3-7 or the kit of claim 8 in detecting pantothenic acid.

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