Hybridoma cell strain secreting monoclonal antibody of rosmarinic acid, carnosol and rosmaridol of rosemary extract and its application

Abstract

This invention provides a hybridoma cell line that secretes monoclonal antibodies against rosemary extracts, including sarsaparillarin, sarsaparillarin, and rosmarinic acid, and its applications, belonging to the field of immunochemistry. The hybridoma cell line has the preservation number CGMCC No. 45475. In this invention, the complete antigen of rosemary extract is emulsified with an equal amount of Freund's adjuvant and immunized BALB / c mice via multiple subcutaneous injections at the nape of the neck. The initial immunization (100 μg / mouse) uses complete Freund's adjuvant for emulsification, multiple booster immunizations (50 μg / mouse) use incomplete Freund's adjuvant, and the final immunization uses complete antigen (25 μg / mouse, without adjuvant) via intraperitoneal injection. High-titer and high-sensitivity mouse spleen cells are fused with mouse myeloma cells. Using a selective culture medium, hybrid cells resulting from the fusion of the two cell lines are screened. Cells are then screened using an indirect competitive enzyme-linked immunosorbent assay (ELISA) and subjected to multiple subcloning processes to finally obtain a monoclonal antibody hybridoma cell line. The monoclonal antibody secreted by this cell line has good detection sensitivity (IC50) for sarsaparilla oxalate, sarsaparilla phenol, and rosmarinic acid. 50 The values ​​were 0.61 ng / mL, 0.73 ng / mL, and 1.36 ng / mL, respectively, which can be used to detect rosemary extract in food.

Description

Technical Field

[0001] This invention relates to the field of immunoassay for food additives, and in particular to a hybridoma cell line that secretes monoclonal antibodies against rosemary extracts, namely sarsaparilla acid, sarsaparilla phenol, and rosmarinic acid, and its applications. Background Technology

[0002] Rosemary (Rosmarinus officinalis) is a spice plant belonging to the genus Rosmarinus L. of the family Lamiaceae, native to the Mediterranean region. Rosemary is rich in diterpenoids, including sagenic acid, carvulin, and rosmarinicol. Oil-soluble rosemary extracts (sagenic acid and carvulin) are mainly used in edible oils, meat products, dairy products, high-fat products, and baked goods, primarily to prevent oxidation and spoilage of oils and foods, exhibiting excellent heat resistance. Water-soluble antioxidants (rosmarinic acid) are mainly used in beverages, seafood, and natural water-soluble pigments, possessing strong antioxidant capabilities and also exhibiting some heat resistance. In 2008, the European Food Safety Authority (EFSA) authorized rosemary extract as a food additive, with safety margins of 100-2000 mg for children and 200-3000 mg for adults (sagenic acid plus carvulin). The European Commission, the Food Standards Australia New Zealand, the Japanese Ministry of Health, Labour and Welfare, and the U.S. Food and Drug Administration all permit its use in food as an antioxidant or food flavoring. According to the assessment of the Joint FAO / WHO Expert Committee on Food Additives, the provisional acceptable daily intake (TDI) for this substance is 0.3 mg / kg. my country issued the "Food Additive Rosemary Extract" (GB 1886.172-2016) on August 31, 2016, setting the maximum permitted level for its use in food at 0.3 g / kg.

[0003] Currently, the detection methods for caryopsisic acid, caryopsisol, and rosmarinic acid are mainly instrumental, with gas chromatography, liquid chromatography, and gas chromatography-mass spectrometry being commonly used. Although these chromatographic methods have high sensitivity and specificity, they also have some drawbacks, such as the need for thorough sample purification, high solvent consumption, expensive equipment, and skilled technicians. Therefore, a rapid and simple method for the detection of caryopsisic acid, caryopsisol, and rosmarinic acid is needed.

[0004] Enzyme-linked immunosorbent assay (ELISA) is an extremely efficient, sensitive, and rapid detection method. It requires simple sample pretreatment, involves few purification steps, has a large analytical capacity, low detection cost, and is easy to operate, making it suitable for rapid on-site detection of large numbers of samples. Therefore, it is widely used in drug residue analysis. However, the prerequisite for using ELISA to detect carobacillus acid, carobol, and rosmarinic acid is obtaining monoclonal antibodies with high specificity and sensitivity to these substances. Therefore, finding a method to prepare monoclonal antibodies with high specificity and sensitivity to carobacillus acid, carobol, and rosmarinic acid is crucial. The inventors attempted to prepare monoclonal antibodies for the simultaneous detection of oxalic acid, caryophyllene, and rosmarinic acid using hybridoma cells. However, further research is needed on how to obtain haptens of oxalic acid, caryophyllene, and rosmarinic acid and prepare complete antigens, as well as how to induce a strong immune response in mice during the preparation of hybridoma cell lines capable of secreting these monoclonal antibodies. Further research is also needed on how to ensure that the prepared hybridoma cell lines can successfully secrete monoclonal antibodies for oxalic acid, caryophyllene, and rosmarinic acid. Finally, further research is needed on how to ensure that the secreted monoclonal antibodies for oxalic acid, caryophyllene, and rosmarinic acid have high specificity and sensitivity. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides a hybridoma cell line that secretes monoclonal antibodies against rosmarinic acid, rosmarinic acid, and rosmarinic acid, as well as its applications. The monoclonal antibodies against rosmarinic acid, rosmarinic acid, and rosmarinic acid secreted by the hybridoma cell line obtained in this invention exhibit good detection sensitivity (IC50) for these three substances. 50 The values ​​were 0.61 ng / mL, 0.73 ng / mL, and 1.36 ng / mL, respectively. This method can be used to establish immunological detection methods for caryopsisic acid, caryopsisol, and rosmarinic acid, and to detect caryopsisic acid, caryopsisol, and rosmarinic acid in edible oils, meat products, dairy products, beverages, etc.

[0006] The first objective of this invention is to provide a hybridoma cell line that secretes monoclonal antibodies against sarsaparillaric acid, sarsaparillarol, and rosmarinic acid. This hybridoma cell line was deposited on February 8, 2023, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 45475. The hybridoma cell line obtained by this invention is a monoclonal cell line.

[0007] A second objective of this invention is to provide a method for preparing the aforementioned hybridoma cell line, comprising the following steps:

[0008] S1: Provide a hapten of oxalic acid, oxalool, and rosmarinic acid; prepare a complete antigen using the hapten of oxalic acid, oxalool, and rosmarinic acid;

[0009] S2: Immunogens are prepared by emulsifying complete antigens with Freund's adjuvant or incomplete Freund's adjuvant.

[0010] S3: The obtained immunogen was injected subcutaneously into BALB / c mice at multiple points on the back of the neck for multiple immunizations; the first immunization used complete Freund's adjuvant, the multiple booster immunizations used incomplete Freund's adjuvant, and the sprint immunization was performed with cyclohexane-BSA complete antigen without adjuvant.

[0011] S4: Take spleen cells and myeloma cells from mice that have undergone sprint immunization in S3, and perform cell fusion to obtain the hybridoma cell line.

[0012] In one embodiment of the present invention, in step S1, the structural formulas of the succinic acid, succinic acid, and rosmarinic acid haptens are as follows:

[0013] In some embodiments of the present invention, in step S1, the structural formula of the complete antigen is: The preparation method of the complete antigen is as follows: The hapten, N-hydroxysuccinimide and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) are dissolved in anhydrous N,N-dimethylformamide and stirred to obtain an activated hapten solution, i.e., solution A; bovine serum albumin is diluted with carbonate buffer solution to obtain solution B; solution A is slowly added to solution B to carry out the reaction to obtain a reaction solution; after the reaction is completed, the reaction solution is dialyzed with phosphate buffer to obtain the complete antigen.

[0014] The specific preparation method of the hybridoma cell line secreting monoclonal antibodies against sarstigmate, sarstigmol, and rosmarinic acid includes the following steps:

[0015] Step 1: Select haptens of oxalic acid, oxalool, and rosmarinic acid and prepare complete antigens of oxalic acid, oxalool, and rosmarinic acid. Emulsify the obtained complete antigens of oxalic acid, oxalool, and rosmarinic acid with Freund's adjuvant or incomplete Freund's adjuvant to prepare immunogens.

[0016] Step 2: The obtained immunogen was injected subcutaneously into BALB / c mice via the back for multiple immunizations. The first immunization used complete Freund's adjuvant, and the booster immunization used incomplete Freund's adjuvant.

[0017] Step 3: Collect blood from mice that have undergone the above immunization process, and detect the serum immune titer and immunosuppressive ability of mice by indirect ELISA, and screen out mice with high sensitivity of serum antibodies against oxalic acid, oxalool, and rosmarinic acid.

[0018] Step 4: The selected mice were immunized by intraperitoneal injection using complete antigens of sarsaparilla acid, sarsaparilla phenol, and rosmarinic acid without Freund's adjuvant.

[0019] Step 5: Fuse spleen cells and myeloma cells from BALB / c mice after sprint immunization. The fused cells are then screened and cultured in HAT medium. Positive cell pores are detected using indirect ELISA, and the inhibitory effect of positive cell pores is further determined using indirect competitive ELISA. The positive cell pores with the best inhibition are subcloned using limiting dilution. Finally, hybridoma cell lines that secrete highly sensitive monoclonal antibodies against succinic acid, succinol, and rosmarinic acid are selected.

[0020] In some embodiments of the present invention, the interval between the first immunization and the booster immunization in steps 2 and 4 is one month, the interval between booster immunizations is 21 days, and the interval between booster immunization and the sprint immunization is 18 to 21 days.

[0021] In some embodiments of the present invention, the initial immunization dose in steps 2 and 4 is 100 μg / animal, the booster immunization dose is 50 μg / animal, and the final immunization dose is 25 μg / animal.

[0022] In some embodiments of the present invention, the immunization process in steps 2 and 4 includes one initial immunization, four booster immunizations and one sprint immunization.

[0023] In some embodiments of the present invention, the blood collection in step 3 is performed on the 7th day after the completion of the 3rd immunization process.

[0024] In some embodiments of the present invention, the cell fusion in step 5 is performed 3 to 5 days after the end of the sprint immunization.

[0025] In some embodiments of the present invention, the cell fusion in step 5 is performed by the polyethylene glycol (PEG4000) method.

[0026] In some embodiments of the present invention, the culture medium in step 5 is RPMI-1640 culture medium.

[0027] In some embodiments of the present invention, the number of subcloning operations in step 5 is 4.

[0028] A third objective of this invention is to provide a monoclonal antibody containing oxalic acid, oxalool, and rosmarinic acid, wherein the monoclonal antibody is secreted by the hybridoma cell line.

[0029] The method for preparing the monoclonal antibodies against oxalic acid, oxalool, and rosmarinic acid is as follows: BALB / c mice are injected intraperitoneally with paraffin oil, followed by an intraperitoneal injection of a hybridoma cell line with preservation number CGMCC No. 45475. Ascites fluid is collected after injection, purified, and the obtained monoclonal antibodies are stored at low temperature.

[0030] In one embodiment of the present invention, the method involves taking 8-10 week old BALB / c mice, injecting 1 mL of paraffin oil into each mouse intraperitoneally, and then injecting 1×10⁻⁶ g of paraffin oil into each mouse intraperitoneally 7 days later. 6 Hybridoma cell line with accession number CGMCC No.45475 was used. Ascites fluid was collected starting from day 7. The ascites fluid was purified by the caprylic acid-ammonium sulfate method, and the obtained monoclonal antibody was stored at -20°C.

[0031] A fourth object of the present invention is to provide a composition comprising the hybridoma cell line described above and / or the monoclonal antibodies of sarsaparilla acid, sarsaparilla phenol, and rosmarinic acid.

[0032] A fifth objective of the present invention is to provide a kit containing one or more of the hybridoma cell line, the sarcopenic acid, sarcopenic acid, rosmarinic acid monoclonal antibody, or the composition thereof.

[0033] A sixth objective of the present invention is to provide a test strip containing one or more of the hybridoma cell line, caryopsisic acid, caryopsisol, rosmarinic acid monoclonal antibody, or the composition thereof.

[0034] A seventh object of the present invention is to provide a chip comprising one or more of the aforementioned hybridoma cell line, the aforementioned oxalic acid, oxalool, rosmarinic acid monoclonal antibody, and the aforementioned composition.

[0035] An eighth object of the present invention is to provide the application of the hybridoma cell line, the monoclonal antibodies against oxalic acid, oxalool, and rosmarinic acid, the composition, the kit, the test strip, or the chip described herein in the detection of oxalic acid, oxalool, and rosmarinic acid.

[0036] The technical solution of the present invention has the following advantages compared with the prior art:

[0037] The monoclonal antibodies against oxalic acid, oxalool, and rosmarinic acid obtained in this invention exhibit good detection sensitivity (IC50) for these three substances. 50 The values ​​were 0.61 ng / mL, 0.73 ng / mL, and 1.36 ng / mL, respectively.

[0038] The monoclonal antibody cell lines containing sarsaparilla acid, sarsaparilla phenol, and rosmarinic acid obtained by this invention can be used for immunoassay detection. Attached Figure Description

[0039] 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, wherein...

[0040] Figure 1 , Figure 2 , Figure 3 The figures show the standard curves of inhibition of oxalic acid, oxalool, and rosmarinic acid by the monoclonal antibodies of the present invention. Detailed Implementation

[0041] 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.

[0042] The culture media involved in the following examples are as follows:

[0043] 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.

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

[0045] Carbonate buffer: 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.

[0046] Phosphate buffer: Dissolve 8.00g NaCl, 0.2g KCl, 0.2g KH2PO4, and 2.9g Na2HPO4·12H2O in 800mL of pure water, adjust the pH to 7.2-7.4 with NaOH or HCl, and bring the volume to 1000mL.

[0047] Phosphate buffer T: Phosphate buffer containing 0.05% Tween 20;

[0048] Antibody dilution buffer: phosphate buffer containing 0.1% gelatin;

[0049] TMB colorimetric solution: Solution A: Na₂HPO₄ 4. 12H₂O 18.43g, citric acid 9.33g, diluted to 1000mL with pure water; Solution B: 60mg TMB dissolved in 100mL ethylene glycol. Mix solutions A and B in a 5:1 ratio to obtain the TMB colorimetric solution, and mix again before use.

[0050] The detection methods involved in the following embodiments are as follows:

[0051] Methods for detecting the inhibition rates of succinic acid, succinol, and rosmarinic acid: The optimal antigen and antibody concentrations for ic-ELISA were selected using a checkerboard assay. The antigen was diluted to 0.01, 0.03, 0.1, and 0.3 μg / mL with carbonate buffer, and the antibody was diluted to 0.03, 0.1, 0.3, and 1 μg / mL with antibody dilution buffer. After selecting the optimal operating point, the succinic acid, succinol, and rosmarinic acid standards were diluted to eight concentrations (0, 0.027, 0.082, 0.247, 0.74, 2.222, 6.667, and 20 ng / mL). Following the ic-ELISA procedure, the results were plotted using OriginPro 8.5 (see results below). Figure 1 , Figure 2 , Figure 3 As shown in the figure, standard inhibition curves for sarsaparilla acid, sarsaparillaol, and rosmarinic acid were obtained, and IC50 was calculated. 50 .

[0052] Example 1: Synthesis of caryopsisic acid, caryopsisol, and rosmarinic acid immunogens

[0053] Weigh 9.2 mg of oxalic acid, oxalool, and rosmarinic acid hapten, and 8 mg of N-hydroxysuccinimide, and dissolve them in 200 μL of N,N-dimethylformamide. Stir at room temperature for 10 min. Then weigh 9.2 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and add it to the oxalic acid, oxalool, and rosmarinic acid hapten solution. Stir at room temperature for 8-10 h to activate the hapten. Take 6 mg of BSA and add it to 3 mL of 0.01 M carbonate buffer. Dissolve the hapten thoroughly. Slowly add the activated hapten to the diluted BSA solution and stir overnight at room temperature. Then dialyze with 0.01 M phosphate buffer to remove unreacted small molecules, obtaining a relatively pure complete antigen, which is then identified by gel electrophoresis.

[0054] Example 2: Synthesis of the coating agents of sarsaparilla acid, sarsaparillaol, and rosmarinicol

[0055] 4.5 mg of caryopsisic acid, caryopsisol, rosmarinic acid hapten, and 3.1 mg of N-hydroxysuccinimide were dissolved in 200 μL of N,N-dimethylformamide and reacted with the solution at room temperature for 10 min. 7.6 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride was dissolved in the above solution and reacted with the solution at room temperature for 8–10 h to obtain the hapten activation solution. 6 mg of OVA was dissolved in carbonate buffer. The hapten activation solution was slowly added to the protein dilution solution and stirred overnight at room temperature. Then, the reaction solution was dialyzed against 0.01 M phosphate buffer to remove unreacted small molecules, yielding the coating antigen.

[0056] Example 3: Preparation of hybridoma cell lines secreting monoclonal antibodies against sarsaparilla acid, sarsaparilla phenol, and rosmarinic acid

[0057] 1. Acquisition of immunity in animals

[0058] The complete antigens of sageric acid, sagerol, and rosmarinic acid were emulsified with an equal amount of Freund's adjuvant and then administered to BALB / c mice via subcutaneous injection at multiple sites on the neck and back (except for sprint immunization). The first immunization used complete Freund's adjuvant at a dose of 100 μg / mouse. For multiple booster immunizations, incomplete Freund's adjuvant was used at a dose halved to 50 μg / mouse. For sprint immunization, no adjuvant was used; the adjuvant was diluted directly with physiological saline and injected intraperitoneally at a dose halved to 25 μg / mouse. The interval between the first and second booster immunizations was one month, the interval between multiple booster immunizations was 21 days, and the interval between sprint immunizations and the last booster immunization was 18-21 days. The immunization effect in mice was observed by indirect competitive enzyme-linked immunosorbent assay (ic-ELISA), i.e., the titer and inhibition of mouse serum were detected.

[0059] 2. Cell fusion

[0060] Three days after the sprint immunization, cell fusion was performed using the standard PEG (polyethylene glycol, molecular weight 4000) method, with the following specific steps:

[0061] a. After euthanizing the mice by cervical dislocation, immediately disinfect them in 75% alcohol for about 5 minutes. Aseptically remove the spleen of the mice, grind it moderately with the rubber tip of a syringe and pass it through a 200-mesh cell sieve to obtain a spleen cell suspension. Collect the suspension, centrifuge (1200 rpm, 8 min), wash the spleen cells three times with RPMI-1640 medium, and after the last centrifugation, dilute the spleen cells to a certain volume, count them, and set them aside for later use.

[0062] b. Collection of SP2 / 0 cells: 7-10 days before fusion, SP2 / 0 tumor cells were expanded in RPMI-1640 medium containing 10% fetal bovine serum in a 5% CO2 incubator. The number of SP2 / 0 tumor cells should reach 1-4 × 10⁶ cells before fusion. 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 for cell counting during fusion.

[0063] c. Fusion process (7 min): At min 1, add 1 mL of PEG 4000 to the cells dropwise, gradually increasing the speed; at min 2, allow to stand; at min 3 and min 4, add 1 mL of RPMI-1640 medium dropwise over 1 min; at min 5 and min 6, add 2 mL of RPMI-1640 medium dropwise over 1 min; at min 7, add 1 mL of RPMI-1640 medium dropwise every 10 s. Except for min 2, continuously agitate the solution. Then incubate at 37°C for 5 min; centrifuge (800 rpm, 8 min), discard the supernatant, and resuspend in RPMI-1640 selection medium containing 20% ​​fetal bovine serum and 2% 50×HAT. Add 200 μL / well to a 96-well cell plate and incubate at 37°C in a 5% CO2 incubator.

[0064] 3. Cell selection and cell line establishment

[0065] On day 3 after cell fusion, the fused cells were partially replaced with RPMI-1640 selection medium. On day 5, the medium was completely replaced with RPMI-1640 transition medium containing 20% ​​fetal bovine serum and 1% 100×HT. On day 7, the cell supernatant was collected for screening.

[0066] The screening process consists of two steps: First, positive cell wells are selected using ic-ELISA. Second, caryopsisic acid, caryopsisol, and rosmarinic acid are used as standards, and the inhibitory effect on positive cells is determined using ic-ELISA.

[0067] Cell wells that showed good inhibition of caryopsisic acid, caryopsisol, and rosmarinic acid standards were selected, and subcloning was performed using the limiting dilution method. The results were then tested using the same method after seven days.

[0068] At least three subcloning operations were performed using the method described above to finally obtain monoclonal antibody cell lines containing sarsaparilla oxalate, sarsaparilla phenol, and rosmarinic acid.

[0069] Example 4: Preparation and identification of monoclonal antibodies against sarsaparillaric acid, sarsaparillarol, and rosmarinic acid

[0070] 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 1×10 6 Ascites was collected from sarcopenic acid, sarcopenic acid, and rosmarinic acid hybridoma cells starting from day 7, and the ascites was purified for antibody using the caprylic acid-saturated ammonium sulfate method.

[0071] Under slightly acidic conditions, octanoic acid can precipitate other proteins in the ascites fluid besides IgG immunoglobulin. After centrifugation, the precipitate is discarded. Then, an equal volume of saturated ammonium sulfate solution is used to precipitate IgG-type monoclonal antibodies. After centrifugation, the supernatant is discarded. The antibody is dissolved in 0.01M phosphate buffer solution (pH 7.4), dialyzed to desalt, and finally purified monoclonal antibodies are obtained and stored at -20℃.

[0072] The IC50 values ​​of monoclonal antibodies against sarsaparilla oxalate, sarsaparilla phenol, and rosmarinic acid were measured using an indirect competitive ELISA. 50 The values ​​were 0.61 ng / mL, 0.73 ng / mL, and 1.36 ng / mL, respectively, indicating that the assay has good sensitivity to carnosic acid, carnosine, and rosmarinic acid, and can be used for immunoassay detection of carnosic acid, carnosine, and rosmarinic acid.

[0073] Cross-reactivity experiments were conducted with oxaliplatin, oxaliplatinol, and rosmarinic acid (estradiol, estriol, deuterated estrone, 3-methoxyestrone, estrone). The results showed that the absorbance values ​​in each well of the ELISA plate corresponding to each structural analog did not differ significantly with the dilution concentration of the standard. The specific results are shown in Table 1.

[0074] These results indicate that the monoclonal antibody has a low cross-reactivity with the other five structural analogs, but good specificity with sarsaparilla acid, sarsaparilla phenol, and rosmarinic acid.

[0075] Cross-reactivity rate (%) = (Ic(saurus oxalate ICc)) 50 ) / (Similar IC 50 )×100%

[0076] Table 1

[0077]

[0078] Example 5: Application of monoclonal antibodies against sarsaparilla acid, sarsaparilla phenol, and rosmarinic acid

[0079] Monoclonal antibodies prepared from hybridoma cell lines via in vivo ascites fluid were used in an ELISA addition and recovery assay for caryopsisic acid, caryopsisol, and rosmarinic acid. The specific steps are as follows:

[0080] (1) Coat a 96-well microplate with 0.1 μg / mL of the coating stock diluted with carbonate buffer, 100 μL per well, dry at 37°C for 2 h, wash the plate three times with phosphate buffer T wash solution, 200 μL per well each time, for 3 min each time, and pat dry.

[0081] (2) Block with carbonate buffer containing 0.2% gelatin, 200 μL per well, dry at 37°C for 2 h, wash the plate three times with phosphate buffer T, 200 μL per well each time, 3 min each time, and pat dry.

[0082] (3) Prepare standard solutions of 0, 0.019, 0.056, 0.17, 0.5, 1, 5 and 10 ng / mL of caryopsisic acid, caryopsisol and rosmarinic acid using phosphate buffer (phosphate buffer). Add the standard solutions and the extract of the sample to be tested to the sealed microplate, 50 μL per well, and repeat each sample in 3 wells. Then add 50 μL of caryopsisic acid, caryopsisol and rosmarinic acid monoclonal antibody diluted to 0.1 μg / mL to each well. After reacting at 37℃ for 30 min, wash the plate and pat dry.

[0083] (4) Add 100 μL of HRP-labeled goat anti-mouse IgG secondary antibody diluted 1:3000 with phosphate buffer containing 0.1% gelatin to each well, react at 37°C for 30 min, then wash the plate and pat dry.

[0084] (5) Add 100 μL of TMB colorimetric solution to each well, develop the color at 37℃ for 15 min, then add 50 μL of 2 M H2SO4 stop solution to each well, and measure the absorbance at 450 nm.

[0085] (6) Add recovery and sample pretreatment:

[0086] Weigh 180 mg of the ham sample to be tested, and weigh three portions of the sample. Add 1 μg / kg, 5 μg / kg, and 10 μg / kg of oxalic acid standard to the sample respectively. Add 20 mL of acetone, vortex to disperse and dissolve, extract by sonication for 15 min, centrifuge at 10000 r / min for 10 min, and after collecting the supernatant, filter it through a 0.22 μm microporous membrane and collect the liquid into a 50 mL centrifuge tube.

[0087] Weigh 180 mg of the ham sample to be tested, and weigh three samples respectively. Add 1 μg / kg, 5 μg / kg and 10 μg / kg of caryophyllene standard to the samples respectively. Add 20 mL of acetone, vortex to disperse and dissolve, extract by sonication for 15 min, centrifuge at 10000 r / min for 10 min, and after collecting the supernatant, filter it through a 0.22 μm microporous membrane and collect the liquid into a 50 mL centrifuge tube.

[0088] Weigh 200mg of the plant protein beverage sample to be tested, and weigh three samples respectively. Add 1μg / kg, 5μg / kg and 10μg / kg of rosmarinic acid standard to the samples respectively. Make up to 100mL with methanol-sulfuric acid solution, vortex to disperse and dissolve, extract by sonication for 15min, centrifuge at 10000r / min for 10min, filter through 0.45μm microporous membrane and collect the liquid into 50mL centrifuge tube.

[0089] Spiking recovery assays were performed using ic-ELISA. The recoveries of oxalic acid were 90.4%, 110.7%, and 93.5%, respectively. The recoveries of oxalool were 102.5%, 91.8%, and 114.2%, respectively. The recoveries of oxalool were 89.7%, 93.5%, and 108.2%, respectively.

[0090] 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 that secretes monoclonal antibodies against sarsaparilla acid, sarsaparillaol, and rosmarinic acid, characterized in that, The hybridoma cell line was deposited on February 8, 2023, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 45475.

2. A monoclonal antibody containing sarsaparilla oxalate, sarsaparilla phenol, and rosmarinic acid, characterized in that, The monoclonal antibodies containing sarsaparilla acid, sarsaparilla phenol, and rosmarinic acid are secreted by the hybridoma cell line described in claim 1.

3. A composition, characterized in that, The composition contains the monoclonal antibodies against caryophyllic acid, caryophyllophenol, and rosmarinic acid as described in claim 2.

4. A reagent kit, characterized in that, The kit contains the monoclonal antibodies against caryophyllic acid, caryophylloxera, and rosmarinic acid as described in claim 2, or the composition as described in claim 3.

5. A test strip, characterized in that, The test strip contains the monoclonal antibodies against caryophyllic acid, caryophylloxera, and rosmarinic acid as described in claim 2, or the composition as described in claim 3.

6. A chip, characterized in that, The chip contains the monoclonal antibody of sarsaparilla acid, sarsaparilla phenol, and rosmarinic acid as described in claim 2, or the composition as described in claim 3.

7. The application of the monoclonal antibodies against oxalic acid, oxalool, and rosmarinic acid as described in claim 2 in the detection of oxalic acid, oxalool, and rosmarinic acid.

8. The application of the composition of claim 3, the kit of claim 4, the test strip of claim 5, or the chip of claim 6 in the detection of caryopsisic acid, caryopsisol, and rosmarinic acid.

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