An artificial antigen for detecting sennoside, an artificial antibody and application thereof

Abstract

This application discloses an artificial antigen and an artificial antibody for detecting sennosides and their applications. The structural formula of the artificial antigen is shown in (I), the amino acid sequence of the heavy chain of the artificial antibody is shown in SEQ ID NO: 1, and the amino acid sequence of the light chain is shown in SEQ ID NO: 2. A kit for detecting sennosides is constructed using the artificial antigen and / or the artificial antibody, and an immunological method for detecting sennosides is established. When sennosides are detected using the kit or the immunological method shown, specific and accurate detection of sennosides can be achieved, with excellent detection sensitivity and fast detection time, which can be used for rapid clinical detection of actual samples.

Description

Technical Field

[0001] This application relates to the field of food safety testing technology, and in particular to an artificial antigen and an artificial antibody for detecting sennosides and their applications. Background Technology

[0002] Senna leaves (Folium sennae) are the dried leaflets of the legume Senna angustifolia or Senna acuminata. They are a traditional Chinese medicine used to lubricate the intestines and relieve constipation. Their core active components are anthraquinone compounds such as sennosides, including sennoside A, sennoside B, sennoside C, and sennoside D. Sennoside A and sennoside B have the most pronounced laxative effect in senna leaves, with sennoside B typically present at 1.2 to 1.4 times the concentration of sennoside A in crude senna leaves. After being hydrolyzed by colonic bacteria, sennosides directly stimulate the nerve plexus of the colonic wall, thereby contracting the large intestine and causing diarrhea. They have a strong stimulant laxative effect, and long-term or excessive use may lead to electrolyte imbalances, intestinal dependence, and even genotoxicity.

[0003] The Ministry of Health’s “Notice on Further Regulating the Management of Raw Materials for Health Foods” (Health Supervision Letter

[2009] No. 326) clearly lists senna leaves as a raw material for health foods. This means that senna leaves cannot be used for ordinary food review, and if used in health foods, it must be indicated on the label.

[0004] However, because senna leaves are inexpensive and have an immediate laxative effect, some unscrupulous merchants, in order to cater to the market's pursuit of effects such as "weight loss," "laxation," and "detoxification," illegally add them to ordinary foods such as herbal teas, solid beverages, and candies. Such behavior not only violates the Food Safety Law, but also poses a serious threat to consumers' health.

[0005] Currently, the detection of sennosides mainly relies on instrumental analysis methods. The national standard method "BJS 201917 Determination of Sennoside A, Sennoside B and Emodin Methyl Ether in Food" and related studies mostly use high-performance liquid chromatography (HPLC) or high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS). Although these methods have high sensitivity, good specificity, and accurate results, they usually require expensive instruments, professional operators, complex sample pretreatment processes, and long analysis times, making it difficult to meet the needs of market supervision departments for rapid on-site screening, large-scale sample initial screening, and rapid quality control of raw materials for enterprises.

[0006] Immunological detection methods, especially those based on antigen-antibody specific reactions (such as enzyme-linked immunosorbent assay, ELISA), have been widely used in the rapid detection of food safety residues, including pesticide residues, veterinary drug residues, and toxins, due to their advantages such as ease of operation, speed, low cost, no need for complex instruments, suitability for on-site and high-throughput screening. However, immunological detection methods for sennosides, a small molecule, suffer from insufficient sensitivity; furthermore, a rapid immunoassay method capable of simultaneously and broadly detecting sennoside A, sennoside B, sennoside C, and sennoside D has not yet been discovered.

[0007] Therefore, there is an urgent need to provide a detection method that can simultaneously and broadly detect sennoside A, sennoside B, sennoside C, and sennoside D, and has excellent sensitivity. Summary of the Invention

[0008] The purpose of this invention is to overcome the above-mentioned shortcomings of the prior art and to provide an artificial antigen and artificial antibody for detecting sennosides and their applications.

[0009] The primary objective of this invention is to provide an artificial antigen for detecting sennosides.

[0010] A second objective of this invention is to provide the application of the above-mentioned artificial antigen in the preparation of artificial antibodies for detecting sennosides.

[0011] A third objective of this invention is to provide an artificial antigen composition for detecting sennosides.

[0012] A fourth objective of this invention is to provide the use of the above-described artificial antigen composition in the preparation of products for detecting sennosides.

[0013] The fifth objective of this invention is to provide an artificial antibody for detecting sennosides.

[0014] The sixth objective of this invention is to provide an antigen-antibody composition for detecting sennosides.

[0015] A seventh objective of the present invention is to provide the use of the above-described artificial antibody and / or the above-described antigen-antibody composition in the preparation of sennoside kits or in the establishment of immunological methods for detecting sennoside.

[0016] The eighth objective of this invention is to provide a kit for detecting sennosides.

[0017] The ninth objective of this invention is to provide a method for detecting sennosides.

[0018] To achieve the above objectives, the present invention is implemented through the following solution: An artificial antigen for detecting sennosides, the structural formula of which is shown in formula (I),

[0019] Formula (I); Z represents a carrier protein; the carrier protein is lactoferrin or chicken ovalbumin.

[0020] Preferably, the sennoside is sennoside A, sennoside B, sennoside C and / or sennoside D.

[0021] More preferably, the sennoside is sennoside B.

[0022] Preferably, the artificial antigen is prepared by coupling a carrier protein to the carboxyl group of sennoside B via an active esterification method, wherein the carrier protein is lactoferrin or chicken egg albumin.

[0023] More preferably, the active ester method specifically involves: uniformly dissolving sennoside B, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide in N,N-dimethylformamide to obtain a sennoside activated solution; mixing the sennoside activated solution with a carrier protein and reacting thoroughly; and then dialyzing to obtain the final product.

[0024] More preferably, the sennoside B, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide are mixed evenly in a molar ratio of 1:1 to 2:1 to 2.

[0025] More preferably, the sennoside B, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide are mixed evenly in a molar ratio of 1:1.6:1.6.

[0026] More preferably, the molar ratio of the carrier protein to sennoside B is 1:60-80.

[0027] More preferably, the complete reaction specifically involves reacting at 4–25°C in the dark for 4–16 hours.

[0028] The present invention also claims protection for the use of the above-mentioned artificial antigen in the preparation of artificial antibodies for detecting sennosides.

[0029] The application of the artificial antigen in the detection of sennosides for non-diagnostic purposes, as well as the application of the artificial antigen in the preparation of a kit for detecting sennosides, are also within the scope of protection of this invention.

[0030] Preferably, the sennoside is sennoside A, sennoside B, sennoside C and / or sennoside D.

[0031] More preferably, the sennoside is sennoside B.

[0032] The present invention also claims protection for an artificial antigen composition for detecting sennosides, comprising a coating antigen and an immunogen, wherein the coating antigen is an artificial antigen in which the carrier protein is chicken ovalbumin, and the immunogen is an artificial antigen in which the carrier protein is lactoferrin.

[0033] This invention also claims protection for the use of the above-described artificial antigen composition in the preparation of products for detecting sennosides.

[0034] The present invention also claims protection for an artificial antibody for detecting sennosides, wherein the amino acid sequence of the CDR1 region of the heavy chain is shown in SEQ ID NO: 3, the amino acid sequence of the CDR2 region of the heavy chain is shown in SEQ ID NO: 4, and the amino acid sequence of the CDR3 region of the heavy chain is shown in SEQ ID NO: 5; the amino acid sequence of the CDR1 region of the light chain is shown in SEQ ID NO: 6, the amino acid sequence of the CDR2 region of the light chain is LVS, and the amino acid sequence of the CDR3 region of the light chain is shown in SEQ ID NO: 7.

[0035] CDR1 region of the heavy chain (SEQ ID NO: 3): GFTFSSFG; CDR2 region of the heavy chain (SEQ ID NO: 4): ISGGSSTI; CDR3 region of the heavy chain (SEQ ID NO: 5): ARSAYFGKFDQRFDY; CDR1 region of the light chain (SEQ ID NO: 6): QSLLYSNGKTY; CDR2 region of the light chain: LVS; CDR3 region of the light chain (SEQ ID NO: 7): VQGTHFPWT.

[0036] Preferably, the amino acid sequence of its heavy chain is as shown in SEQ ID NO: 1, and the amino acid sequence of its light chain is as shown in SEQ ID NO: 2.

[0037] The amino acid sequence of the heavy chain (SEQ ID NO: 1): EVKLEESGGGLVQPGGSRKLSCAASGFTFSSFGMHWVRQAPEKGLEWVAYISGGSSTIHYADTVKGRFTISRDNPKNTLFLQLTSLRSEDTAMYYCARSAYFGKFDQRFDYWGQGTTLTVSS; The amino acid sequence of the light chain (SEQ ID NO: 2): DIVMTQSTLTLSVTIGQPASISCKSSQSLLYSNGKTYLNWLLQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAADLGLYYCVQGTHFPWTFGGGTKLEIK.

[0038] Preferably, the artificial antibody is prepared by immunizing animals with an artificial antigen containing lactoferrin, which is the carrier protein described above.

[0039] The present invention also claims protection for an antigen-antibody composition for detecting sennosides, comprising a coating antigen and the aforementioned artificial antibody, wherein the coating antigen is an artificial antigen whose carrier protein is chicken ovalbumin.

[0040] The present invention also claims protection for the use of the above-described artificial antibody and / or the above-described antigen-antibody composition in the preparation of kits for detecting sennosides or in the establishment of immunological methods for detecting sennosides.

[0041] The present invention also claims protection for a kit for detecting sennosides, wherein the kit contains the above-described artificial antigen composition, the above-described artificial antibody, or the above-described antigen-antibody composition.

[0042] Preferably, the kit contains ELISA reagents.

[0043] More preferably, the kit further includes an enzyme-labeled plate and a substrate chromogenic solution.

[0044] More preferably, the enzyme-labeled plate is coated with the coating antigen, which is an artificial antigen of chicken ovalbumin, the carrier protein of which is described above.

[0045] More preferably, the substrate colorimetric solution contains urea peroxide and tetramethylbenzidine.

[0046] More preferably, the product further includes a stop solution, a blocking solution, and an enzyme-labeled secondary antibody.

[0047] More preferably, the terminating liquid is H2SO4 with a volume fraction of 7% to 12%.

[0048] More preferably, the terminating liquid is 10% H2SO by volume.

[0049] More preferably, the blocking solution is a phosphate buffer containing 6% casein and 0.2 mol / L, with a pH of 7.4.

[0050] More preferably, the enzyme-labeled secondary antibody is a horseradish peroxidase-labeled goat anti-mouse secondary antibody.

[0051] Preferably, the kit contains an immunoprobe and an immunochromatographic test strip, wherein the immunoprobe contains the aforementioned artificial antibody, and the immunochromatographic test strip includes a base plate and a sample pad, an NC membrane, and absorbent paper disposed on the base plate; The NC membrane is equipped with a detection line and a control line. The detection line is coated with an artificial antigen of chicken ovalbumin, which is the carrier protein mentioned above, and the control line is coated with goat anti-mouse secondary antibody.

[0052] More preferably, the method for preparing the immune probe is as follows: The pH of the colloidal gold solution was adjusted to 8. The colloidal gold solution was mixed with the above artificial antibody at a volume ratio of 0.8-1.2 mL: 80-120 μL. After thorough incubation, the mixture was blocked with BSA. The solid was then collected by solid-liquid separation and resuspended in the gold-labeled antibody reconstitution solution to obtain the final product. The gold-labeled antibody reconstitution solution is prepared by adding 0.191g of Na2B4O7·10H2O, 0.495g of H3BO4, 2.5g of BSA, 25g of sucrose, 0.05g of NaN3, 15mL of 10wt% PVP and 25mL of 10% Tween-20 to 400mL of distilled water, mixing thoroughly, and then bringing the volume to 500mL.

[0053] More preferably, the concentration of the artificial antigen of chicken ovalbumin as the carrier protein on the detection line is 0.3 to 0.6 mg / mL.

[0054] More preferably, the concentration of the artificial antigen of chicken ovalbumin as the carrier protein on the detection line is 0.5 mg / mL.

[0055] More preferably, the coating concentration of the goat anti-mouse secondary antibody on the quality control line is 0.08–0.12 mg / mL.

[0056] More preferably, the coating concentration of the goat anti-mouse secondary antibody on the quality control line is 0.1 mg / mL.

[0057] More preferably, the sample pad is prepared by immersing a glass fiber membrane in a PB solution containing PVP, sucrose, bovine serum albumin and Tween-20, and then drying it.

[0058] More preferably, the concentration of PVP in the PB solution is 5 mg / mL, the concentration of sucrose in the PB solution is 5 mg / mL, the concentration of bovine serum albumin in the PB solution is 5 mg / mL, and the volume concentration of Tween-20 in the PB solution is 0.75%.

[0059] This invention also provides a method for using any of the above-described reagent kits, as follows: Mix the sample to be tested with the immunoprobe in any of the above-mentioned kits, incubate thoroughly to obtain a mixture, then insert the immunochromatographic test strip in any of the above-mentioned kits into the reaction solution to perform a chromatographic reaction. After the chromatographic reaction is completed, remove the immunochromatographic test strip and observe the color of the detection line and the control line. If red bands appear on the test line and the control line, the sample to be tested does not contain sennoside. If no red band appears on the test line but a red band appears on the quality control line, then the sample contains sennoside. If no red band appears on the quality control line, the test result is invalid and the test must be repeated.

[0060] The present invention also claims protection for a method for detecting sennosides, using the above-described artificial antibody, the above-described antigen-antibody composition, and / or any of the above-described kits.

[0061] Compared with the prior art, the present invention has the following beneficial effects: This invention provides an artificial antigen and an artificial antibody for detecting sennosides and their applications. The structural formula of the artificial antigen is shown in (I), the amino acid sequence of the heavy chain of the artificial antibody is shown in SEQ ID NO: 1, and the amino acid sequence of the light chain is shown in SEQ ID NO: 2. A kit for detecting sennosides is constructed using the artificial antigen and / or the artificial antibody. When using the aforementioned artificial antigens and antibodies in conjunction with the ELISA immunoassay method, simultaneous quantitative detection of sennoside A, sennoside B, sennoside C, and sennoside D can be achieved. The detection limits for sennoside A reach 41.404 ng / mL, for sennoside B 0.093 ng / mL, for sennoside C 80.049 ng / mL, and for sennoside D 0.266 ng / mL. Furthermore, when using the aforementioned artificial antigens and antibodies in conjunction with colloidal gold immunochromatography to detect sennosides, the detection time is fast, enabling rapid on-site detection of actual samples. The cut-off values ​​for sennoside B in herbal tea beverages, enzyme jellies, and solid substitutes are less than 50 ng / mL, 50 ng / g, and 500 ng / g, respectively. Attached Figure Description

[0062] Figure 1 This is a graph showing the ultraviolet wavelength scanning results from Example 1; Figure 1 In the diagram, A represents the ultraviolet wavelength scan results for SB, LF, SB-LF, OVA, and SB-OVA. Figure 1 In the diagram, B represents the ultraviolet wavelength scan results of SB, BSA, SB-BSA, HSA, and SB-HSA. Figure 2The following are standard curves of the FXG antibody against each sennoside in Example 3; A is the standard curve against sennoside A; B is the standard curve against sennoside B; C is the standard curve against sennoside C; and D is the standard curve against sennoside D. Figure 3 The graphs show the sensitivity test results for the herbal tea sample, jelly sample, and tea sample in Example 4; A is the sensitivity test result for the herbal tea sample; B is the sensitivity test result for the jelly sample; and C is the sensitivity test result for the tea sample. Detailed Implementation

[0063] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods; the materials and reagents used, unless otherwise specified, are commercially available.

[0064] The lactoferrin (LF), ovalbumin (OVA), bovine serum albumin (BSA), and human serum albumin (HSA) used in the embodiments of this invention were all purchased from Sigma-Aldrich, Inc., USA, with the following catalog numbers: LF: L9507-50MG; OVA: A2512-250MG; BSA: V900933-100G; HSA: A1653-500MG.

[0065] Example 1: Synthesis and Identification of Artificial Sennoside B Antigen I. Synthesis of Sennoside B Artificial Antigen Accurately weigh 0.01 mmol of sennoside B (SB, CAS No.: 128-57-4), 0.016 mmol of N-hydroxysuccinimide (NHS), and 0.016 mmol of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and dissolve them together in 500 μL of anhydrous N,N-dimethylformamide (DMF). The solution is stirred at 40 °C (activation temperature) in the dark for 4 h to obtain the SB activation solution. Then, under continuous stirring, the SB activation solution is added dropwise to a solution containing 10... 1 mL of 10 mM carbonate buffer (pH 7.4) containing 1 mg of lactoferrin (LF) (equivalent to a molar ratio of 1:75 with sennoside B) was placed at 4 °C in the dark and reacted for 12 h to obtain a mixture. After the reaction was completed, the mixture was transferred to a dialysis bag with a molecular cutoff of 10 kDa and dialyzed with carbonate buffer (10 mM, pH 7.4) for 3 days, with the dialysate changed daily. After dialysis, the product collected was SB-LF (i.e., sennoside B artificial antigen with lactoferrin coupled to the hydroxyl group of sennoside B).

[0066] The 10mM carbonate buffer solution is prepared by dissolving 8.45g of Na2CO3 and 14.7g of NaHCO3 in distilled water and bringing the volume up to 5000mL.

[0067] Next, following the synthesis method shown in SB-LF, lactoferrin (LF) was replaced with chicken ovalbumin (OVA), bovine serum albumin (BSA), and human serum albumin (HSA), respectively, while keeping the other conditions unchanged, to synthesize SB-OVA, SB-BSA, and SB-HSA.

[0068] II. Identification of sennoside B artificial antigen 1. Experimental Methods Sennoside B (SB), lactoferrin (LF), ovalbumin (OVA), bovine serum albumin (BSA), human serum albumin (HSA), and SB-LF, SB-OVA, SB-BSA, and SB-HSA synthesized in "I. Synthesis of Artificial Antigen of Sennoside B" were identified by ultraviolet wavelength scanning (190-500 nm), and the ultraviolet wavelength scanning results were recorded.

[0069] 2. Experimental Results The ultraviolet wavelength scanning results are shown in the figure below. Figure 1 As shown, Figure 1 In the image, A represents the ultraviolet wavelength scan results for SB, LF, SB-LF, OVA, and SB-OVA. Figure 1 In the diagram, B represents the ultraviolet wavelength scan results of SB, BSA, SB-BSA, HSA, and SB-HSA.

[0070] The results showed that the absorption curves of each artificial antigen were significantly different from the UV wavelength absorption curves of each carrier protein (LF, OVA, BSA, HSA), with different shifts at 280nm. This indicates that the absorption curves of SB-LF, SB-BSA, SB-OVA, and SB-HSA are the cumulative absorption peaks of LF and SB, OVA and SB, BSA and SB, and HSA and SB, respectively. The results show that sennoside B was successfully coupled with each carrier protein, and the artificial antigens SB-LF, SB-OVA, SB-BSA, and SB-HSA were successfully constructed.

[0071] III. Determination of the optimal artificial antigen for sennoside B 1. Experimental Methods Using SB-LF / SB-BSA synthesized in "I. Synthesis of Artificial Sennoside B Antigen" as the immunogen, SB-LF / SB-BSA was diluted to 5 mg / mL with 0.01 mol / L PBS, then mixed with an equal volume of Freund's complete adjuvant and thoroughly emulsified. This mixture was then used to immunize 8-week-old female BALB / c mice. For the first immunization, three female BALB / c mice were subcutaneously inoculated at multiple sites in the abdomen, with an antigen dose of 100 μg / mouse and 0.1 ml per mouse. A second immunization was performed on day 14, emulsified with an equal volume of Freund's incomplete adjuvant, using the same dose as the first immunization. A third immunization was performed on day 28, following the same procedure as the second immunization. After immunization, tail serum was collected to determine its titer and inhibition rate. Mice with the best results were selected for a pulse immunization, with an antigen dose of 100 μg / mouse.

[0072] One week after the third immunization, blood was collected from the tail vein of each immunized mouse, centrifuged, and mouse serum was collected. The antibody titer and inhibition rate in the mouse serum were detected by ic-ELISA. The specific steps of ic-ELISA are as follows: (1) The coating agent (SB-OVA) was diluted to 1000 ng / mL using coating buffer (0.05 M carbonate buffer, pH 9.6), and 100 μL / well was used to coat a 96-well microplate. The plate was incubated in a constant temperature water bath at 37°C for 12 h. The plate was washed with ic-ELISA washing buffer (pH 7.4) by adding 32 g NaCl, 12 g Na2HPO4·12H2O and 2 g NaCl.(1) Mix 4 mL of Tween-20 and dilute to 5000 mL with distilled water to obtain a microplate containing the coating agent; (2) Add 5 wt% skim milk powder to the microplate containing the coating agent at 120 μL / well, block at 37°C for 3 h, discard the liquid, and dry in a drying oven at 37°C to obtain a coated microplate; (3) Use PBST to perform serial dilution of mouse serum to obtain serum diluents of various dilutions, including 1:2000, 1:4000, 1:8000, 1:16000, 1:32000, 1:640 00 and 1:128000; sennoside A (CAS No.: 81-27-6), sennoside B (CAS No.: 128-57-4), sennoside C (CAS No.: 37271-16-2) and sennoside D (CAS No.: 37271-17-3) were dissolved in PBST to obtain standard solutions of sennoside A, sennoside B, sennoside C and sennoside D with a concentration of 1 μg / mL; (4) titer column addition: In one column (titer column) of the coated microplate, 50 μL of PBST was added to each well, and then the serum diluent of each dilution was added according to the following ratio. Add 50 μL of serum to each well, and replace the serum in the last well with an equal amount of PBST as a blank control well; (5) Add the inhibitory column: Add 50 μL of sennoside A standard solution to each well in one of the columns (sennoside A inhibition column) of the coated microplate, and then add 50 μL of serum dilution at each dilution to each well, and replace the serum in the last well with an equal amount of PBST as a blank control well; At the same time, obtain the sennoside B inhibition column, sennoside C inhibition column and sennoside D inhibition column according to the above method; (6) Incubate the microplate at 37°C for 40 min, and then... (7) Add horseradish peroxidase-labeled goat anti-mouse secondary antibody IgG (5000-fold dilution, Beijing TransGen Biotech, HS201-01) at 100 μL / well, incubate at 37℃ for 30 min, and wash 5 times with ic-ELISA washing buffer; (8) Add TMB chromogenic solution (Sigma-Aldrich, T0440-100 mL), incubate at 37℃ for 10 min, then add 10% (v / v) H2SO4 to stop the reaction, and read the OD of each well at 450 nm. 450 .

[0073] OD in valence 450 Wells with a diameter between 0.8 and 1.2 were used as titer wells. The dilution factor of the serum diluent corresponding to the titer wells was recorded, and the inhibition rate of each sennoside in the immunized mice was calculated according to Formula I.

[0074] Formula I: Inhibition rate (%) = (OD value of potent pore - OD value of inhibited pore) / OD value of inhibited pore × 100%; The potency wells are those with an OD450 between 0.8 and 1.2 in the potency column, and the inhibition wells are those with the same serum dilution as the potency wells.

[0075] 2. Experimental Results The inhibition rate of each sennoside in each immunized mouse is shown in Table 1.

[0076] Table 1. Inhibition rate test results of various sennosides in immunized mice.

[0077] The results showed that, according to the "I. Synthesis of Artificial Antigens of Sennoside B", SB-LF and SB-BSA, as immunogens and SB-OVA and SB-HSA, as coating antigens, could induce antibodies of certain titers in mice, and all four sennosides showed varying degrees of inhibitory effects. Among them, SB-LF and SB-OVA, as immunogens and coating antigens respectively, showed the best antiserum titer (1:64000) and the highest inhibition rate against the four sennosides. Therefore, the combination of artificial antigens of SB-LF and SB-OVA was subsequently used to prepare anti-sennoside antibodies and establish a rapid immunoassay method for sennosides. The structural formula of the artificial antigen of sennosides is shown in formula (I).

[0078] Formula (I); Where Z represents the carrier protein; when Z represents lactoferrin (LF), the corresponding structural formula is SB-LF; when Z represents ovalbumin (OVA), the corresponding structural formula is SB-OVA.

[0079] The DMF activation and low-temperature drop reaction operation further improves the coupling efficiency and protects the hapten structure, making the coupling between the hapten and the carrier protein more uniform and efficient. It also preserves the key epitopes of the glycosylated hapten, which can induce mice to produce antibodies that specifically recognize the four target analytes. Moreover, the antibodies have high titers and good sensitivity.

[0080] Based on the results shown in Table 1, under the same coating antigen (SB-OVA), the titer and inhibition rate of SB-LF as the immunogen were much higher than those of SB-BSA as the immunogen, indicating that lactoferrin (LF) is a better carrier protein for the immunogen. Under the same immunogen SB-LF, the summation and inhibition rate of SB-OVA as the coating antigen were significantly better than those of SB-HSA as the coating antigen, indicating that ovalbumin (OVA) is more suitable as a carrier protein for the coating antigen.

[0081] Example 2: Preparation of artificial antibody against sennoside B I. Experimental Methods 1. Preparation of myeloma cells Bone marrow granulocytes (SP2 / 0 cells) were revived and mixed evenly with complete culture medium, and cultured in 100 mm culture dishes. The culture was expanded to 4 dishes. Myeloma cells were collected into 50 mL centrifuge tubes, sealed, and centrifuged at 1000 r / min for 8 min. The supernatant was discarded to obtain myeloma cells. The complete culture medium is prepared as follows: Add 6 mL of penicillin-streptomycin solution (Life Technologies Corporation, 15140-122, 100 mL), 6 mL of glutamine additive, and 100 mL of fetal bovine serum to 500 mL of RPM-1640 basal culture medium, then filter through a 0.22 μm filter membrane and store at 4 °C.

[0082] 2. Preparation of immune spleen cells Using the SB-LF prepared in Example 1 as an immunogen, Balb / C mice were immunized according to the immunization protocol in Example 1. After sprint immunization, the mice were euthanized and soaked in 75% (v / v) alcohol for 4 min. The mice were then transferred to a clean bench, and their spleens were removed and placed in a disposable cell grinding mesh. The pulp was ground using the plunger of a syringe. The pulped material was collected in a 50 mL centrifuge tube, sealed, and centrifuged at 1000 r / min for 7 min. The supernatant was discarded and the precipitate was collected to obtain immune spleen cells.

[0083] 3. Cell fusion The myeloma cells obtained in step 1 and the immune spleen cells obtained in step 2 were mixed in a centrifuge tube, loosened and precipitated, and placed in 37°C warm water. 1 mL of PEG1500 was added to the centrifuge tube within 1 min, followed by 1 mL of RPMI-1640 basal medium (Gibco, USA, 12633012) within 1 min. RPMI-1640 basal medium was added to 20 mL within 5 min, and the mixture was stirred until homogeneous. The mixture was centrifuged at 1000 rpm for 7 min, and the supernatant was discarded to obtain fused cells. The fused cells were resuspended in 200 mL of HAT medium and seeded into 10 96-well plates at 2 mL / well to obtain 96-well plates seeded with fused cells. The preparation method for HAT medium is as follows: add 2g of HAT (Sigma, USA, H0262-10VL) to 100mL of complete medium.

[0084] 4. Monoclonal hybridoma cells The 96-well plates containing fusion cells obtained in step 3 were cultured in a carbon dioxide incubator containing 5% CO2 at 37°C. On day 5 of culture, the medium was partially replaced with HAT medium. On day 8 of culture, the medium was partially replaced with HT medium, and 100 μL of the supernatant of the fusion cells was collected for the first ic-ELISA test. On day 12 of culture, the medium was completely replaced with HT medium, and the supernatant of the fusion cells was collected for the second ic-ELISA test. The HT medium is prepared by adding 2g of HT (HT: Sigma-Aldrich, H0137-10VL) to 100mL of complete medium.

[0085] The ic-ELISA detection method is as described in Example 1; during the ic-ELISA detection process, titer and inhibition rate are used as indicators to select positive wells (wells with undiluted OD of the supernatant). 450 After reaching ≥0.5, the supernatant in the positive wells was discarded, and the cells in the positive wells were resuspended in 40 mL of complete culture medium and distributed to two 96-well cell plates for culture. After 10 days of culture, the cell supernatant was collected for ic-ELISA detection. Cells in the positive wells were screened again for culture until each well of each plate was positive and the titers and inhibition were similar. Hybridoma cells were obtained. The obtained hybridoma cells (monoclonal cells) can secrete anti-sennoside monoclonal antibodies (sennoside B artificial antibodies), which are the FXG cell line.

[0086] 5. Preparation and purification of monoclonal antibodies Ascites fluid was prepared from 6-week-old magnetic BALB / c mice, as detailed below: Hybridoma cells were loaded at a rate of 1×10 7 The cells were resuspended at 1 mL in 1 mL of basal culture medium to obtain a hybridoma cell suspension.

[0087] One week prior to ascites preparation, liquid paraffin was injected into the peritoneal cavity of mice. Then, the hybridoma cell suspension obtained in step 4 was injected into the peritoneal cavity of each mouse at a rate of 1 mL. Ten days later, the mice were dissected, and the ascites was collected. The ascites was centrifuged at 12000 rpm for 15 min at 4°C, and the middle layer was collected. The ascites was purified for antibody removal by immunoaffinity chromatography. Under neutral conditions, Protein G bound to IgG immunoglobulins in the ascites, causing other impurities to flow out and be discarded. Then, 0.01 M glycine buffer was used to desorb IgG from Protein G under acidic conditions to obtain high-purity IgG immunoglobulins. 0.01 M Tris... After adjusting the pH of the IgG immunoglobulin solution to neutral with HCl buffer, the solution was dialyzed to desalt, ultimately yielding a high-purity sennoside B artificial antibody (denoted as FXG antibody), which was then sequenced.

[0088] II. Experimental Results The artificial sennoside B antibody (FXG antibody) prepared according to the above method has the following amino acid sequences: heavy chain as shown in SEQ ID NO: 1, light chain as shown in SEQ ID NO: 2; amino acid sequences of the CDR1 region of the heavy chain as shown in SEQ ID NO: 3, CDR2 region as shown in SEQ ID NO: 4, and CDR3 region as shown in SEQ ID NO: 5; amino acid sequences of the CDR1 region of the light chain as shown in SEQ ID NO: 6, CDR2 region of the light chain as LVS, and CDR3 region of the light chain as shown in SEQ ID NO: 7.

[0089] The amino acid sequence of the heavy chain (SEQ ID NO: 1): EVKLEESGGGLVQPGGSRKLSCAASGFTFSSFGMHWVRQAPEKGLEWVAYISGGSSTIHYADTVKGRFTISRDNPKNTLFLQLTSLRSEDTAMYYCARSAYFGKFDQRFDYWGQGTTLTVSS; CDR1 region of the heavy chain (SEQ ID NO: 3): GFTFSSFG; CDR2 region of the heavy chain (SEQ ID NO: 4): ISGGSSTI; CDR3 region of the heavy chain (SEQ ID NO: 5): ARSAYFGKFDQRFDY; The amino acid sequence of the light chain (SEQ ID NO: 2): DIVMTQSTLTLSVTIGQPASISCKSSQSLLYSNGKTYLNWLLQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAADLGLYYCVQGTHFPWTFGGGTKLEIK; CDR1 region of the light chain (SEQ ID NO: 6): QSLLYSNGKTY; CDR2 region of the light chain: LVS; CDR3 region of the light chain (SEQ ID NO: 7): VQGTHFPWT.

[0090] Example 3: Establishment of an indirect competitive ELISA method for detecting sennosides and performance evaluation of the artificial antibody against sennoside B. I. Establishment and Sensitivity Evaluation of an Indirect Competitive ELISA Method for Detecting Sennosides 1. Experimental Methods An indirect competitive ELISA method for detecting sennosides includes the following steps: S1. Using the SB-OVA prepared in Example 1 as the coating agent, dilute it to a concentration of 156.25 ng / mL with coating buffer (0.05 M carbonate buffer, pH 9.6). Then, coat each well of a 96-well microplate with 100 μL of the coating agent and incubate at 37°C for 12 h. Wash twice with ic-ELISA washing buffer, blot dry, then add 120 μL of blocking buffer (PBST containing 5% skim milk powder, w / v) to each well and block at 37°C for 3 h. Discard the blocking buffer, blot the plate, and dry at 37°C for 30 min to obtain the coated microplate. S2. The FXG antibody obtained in Example 2 (corresponding to OD) was diluted 1:256000 using PBST. 450 With a value of 1.0, the FXG antibody dilution was obtained; Sennoside A was then dissolved in PBST to obtain standard solutions of sennoside A with concentrations of 10000 ng / mL, 5000 ng / mL, 2000 ng / mL, 285.71429 ng / mL, 40.81633 ng / mL, 5.8309 ng / mL, 0.83299 ng / mL, 0.119 ng / mL, 0.017 ng / mL, 0.00243 ng / mL, and 0.00034714 ng / mL. Sennoside B, sennoside C, and sennoside D were treated in the same way to obtain standard solutions of sennoside B, sennoside C, and sennoside D at various concentrations. S3. In the coated microplate obtained in step S1, add 50 μL of sennoside A standard solution of each concentration to each well (set 4 parallel wells for each concentration), and then add 50 μL of FXG antibody dilution solution obtained in step S2 to each well. Incubate at 37°C for 40 min. After incubation, wash 5 times with ic-ELISA washing buffer and pat dry. The sennoside B standard solution, sennoside C standard solution, and sennoside D standard solution were treated in the same manner as shown above. At the same time, a blank control well was set up, specifically by replacing the sennoside A standard solution with an equal volume of PBST solution. S4. Next, add 100 μL / well of goat anti-mouse secondary antibody-HRP (5000-fold dilution), incubate at 37°C for 30 min, wash 5 times with ic-ELISA washing buffer, and pat dry. S5. Next, add 100 μL of colorimetric reagent to each well and incubate for 10 min. After incubation, add 50 μL of 10% (v / v) H2SO4 to terminate the reaction and read the OD value at 450 nm using a microplate reader (Multiskan MK3 microplate reader, Thermo Scientific, USA). The OD value of the blank control well at 450 nm is recorded as B0. S6. Plot B / B0 as the ordinate (where B0 is the absorbance OD of the blank control well). 450 B represents the absorbance (OD) of the corresponding well for each sennoside standard solution of different concentrations. 450 The standard curves and formulas for the FXG antibody against each sennoside (sennoside A, sennoside B, sennoside C, and sennoside D) were obtained by using the logarithm of the concentration of each sennoside standard solution as the abscissa and performing curve fitting with the Logistic function.

[0091] 2. Experimental Results The standard curves of FXG antibody against various sennosides are shown below. Figure 2 As shown, Figure 2 In the figure, A represents the standard curve for sennoside A. Figure 2 In the figure, B represents the standard curve for sennoside B. Figure 2 C in the figure represents the standard curve for sennoside C. Figure 2 D in the figure represents the standard curve for sennoside D.

[0092] The standard curve formula for FXG antibody against sennoside A is: Y = -3.703 × lgX + 0.66433; The standard curve formula for FXG antibody against sennoside B is: Y = -0.4813 × lgX + 0.4459; The standard curve formula for FXG antibody against sennoside C is: Y = -7.8144 × lgX + 0.6468; The standard curve formula for FXG antibody against sennoside D is: Y = -0.03411 × lgX + 0.71012; Where X is the concentration and Y is the B / B0 value.

[0093] The results showed that the FXG antibody detection of the half-inhibitory concentration (IC50) of sennoside A (IC50) was effective. 50The half-maximal inhibitory concentration (WMC) of sennoside B detected by FXG antibody was 301.457 ng / mL, with a quantitative detection range of 86.148 ng / mL to 1054.884 ng / mL and a limit of detection (LOD) of 41.404 ng / mL. The half-maximal inhibitory concentration (WMC) of sennoside B detected by FXG antibody was 0.793 ng / mL, with a quantitative detection range of 0.205 ng / mL to 3.065 ng / mL and a LOD of 0.093 ng / mL. The half-maximal inhibitory concentration (WMC) of sennoside C detected by FXG antibody was 1160.56 ng / mL, with a quantitative detection range of 214.764 ng / mL to 6271.54 ng / mL and a LOD of 80.049 ng / mL. The half-maximal inhibitory concentration (WMC) of sennoside D detected by FXG antibody was 3.077 ng / mL, with a quantitative detection range of 0.657 ng / mL to 14.413 ng / mL and a LOD of 0.266 ng / mL.

[0094] When the FXG antibody prepared in Example 2 is used in conjunction with an indirect competitive ELISA method to detect sennosides, it exhibits excellent sensitivity.

[0095] II. Specificity Evaluation of the Indirect Competitive ELISA Method for Detecting Sennosides 1. Experimental Methods Following the indirect competitive ELISA method for detecting sennosides described in "I. Establishment and Sensitivity Evaluation of the Indirect Competitive ELISA Method for Detecting Sennosides," sennoside A was successively replaced with rhein (CAS No.: 478-43-3), emodin (CAS No.: 518-82-1), aloe-emodin (CAS No.: 481-72-1), buthiazide (CAS No.: 2043-38-1), bisacodyl (CAS No.: 603-50-9), sibutramine (CAS No.: 84485-00-7), orlistat (CAS No.: 96829-58-2), and fenfluramine (CAS No.: 458-24-2). The remaining methods remained unchanged. The half-maximal inhibitory concentration (IC50) of the FXG antibody against each sennoside analogue was determined. 50类似物 And calculate the cross-reactivity rate (CR) of sennoside B according to Formula II. CR (%) = IC 50番泻苷B / IC 50类似物 ×100%; IC 50番泻苷B The half-maximal inhibitory concentration (IC50) of FXG antibody against sennoside B is given. 50类似物 The half-maximal inhibitory concentrations (WMCs) of FXG antibody against each sennoside analogue are given.

[0096] 2. Experimental Methods The half-maximal inhibitory concentrations (WMCs) of FXG antibodies against various sennoside analogues and the cross-reactivity of sennoside B are shown in Table 2.

[0097] Table 2. Results of the half-maximal inhibitory concentrations (WMCs) of FXG antibodies against various sennoside analogues and the cross-reactivity of sennoside B.

[0098] The results showed that the cross-reactivity of the FXG antibody obtained in Example 2 with sennoside B of rhein, emodin, aloe-emodin, buthiazine, bisacodyl, sibutramine, orlistat, and fenfluramine was all <0.005%, indicating that the FXG antibody prepared in Example 2 did not cross-react with these eight structural analogs or other synthetic laxatives that may exist in food; the cross-reactivity of the FXG antibody with sennoside B of sennoside A was 0.263%, and the specificity for sennoside B was 9 times higher than that of antibodies prepared with non-glycoproteins as carrier proteins (compared to the prior art). 1372-1376.); FXG antibodies show no cross-reactivity with non-sennoside compounds and exhibit superior specificity for recognizing sennoside compounds compared to existing antibodies prepared using non-glycoprotein carrier proteins.

[0099] The above results demonstrate that the FXG antibody (sennoside B artificial antibody) prepared in Example 2 has high recognition ability, strong specificity, and high detection sensitivity for sennoside compounds, meeting the detection requirements.

[0100] Example 4: Establishment and validation of a colloidal gold immunochromatographic method for detecting sennosides I. Establishment of a colloidal gold immunochromatographic method 1. Preparation of colloidal gold probes Take 1 mL of colloidal gold solution (prepared from 0.04% (w / v) chloroauric acid solution), add 4 μL of K2CO3 solution (0.2 mol / mL) to adjust the pH to 8.0, then add 100 μL of FXG antibody with a concentration of 20 μg / mL (prepared in Example 2), incubate at 25 °C for 30 min, then add 50 μL of BSA solution (10%, v / v) to block the reaction for 30 min, then centrifuge at 1000 rpm for 10 min at 4 °C, collect the precipitate, and resuspend the precipitate in 200 μL of gold-labeled antibody reconstitution solution to obtain the colloidal gold probe; The antibody diluent was prepared by mixing 2.15g of Na2HPO4·12H2O, 0.45g of Na2HPO4·2H2O, and 5g of BSA and bringing the volume to 1000mL. The gold-labeled antibody reconstitution solution was prepared by adding 0.191g of Na2B4O7·10H2O, 0.495g of H3BO4, 2.5g of BSA, 25g of sucrose, 0.05g of NaN3, 15mL of 10% PVP, and 25mL of 10% Tween-20 to 400mL of distilled water, mixing thoroughly, and bringing the volume to 500mL.

[0101] 2. Preparation of colloidal gold chromatography test strips (1) Scratching membrane Using an XYZ 3060 coating instrument (BioDot, USA), a detection line (T line) and a control line (C line) were sprayed onto a nitrocellulose membrane (NC membrane) at a rate of 8 μL / cm, spaced 10 mm apart. The T line was coated with 0.5 mg / mL of SB-OVA prepared in Example 1, and the C line was coated with 0.1 mg / mL of horseradish peroxidase-labeled goat anti-mouse IgG (Beijing TransGen Biotech, HS002-01). The coated NC membrane was then dried in a 37°C oven for 12 h to obtain the final coated NC membrane.

[0102] (2) Preparation of sample pad The glass fiber membrane (SB08, Shanghai Liangxin Technology Co., Ltd.) was immersed in a 0.05M PB solution containing 5 mg / mL PVP, 5 mg / mL sucrose, 5 mg / mL bovine serum albumin and 0.75% Tween-20 (v / v). After being fully immersed, it was dried in a 60℃ oven to obtain the sample pad.

[0103] (3) Assembly The sample pad, coated NC membrane, and absorbent paper are sequentially pasted onto a PVC base plate, with the sample pad, coated NC membrane, and absorbent paper overlapping by 2 mm. The strips are then cut into 2.5 mm wide strips using a strip cutter to obtain colloidal gold chromatography test strips.

[0104] 3. Colloidal gold immunochromatography method A colloidal gold immunochromatographic method for detecting sennoside B is as follows: Take 2 μL of the above-obtained colloidal gold probe and 150 μL of the sample solution to be tested and mix them in the enzyme-labeled microwell to obtain the reaction solution. Incubate at 25℃ for 5 min. Then insert the above-obtained colloidal gold chromatography test strip into the reaction solution and perform chromatography reaction for 5 min. After the chromatography reaction is completed, take out the colloidal gold chromatography test strip, remove the sample pad, and take a picture under natural light to record.

[0105] If red bands appear on both the T and C lines of the colloidal gold chromatography test strip, the sample does not contain sennoside B; if no red bands appear on the T line but red bands appear on the C line, the sample contains sennoside B; if no red bands appear on the C line, the test result is invalid and the test should be repeated.

[0106] II. Validation of the colloidal gold immunochromatography method 1. Sample pretreatment Three representative weight-loss food bases—liquid herbal tea, gel-like enzyme jelly, and solid substitute tea—were used as samples and subjected to the following processing: Weigh 1.0g of sample liquid herbal tea, add 4.0mL of sample extract (obtained by mixing methanol and 0.002mol / L ammonium formate solution at a volume ratio of 9:1), vortex mix for 1min, then centrifuge at 5000r / min for 1min, take the supernatant and filter it through a 0.22μm organic filter membrane, then dilute it 4 times with 0.02mol / mL PB solution to obtain the herbal tea sample to be tested.

[0107] After homogenizing the gel-like enzyme jelly, it was processed according to the above method to obtain the jelly sample to be tested.

[0108] After grinding the solid substitute tea, it was processed according to the method shown above, and then diluted 10 times with 0.02mol / mL PB solution to obtain the tea sample to be tested.

[0109] 2. Sensitivity Test (1) Experimental methods Add 0, 10, 20, 30, 40, 50, and 60 ng / g of sennoside B standard solution to the herbal tea and jelly samples to be tested, respectively. Add 0, 100, 200, 300, 400, 500, and 600 ng / g of sennoside B standard solution to the tea samples to be tested, respectively.

[0110] Next, for the herbal tea samples, jelly samples, and tea samples containing different concentrations of sennoside B standard solution, the above-mentioned "colloidal gold immunochromatography method" was used for detection, and the concentration of sennoside B at which the red band on the T line of the colloidal gold chromatography test strip disappeared visually was used as the visual judgment threshold (cut-off value) for the corresponding sample.

[0111] (2) Experimental results The cut-off value test results for the herbal tea sample, the jelly sample, and the tea sample are shown in the figure below. Figure 3 As shown, Figure 3 In the figure, A represents the cut-off value test results for the herbal tea sample to be tested. Figure 3In the graph, B represents the cut-off value test results for the jelly sample to be tested. Figure 3 C in the figure represents the cut-off value test results for the tea sample to be tested. The results show that when the amount of SB added in the herbal tea sample is 0.05 mg / kg, the amount of SB added in the jelly sample is 0.05 mg / kg, and the amount of SB added in the solid substitute tea sample is 0.5 mg / kg, the detection line (T line) on the colloidal gold chromatography test strip in the colloidal gold immunochromatographic method completely disappears. Therefore, this concentration can be used as the visual judgment threshold (cut-off value).

[0112] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description and ideas, and it is neither necessary nor possible to exhaustively describe all implementation methods here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. An artificial antigen for detecting sennosides, characterized in that, Its structural formula is shown in equation (I). Formula (I); Z represents a carrier protein; the carrier protein is lactoferrin or chicken ovalbumin.

2. The use of the artificial antigen according to claim 1 in the preparation of artificial antibodies for detecting sennosides.

3. An artificial antigen composition for detecting sennosides, characterized in that, It includes a coating antigen and an immunogen, wherein the coating antigen is an artificial antigen in which the carrier protein is chicken ovalbumin as described in claim 1, and the immunogen is an artificial antigen in which the carrier protein is lactoferrin as described in claim 1.

4. The use of the artificial antigen composition of claim 3 in the preparation of products for detecting sennosides.

5. An artificial antibody for detecting sennosides, characterized in that, The amino acid sequence of the CDR1 region of the heavy chain is shown in SEQ ID NO: 3, the amino acid sequence of the CDR2 region of the heavy chain is shown in SEQ ID NO: 4, and the amino acid sequence of the CDR3 region of the heavy chain is shown in SEQ ID NO: 5; the amino acid sequence of the CDR1 region of the light chain is shown in SEQ ID NO: 6, the amino acid sequence of the CDR2 region of the light chain is LVS, and the amino acid sequence of the CDR3 region of the light chain is shown in SEQ ID NO:

7.

6. The artificial antibody according to claim 5, characterized in that, The amino acid sequence of its heavy chain is shown in SEQ ID NO: 1, and the amino acid sequence of its light chain is shown in SEQ ID NO:

2.

7. An antigen-antibody composition for detecting sennosides, characterized in that, It contains a coating antigen and the artificial antibody of claim 5, wherein the coating antigen is an artificial antigen in claim 1 in which the carrier protein is chicken ovalbumin.

8. The use of the artificial antibody of claim 5 and / or the antigen-antibody composition of claim 7 in the preparation of a kit for detecting sennosides or in the establishment of an immunological method for detecting sennosides.

9. A kit for detecting sennosides, characterized in that, It contains the artificial antigen composition of claim 3, the artificial antibody of claim 5, or the antigen-antibody composition of claim 7.

10. A method for detecting sennosides, characterized in that, The detection is performed using the artificial antibody of claim 5, the antigen-antibody composition of claim 7, and / or the kit of claim 9.

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