A universal monoclonal antibody HOPE1 against natural capsaicin, dihydrocapsaicin and synthetic capsaicin and its application

Abstract

The present invention discloses a universal monoclonal antibody HOPE1 against natural capsaicin, dihydrocapsaicin, and synthetic capsaicin, and its application. The heavy chain variable region sequence of the antibody is shown in SEQ ID NO. 1, and the light chain variable region sequence is shown in SEQ ID NO. 2. The monoclonal antibody HOPE1 has a half-inhibitory concentration of 0.32 ng / mL for natural capsaicin, 0.07 ng / mL for synthetic capsaicin, 0.21 ng / mL for dihydrocapsaicin, and a mixture of natural capsaicin, synthetic capsaicin, and dihydrocapsaicin, respectively, and has a strong affinity for capsaicinoids. The IC-ELISA immunoassay method and time-resolved fluorescence immunochromatography assay for detecting the content of capsaicinoids established using the monoclonal antibody HOPE1 provided by the present invention have the advantages of high sensitivity and low detection limit.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to a universal monoclonal antibody HOPE1 against natural capsaicin, dihydrocapsaicin and synthetic capsaicin and an application thereof. Background Art

[0002] Capsaicinoids are small molecule compounds with a vanillin structure as the skeleton and long side chain groups. The three representative capsaicinoids are natural capsaicinoids (CPC), synthetic capsaicinoids (NV) and dihydrocapsaicinoids (DCPC). Their structures are as follows: a. Natural capsaicinoids , b. Synthetic capsaicin , c. Dihydrocapsaicin .

[0003] Capsaicinoids have been widely used, for example, as flavorings, preparations for medicines, pesticides, antifouling coatings, and cable materials. Establishing a highly sensitive detection method for capsaicinoids is of great significance for the application of capsaicinoids. Natural capsaicinoids and dihydrocapsaicinoids account for 90% of the total amount of capsaicinoids used. Synthetic capsaicinoids have similar structures and functions to natural capsaicinoids. Since synthetic capsaicinoids have lower production costs than natural capsaicinoids, they are likely to replace natural capsaicinoids and dihydrocapsaicinoids in the future and be widely used. Therefore, natural capsaicinoids, dihydrocapsaicinoids, and synthetic capsaicinoids can be used as representatives of capsaicinoids. Establishing a detection method suitable for natural capsaicinoids, dihydrocapsaicinoids, and synthetic capsaicinoids can meet the existing detection needs for capsaicinoids.

[0004] Existing analytical methods for capsaicinoids include sensory identification, Kjeldahl nitrogen determination, chemical detection (including the furfural, ferric chloride, and vanadium oxychloride methods), spectrophotometry, spectrometry, thin-layer chromatography, high-performance liquid chromatography, chromatography-mass spectrometry, and immunoassays. Immunoassays are widely used for the detection of capsaicinoids due to their high specificity, high sensitivity, large analytical capacity, convenience, speed, low cost, and suitability for on-site batch testing. Antibodies are the core reagents of immunoassays, and the preparation of highly sensitive and versatile capsaicinoid antibodies is a prerequisite for establishing a robust immunoassay for capsaicinoids. Because capsaicinoids are small molecules that can be specifically recognized by antibodies but cannot elicit an immune response, the preparation of monoclonal antibodies against capsaicinoids requires immunization with synthetic antigens.

[0005] In the study of immunoassay technology for capsaicin and aflatoxins as exogenous pollutants in edible vegetable oils, Yang Qingqing (2016) prepared a capsaicinoid hapten HaptenC, which was then coupled with a carrier protein to make an artificial complete antigen, and immunized mice. Through monoclonal cloning, the monoclonal antibody YQQD8 against capsaicinoids was prepared for the first time, and a series of immunoassay methods (ELISA, fluorescence polarization and capillary electrophoresis immunoassay, colloidal gold lateral flow immunochromatography rapid detection method, time-resolved fluorescence immunochromatography, immunoaffinity column-HPLC-MS / MS detection method) were established based on this antibody. However, the IC values ​​of this antibody for natural capsaicin, dihydrocapsaicin, and synthetic capsaicin were low. 50 The values ​​were only 8.5 ng / mL, 5.0 ng / mL, and 13.5 ng / mL, respectively. Through the 3D model docking of antibodies and capsaicinoids, it was found that only one amino acid residue of the monoclonal antibody YQQD8 was involved in the hydrophobic interaction with capsaicinoids, and the affinity was not strong. The affinity between antibodies and antigens in immunoassay methods has a great influence on the sensitivity of the analytical method. Therefore, it is quite necessary to find antibodies with higher affinity for capsaicinoids. Summary of the Invention

[0006] In view of the shortcomings of the existing technology, the present invention aims to provide a universal monoclonal antibody HOPE1 against natural capsaicin, dihydrocapsaicin and synthetic capsaicin and its application, and to establish a more sensitive analysis method for capsaicinoids.

[0007] The primary purpose of the present invention is to provide a monoclonal antibody HOPE1 that is resistant to one or more of natural capsaicin, dihydrocapsaicin and synthetic capsaicin.

[0008] Another object of the present invention is to provide the use of the monoclonal antibody HOPE1 in detecting the content of one or more of natural capsaicin, dihydrocapsaicin, and synthetic capsaicin and / or in preparing a kit for detecting the content of one or more of natural capsaicin, dihydrocapsaicin, and synthetic capsaicin.

[0009] Another object of the present invention is to provide a kit for detecting the content of one or more of natural capsaicin, dihydrocapsaicin and synthetic capsaicin.

[0010] Another object of the present invention is to provide an ic-ELISA immunoassay method for detecting the content of one or more of natural capsaicin, dihydrocapsaicin, and synthetic capsaicin.

[0011] Another object of the present invention is to provide a time-resolved fluorescence immunochromatography method for detecting the content of one or more of natural capsaicin, dihydrocapsaicin, and synthetic capsaicin.

[0012] Another object of the present invention is to provide a nucleotide sequence encoding the monoclonal antibody HOPE1.

[0013] Another object of the present invention is to provide a vector of nucleotide sequences.

[0014] Another object of the present invention is to provide a recombinant cell.

[0015] The present invention achieves the above-mentioned purpose through the following technical means:

[0016] The present invention provides a universal monoclonal antibody HOPE1 against natural capsaicin, dihydrocapsaicin and synthetic capsaicin, the heavy chain variable region sequence is shown in SEQ ID NO.1, and the light chain variable region sequence is shown in SEQ ID NO.2.

[0017] The universal monoclonal antibody HOPE1 against natural capsaicin, dihydrocapsaicin and synthetic capsaicin is a monoclonal antibody HOPE1 against one or more of natural capsaicin, dihydrocapsaicin and synthetic capsaicin.

[0018] Preferably, the subtype of the universal monoclonal antibody HOPE1 against natural capsaicin, dihydrocapsaicin and synthetic capsaicin is IgG1.

[0019] The monoclonal antibody HOPE1 is secreted and produced by the hybridoma cell line HOPE1. The specific steps are as follows:

[0020] S1. Synthesis of Hapten C-BSA, a universal artificial complete antigen for natural capsaicin, dihydrocapsaicin, and synthetic capsaicin

[0021] The hapten Hapten C was synthesized by reacting vanillinamine hydrochloride with succinic anhydride, and the hapten Hapten C was coupled with bovine serum albumin (BSA) to obtain Hapten C-BSA, a universal artificial complete antigen for capsaicinoids.

[0022] S2. Animal Immunization and Establishment of Hybridoma Cell Line HOPE1

[0023] Balb / c mice were immunized with the artificial complete antigen Hapten C-BSA prepared in S1, and mouse spleen cells were collected and fused with mouse myeloma cells. The cell line with the best antibody titer and inhibition rate was screened by ic-ELISA, which was the hybridoma cell line HOPE1. This cell line was expanded and cultured, and then collected and frozen for establishment.

[0024] S3. The hybridoma cell line HOPE1 was injected into the peritoneal cavity of Balb / c mice, and the ascites was collected. The ascites was purified by the octanoic acid-ammonium sulfate method to obtain the monoclonal antibody HOPE1.

[0025] Wherein, the structural formula of the hapten Hapten C in step S1 is shown in formula (II): .

[0026] The half inhibitory concentration (IC50) of the monoclonal antibody HOPE1 provided by the present invention on natural capsaicin, synthetic capsaicin and dihydrocapsaicin was detected by IC-ELISA. 50 ) were 0.32 ng / mL, 0.07 ng / mL, and 0.21 ng / mL, respectively, and the half-inhibitory concentration (IC50) of the mixture of natural capsaicin, synthetic capsaicin, and dihydrocapsaicin was 0. 50 ) was 103.8 pg / mL. This is significantly lower than the YQQD8 monoclonal antibody against natural capsaicin, dihydrocapsaicin, and synthetic capsaicin provided in the prior art CN201610079095.X (monoclonal antibody YQQD8 has a 50% inhibitory concentration of 8.5 ng / mL for natural capsaicin, 13.5 ng / mL for synthetic capsaicin, and 5.0 ng / mL for dihydrocapsaicin, respectively, and a 50% inhibitory concentration of 50% for a mixture of natural capsaicin, synthetic capsaicin, and dihydrocapsaicin of 50%. This indicates that the monoclonal antibody HOPE1 provided by the present invention has a higher affinity for capsaicinoids.

[0027] The present invention establishes 3D antibody models of monoclonal antibody HOPE1 and monoclonal antibody YQQD8, as well as 3D molecular models of natural capsaicin, dihydrocapsaicin, and synthetic capsaicin. Through model docking, it is found that 3-4 amino acids of monoclonal antibody HOPE1 participate in hydrophobic interactions with the three capsaicin molecules, while only one amino acid participates in monoclonal antibody YQQD8. This indicates that the monoclonal antibody HOPE1 provided by the present invention has a stronger hydrophobic interaction with capsaicinoid substances, and the monoclonal antibody HOPE1 provided by the present invention has a higher affinity for capsaicinoid substances.

[0028] Based on the above experimental results, the use of monoclonal antibody HOPE1 in detecting the content of one or more of natural capsaicin, dihydrocapsaicin, and synthetic capsaicin and / or in preparing a kit for detecting the content of one or more of natural capsaicin, dihydrocapsaicin, and synthetic capsaicin should also fall within the scope of protection of the present invention.

[0029] The present invention also provides a kit for detecting the content of one or more of natural capsaicin, dihydrocapsaicin and synthetic capsaicin, comprising the monoclonal antibody HOPE1.

[0030] More preferably, the kit further comprises reagents required for the ic-ELISA immunoassay method.

[0031] Most preferably, the reagents required for the ic-ELISA immunoassay method are artificial complete antigen HaptenC-KLH, 0.01M PBS buffer, CB buffer, 0.01M PBST solution, 0.05M CB buffer containing 5% skim milk powder, goat anti-mouse IgG, TMB color development solution, and 10% (v / v) H2SO4 solution.

[0032] More preferably, the kit further comprises reagents required for time-resolved fluorescence immunochromatography.

[0033] Most preferably, the reagents required for the time-resolved fluorescent immunochromatography are a time-resolved fluorescent probe coupled with the monoclonal antibody HOPE1, a test strip coated with the monoclonal antibody HOPE1 on the T line of the chromatography membrane, and a secondary antibody on the C line of the chromatography membrane.

[0034] Preferably, the secondary antibody is goat anti-mouse IgG.

[0035] The present invention also provides an IC-ELISA immunoassay method for detecting the content of one or more of natural capsaicin, dihydrocapsaicin, and synthetic capsaicin, characterized in that it comprises the following steps:

[0036] S1. Add the sample extract to an ELISA plate coated with natural capsaicin, dihydrocapsaicin, and the universal artificial complete antigen HaptenC-KLH for synthetic capsaicin (structural formula (I)), along with the monoclonal antibody HOPE1. Incubate in a water bath at 35-40°C for 10-20 minutes, then wash the plate.

[0037] S2. Add secondary antibody and incubate in a 35-40°C water bath for 10-20 minutes. Wash the plate after the water bath.

[0038] S3. Add color developing solution and incubate in a 35-40°C water bath for 10-20 minutes. Wash the plate after the water bath.

[0039] S4. Add the stop solution, measure the absorbance of the solution at 420-480 nm, and substitute the absorbance value into the standard curve to calculate the content of one or more natural capsaicin, dihydrocapsaicin, and synthetic capsaicin in the sample;

[0040] .

[0041] Preferably, the water bath in S1 is a water bath at 37° C. for 15 minutes.

[0042] Preferably, the water bath in S2 is a water bath at 37° C. for 20 minutes.

[0043] Preferably, the secondary antibody in S2 is goat anti-mouse IgG.

[0044] Preferably, the water bath in S3 is a water bath at 37° C. for 10 minutes.

[0045] Preferably, S3 is TMB color developing solution.

[0046] Preferably, the stop solution in S4 is 10% (v / v) H2SO4 solution.

[0047] Preferably, S4 reads the absorbance at 450 nm.

[0048] The present invention also provides a time-resolved fluorescence immunochromatographic method for detecting the content of one or more of natural capsaicin, dihydrocapsaicin, and synthetic capsaicin, which specifically comprises the following steps:

[0049] S1. Add the time-resolved fluorescent probe conjugated to the monoclonal antibody HOPE1 to the sample solution and react for 1 to 5 minutes;

[0050] S2. Add the test strip to the sample solution and run the test for 3-7 minutes;

[0051] S3. Detect the fluorescence intensity of the test strip T line and C line under 320-400nm ultraviolet light, calculate the ratio of the fluorescence intensity of the T line to the C line, and calculate the content of one or more natural capsaicin, dihydrocapsaicin, and synthetic capsaicin in the sample based on the ratio of the fluorescence intensity of the T line to the C line;

[0052] Among them, the T line in S3 is coated with monoclonal antibody HOPE1, and the C line is coated with secondary antibody.

[0053] Preferably, the preparation method of the time-resolved fluorescent probe coupled to the monoclonal antibody HOPE1 in S1 is: activating the time-resolved fluorescent microspheres with EDC and NHS and then coupling them with the monoclonal antibody HOPE1.

[0054] Preferably, the preparation method of the test strip described in S2 is: coating the monoclonal antibody HOPE1 on the T line of the chromatography membrane, and coating the secondary antibody on the C line of the chromatography membrane. After the coating is completed, the chromatography membrane is attached to the adhesive base plate, and a sample pad is attached to the end close to the T line, and an absorbent pad is attached to the end close to the C line, thereby completing the assembly preparation of the test strip.

[0055] More preferably, the secondary antibody is goat anti-mouse IgG.

[0056] Preferably, the reaction time in S1 is 3 minutes.

[0057] Preferably, the sample running time in S2 is 5 minutes.

[0058] Preferably, the detection of fluorescence intensity in S3 is detection of fluorescence intensity under 360 nm ultraviolet light.

[0059] The present invention also provides a nucleotide sequence encoding the monoclonal antibody HOPE1.

[0060] The present invention also provides a vector containing a nucleotide sequence encoding the monoclonal antibody HOPE1.

[0061] The present invention also provides a recombinant cell containing the above vector.

[0062] The present invention also provides a recombinant cell expressing the monoclonal antibody HOPE1.

[0063] The present invention has the following advantages and beneficial effects compared to the prior art:

[0064] The present invention provides a universal monoclonal antibody, HOPE1, for natural capsaicin, synthetic capsaicin, and dihydrocapsaicin. The half-inhibitory concentrations of HOPE1 for natural capsaicin, synthetic capsaicin, dihydrocapsaicin, and a mixture of natural capsaicin, synthetic capsaicin, and dihydrocapsaicin are 0.32 ng / mL, 0.07 ng / mL, 0.21 ng / mL, and 103.8 pg / mL, respectively. Three to four amino acid residues participate in hydrophobic interactions with capsaicinoids, demonstrating strong affinity for them. The IC-ELISA immunoassay and time-resolved fluorescence immunochromatography assay for detecting capsaicinoids, established using the monoclonal antibody HOPE1 provided by the present invention, offer the advantages of high sensitivity and low detection limits. BRIEF DESCRIPTION OF THE DRAWINGS

[0065] Figure 1 Figure A shows the UV absorption spectra of hapten Hapten C, BSA and the coupling product Hapten C-BSA; Figure 1 Figure B shows the UV absorption spectra of hapten Hapten C, KLH and the coupled product Hapten C-KLH; Figure 1 Figure C shows the UV absorption spectra of hapten Hapten C, LF and the coupling product Hapten C-LF.

[0066] Figure 2 This is the microplate reader reading result of Example 6.

[0067] Figure 3 This is the standard curve of the mixture of natural capsaicin, dihydrocapsaicin and synthetic capsaicin in Example 7.

[0068] Figure 4 This is the standard curve of natural capsaicin, dihydrocapsaicin, and synthetic capsaicin in Example 7.

[0069] Figure 5 Schematic diagram of the preparation principle of the time-resolved fluorescent probe in Example 8.

[0070] Figure 6 Schematic diagram of the structure of the time-resolved fluorescence immunochromatography test strip in Example 8.

[0071] Figure 7 This is the standard curve of the mixture of natural capsaicin, dihydrocapsaicin and synthetic capsaicin in Example 8. DETAILED DESCRIPTION

[0072] The present invention will be further described below with reference to the accompanying drawings and specific examples, but the examples do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0073] Unless otherwise specified, the reagents and materials used in the following examples were commercially available.

[0074] Example 1 Preparation of universal artificial complete antigen for capsaicinoids

[0075] 1. Synthesis of Hapten C

[0076] The hapten Hapten C was synthesized with reference to the synthesis method of hapten Hapten C in the literature "Yang Qingqing. Research on immunoassay technology of capsaicin and aflatoxins as exogenous pollutants in edible vegetable oils [D]. Chinese Academy of Agricultural Sciences, 2016."

[0077] The specific steps are as follows:

[0078] S1. Weigh 0.28 g (0.0015 mol) of vanillinamine hydrochloride and dissolve it in 6 mL of tetrahydrofuran. Then, add 0.15 g (0.0015 mol) of triethylamine dropwise while stirring. After the addition is complete, stir at room temperature for 30 min.

[0079] S2. 0.15 g (0.0015 mol) of succinic anhydride was added, and the mixture was stirred at room temperature for 4.5 h. The disappearance of the starting material spot was confirmed by TLC (developing solvent: a mixture of ethyl acetate and methanol in a volume ratio of 2:1).

[0080] S3. Add 10mL of water and 10mL of ethyl acetate and stir at room temperature for 3min, then add 5mL of saturated sodium bicarbonate and continue stirring until the white substance is completely dissolved and the liquids are separated to give an aqueous phase and an ethyl acetate phase;

[0081] S4. Adjust the pH of the aqueous phase to 2-3, extract twice with 10 mL of ethyl acetate each time, combine the ethyl acetate phases and evaporate the solvent, and finally recrystallize with 3-4 mL of methanol to obtain pure hapten C, with the molecular formula C 12 H 15NO5, relative molecular mass is 253.2. The structural formula of hapten Hapten C is shown in formula (II):

[0082] .

[0083] 2. Preparation of universal artificial complete antigen for capsaicinoids

[0084] Hapten C has a carboxyl group, which is coupled to the amino group on the carrier protein through the active lipid method to prepare an artificial complete antigen. The specific steps are as follows:

[0085] S1. Weigh 23.5 mg, 45.6 mg, and 25.5 mg of the synthesized hapten Hapten C described above and add them to three vials A, B, and C, respectively. Add 600 μL of DMF to each vial to dissolve the hapten.

[0086] S2. Add 27 mg, 54 mg, and 29.2 mg of EDC powder and 16 mg, 32 mg, and 17.2 mg of NHS powder to the three small bottles A, B, and C, respectively. Stir at room temperature in the dark for 4 h to obtain the activation solution.

[0087] S3. Weigh 200 mg of bovine serum albumin (BSA), hemocyanin (KLH), and lactoferrin (LF) into three bottles respectively. Add 0.05 mol / L sodium carbonate (CB) buffer with a pH of 9.6 to dissolve the proteins to obtain the corresponding protein solutions, maintaining the concentration at 10 mg / mL. Then cool in an ice bath.

[0088] S4. While stirring in an ice bath, add the activation solution in bottle A to the BSA solution, add the activation solution in bottle B to the KLH solution, and add the activation solution in bottle C to the LF solution. After the additions are complete, measure the pH. If it is alkaline, remove the ice bath and allow the coupling to proceed overnight at room temperature. Start dialysis the next day, changing the dialysate three times a day. Continue dialysis for three days to obtain the purified coupling products (Hapten C-BSA, Hapten C-KLH, Hapten C-LF).

[0089] S5. Perform UV absorption spectroscopic scanning on the hapten HaptenC, three carrier proteins (BSA, KLH, LF), and three coupling products.

[0090] The UV absorption spectrum results are as follows Figure 1 As shown, Figure 1 Figure A shows the UV absorption spectra of hapten Hapten C, BSA and the coupling product Hapten C-BSA; Figure 1 Figure B shows the UV absorption spectra of hapten Hapten C, KLH and the coupled product Hapten C-KLH; Figure 1Figure C shows the UV absorption spectra of hapten Hapten C, LF and the coupling product Hapten C-LF.

[0091] It can be seen that the absorption peaks of the three coupling products are similar to those of Hapten C and its carrier protein, and there is a certain offset. Therefore, it can be judged that the three artificial complete antigens are successfully coupled, and the artificial complete antigens Hapten C-BSA, Hapten C-KLH, and Hapten C-LF are prepared respectively.

[0092] Example 2 Animal Immunization and Establishment of Hybridoma Cell Line HOPE1

[0093] 1. Animal Immunization

[0094] The three artificial complete antigens Hapten C-BSA, Hapten C-KLH, and Hapten C-LF prepared in Example 1 were diluted to 1 mg / mL with 0.01 M PBS solution to obtain artificial complete antigen solutions.

[0095] The three artificial complete antigen solutions were mixed with Freund's complete or Freund's incomplete adjuvant to obtain emulsions. During the first immunization, the artificial complete antigen solution was mixed with Freund's complete adjuvant in a ratio of 1:1. During subsequent booster immunizations, the artificial complete antigen solution was mixed with Freund's incomplete adjuvant in a ratio of 1:1.

[0096] The obtained emulsion was injected intraperitoneally into female Balb / c mice at a volume of 100 μL per mouse. Each group (i.e., Hapten C-BSA experimental group, Hapten C-KLH experimental group, and Hapten C-LF experimental group) consisted of 3 female Balb / c mice, and the mice were injected once every other week for a total of 4 injections. The first injection was the initial immunization, and the subsequent injections were booster immunizations.

[0097] Starting with the third injection, serum ic-ELISA testing was performed one week after injection to observe changes in capsaicinoid antibody titer and affinity in each mouse's serum. After the fourth injection and serum ic-ELISA testing, mice with the highest serum titer and highest inhibition rate were selected from the three experimental groups based on the serum ic-ELISA test results after the fourth injection. After verification, these mice were immunized with the artificial complete antigen Hapten C-BSA. The selected mice were subjected to shock immunization, specifically by injecting 100μg of the artificial complete antigen Hapten C-BSA directly into the mouse's peritoneal cavity without the addition of an adjuvant. One week after the shock immunization, spleen cells from these mice were harvested for cell fusion experiments.

[0098] 2. Cell Fusion

[0099] 1. Collect spleen cells from immunized female Balb / c mice

[0100] The mice that had undergone shock immunization were asphyxiated with carbon dioxide and then immersed in a 75% ethanol aqueous solution. The spleens were then dissected out in a clean bench and crushed in RPMI-1640 basal medium. The spleen cells were then collected into a 50 mL centrifuge tube.

[0101] 2. Cell fusion experiment

[0102] S1. Mix mouse myeloma cells (SP2 / 0 cells) and spleen cells collected above at a ratio of 1:5 in a centrifuge tube. Add 20 mL of RPMI-1640 basal medium, centrifuge at 1000 rpm for 7 minutes, and remove the supernatant.

[0103] S2. Slowly add 1 mL of PEG solution incubated at 37°C over 1 min while rotating the centrifuge tube, and then add RPMI-1640 basal medium.

[0104] S3. Centrifuge at 800 rpm for 7 min, remove the supernatant, transfer the cell pellet to 200 mL of HAT medium, mix well, and plate the HAT medium in a 96-well ELISA plate for incubation.

[0105] 3. Screening and establishment of hybridoma cells

[0106] On the 5th day of culture in the 96-well cell culture plate, the HAT medium was half-replaced with HT medium, and on the 8th day, the medium was fully replaced with HT medium. During the full medium replacement, the cell supernatant (original culture medium) of the original culture plate was transferred to a new 96-well plate, and capsaicinoids were detected by IC-ELISA. The titer and inhibition rate of the supernatant of each well were calculated based on the results. The fusion cell line in the 96-well enzyme-labeled plate corresponding to the positive well with a titer greater than 1.0 and an inhibition rate greater than 80% was selected and subcloned by limiting dilution. After 4 subcloning, the cell supernatant was taken for detection, and the cell line with the best titer and inhibition rate was selected for expansion culture. The cell line was then collected and frozen to obtain the hybridoma cell HOPE1 cell line, which was frozen at -80°C.

[0107] Example 3 Preparation of universal monoclonal antibody HOPE1 against natural capsaicin, dihydrocapsaicin and synthetic capsaicin

[0108] 1. Preparation of Mouse Ascites

[0109] 10-week-old Balb / c mice were intraperitoneally injected with 0.5 mL of paraffin wax, and hybridoma cells were injected 7 days later.

[0110] Resuscitate frozen cell lines and expand them in RPMI-1640 complete medium (RPMI-1640 basal medium plus 200 mL of fetal bovine serum). Once hybridoma cells have spread to the bottom of the culture dish, collect the cell suspension into two 1 mL syringes, aspirating 1 mL of cell suspension into each syringe. Inject the cells intraperitoneally into the abdomen of mice (one injection per mouse). After 10-15 days, when the abdomen becomes noticeably enlarged, sacrifice the mice, dissect them, and collect ascites.

[0111] The ascites was centrifuged at 2000 rpm for 10 min to remove the upper fat layer and the lower fibrin and cells. The middle layer was collected and a portion was used to determine its potency against capsaicinoids by IC-ELISA. After confirming that the potency inhibition was no significantly different from the results of hybridoma cell establishment in Example 2, the remainder was aliquoted and frozen at -80°C for future use.

[0112] 2. Purification of Mouse Ascites

[0113] Mouse ascites was purified using the caprylic acid-ammonium sulfate precipitation method. The specific steps are as follows:

[0114] S1. Take 3 mL of ascites and add 2 volumes of 0.06 mol / L sodium acetate buffer (pH 4.5). Add octanoic acid dropwise at 33 μg / mL of ascites. Continue stirring for 30 minutes after addition. Then, centrifuge at 12,000 rpm for 30 minutes at 4°C.

[0115] S2. Remove the supernatant, filter it through a 0.45 μm microporous membrane, and mix it with 1 / 10 the volume of 10× PBS. Adjust the pH to 7.4 with 1 M NaOH solution. Cool the supernatant to 4°C, then add 0.277 g / mL of ammonium sulfate to a final saturation of 45%. Stir continuously for 30 minutes, then centrifuge at 12,000 rpm at 4°C for 30 minutes and discard the supernatant.

[0116] S3. Dissolve the precipitate in a small amount of 0.01M PBS solution and dialyze against 50-100 volumes of 0.01M PBS for one day, changing the solution three times. The dialyzed solution is concentrated using an ultrafiltration tube to obtain HOPE1, a universal monoclonal antibody against natural capsaicin, dihydrocapsaicin, and synthetic capsaicin. Store it at -80°C until use.

[0117] Example 4 Determination of the variable region sequence of the monoclonal antibody HOPE1

[0118] (1) Total RNA extraction: The total RNA of the hybridoma cell HOPE1 obtained in Example 2 was extracted using the total RNA extraction kit of Tiangen Company according to the instructions.

[0119] (2) cDNA synthesis: cDNA was synthesized by reverse transcription using the cDNA first-strand synthesis kit from PROMEGA, USA.

[0120] (3) PCR cloning of variable region genes: Primers were designed based on the conserved sites of mouse antibody gene sequences in GENEBANK (sequences shown in Table 1). The cDNA synthesized in step (2) was used as a template to amplify the variable region genes of the light and heavy chains of the monoclonal antibody HOPE1. The PCR program was as follows: 94°C for 30 seconds, 55°C for 45 seconds, and 72°C for 1 minute, for 30 cycles of amplification, and finally extension at 72°C for 10 minutes.

[0121] After PCR products were separated by 1% (weight percent) agarose gel electrophoresis, the monoclonal antibody HOPE1 DNA fragment was purified and recovered using a kit, ligated into the vector pEASY-T3, and transformed into E. coli DH5a competent cells. Positive clones were picked and sent to Guangzhou Tianyi Huiyuan Gene Technology Co., Ltd. for sequencing. The sequencing primer sequences are shown in Table 1:

[0122] Table 1 Sequencing primer sequences

[0123]

[0124] Among them, Y and K are degenerate bases, Y=C / T, K=G / T.

[0125] The amino acid sequences of the heavy chain and light chain of the universal monoclonal antibody HOPE1 against natural capsaicin, dihydrocapsaicin and synthetic capsaicin were determined to be shown in SEQ ID NO.1 and SEQ ID NO.2.

[0126] SEQ ID NO.1:

[0127] GPGILQPSQTLSLACTFSGISLSTSMGGLSWLRKPSGKGLEWLASIWNNDKYYIPSLKSRLTISKETSNNQAFLKLTSVDTADSATYYCAWVYFGLGFAYWGQG

[0128] SEQ ID NO.2:

[0129] LPLSSGINAEYMGDPLFQLSEMETDTLLLWVLLLWVPGSTGDIVLTQSPASLAVSLGQRATISYRASKSVSTSGYSYMHWNQQKPGQPPRLLIYLVSNLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHIRELTRSEGGPSWK

[0130] Example 5 Amino acids and interaction types of universal monoclonal antibodies against natural capsaicin, dihydrocapsaicin and synthetic capsaicin interacting with capsaicinoid molecules

[0131] According to the sequence of the universal monoclonal antibody YQQD8 against natural capsaicin, dihydrocapsaicin, and synthetic capsaicin provided by CN201610079095.X and the sequence of the monoclonal antibody HOPE1 obtained by sequencing in Example 4, 3D antibody models of monoclonal antibody YQQD8 and monoclonal antibody HOPE1 were respectively established using the SWISS-MODEL model. At the same time, 3D molecular models of natural capsaicin, dihydrocapsaicin and synthetic capsaicin were established using KINGDRAW software. Subsequently, the 3D molecular models of the three capsaicin molecules were molecularly docked with the 3D antibody models of monoclonal antibody YQQD8 and monoclonal antibody HOPE1, respectively. The key amino acids that interacted in the docking results and the corresponding interaction forces generated by the key amino acids were sorted out. The results are shown in Table 2.

[0132] Table 2 Key amino acids and interaction types of monoclonal antibody HOPE1 and monoclonal antibody YQQD8 with capsaicinoids

[0133]

[0134] The results showed that the hydrophobic interactions between monoclonal antibody HOPE1 and the three capsaicinoid molecules involved 3-4 amino acids, while monoclonal antibody YQQD8 involved only 1 amino acid. Hydrophobic interactions are one of the primary interactions between capsaicinoids and monoclonal antibodies and have a significant impact on the affinity of antibodies for capsaicinoids (Bai et al., 2021). The universal monoclonal antibodies prepared in this invention against natural capsaicin, dihydrocapsaicin, and synthetic capsaicin exhibit stronger hydrophobic interactions with capsaicinoids. Therefore, the monoclonal antibody HOPE1 of the present invention has a stronger affinity for capsaicinoids, further improving the detection sensitivity of immunoassays developed using this antibody.

[0135] Example 6 Identification of subtypes and properties of monoclonal antibody HOPE1

[0136] The Sigma subtype determination kit (batch number ISO2) was used to determine the subtype of monoclonal antibody HOPE1. This kit can identify six subtypes: IgG1, IgG3, IgG2a, IgM, IgG2b, and IgA. The specific steps are as follows:

[0137] S1. Dilute the monoclonal antibody HOPE1 solution prepared in Example 3 to obtain a 2-fold gradient series of solutions (1000 ng / mL, 500 ng / mL, 250 ng / mL, 125 ng / mL, 62.5 ng / mL, 31.25 ng / mL, 15.625 ng / mL, 0 ng / mL).

[0138] S2. ELISA plate preparation: Dilute the artificial complete antigen Hapten C-BSA to 1 μg / mL with 0.05 M CB buffer and add 100 μL per well to a 96-well ELISA plate. Incubate at 4°C overnight. The next day, remove the coated plate and wash twice with wash buffer (0.01 M PBST). Then, add 120 μL per well of blocking buffer (0.01 M carbonate buffer containing 5% skim milk powder) to each well of the ELISA plate. Block at 37°C for 3 hours. Remove the plate, shake off the blocking buffer, blot dry, and dry in a 37°C oven for 1 hour. Remove the plate and store in a dry place at 2-8°C until ready for use.

[0139] S3. Add 50 μL of 0.01 M PBST and 50 μL of diluted antibody solution (n = 3) to the microwells of the ELISA plate. Incubate in a 37°C water bath for 40 min. Wash the plate five times with 0.01 M PBST and blot dry.

[0140] S4. Add 100 μL of the reagents for detecting six subtypes (provided in the kit) diluted 4000-fold with 0.01 M PBST to each well, incubate for 30 minutes, then remove the plate, wash it five times, and dry it.

[0141] S5. Add 5000-fold diluted rabbit anti-goat antibody at 100 μL per well, incubate at 37°C for 30 min, then remove the plate, wash it 5 times, and tap it dry.

[0142] S6. Add 100 μL of TMB colorimetric solution to each well and incubate in a 37°C water bath for 10 minutes. Then, add 50 μL of stop solution (10% (v / v) H2SO4 solution) to each well and read the absorbance of each well at 450 nm using a microplate reader.

[0143] The results of the enzyme-labeled instrument readings are as follows Figure 2 As shown, the results show that the subtype of the monoclonal antibody HOPE1 prepared by the present invention is IgG1.

[0144] Example 7 Construction of ic-ELISA immunoassay based on monoclonal antibody HOPE1

[0145] The specific steps of the ic-ELISA immunoassay based on the monoclonal antibody HOPE1 are as follows:

[0146] (1) Preparation of antibody solution: The monoclonal antibody HOPE1 prepared in Example 3 at a concentration of 2.98 mg / mL was diluted 32,000 times with 0.01 M PBS buffer and set aside.

[0147] (2) Preparation of ELISA plate: Dilute natural capsaicin, dihydrocapsaicin, and synthetic capsaicin universal artificial complete antigen HaptenC-KLH with 0.05M CB buffer to 1μg / mL, add 100μL per well to a 96-well ELISA plate, and place it at 4℃ for overnight coating. The next day, remove the coated ELISA plate and wash it twice with 0.01M PBST solution. Then add 120μL of blocking solution (0.05M CB buffer containing 5% skim milk powder) to each well and block it at 37℃ for 3h. Then remove it, shake off the blocking solution, dry it and place it in a 37℃ oven to dry for 1h. Then remove it and store it in a dry environment at 2-8℃ for use.

[0148] (3) Preparation of negative / positive solutions: Dilute the capsaicin mixed standard solution (natural capsaicin solution, dihydrocapsaicin solution, and synthetic capsaicin solution in a volume ratio of 1:1:1, and all three capsaicin standard solution solutions are 4 mg / mL) with 0.01 M PBS buffer to 1250 pg / mL, 625 pg / mL, 312.50 pg / mL, 156.25 pg / mL, 78.13 pg / mL, 39.06 pg / mL, 19.53 pg / mL, 9.77 pg / mL, and 4.89 pg / mL, respectively. Separately, dihydrocapsaicin (DCPC), natural capsaicin (CPC), and synthetic capsaicin (NV) were diluted with 0.01 M PBS buffer. PBS was diluted into a series of gradient solutions of 100 ng / mL, 25 ng / mL, 6.25 ng / mL, 1.562 ng / mL, 0.391 ng / mL, 0.098 ng / mL, 0.024 ng / mL, and 0.006 ng / mL as positive solutions; 0.01 M PBS was taken as negative solution.

[0149] (4) Sample addition: Add 50 μL of the test solution (the above-mentioned negative / positive solution) to each well of the coated ELISA plate, and add 50 μL of the diluted monoclonal antibody HOPE1 solution to each well, and then heat the reaction in a water bath at 37°C for 15 minutes.

[0150] (5) Adding secondary antibody: Take out the ELISA plate after water bath in step (4), wash it 5 times with 0.01M PBST, blot it dry, add 100μL of 7K diluted Goat Anti-mouse IgG-HRP (secondary antibody) solution to each well, and place it in a 37℃ water bath for 20 minutes.

[0151] (6) Color development: Take out the ELISA plate after the water bath in step (5), wash it 5 times with 0.01M PBST, blot it dry, add 100 μL TMB color development solution to each well, and then water bath it at 37°C for 10 minutes.

[0152] (7) Termination: Take out the ELISA plate. The wells will now be blue. Add 50 μL of stop solution (10% (v / v) H2SO4 solution) to each well to terminate the reaction. The color of the wells should change from blue to yellow.

[0153] (8) Reading: Use an enzyme-labeled plate reader to read the absorbance value of each well of the plate at 450 nm and record it.

[0154] According to the reading results, Figure 3 The standard curves of natural capsaicin, dihydrocapsaicin and synthetic capsaicin mixtures are shown as follows Figure 4 Standard curves of natural capsaicin, dihydrocapsaicin, and synthetic capsaicin are shown.

[0155] The figure shows the half inhibitory concentration (IC) of the monoclonal antibody HOPE1 prepared by the present invention on the mixture of natural capsaicin, dihydrocapsaicin and synthetic capsaicin in ELISA. 50 ) was 103.76 pg / mL, and the IC values ​​for natural capsaicin, synthetic capsaicin, and dihydrocapsaicin were 50 The values ​​were 0.32 ng / mL, 0.07 ng / mL, and 0.21 ng / mL, respectively, indicating that monoclonal antibody HOPE1 has good affinity for natural capsaicin, synthetic capsaicin, and dihydrocapsaicin.

[0156] Example 8 Construction of a time-resolved fluorescence immunochromatography assay based on the monoclonal antibody HOPE1

[0157] The process of the time-resolved fluorescence immunochromatography method based on the monoclonal antibody HOPE1 is as follows: the monoclonal antibody HOPE1 is coupled to the time-resolved fluorescent microspheres to obtain a time-resolved fluorescent probe (TRFMs-LFIA), and then the time-resolved fluorescent probe is placed in the microwells of the ELISA plate to react with the sample liquid. After the reaction is completed, the prepared test strip is inserted into the microwells of the ELISA plate, and the test strip is read using a fluorescent immunoassay quantitative analyzer after 5 minutes.

[0158] The establishment of this method consists of two main parts: the preparation of time-resolved fluorescent probes and the detection of test strips.

[0159] (1) Preparation of TRFMs-LFIA

[0160] The preparation process of TRFMs-LFIA is as follows Figure 5As shown in Figure 2, time-resolved fluorescent microspheres (TRFMs) were activated by EDC and NHS, and then the monoclonal antibody HOPE1 was coupled to TRFMs. The specific steps are as follows:

[0161] S1. Dissolution: Dissolve 10 μL of TRFMs in 1 mL of 0.05 M MES solution in a 1.5 mL centrifuge tube.

[0162] S2. Activation: Add 15 μL of freshly prepared 0.5 mg / mL EDC and 20 μL of freshly prepared 0.5 mg / mL NHS to the above MES solution. Mix by shaking for 10 seconds, and then activate by shaking on a shaker at 200 rpm / min at 25°C for 1 hour.

[0163] S3. Labeling: Centrifuge the activated TRFMs solution at 14,000 rpm / min for 15 minutes, discard the supernatant, and reconstitute with 1 mL of 0.01 M borate buffer (pH 8.0). Add 25 μL of a 2.98 mg / mL monoclonal antibody HOPE1 solution to the reconstituted solution and activate with shaking at 200 rpm / min at 25°C for 1 hour.

[0164] S4. Blocking: Add 20 μL of 20% BSA blocking solution to the solution and continue shaking at 200 rpm / min at 25°C for 2 h.

[0165] S5. Centrifugation: Centrifuge the blocked solution at 14,000 rpm / min for 15 minutes and discard the supernatant.

[0166] S6. Resuspend: Resuspend the sample in 200 μL of time-resolved fluorescent microsphere reconstitution solution (20 mL of 0.5 mol / L PB, 2.5 g of BSA, 15 mL of 10% PVP, 25 mL of 10% Tween-20, 25 g of trehalose / sucrose, 5 mL of 3% Procline-300, and add first-grade water to 500 mL). Store at 4°C in the dark to obtain TRFMs-LFIA.

[0167] (2) Preparation of test strips

[0168] The structure of the test strip is as follows Figure 6 As shown, the test strip consists of a chromatographic membrane, a sample pad, an absorbent pad, and a PVC base. The T and C lines are located on the chromatographic membrane, with the T line close to the sample pad and the C line close to the absorbent pad. The specific production steps are as follows:

[0169] Use a membrane stripper to streak the diluted T and C line coating solutions (the T line coating solution is a monoclonal antibody HOPE1 solution diluted to a concentration of 0.2 mg / mL in 0.2 M PB buffer; the C line coating solution is a goat anti-mouse IgG solution diluted to a concentration of 4.8 mg / mL in 0.2 M PB buffer) onto the chromatographic membrane, streaking 30 cm at a time. The membrane is then oven-dried at 37°C for 24 hours. After drying, the membrane is attached to an adhesive backing and then mounted with an absorbent pad and a prepared sample pad (soaked in sample pad treatment solution (PBS (pH = 7.4) 0.05 M, Tween-20 0.5% (v / v), PVP 0.3% by weight) and then dried). The absorbent pad and sample pad overlap the membrane by 2 mm. The assembled test strips are cut into 3 mm wide strips using a strip cutter and stored for future use.

[0170] (4) TRFMs-LFIA detection steps

[0171] With reference to the document "Study on dual-detection mode mycotoxin multi-residue immunochromatographic technology based on smartphone", the present invention defines the following detection steps:

[0172] Sampling: Place 150 μL of blank buffer standard dilution solution into each microwell. The standard dilution solution is diluted in 0.01 M PBS to a series of concentrations of 50 ng / mL, 12.5 ng / mL, 3.125 ng / mL, 0.78125 ng / mL, 0.19531 ng / mL, 0.04883 ng / mL, 0.01221 ng / mL, and 0.00305 ng / mL in a CPC:DCPC:NV ratio of 1:1:1.

[0173] Reaction: Add 5 μL TRFMs-LFIA probe, quickly pipette and mix several times, and let it stand for 3 minutes.

[0174] Running the sample: Insert the prepared test strip and let it stand (run the sample) for 5 minutes.

[0175] Stop: Pull out the test strip and remove the sample pad to stop color development.

[0176] Result determination: Use the test strip to detect the absorbance values ​​of T line and C line at 360nm using a fluorescent immunoassay and record the data.

[0177] The standard curve of mixed capsaicin was drawn based on the data measured by the standard. Figure 7 shown.

[0178] As shown in the figure, the half-inhibitory concentration (IC50) of the monoclonal antibody HOPE1 prepared by the present invention on the time-resolved fluorescence immunochromatography of natural capsaicin, dihydrocapsaicin and synthetic capsaicin mixed standard 50) was 0.57 ng / mL, and the minimum detection content was 0.14 ng / mL. The time-resolved fluorescence immunochromatography based on HOPE1 has the advantages of high sensitivity and low detection limit.

[0179] When the sample to be tested is edible oil, the edible oil is pretreated by the following method:

[0180] Weigh 10g of edible vegetable oil sample into a 15mL centrifuge tube. Add 5mL of methanol, vortex for 2 minutes, then centrifuge at 5500rpm / min for 20 minutes. Collect the supernatant and repeat twice. Mix the upper organic phase, take 2mL of the sample, and purge with nitrogen until nearly dry. Then, add 1mL of 0.01M methanol-PBS solution to reconstitute the sample. Dissolve 50μL of the reconstituted solution in 950μL of 0.01M PBS to obtain the test solution, which is set aside.

[0181] Example 9 Kit for Detecting the Content of One or More of Natural Capsaicin, Dihydrocapsaicin, and Synthetic Capsaicin

[0182] A kit for detecting the content of one or more of natural capsaicin, dihydrocapsaicin, and synthetic capsaicin, comprising the monoclonal antibody HOPE1, the artificial complete antigen HaptenC-KLH, 0.01M PBS buffer, CB buffer, 0.01M PBST solution, 0.05M CB buffer containing 5% skim milk powder, Goat Anti-mouse IgG-HRP solution, TMB colorimetric solution, and 10% (v / v) H2SO4 solution.

[0183] The kit was used according to the ic-ELISA immunoassay method described in Example 7.

[0184] Example 10 Kit for Detecting the Content of One or More of Natural Capsaicin, Dihydrocapsaicin, and Synthetic Capsaicin

[0185] A kit for detecting the content of one or more of natural capsaicin, dihydrocapsaicin, and synthetic capsaicin comprises the monoclonal antibody HOPE1, a time-resolved fluorescent probe coupled to the monoclonal antibody HOPE1, a test strip coated with a monoclonal antibody HOPE1 solution on the T line of a chromatographic membrane, and a goat anti-mouse IgG solution on the C line of the chromatographic membrane.

[0186] The kit was used according to the time-resolved fluorescence immunochromatography method described in Example 8.

[0187] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A universal monoclonal antibody against natural capsaicin, dihydrocapsaicin and synthetic capsaicin, characterized in that: The heavy chain variable region sequence is shown in SEQ ID NO.1, and the light chain variable region sequence is shown in SEQ ID NO.

2.

2. Use of the monoclonal antibody according to claim 1 in detecting the content of one or more of natural capsaicin, dihydrocapsaicin, and synthetic capsaicin and / or in preparing a kit for detecting the content of one or more of natural capsaicin, dihydrocapsaicin, and synthetic capsaicin.

3. A kit for detecting the content of one or more of natural capsaicin, dihydrocapsaicin, and synthetic capsaicin, characterized in that: Comprising the monoclonal antibody according to claim 1.

4. The kit according to claim 3, wherein Also includes reagents required for ic-ELISA immunoassay.

5. The kit according to claim 3, wherein Also includes the reagents required for time-resolved fluorescence immunochromatography.

6. An ic-ELISA immunoassay method for detecting the content of one or more of natural capsaicin, dihydrocapsaicin, and synthetic capsaicin, characterized in that: The following steps are involved: S1. Add the sample extract to an ELISA plate coated with natural capsaicin, dihydrocapsaicin, or Hapten C-KLH, a universal artificial complete antigen for synthetic capsaicin, as shown in structural formula (I), and simultaneously add the monoclonal antibody described in claim 1. Incubate in a water bath at 35-40°C for 10-20 minutes, and then wash the plate. S2. Add secondary antibody and incubate in a 35-40°C water bath for 10-20 minutes. Wash the plate after the water bath. S3. Add color developing solution and incubate in a 35-40°C water bath for 10-20 minutes. Wash the plate after the water bath. S4. Add the stop solution, measure the absorbance of the solution at 420-480 nm, and substitute the absorbance value into the standard curve to calculate the content of one or more natural capsaicin, dihydrocapsaicin, and synthetic capsaicin in the sample; 。 7. A time-resolved fluorescence immunochromatographic method for detecting the content of one or more of natural capsaicin, dihydrocapsaicin, and synthetic capsaicin, characterized in that: The following steps are involved: S1. Add the time-resolved fluorescent probe coupled with the monoclonal antibody according to claim 1 to the sample solution and react for 1 to 5 minutes; S2. Add the test strip to the sample solution and run the test for 3-7 minutes; S3. Detect the fluorescence intensity of the test strip T line and C line under 320-400nm ultraviolet light, calculate the ratio of the fluorescence intensity of the T line to the C line, and calculate the content of one or more natural capsaicin, dihydrocapsaicin, and synthetic capsaicin in the sample based on the ratio of the fluorescence intensity of the T line to the C line; Among them, the T line in S3 is coated with the monoclonal antibody according to claim 1, and the C line is coated with the secondary antibody.

8. A nucleic acid molecule encoding the monoclonal antibody of claim 1.

9. A vector comprising the nucleic acid molecule according to claim 8.

10. A recombinant cell, characterized in that Contains the vector according to claim 9 or expresses the monoclonal antibody according to claim 1.

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