Gallic acid hapten, artificial antigen, monoclonal antibody and application

Abstract

The present invention relates to the technical field of biochemistry, and in particular to gallic acid hapten, artificial antigen, monoclonal antibody and applications. On the basis of retaining the basic structure of gallic acid, the hapten of the present invention introduces a linker arm and an active group for coupling with macromolecules, which is highly consistent with the structure of gallic acid and is conducive to the stable induction of highly specific antibodies. Further, the present invention couples the hapten with carrier protein by glutaraldehyde method to prepare artificial antigen. The anti-gallic acid antibody prepared by using the complete antigen of gallic acid has good specificity, high sensitivity, and no cross-reaction with structural analogs, and can be used to prepare reagents or kits for detecting gallic acid, establish an immunological detection method for gallic acid, and realize the accurate detection of gallic acid.

Description

Technical Field

[0001] The present invention relates to the technical field of biochemistry, and in particular to gallic acid hapten, artificial antigen, monoclonal antibody and application thereof. Background Art

[0002] Gallic acid (GA) is a polyphenolic compound with pharmacological effects such as antibacterial, anti-inflammatory, antiviral, antitumor, hypoglycemic, and neuroprotective effects. GA is the main phenolic acid in mangoes and exhibits good antioxidant activity. Studies have shown that there are significant differences in the GA content in mangoes at different maturity stages: the GA content is relatively high during the immature period, but significantly decreases in fully ripe mangoes. GA also widely exists in tea and wine. Research has shown that with the increase of years, the GA content in Scotch whisky also increases significantly. The gallic acid contents in 12-year, 17-year, and 30-year Scotch whiskies are 2.4, 7.0, and 37.6 mg / L respectively. Therefore, some scholars believe that gallic acid can be used as an index for evaluating the quality of Scotch whisky. In addition, some scholars have found that the GA content is positively correlated with the quality grade of green tea. For example, the gallic acid content in the 1-2 grades of Pingchaoqing is 0.1886%, while that in the 4-8 grades is 0.1475%. Therefore, gallic acid can be used as an index for evaluating the quality of green tea. At the same time, the "Chinese Pharmacopoeia" (2020 Edition) lists GA as an effective active ingredient in traditional Chinese medicines such as Chinese gall, bistort rhizome, sanguisorba root, and phyllanthus emblica fruit. Therefore, developing rapid and accurate detection methods for gallic acid is of great significance for the quality evaluation and quality control of agricultural products and foods.

[0003] Immunoassay is an analytical method based on the specific recognition and binding reaction of antigen and antibody, with the advantages of high sensitivity, simple operation, low requirements for instruments and low cost. Gallic acid belongs to a small molecule compound with a molecular weight less than 1000 and does not have reactivity and immunogenicity by itself. In order to achieve the immunoassay of gallic acid, it is not only necessary to synthesize a gallic acid antigen with a high titer, but also to screen a gallic acid monoclonal antibody with strong specificity and high sensitivity. However, there is currently no relevant report on gallic acid monoclonal antibody, and for technicians, the design of a gallic acid antigen with a high titer is still a difficult point. Summary of the Invention

[0004] To overcome the above-mentioned defects existing in the prior art, the present invention provides the following technical solutions:

[0005] In the first aspect, the present invention provides a gallic acid hapten, and its structural formula is shown in Formula I:

[0006]

[0007] Second aspect, the present invention provides a gallic acid artificial antigen, whose structural formula is shown in Formula II:

[0008]

[0009] Preferably, the protein is at least one of bovine serum albumin, ovalbumin or keyhole limpet hemocyanin.

[0010] The present invention uses gallic acid as a raw material, reacts with N-(3-bromopropyl) phenylenediamine to obtain an amino-functionalized gallic acid hapten. On the basis of retaining the basic structure of gallic acid, this hapten introduces a linker arm and a reactive group for coupling with macromolecules. This not only facilitates its coupling with macromolecules, but also fully exposes the basic structure of gallic acid itself with a small molecular structure and molecular weight (170) after coupling, avoiding its being masked by macromolecules and affecting the recognition of the animal body, thus efficiently inducing the production of antibodies.

[0011] Among them, the present invention finds that for the basic structure of gallic acid, it is necessary to strictly control the distance between the basic structure of gallic acid and the protein. If the distance between the two is too small, the protein macromolecule is very likely to mask the basic structure of gallic acid, affecting the recognition of the antigen by the animal body; if the distance between the two is too large, the chain will fold due to the hydrophobic effect of the linker arm, resulting in the basic structure of gallic acid still being covered by the protein macromolecule, which is not conducive to the recognition of the antigen by the animal body.

[0012] Furthermore, the artificial antigen is prepared by coupling the hapten with the protein through the glutaraldehyde method, and the coupling ratio can reach 15, which can be used for the immunoassay of gallic acid.

[0013] Third aspect, the present invention provides a preparation method of the gallic acid hapten, including:

[0014] Mix gallic acid with N-(3-bromopropyl) phenylenediamine and react to obtain the gallic acid hapten.

[0015] Preferably, the mass ratio of gallic acid to N-(3-bromopropyl) phenylenediamine is 1:0.5 - 2.

[0016] Preferably, the solvent for the reaction is DMF; and / or, the reaction temperature is 30 - 50 °C.

[0017] More preferably, the reaction time is 1 - 3 h.

[0018] Fourth aspect, the present invention provides a preparation method of the gallic acid artificial antigen, including:

[0019] Mix the gallic acid hapten with glutaraldehyde and react to obtain an intermediate reaction product; then mix the protein with the intermediate reaction product and perform a secondary reaction to obtain the gallic acid artificial antigen; preferably, the reaction and the secondary reaction are carried out in a phosphate buffer solution.

[0020] Preferably, the method for preparing the gallic acid artificial antigen includes:

[0021] (1) React the gallic acid hapten with glutaraldehyde in a phosphate buffer solution to obtain solution A;

[0022] (2) Mix the carrier protein with a phosphate buffer solution to obtain solution B;

[0023] (3) Mix solution A and solution B and react to obtain the gallic acid artificial antigen.

[0024] Preferably, the reaction temperature in step (1) is 2 - 30 °C, and more preferably, it is stirred and reacted for 2 - 28 h under light-shielded conditions.

[0025] Preferably, in step (3), drop solution A into solution B, and stir and react for 2 - 6 h at 2 - 10 °C under light-shielded conditions.

[0026] More preferably, in step (3), add solution A dropwise to solution B at a rate of 4 seconds per drop, and stir and react for 6 h at 4 °C under light-shielded conditions.

[0027] Preferably, the mass ratio of the gallic acid hapten to glutaraldehyde is 1:10 - 15; more preferably 1:13.

[0028] Preferably, the mass ratio of the carrier protein to the gallic acid hapten is 1:12 - 16.

[0029] Preferably, the carrier protein is bovine serum albumin (BSA).

[0030] Fifthly, the present invention provides a hybridoma cell line with a preservation number of CGMCC No. 45607.

[0031] The present invention immunizes a host animal with the above-mentioned gallic acid complete antigen, then isolates spleen cells from the host animal, fuses the spleen cells with SP2 / 0 tumor cells in vitro, and screens out a hybridoma cell line capable of secreting anti-gallic acid antibodies.

[0032] This hybridoma cell line was preserved in the China General Microbiological Culture Collection Center on May 16, 2023. Preservation unit address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing; Postcode: 100101, Classification name: Hybridoma cell line, Preservation number: CGMCC No. 45607.

[0033] The anti-gallic acid antibody (or serum against gallic acid) prepared based on this hybridoma cell line can specifically bind to gallic acid and has good immune effects.

[0034] There is no special limitation on the type of the above host animal, and any host animal well-known to those skilled in the art can be used.

[0035] The present invention has no special limitation on the specific method of separating the spleen cells, fusing the spleen cells with SP2 / 0 tumor cells, screening the hybridoma cell line capable of secreting anti-gallic acid antibody, and preparing the anti-gallic acid antibody based on the hybridoma cell line. Any method well-known to those skilled in the art can be adopted. The present invention preferably obtains different anti-gallic acid antibodies by using different host animals. Specifically, monoclonal antibodies can be obtained by using mice as host animals; polyclonal antibodies can be obtained by using rabbits or goats as host animals; and nanobodies can be obtained by using camels or alpacas as host animals. In the examples of the present invention, mice are specifically used as host animals, and finally monoclonal antibodies are obtained.

[0036] In the sixth aspect, the present invention provides an antibody secreted by the above hybridoma cell line; or, prepared by immunizing a host with the above gallic acid artificial antigen.

[0037] Preferably, the amino acid sequence of HCDR1 in the heavy chain variable region of the antibody is GYTFTSYW (SEQ ID NO.1), the amino acid sequence of HCDR2 is INPSNGRT (SEQ ID NO.2), and the amino acid sequence of HCDR3 is ARGGFDY (SEQ ID NO.3);

[0038] and / or, the amino acid sequence of LCDR1 in the light chain variable region of the antibody is ESVDNYGISF (SEQ ID NO.4), the amino acid sequence of LCDR2 is AAS, and the amino acid sequence of LCDR3 is QQSKEVPFT (SEQ ID NO.5).

[0039] In the seventh aspect, the present invention provides a reagent or kit for detecting gallic acid, which contains the above gallic acid hapten, or the above gallic acid artificial antigen, or the above hybridoma cell line, or the above antibody.

[0040] In the eighth aspect, the present invention further provides a method for detecting gallic acid, including: detecting gallic acid by using the above gallic acid hapten, or the above gallic acid artificial antigen, or the above hybridoma cell line, or the above antibody, or the above reagent or kit.

[0041] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0042] On the basis of retaining the basic structure of gallic acid, the hapten of the present invention introduces a linker arm and an active group for conjugating macromolecules, which is highly consistent with the structure of gallic acid, facilitating the stable induction of highly specific antibodies. Further, the present invention conjugates the hapten with carrier protein by glutaraldehyde method to prepare an artificial antigen. The anti-gallic acid antibody prepared by using the complete antigen of gallic acid has good specificity, high sensitivity, and no cross-reaction with structural analogs, and can be used to prepare reagents or kits for detecting gallic acid, establish an immunological detection method for gallic acid, and achieve accurate detection of gallic acid.

[0043] After testing, the IC 50 of the anti-gallic acid antibody of the present invention is 849.18 ng / mL, the lowest detection limit is 297.17 ng / mL, the linear range is 297.17 - 2426.61 ng / ml, and there is no cross-reaction with structural analogs of gallic acid, providing the core raw material for establishing an immunological analysis method for gallic acid and having broad application prospects. Description of the Drawings

[0044] Figure 1 It is the identification diagram of high-resolution mass spectrometry HRMS[M + H]+ of gallic acid hapten (GA-NH2).

[0045] Figure 2 It is the identification diagram of high-resolution mass spectrometry HRMS[M + H]- of gallic acid hapten (GA-NH2).

[0046] Figure 3 It is the full-spectrum scanning diagram of ultraviolet spectrophotometer of gallic acid artificial antigen (GA-NH2-BSA) and carrier protein (BSA).

[0047] Figure 4 It is the identification diagram of MALDI-TOF-MS of gallic acid artificial antigen (GA-NH2-BSA) and carrier protein (BSA).

[0048] Figure 5 It is the identification diagram of MALDI-TOF-MS of gallic acid artificial antigen (GA-NH2-OVA) and carrier protein (OVA).

[0049] Figure 6 It is the standard curve diagram of indirect competitive ELISA established based on monoclonal antibody of gallic acid. Detailed Embodiments

[0050] To make the objectives, technical solutions and advantages of the present invention clearer, the technical solutions in the present invention will be clearly and completely described below. Apparently, the described embodiments are some but not all of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without any creative effort fall within the scope of protection of the present invention.

[0051] For those not specified with specific technologies or conditions in the embodiments, they are all conventional methods or carried out according to the technologies or conditions described in the literature in this field, or according to the product specifications. For those reagents and instruments not specified with the manufacturer, they are all conventional products that can be obtained through regular channels.

[0052] Example 1: Preparation of gallic acid hapten

[0053] The synthesis route is shown as follows:

[0054]

[0055] Weigh 1.37 g of gallic acid, 1.115 g of N-(3-bromopropyl) phenylenediamine, and 1 g of potassium carbonate, dissolve them in 35 mL of DMF, heat in a water bath at 40 °C for 2 h, extract the reaction solution three times with 100 mL of ethyl acetate and 200 mL of water, then wash with saturated brine, dry with anhydrous magnesium sulfate, after rotary evaporation and drying, add 50 mL of water and 25 mL of concentrated sulfuric acid, hydrolyze under acidic conditions, stir at room temperature for 3 h, and recrystallize to precipitate a white powder solid, which is gallic acid hapten (GA-NH2).

[0056] The high-resolution mass spectrometry characterization of gallic acid hapten (GA-NH2) is as follows:

[0057] HRMS[M+H]+: The theoretical value is 226.07210, and the measured value is 226.07158( Figure 1 )

[0058] HRMS[M+H]−: The theoretical value is 228.08665, and the measured value is 228.08630( Figure 2 ).

[0059] The above high-resolution mass spectrometry detection results show that the preparation method provided by the present invention can effectively prepare gallic acid hapten (GA-NH2).

[0060] Example 2: Preparation of gallic acid artificial antigen

[0061] The synthesis route is shown as follows:

[0062]

[0063] This embodiment provides a method for preparing gallic acid artificial antigen, including the synthesis of immunogen and coating antigen. The difference between the preparation of immunogen and coating antigen lies in the type of carrier protein. The carrier protein used for the immunogen is bovine serum albumin (BSA); the carrier protein used for the coating antigen is ovalbumin (OVA). The method for synthesizing immunogen / coating antigen is the glutaraldehyde method. The specific steps are as follows:

[0064] Equilibrate the hapten (GA-NH2) shown in Formula I, glutaraldehyde, and PBS solution to room temperature; weigh 9.4 mg of GA-NH2 and dissolve it in 2 mL of PBS solution, add 50 μL of 50% glutaraldehyde, and react at room temperature for 4 h (Solution 1). Weigh 40 mg of BSA and dissolve it in 4 mL of PBS solution (Solution 2). Dropwise add Solution 1 to Solution 2. After dropping, control the ratio of carrier protein to hapten compound to be 1:15, place it in the dark at 4 °C and react for 24 h. Centrifuge the reaction solution, and place the supernatant in a dialysis bag with a molecular cut-off of 20,000 - 30,000, and dialyze it in 0.01 mol / L PBS buffer (pH = 7.4) at 4 °C for 72 h, changing the dialysis solution every 8 h. The solution obtained in the dialysis bag after dialysis is freeze-dried to obtain a white solid, which is the artificial antigen (GA-NH2-BSA) and is used as an immunogen; replace the carrier protein with ovalbumin OVA and couple to obtain the artificial antigen (GA-NH2-OVA), which is used as a coating antigen.

[0065] Example 3. Performance determination of gallic acid artificial antigen

[0066] (1) Full-spectrum scanning of gallic acid artificial antigen and BSA by ultraviolet spectrophotometer

[0067] Prepare a 1 mg / mL gallic acid artificial antigen (GA-NH2-BSA) solution and a 1 mg / mL carrier protein (BSA) solution, and perform ultraviolet scanning on the gallic acid artificial antigen (GA-NH2-BSA) solution and the carrier protein (BSA) solution in the range of 200 - 400 nm to obtain an ultraviolet absorption spectrum, and analyze and determine whether the gallic acid hapten (GA-NH2) and the carrier protein (BSA) are successfully coupled( Figure 3 ).

[0068] (2) Determination of coupling ratio

[0069] Use MALDI-TOF-MS to detect the coupling ratio of the carrier protein (BSA) and the gallic acid hapten (GA-NH2-BSA). Calculate the coupling ratio according to the change in the mass-to-charge ratio of the molecular ion peak before and after coupling.

[0070] Coupling ratio calculation formula: Coupling ratio = Mp - Mstd / Mh.

[0071] Where: Mp, Mstd, and Mh are the relative molecular masses of the gallic acid artificial antigen, carrier protein, and gallic acid hapten, respectively.

[0072] According to the above formula, and substituting Figure 4 the data, the coupling ratio was calculated to be 15.

[0073] Figure 4 are the MALDI-TOF-MS identification diagrams of the gallic acid artificial antigen (GA-NH2-BSA) and the carrier protein (BSA).

[0074] Figure 5 are the MALDI-TOF-MS identification diagrams of the gallic acid artificial antigen (GA-NH2-OVA) and the carrier protein (OVA).

[0075] Example 4. Preparation of Gallic Acid Monoclonal Antibody

[0076] 1. Take 6- to 8-week-old Balb / c mice as experimental animals

[0077] 2. Primary immunization: Dissolve the gallic acid complete antigen (GA-NH2-BSA) obtained in Example 2 in PBS to a solution with a concentration of 1 mg / mL. After filtering through a sterile filter, add an equal volume of Freund's complete adjuvant and stir well until emulsified, until it does not disperse when dropped into water, to obtain the gallic acid complete antigen emulsion. The immunization strategy of the emulsion is to inject 0.05 mg intraperitoneally into the mice and 0.05 mg by multiple subcutaneous injections in the back. The total injection dose is 0.1 mg of the gallic acid complete antigen emulsion per mouse.

[0078] 3. Booster immunization: Two weeks after the primary immunization, the gallic acid complete antigen (GA-NH2-BSA) obtained in Example 2 was dissolved in PBS to a solution with a concentration of 1 mg / mL. After filtration through a sterile filter, an equal volume of Freund's incomplete adjuvant was added and stirred thoroughly until emulsified, that is, it did not disperse when dropped into water, to obtain the gallic acid complete antigen emulsion. The immunization strategy for the emulsion was to inject 0.05 mg intraperitoneally into the mice and 0.05 mg by multiple subcutaneous injections on the back, with a total injection dose of 0.1 mg of the gallic acid complete antigen emulsion per mouse; the booster immunization was carried out every 14 days. Starting from the third booster immunization, three days after each immunization, blood was collected from the mouse orbital cavity to measure the antibody titer. The coating antigen was the gallic acid complete antigen (GA-NH2-OVA) prepared in Example 2, with a concentration of 1 mg / mL. The mouse eyeballs were removed for blood collection, and the mouse spleens were removed. The spleen cells were isolated for cell fusion; the culture medium was taken from the fused hybridoma cell line for detection. After screening out the positive monoclonal cell lines, they were preserved, cultured in large quantities, and the monoclonal cell line with the best titer and specificity was selected for the preparation of ascites, and the gallic acid monoclonal antibody was purified; the titer and specificity of the gallic acid monoclonal antibody were detected; the blood of the fused mice was left standing in a 37 °C constant temperature incubator for 30 min, then left standing in a 4 °C refrigerator for 2 h, and then centrifuged at 4 °C and 10,000 rpm / min for 10 min in a centrifuge to separate the gallic acid antiserum for the following experiments.

[0079] Evaluation of the effect of antibody serum: Using the gallic acid coating antigen prepared in Example 2, and taking the above-mentioned collected mouse serum as the detected antibody, the antiserum titer of the mouse serum was measured by the indirect ELISA method, and the antiserum was evaluated by comprehensively considering the titers of each antiserum. The specific operation steps are as follows:

[0080] I. The various buffer solutions used in the following experiments are as follows:

[0081] Coating buffer (PH = 9.6, 0.05 M carbonate buffer): Weigh 1.5 g of Na2CO3 and 2.94 g of NaHCO3, and make up to 1000 mL with ultrapure water;

[0082] Phosphate buffer: (0.01 M, PH = 7.4): Weigh 0.2 g of KH2PO4, 8 g of NaCl and 2.92 g of NaH2PO4·12H2O, and make up to 1000 mL with ultrapure water;

[0083] Washing buffer PBST: Add Tween-20 to the prepared phosphate buffer to make the volume fraction of Tween-20 0.1%;

[0084] Sample diluent: Add 10 mL of Tween-20 and 1 g of gelatin to the prepared phosphate buffer solution, melt it by microwave heating, and make up the volume to 1 L with ultrapure water.

[0085] Substrate buffer solution (pH = 5.5): Weigh 9.22 g of NaH2PO4·12H2O, 2.55 g of citric acid monohydrate, measure 0.5 mL of Tween-20, and make up the volume to 1 L with ultrapure water.

[0086] Stop solution: (2M H2SO4): Measure 445.6 mL of distilled water, and gradually add 54.4 mL of 98% (volume / volume) concentrated sulfuric acid dropwise under stirring.

[0087] II. Inhibition test of antibodies in gallic acid antiserum

[0088] 1. Gradiently dilute the gallic acid complete antigen (GA-NH2-OVA) prepared in Example 2 above with the coating buffer at a ratio of 4000.

[0089] 2. Preparation of gallic acid antiserum diluent

[0090] Gradually dilute the gallic acid antiserum prepared in the above steps with the sample diluent to obtain gallic acid antiserum diluents with different dilution factors.

[0091] 3. Checkerboard experiment of antigen and antiserum

[0092] (1) Coating: Add 100 μL of the gallic acid complete antigen (GA-NH2-OVA) coating antigen solution prepared in step 1 to each well of a 96-well enzyme-linked immunosorbent assay (ELISA) plate, incubate at 37 °C for 3 h, wash three times with the washing buffer, and drain.

[0093] (2) Blocking: Add 100 μL of 10 mg / mL gelatin solution to each well, incubate at 37 °C for 30 min, wash the plate 3 times with the washing buffer, and drain. Add the gallic acid antiserum diluent prepared in step 2 to the ELISA plate (50 μL / well), incubate at 37 °C for 30 min, wash the plate 3 times with the washing buffer, and drain.

[0094] (3) Add enzyme-labeled secondary antibody: Dilute the goat anti-mouse enzyme-labeled secondary antibody (IgG-HRP, Jackson) 10,000 times with a diluent of 0.1 M sample diluent with pH = 9.6. Add 100 μL to each well, incubate at 37 °C for 30 min, wash the plate 3 times with the washing buffer, and drain.

[0095] (4) Color development: The color development solution is prepared immediately before use. For each plate, add 200 μL of TMB stock solution and 11 mL of buffer. Add 101 μL of 1% hydrogen peroxide before use. Add the color development solution to the ELISA plate for color development reaction, 100 μL per well, and develop color for 15 min.

[0096] (5) Termination: After the color development reaction is completed, add 2M hydrochloric acid solution to terminate the color development, 50 μL per well, and use an ELISA reader to detect the OD value at 450 nm.

[0097] (6) Record the reading (when the ratio of P (positive serum A450) to N (negative serum A450) is greater than 2.1, the maximum dilution factor of the positive serum is the titer of the antibody)

[0098] The experimental results are as follows:

[0099] Table 1: Detection of serum titer of gallic acid in mice (P / N)

[0100]

[0101] The results in Table 1 show that after the fifth immunization, the titer of mouse No. MSZ2-2 reached over 9000. There is literature indicating that a titer of 8000 can be used for fusion, indicating that the complete antigen of gallic acid (GA-NH2-BSA) prepared in Example 2 above can be used as an immunogen to prepare gallic acid antibody.

[0102] Table 2: Detection results of monoclonal antibody titer of anti-gallic acid mice

[0103]

[0104] The results in Table 2 show that the antibody titer is higher than 1:40500, indicating that the complete antigen of gallic acid (GA-NH2-BSA) prepared in Example 2 above can be used as an immunogen to prepare gallic acid monoclonal antibody.

[0105] III. Establishment of the standard curve of gallic acid monoclonal antibody

[0106] Dilute the gallic acid standard product with the sample diluent to the following different concentrations: 4000 ng / mL, 2000 ng / mL, 1000 ng / mL, 500 ng / mL, 250 ng / mL, 200 ng / mL, 150 ng / mL, 100 ng / mL.

[0107] (1) Coating of antigen: Dilute the complete antigen of gallic acid (GA-NH2-OVA) prepared in Example 2 with the sample diluent at a ratio of 1:2000, add 100 μL per well to the ELISA plate, place it in a wet box and incubate at 37 °C in a constant temperature incubator for 3 h; pour out the solution in the ELISA plate, wash the plate 3 times with the washing buffer, and spin dry;

[0108] (2) Add 50 μL of the above gallic acid standard solutions with different concentrations (inhibitory wells) to the enzyme-linked immunosorbent assay (ELISA) plates in step (1), and add 50 μL of sample diluent to the control wells without adding gallic acid standard solution;

[0109] (3) Add 50 μL of gallic acid antibody diluent with a dilution factor of 1500 to the above experimental wells and control wells respectively, incubate at 37 °C for 30 min, pour out the solution in the ELISA plates, wash the plates 3 times with washing buffer, and shake dry;

[0110] (4) Add 100 μL of IgG-HRP secondary antibody with a dilution factor of 1000 to the experimental wells and control wells respectively, incubate at 37 °C for 30 min, pour out the solution in the ELISA plates, wash the plates 3 times with washing buffer, and shake dry;

[0111] (5) Add 100 μL of the prepared substrate buffer to the experimental wells and control wells respectively. After reacting at room temperature for 15 min, add 50 μL of 1 M hydrochloric acid to each well to terminate the reaction;

[0112] (6) Measure the absorbance at 450 nm;

[0113] (7) Plot the standard curve: Use the logarithm of the different concentrations (ng / mL) of the gallic acid standard solution as the X-axis, and use the ratio of absorbance values (B / B0×100%, where B is the average absorbance value of the gallic acid standard, and B0 is the average absorbance value of the control well) as the Y-axis to plot the standard curve. The experiment is set up with 3 replicates, and the average value of the 3 experimental results is taken to obtain the standard curve as Figure 6 shown.

[0114] The results show that its sensitivity (expressed by the median inhibitory concentration IC 50 , the standard sample concentration value at which the inhibition rate reaches 50%) is 849.18 ng / mL, and the detection range (IC 20 ~IC 80 ) is 297.17 - 2426.61 ng / mL. It shows that the antibody obtained by immunizing mice with the complete antigen of gallic acid (GA-NH2-BSA) prepared in Example 2 above has a good detection effect and a low detection limit.

[0115] IV. Antibody Specificity Detection

[0116] According to the preparation of gallic acid analog standard samples, referring to the preparation method of gallic acid standard samples, dilute them to the following concentrations: 4000 ng / mL, 2000 ng / mL, 1000 ng / mL, 500 ng / mL, 250 ng / mL, 200 ng / mL, 150 ng / mL, 100 ng / mL.

[0117] Cross - reaction rate (%) = (Gallic acid IC 50 / Analogue IC 50 ) × 100%

[0118] Table 3 Specificity of Monoclonal Antibody

[0119]

[0120] The experimental results are shown in Table 3. The cross - reaction rate of gallic acid structural analogues and gallic acid monoclonal antibody is < 0.09, indicating that the gallic acid monoclonal antibody of the present invention has high specificity.

[0121] In summary, the gallic acid hapten provided by the present invention has good solubility and stability, and can meet the requirements of carrier protein conjugation; using the gallic acid hapten conjugated with carrier protein, the gallic acid complete antigen can be obtained. After immunizing host animals with the gallic acid complete antigen, it can stimulate the body to produce anti - gallic acid antibodies with high specificity and sensitivity. The IC 50 of the anti - gallic acid antibody is 849.18 ng / mL, the linear range is 297.17 - 2426.61 ng / mL, and there is no cross - reaction with gallic acid structural analogues.

[0122] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements for some of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A gallic acid hapten, characterized in that, Its structural formula is as shown in Formula I:

2. An artificial antigen of gallic acid, characterized in that, Its structural formula is as shown in Formula II:

3. The gallic acid artificial antigen according to claim 2, wherein, The protein is at least one of bovine serum albumin, ovalbumin or keyhole limpet hemocyanin.

4. The preparation method of the gallic acid hapten according to claim 1, characterized in that, Including: The gallic acid hapten is prepared by mixing gallic acid with N-(3-bromopropyl) phenylenediamine and then reacting.

5. The preparation method according to claim 4, characterized in that, The solvent for the reaction is DMF; and / or, the temperature of the reaction is 30-50 °C.

6. The preparation method of the gallic acid artificial antigen according to claim 2, characterized in that, Including: The gallic acid hapten described in claim 1 is mixed with glutaraldehyde and then reacted to obtain an intermediate reaction product; then the protein is mixed with the intermediate reaction product and subjected to a secondary reaction to obtain the gallic acid artificial antigen.

7. The preparation method according to claim 6, characterized in that, The reaction and the secondary reaction are carried out in phosphate buffer.

8. A hybridoma cell line, characterized in that, Its preservation number is CGMCC No. 45607.

9. An antibody, characterized in that, It is secreted and produced by the hybridoma cell line described in claim 8.

10. The antibody according to claim 9, wherein, The amino acid sequence of HCDR1 in the heavy chain variable region of the antibody is as shown in SEQ ID NO.1, the amino acid sequence of HCDR2 is as shown in SEQ ID NO.2, and the amino acid sequence of HCDR3 is as shown in SEQ ID NO.3; And / or, the amino acid sequence of LCDR1 in the light chain variable region of the antibody is as shown in SEQ ID NO.4, the amino acid sequence of LCDR2 is AAS, and the amino acid sequence of LCDR3 is as shown in SEQ ID NO.

5.

11. A reagent or kit for detecting gallic acid, characterized in that, It contains the antibody described in claim 9 or 10.

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