Melamine hapten, artificial antigen and antibody as well as preparation method and application thereof

Abstract

The invention discloses a melamine hapten, an artificial antigen and an antibody as well as a preparation method and application thereof, and relates to the technical field of immunochemistry and food safety detection. The hapten is prepared from 2-chloro-4, 6-diamino-1, 3, 5-triazine and 8-amino caprylic acid through a reaction, the artificial antigen is prepared from the hapten, N, N '-dicyclohexylcarbodiimide (DCC) and N-hydroxysuccinimide (NHS), and the antibody is prepared from an animal immunized by the artificial antigen. The antigen and the antibody can be applied to preparation of a melamine colloidal gold immunochromatography test strip, and can be used for rapidly and sensitively detecting melamine residues in animal-derived food.

Description

Technical Field

[0001] The present invention relates to the technical field of immunochemistry and food safety detection, and in particular to a melamine hapten, an antigen and an antibody, and a preparation method and application thereof. Background Art

[0002] Melamine, English name (Melamine), has a molecular formula of C3H6N6. Melamine is a widely used organic chemical intermediate, and its main use is in the preparation of melamine formaldehyde resin. Studies have shown that excessive exposure to melamine in the diet can cause kidney stones and also increase the risk of nephritis. After the "Sanlu Milk Powder Incident", countries such as the European Union, Australia, New Zealand, and Canada have taken corresponding control measures on melamine in food. However, melamine is a white crystalline powder with no special smell, and it is not easy to detect melamine mixed in food. For this reason, melamine is often used as a food additive by unscrupulous businessmen to increase the protein content index in food testing. Therefore, it is necessary to develop a sensitive, accurate and rapid detection method.

[0003] At present, the main methods for detecting melamine include liquid chromatography (HPLC), gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS), etc. However, these methods often require expensive instruments and professional technicians, and have strict requirements for sample purification and analysis operations, which can no longer meet the requirements of modern detection for convenience, speed and accuracy. The immunoassay technology has a simple operation method and high sensitivity and accuracy, which just makes up for the shortcomings of the above detection methods. The key to immunoassay technology is to obtain high-quality antibodies, and the synthesis of antigens is particularly important.

[0004] Melamine is a small molecule with a simple structure. If it is directly coupled to a protein, the characteristic structure of the molecule itself will be obscured. In order to prevent the characteristic groups of the hapten from being covered by the protein, a linker with a certain number of carbon atoms is introduced to meet the immune requirements. There have been studies in China on the introduction of 3-6 carbon atoms in the linker between melamine and carrier proteins, but there have been no studies reported on the introduction of more carbon linkers for body immunity. Patent CN 110938040 A discloses a melamine hapten and a method for preparing it. The method uses 3,4-diaminobutyric acid and 2-chloro-4,6-diamino-1,3,5-triazine to prepare the melamine hapten, but the raw material is expensive and the preparation cost is high. Patent CN 109633159 A discloses a method for detecting melamine content and a special colloidal gold test paper thereof. The test strip is composed of a water-absorbing pad, a nitrocellulose membrane, a quenching antibody binding pad, a sample pad and a fluorescent backplane. During the assembly process, the two ends of the quenching antibody binding pad need to be respectively pasted on the sample pad and the nitrocellulose membrane, which complicates the steps and increases the cost. Summary of the invention

[0005] The purpose of the present invention is to provide a melamine hapten, an artificial antigen and an antibody and their application to solve the shortcomings of the existing melamine detection technology in the above-mentioned background technology.

[0006] In order to achieve the above object, the present invention provides the following technical solutions:

[0007] In a first aspect, the present invention provides a melamine hapten having a structure shown in Formula I:

[0008]

[0009] 8-aminocaprylic acid is used as the spacer arm between melamine and protein, which enriches the characteristic structure of the hapten shown in formula I, thereby achieving the purpose of strengthening the antigenic determinant cluster.

[0010] In a second aspect, the method for preparing the above hapten of the present invention uses 2-chloro-4,6-diamino-1,3,5-triazine and 8-aminooctanoic acid as raw materials, reacts at a reflux temperature of (100-110)°C in an alkaline environment to obtain the hapten shown in formula I.

[0011] The synthesis route of the hapten is shown in Formula II:

[0012]

[0013] In a third aspect, the present invention provides a melamine artificial antigen having a structure shown in Formula III:

[0014]

[0015] In a fourth aspect, the method for preparing the above-mentioned artificial antigen of the present invention uses the hapten shown in formula I, N,N'-dicyclohexylcarbodiimide (DCC) and N-hydroxysuccinimide (NHS) as raw materials, reacts at room temperature, adds phosphate buffer (PBS) dissolved with bovine serum albumin (BSA), and reacts at 20°C to obtain the artificial antigen shown in formula III.

[0016] The synthetic route of the artificial antigen is shown in Formula IV:

[0017]

[0018] DCC and NHS act as coupling agents to promote the coupling reaction between melamine derivatives and bovine serum albumin (BSA).

[0019] BSA is used as a carrier protein to couple with melamine hapten to generate an immunogenic complex.

[0020] Fifthly, the artificial antigen prepared by the method of the present invention can effectively stimulate the body to produce an immune response after immunizing animals, has strong specificity, and the obtained antiserum has a high titer, and produces specific antibodies with high affinity for melamine, and the above antibodies are monoclonal antibodies.

[0021] The above-mentioned monoclonal antibody preparation method comprises the following steps:

[0022] (1) Immunization of mice: Injecting antigens into mice activates their immune system and causes them to produce antibodies. Multiple immunizations are required to enhance the immune response.

[0023] (2) Obtaining B lymphocytes: Obtain B lymphocytes from the spleen of mice. These cells include unstimulated B lymphocytes, plasma cells produced after stimulation with specific antigens, and plasma cells formed after infection with other pathogens.

[0024] (3) Cultivation of SP2 / 0 myeloma cells: Shake the cryotube containing the SP2 / 0 myeloma cell suspension rapidly in a 37°C water bath to thaw, add it to a centrifuge tube containing complete culture medium and mix well. Discard the supernatant by centrifugation, resuspend the cells in complete culture medium, add the cell suspension to a culture flask containing complete culture medium and culture at 37°C, observe the cell growth and cell density, and perform subculture.

[0025] (4) Cell fusion: The obtained B lymphocytes are fused with myeloma cells. Myeloma cells have the ability to proliferate indefinitely, while B lymphocytes have the ability to secrete specific antibodies. Through cell fusion, hybridoma cells are formed.

[0026] (5) Screening of hybridoma cells: Use selective culture medium for the first screening to eliminate unfused cells and self-fused cells and select hybridoma cells.

[0027] (6) Clonal culture and antibody collection: A second screening is performed through clonal culture and antibody detection to select hybridoma cells that can produce specific antibodies. The culture medium is collected and centrifuged to obtain a supernatant with a high antibody content.

[0028] (7) Identification of monoclonal antibodies.

[0029] In a sixth aspect, the present invention provides the use of the above-mentioned melamine monoclonal antibody in the preparation of a colloidal gold test strip for detecting melamine using competitive immunochromatography technology.

[0030] The beneficial effects of the present invention are:

[0031] The present invention provides a melamine hapten, an artificial antigen and an antibody and their application. Melamine is used as a synthetic starting material and synthesized with 8-aminooctanoic acid. The obtained hapten retains the complete structure of melamine and introduces an active group carboxyl, so as to achieve the purpose of strengthening the antigenic determinant. The artificial antigen prepared by the reaction of the hapten with N, N'-dicyclohexylcarbodiimide and N-hydroxysuccinimide has good antigenic immunity and can effectively stimulate the body to produce an immune response. The obtained antiserum has a high titer and produces a monoclonal antibody with high specificity and affinity for melamine. The synthesis method provided by the present invention has simple steps, is easy to operate, has low cost, and has a wide range of applications. The antigen and antibody of the present invention are applied to competitive immunochromatographic technology to detect products, so as to quickly and sensitively detect melamine residues in animal-derived foods, optimize the detection steps, and save detection costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 The chemical formula molecular diagram of melamine hapten;

[0033] Figure 2 The synthetic route diagram of melamine hapten;

[0034] Figure 3 This is the chemical formula molecular diagram of melamine artificial antigen;

[0035] Figure 4 This is the synthetic route diagram of melamine artificial antigen;

[0036] Figure 5 This is the result diagram of serum titer of immunized mice;

[0037] Figure 6 This is the SDS-PAGE image of melamine monoclonal antibody;

[0038] Figure 7 It is a schematic diagram of the side cross-sectional structure of a melamine colloidal gold test strip. DETAILED DESCRIPTION

[0039] To explain the purpose, technical solution and effect of the embodiments of the present invention in detail, Figure 1-Figure 7 Tables 1 to 4 describe the technical solutions of the embodiments of the present disclosure.

[0040] Example 1: Melamine hapten and preparation method thereof

[0041] The molecular structure of melamine hapten is as follows:

[0042]

[0043] The synthesis of melamine hapten uses 2-chloro-4,6-diamino-1,3,5-triazine and 8-aminooctanoic acid as starting materials. The synthetic route is as follows Figure 2 shown.

[0044] The method for preparing melamine hapten comprises the following steps:

[0045] (1) Take 2-chloro-4,6-diamino-1,3,5-triazine (3 g, 20.61 mmol) and 8-aminooctanoic acid (3.1 g, 19.47 mmol), add 200 ml of deionized water, heat and stir to dissolve, and reflux for 3 h;

[0046] (2) Add NaOH solution (20 ml, 2 mol / L) and continue to reflux for 5 h;

[0047] (3) After cooling to room temperature, HCl solution (1 mol / L) was added to adjust the pH to 6.0, and then cooled in an ice-water bath, and a large amount of white solid precipitated;

[0048] (4) The precipitate was filtered, washed with triple distilled water, and filtered three times, and dried to obtain a white powder (1.8 g), i.e., melamine hapten.

[0049] Example 2: Melamine artificial antigen and its preparation method

[0050] The molecular structure of the melamine artificial antigen is as follows:

[0051]

[0052] The synthetic route of melamine artificial antigen is as follows Figure 4 shown.

[0053] The method for preparing melamine artificial antigen comprises the following steps:

[0054] (1) dissolving melamine hapten in N,N-dimethylformamide (DMF);

[0055] (2) adding N,N'-dicyclohexylcarbodiimide (DCC) and N-hydroxysuccinimide (NHS) respectively, and the molar ratio of melamine hapten, DCC and NHS is 1:1:1;

[0056] (3) Stirring at room temperature for 18 h, centrifuging to remove the precipitate, and obtaining Solution No. 1;

[0057] (4) adding phosphate-buffered saline (PBS) buffer containing bovine serum albumin (BSA) to solution No. 1, wherein the ratio of the amount of melamine derivative to the amount of BSA is 10:1;

[0058] (5) Stirring at 20°C for 12 h and dialyzing for 3 days to obtain the melamine artificial antigen.

[0059] Example 3: Preparation of monoclonal antibodies against melamine

[0060] (1) Immunization of mice. Four female Balb / c mice aged 6-8 weeks were selected as experimental subjects. Each mouse was immunized with an antigen, and the mice were immunized once every two weeks by subcutaneous multi-point injection, for a total of three immunizations. Except for the first immunization in which the melamine artificial antigen was emulsified with equal volumes of Freund's complete adjuvant, the second and third immunizations used Freund's incomplete adjuvant for antigen emulsification, and the dose and method of each immunization remained unchanged. One week after the three immunizations, blood was collected from the tail vein of the mice, and the serum was taken to determine its titer by the indirect ELISA method. The titer results of the serum of mice after immunization are shown in Figure 5 The results showed that the serum titer could reach up to 1:1.024×10 6 Then, the melamine artificial antigen was diluted to 200uL with 1×PBS and injected intraperitoneally into the mice for shock immunization. Three days after shock immunization, the cell fusion experiment can be carried out.

[0061] (2) Obtaining B lymphocytes. Place the mouse spleen in a 15 mL sterile centrifuge tube containing 10 mL DMEM medium. After the spleen is moistened, carefully discard the excess medium. Pipette 10 mL DMEM medium and place it in a sterile plate. Grind the spleen with a frosted glass slide to make a single cell suspension and filter it through a 200-mesh nylon mesh into a sterile centrifuge tube. Add 30 mL DMEM to a 50 mL sterile centrifuge tube. Rinse the nylon mesh with a pipette and centrifuge the centrifuge tube containing the spleen cell suspension at 1500 rpm for 5 min. Discard the supernatant, gently break up the cell clumps by hand, add 30 mL DMEM medium to resuspend, and centrifuge again. Discard the supernatant, gently break up the cell clumps by hand, and add 10 mL DMEM medium to resuspend.

[0062] (3) Cultivate SP2 / 0 myeloma cells. Shake the SP2 / 0 myeloma cell cryopreservation tube rapidly in a 37°C constant temperature water bath to thaw. Add the cell culture tube to a 50 mL sterile centrifuge tube containing 10 mL complete culture medium under sterile conditions. Centrifuge at 1000 rpm for 5 min. Discard the supernatant and resuspend the cells in complete culture medium. Take 5 mL of culture medium to resuspend the cells and add it to a cell culture flask containing 5 mL of culture medium. Place the tube in a CO flask. 2 The cells were cultured in a cell culture incubator at 37°C. The cell growth and cell status were observed under a microscope. When the density was about 80%, the SP2 / 0 myeloma cells were subcultured.

[0063] (4) Cell fusion. Blood was collected from the orbits of the experimental mice after shock immunization and placed in EP tubes. After standing at 37°C for 2 hours, the blood was centrifuged at 4000 rpm for 10 minutes. The serum was collected and used as a positive control for subsequent screening of monoclonal antibodies.

[0064] Centrifuge at 1000rpm for 5min, collect well-growing SP2 / 0 myeloma cells, add 30mL DMEM medium to resuspend, centrifuge twice, add 10mL DMEM medium to resuspend, mix B lymphocyte suspension with SP2 / 0 myeloma cell suspension, centrifuge at 1000rpm for 5min, discard the supernatant, and gently break up the cell mass. Place in a 37℃ water bath environment, and within 1min, absorb 1mL PEG fusion agent and drop it into the centrifuge tube. At this time, the cell state is red homogenous quicksand.

[0065] Add 1 mL of preheated DMEM medium within 1 min, add 1 mL of preheated DMEM medium again within 1 min, and then add 7 mL of preheated DMEM medium within 3 min. Let it stand in a 37°C water bath for 5 min, and centrifuge at 800 rpm for 5 min to terminate cell fusion.

[0066] The supernatant was discarded, HAT medium was added, and after mixing the cells, the fused cell suspension was evenly spread in a 96-well cell plate to which feeder cells had been added, and the plate was placed in a CO2 cell culture incubator and cultured at 37°C.

[0067] (5) Screening hybridoma cells. Seven days after cell fusion, when the cell clusters are relatively large, the cell supernatant is tested by indirect ELISA. 1 μg / mL melamine-BSA is used as the antigen for detection, the positive control is the serum of the fused mice, and the negative control is the serum of the PBS-immunized mice. The inhibition rate of the positive wells against the melamine standard is retested.

[0068] Table 1 Hybridoma cell screening (cell supernatant within 24 hours)

[0069]

[0070] Table 2 Limiting dilution test results (cell supernatant within 24 hours)

[0071]

[0072] (6) Cloning culture and antibody collection. The positive hybridoma cells screened were subcloned by limiting dilution method. The hybridoma cell lines that can stably secrete monoclonal antibodies were transferred to cell bottles for expansion culture. When the cell number was about 80%, 10mL of sterile 1×PBS was added, the cell layer was blown off, and the cells were resuspended and transferred to a 15mL centrifuge tube. Centrifuge at 1000r / min for 10min, the supernatant was discarded, and the precipitate was resuspended and mixed with 1mL of sterile 1×PBS. Each mouse was injected with 500μL of cell suspension, and the growth status of the mice was observed. After one week, the ascites was collected in a centrifuge tube when the abdomen of the mouse swelled. Centrifuge at 8000r / min for 20min, the middle ascites layer was aspirated, and crude purification was performed by the caprylic acid-ammonium sulfate method. The crude purified monoclonal antibody was secondary purified using a Protein G pre-loaded column to obtain melamine monoclonal antibody.

[0073] (7) Identification of monoclonal antibodies. Melamine monoclonal antibodies were identified based on concentration, purity, sensitivity, and specificity.

[0074] The concentration of the monoclonal antibody was measured using a micro-spectrophotometer, and the result showed that the concentration of the monoclonal antibody was 4.17 mg / mL.

[0075] The monoclonal antibody was analyzed by SDS-PAGE electrophoresis. Figure 6 As shown, two clear bands appeared near 25 kDa and 50 kDa, 25 kDa was the antibody light chain, 50 kDa was the antibody heavy chain, there were no other mixed bands, and the purity reached 95%.

[0076] The initial concentration of monoclonal antibodies was 1 mg / mL and diluted to 10 2 μg / mL, 10μg / mL, 1ng / mL, 10 2 ng / mL, 25ng / mL, and 6.25ng / mL, and the sensitivity of the monoclonal antibody was determined by indirect ELISA. The results are shown in Table 3. When diluted to 25ng / mL, it was still positive, and the sensitivity was high.

[0077] Table 3 Monoclonal antibody sensitivity identification

[0078]

[0079] The monoclonal antibody was specifically identified by reacting with melamine artificial antigen, tetracycline artificial antigen, gentamicin artificial antigen and chloramphenicol artificial antigen respectively by indirect ELISA method. The results are shown in Table 4. The monoclonal antibody can react specifically with melamine artificial antigen, but not with the other three artificial antigens, which proves that the purified monoclonal antibody has good specificity.

[0080] Table 4 Monoclonal antibody specificity identification

[0081]

[0082] Example 4: Preparation and application of melamine colloidal gold test strips

[0083] 1. Preparation of melamine colloidal gold test strips

[0084] (1) Preparation of microporous reagent. Add chloroauric acid (8 mL, 1%) to ultrapure water, stir rapidly after the water boils, add reducing agent trisodium citrate (1 mL, 1.2%) to react and prepare colloidal gold. Add the melamine monoclonal antibody prepared in Example 3 to the colloidal gold to obtain a microporous reagent of melamine monoclonal antibody-colloidal gold marker.

[0085] (2) Preparation of sample absorption pad: The sample absorption pad was soaked in 0.5% BSA, pH=7.2, 0.1 mol / L phosphate buffer for 2 h, and dried at 37°C for 2 h to obtain the sample absorption pad.

[0086] (3) Preparation of chromatographic membrane. Melamine artificial antigen was coated on the test line (T line) on the chromatographic membrane using a film spotter, and goat anti-mouse IgG antibody was coated on the quality control line (C line) on the chromatographic membrane using a film spotter. After coating, the chromatographic membrane was dried at 37°C for 2 hours to obtain a chromatographic membrane.

[0087] (4) Assembly of test strips. Figure 7 As shown, the sample absorption pad (No. 1), the chromatography membrane (No. 2), and the water absorption pad (No. 3) are pasted on the PVC backing plate (No. 4) in sequence. The beginning of the sample absorption pad is aligned with the beginning of the PVC backing plate, the end of the sample absorption pad is connected to the beginning of the chromatography membrane, the end of the chromatography membrane is connected to the beginning of the water absorption pad, and the end of the water absorption pad is aligned with the end of the PVC backing plate.

[0088] 2. Application of melamine colloidal gold test strips

[0089] (1) Allow the sample to be tested and the test strip to return to room temperature (20-25°C).

[0090] (2) Use a micropipette to draw 200 μL of the sample solution into the microwell reagent, slowly aspirate and mix thoroughly with the microwell reagent.

[0091] (3) After the microporous reagent is incubated at 40°C for 3 min, the test strip is inserted into the microporous reagent so that the test strip is fully immersed in the microporous reagent.

[0092] (4) After incubation at 40°C for 5 min, remove the test strip and determine the result. Determinations made at other times are invalid.

[0093] (5) Result determination:

[0094] Negative: Both the C line and the T line are colored and the color of the T line is stronger than that of the C line, indicating that the melamine concentration in the sample is lower than the detection limit.

[0095] Positive: The C line develops color, the color of the C line is the same as that of the T line, the color of the T line is weaker than that of the C line, or the T line does not develop color, indicating that the melamine concentration in the sample is equal to or higher than the detection limit.

[0096] Invalid: No C line appears, indicating incorrect operation process or the test strip has deteriorated and become invalid.

[0097] The above describes in detail some exemplary embodiments of the present invention by way of illustration. For those skilled in the art, some modifications and improvements may be made to the described embodiments in other different ways without departing from the spirit and scope of the present invention. Therefore, the above drawings and descriptions are illustrative in nature and should not be construed as limiting the scope of protection of the claims of the present invention.

Claims

1. A melamine hapten, characterized in that: The hapten has the structure shown in Formula I:

2. The method for preparing a hapten according to claim 1, characterized in that: The synthesis route of the hapten is shown in Formula II: 2-Chloro-4,6-diamino-1,3,5-triazine and 8-aminooctanoic acid are used as raw materials, and reacted at a reflux temperature of (100-110)°C in an alkaline environment to obtain a hapten shown in formula I.

3. A melamine artificial antigen, characterized in that: The artificial antigen has a structure shown in Formula III:

4. The method for preparing an artificial antigen according to claim 2, characterized in that: The synthetic route of the artificial antigen is shown in Formula IV: The hapten shown in formula I, N,N'-dicyclohexylcarbodiimide (DCC) and N-hydroxysuccinimide (NHS) are used as raw materials, reacted at room temperature, and a phosphate buffer solution (PBS) containing bovine serum albumin (BSA) is added and reacted at 20°C to obtain an artificial antigen shown in formula III.

5. A melamine monoclonal antibody, characterized in that: The method is prepared by immunizing an animal with the artificial antigen according to claim 3 or 4.

6. Use of the artificial antigen according to claim 3 or 4 and the monoclonal antibody according to claim 5 in the preparation of colloidal gold immunochromatographic test strips.

7. A method for detecting melamine residues in animal-derived food, characterized in that: Detection is performed using the test strip described in claim 6.

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