Metronidazole hapten, complete antigen and their synthesis and application

Abstract

The present invention discloses a metronidazole hapten, a complete antigen, and the synthesis and application thereof. The structure of the metronidazole hapten is shown in Formula I. The metronidazole complete antigen provided by the present invention can fully expose the antigenic determinant of metronidazole and maintain the spatial conformation of metronidazole, and has good immunogenicity. The metronidazole colloidal gold detection product provided by the present invention has the characteristics of high sensitivity, good specificity, and strong stability, and can realize the rapid detection of metronidazole in a sample. Formula I

Description

Technical Field

[0001] The invention belongs to the technical field of immunology, and specifically relates to a metronidazole hapten, a complete antigen and the synthesis and application thereof. Background Art

[0002] As a nitroimidazole drug, metronidazole has a strong anti-anaerobic effect and is widely used in livestock and poultry breeding to prevent and treat coccidiosis, histomoniasis and hexapodosis. Metronidazole residues in animal-derived foods can cause toxic and side effects in the digestive system, nervous system, cardiovascular system, and urinary tract, leading to skin allergies and anaphylactic shock; animal test results show that metronidazole is potentially carcinogenic. Therefore, the detection of metronidazole residues is imminent.

[0003] At present, the main detection methods for metronidazole are chemiluminescent immunoassay (CLIA), enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA) and colloidal gold immunochromatographic assay (GICT). CLIA and ELISA methods have lower detection limits, but the instrument detection operation is complicated and expensive, and cannot meet the requirements of rapid on-site detection. The RIA method is prone to radioactive contamination. The colloidal gold immunochromatographic method has the advantages of low cost, high efficiency, high sensitivity, and relatively low requirements for technicians, and is suitable for rapid detection of a large number of samples. Therefore, the colloidal gold immunochromatographic method can achieve efficient and rapid detection of metronidazole. Summary of the invention

[0004] The purpose of the present invention is to provide a metronidazole hapten, a complete antigen and their synthesis and application, so as to realize accurate and rapid detection of metronidazole.

[0005] To achieve the above object, the present invention is implemented through the following technical solutions:

[0006] One of the purposes of the present invention is to provide a metronidazole hapten, the structure of which is shown in Formula I:

[0007] Formula I.

[0008] The second object of the present invention is to provide a method for preparing the above-mentioned metronidazole hapten, the steps are as follows:

[0009] S1. Accurately weigh metronidazole, dissolve it in tetrahydrofuran, slowly add glycolic acid, and stir for 10 minutes;

[0010] S2, add trifluoroacetic acid, react at 25°C for 5h;

[0011] S3, LCMS monitoring, spin-drying, adding purified water to dissolve, adjusting the pH of the solution, extracting with ethyl acetate, and combining and collecting the organic phase;

[0012] S4. Wash the organic phase obtained in step S3 twice with purified water and dry it to obtain the hapten.

[0013] Preferably, the steps are as follows:

[0014] S1. Accurately weigh 1 g of metronidazole, dissolve it in 25 mL of tetrahydrofuran, slowly add 500 mg of glycolic acid, and stir for 10 min;

[0015] S2, add 4 mL of trifluoroacetic acid and react at 25 °C for 5 h;

[0016] S3, LCMS monitoring, spin-drying and adding purified water to dissolve, adjusting the solution pH to 3-5, extracting with ethyl acetate, and combining and collecting the organic phase;

[0017] S4. Wash the organic phase obtained in step S3 twice with purified water and dry it to obtain 500 mg of hapten.

[0018] The third object of the present invention is to provide a complete metronidazole antigen, which is obtained by coupling the above-mentioned metronidazole hapten with a carrier protein. The structure of the complete metronidazole antigen is shown in Formula II:

[0019] Formula II.

[0020] Herein, Protein represents a carrier protein, and the carrier protein is any one of bovine serum albumin (BSA) and chicken ovalbumin (OVA).

[0021] A fourth object of the present invention is to provide a method for preparing the above-mentioned metronidazole complete antigen.

[0022] Specifically, when the carrier protein is bovine serum albumin, the preparation method of metronidazole complete antigen is as follows:

[0023] Dissolve the above metronidazole hapten and EDC / NHS in N,N-dimethylformamide solution, cool to 0°C, stir for 5-8h, and obtain metronidazole hapten activation solution; weigh bovine serum albumin and dissolve it in CB buffer, then slowly drop the metronidazole hapten activation solution, and stir at room temperature for 12h. Dialyze the obtained reaction solution with CB buffer, replace the buffer every two hours, and dialyze at 4°C for 48h to obtain the complete metronidazole antigen with bovine serum albumin as the carrier protein.

[0024] Specifically, when the carrier protein is chicken ovalbumin, the preparation method of metronidazole complete antigen is as follows:

[0025] Dissolve the above metronidazole hapten in N,N-dimethylformamide solution, slowly add triethylamine, stir for 30 minutes, then add isobutyl chloroformate, and stir for 2 hours. Weigh chicken egg albumin, add it to the above reaction solution, and react for 4 hours. Dialyze the obtained reaction solution with PBS buffer, replace the buffer every two hours, and dialyze at 4°C for 48 hours to obtain the complete metronidazole antigen with chicken egg albumin as the carrier protein.

[0026] A fifth object of the present invention is to provide a metronidazole colloidal gold detection product, characterized in that the above-mentioned metronidazole complete antigen is coated on a nitrocellulose membrane.

[0027] The present invention has the following beneficial effects:

[0028] 1. The present invention provides a linker arm having a specific length and containing an active group -COOH derived from the structure of metronidazole. After the hapten is connected to the carrier protein, the resulting complete antigen can fully expose the antigenic determinant of metronidazole and maintain the spatial conformation of metronidazole, thus having good immunogenicity;

[0029] 2. The metronidazole colloidal gold detection product provided by the present invention has the characteristics of strong stability, good specificity and high sensitivity, and can realize rapid and accurate detection of metronidazole. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] In order to more clearly illustrate the implementation methods of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for the implementation methods or the description of the prior art. Obviously, the drawings in the following description are only exemplary, and for ordinary technicians in this field, other implementation drawings can be derived from the provided drawings without creative work.

[0031] The structures, proportions, sizes, etc. illustrated in this specification are only used to match the contents disclosed in the specification so as to facilitate understanding and reading by persons familiar with the technology. They are not used to limit the conditions under which the present invention can be implemented, and therefore have no substantial technical significance. Any structural modification, change in proportion or adjustment of size shall still fall within the scope of the technical contents disclosed in the present invention without affecting the effects and purposes that can be achieved by the present invention.

[0032] Figure 1 This is the LCMS spectrum of metronidazole hapten, which shows a positive signal of 248.12, a molecular weight of metronidazole hapten of 247, and a purity of hapten of more than 90%;

[0033] Figure 2 The ultraviolet full wavelength scanning results of metronidazole hapten, BSA, and metronidazole-BSA provided in the embodiments of the present invention;

[0034] Figure 3 The ultraviolet full-wavelength scanning results of metronidazole hapten, OVA, and metronidazole-OVA provided in the embodiments of the present invention;

[0035] Figure 4 is the detection limit of metronidazole in egg samples;

[0036] Figure 5 This is the specific detection result of the metronidazole colloidal gold test strip. DETAILED DESCRIPTION

[0037] The following is a description of the implementation of the present invention by specific embodiments. People familiar with the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0038] Example 1. Preparation of metronidazole hapten

[0039] The structure of the metronidazole hapten is shown in Formula I:

[0040] Formula I.

[0041] The preparation method of the metronidazole hapten described in this embodiment, its specific operation process is as follows:

[0042] S1. Accurately weigh 1 g of metronidazole, dissolve it in 25 mL of tetrahydrofuran, slowly add 500 mg of glycolic acid, and stir for 10 min;

[0043] S2, add 4 mL of trifluoroacetic acid and react at 25 °C for 5 h;

[0044] S3, LCMS monitoring, spin-drying and adding purified water to dissolve, adjusting the solution pH to 3-5, extracting with ethyl acetate, and combining and collecting the organic phase;

[0045] S4, washing the organic phase obtained in step S3 twice with purified water, and drying to obtain 500 mg of hapten, Figure 1 This is the LCMS spectrum of metronidazole hapten. The spectrum shows that the positive signal is 248.12, the molecular weight of metronidazole hapten is 247, and the purity of the hapten is as high as over 90%.

[0046] Its synthetic route is as follows:

[0047]

[0048] Example 2. Preparation of complete metronidazole antigen

[0049] 1. Preparation of metronidazole hapten-bovine albumin conjugate (metronidazole-BSA)

[0050] S1. Weigh 100 mg of the metronidazole hapten of Example 1, dissolve it in 1 ml of N,N-dimethylformamide, add 160 mg / 90 mg of EDC / NHS, cool to 0°C, and stir at room temperature for 5-8 hours to obtain a metronidazole hapten activation solution;

[0051] S2. Weigh 200 mg BSA and dissolve it in 10 ml CB buffer (0.5 mol / L, pH 7.4);

[0052] S3, slowly adding the activated metronidazole hapten activation solution obtained in step S1 to the reaction solution in step S2, and stirring at room temperature for 12 hours;

[0053] S4. Dialyze with 0.01 mol / L, pH=7.2 CB buffer, change the buffer solution every two hours, and dialyze at 4 degrees for 48 hours to obtain the complete metronidazole antigen with bovine serum albumin as the carrier protein.

[0054] 2. Preparation of metronidazole hapten-chicken ovalbumin conjugate (metronidazole-OVA)

[0055] S1. Weigh 100 mg of the metronidazole hapten of Example 1, dissolve it in 5000 μl of N,N-dimethylformamide, slowly drop 800 μl of triethylamine, stir for 30 min, add 1200 μl of isobutyl chloroformate, and react for 2 h;

[0056] S2, weigh 200 mg of OVA, add it to the reaction solution obtained in step S1, and react for 4 hours;

[0057] S3. Purification and dialysis: dialyze with 0.01 mol / L PBS buffer solution, pH=7.4, change the buffer solution every two hours, and dialyze at 4 degrees for 48 hours to obtain the complete metronidazole antigen with chicken ovalbumin (OVA) as the carrier protein.

[0058] Identification of complete metronidazole antigen

[0059] Metronidazole hapten, BSA, metronidazole hapten-BSA, metronidazole hapten, OVA, metronidazole hapten-OVA were grouped and tested for UV absorption. Figure 2 , 3It can be seen that at 280nm, the ultraviolet absorption spectra of metronidazole hapten-BSA and metronidazole hapten-OVA are obviously different from the absorption spectra of metronidazole hapten, BSA, and OVA, proving that both metronidazole hapten-BSA and metronidazole hapten-OVA are successfully coupled, and through simple calculation, it is concluded that the coupling ratio of metronidazole hapten-BSA is about 20:1, and the coupling ratio of metronidazole hapten-OVA is about 15:1.

[0060] Example 3: Rapid detection of metronidazole residues in eggs by colloidal gold immunochromatography

[0061] 1. Preparation of Metronidazole Colloidal Gold Detection Reagent

[0062] (1) Preparation of colloidal gold

[0063] Take 1 mL of 1% (mass percentage) chloroauric acid solution, add 99 mL of ultrapure water to make a final concentration of 0.01% (mass percentage) chloroauric acid solution, heat to boiling, then take 1.6 mL of 1% (mass percentage) trisodium citrate and quickly add it to the boiling chloroauric acid solution at one time, continue heating until the solution changes from light yellow to blue-black and finally to bright red, continue heating for 5 minutes after the color stabilizes, cool to room temperature, and add pure water to the original volume.

[0064] (2) Preparation of colloidal gold-labeled metronidazole monoclonal antibody

[0065] The colloidal gold solution was adjusted to a pH value of 8.0, stirred evenly with a constant speed stirrer, and a metronidazole monoclonal antibody (prepared by the complete metronidazole antigen described in the present invention) was added dropwise at the same time. After 1 hour, PEG equivalent to the antibody was added. After a full reaction for 30 minutes, BSA equivalent to the antibody was added. After the addition, stirring was continued for 30 minutes. Centrifuged at 9000 rpm for 30 minutes to obtain a uniform gold-labeled antibody precipitate, and then resuspended in PBS for standby use to obtain a colloidal gold-labeled metronidazole monoclonal antibody.

[0066] (3) Preparation of colloidal gold detection reagent

[0067] The metronidazole-coated antigen solution was sprayed on the nitrocellulose membrane to form a detection line T line, and the goat anti-mouse IgG was sprayed to form a control line C line. The detection line and the control line were parallel to each other, and the distance between the two was 0.65 cm. Then, on the bottom plate, the sample pad, the detection line T line sprayed with the metronidazole immune antigen, and the control line C line sprayed with the goat anti-mouse IgG were overlapped and bonded in sequence on the nitrocellulose membrane and absorbent paper in the same direction.

[0068] The detection line is close to the sample pad, the control line is close to the absorbent paper, the stuck detection test paper is cut into test strips of equal width; colloidal gold-labeled metronidazole monoclonal antibody is added into the micropores and freeze-dried; the micropores and test strips are sealed and dried for storage.

[0069] 2. Detection

[0070] Competitive immunochromatography technology is used to qualitatively detect metronidazole residues in samples. The test line (T line) on the nitrocellulose membrane is coated with metronidazole antigen, and the corresponding metronidazole monoclonal antibody is labeled on colloidal gold. During the test, when there is no metronidazole in the sample to be tested, the colloidal gold particles will bind to the metronidazole antigen coated on the membrane when they are chromatographed to the test line position, and the T line will develop color. When metronidazole is present in the sample to be tested, it will bind to the gold-labeled specific monoclonal antibody to form an immune complex. When the complex is chromatographed to the test line position, it will not bind to the metronidazole antigen coated on the membrane, and the T line will not develop color or the color will become lighter.

[0071] 1. Sample pretreatment

[0072] Crack the fresh egg into a beaker and mix the egg white and yolk thoroughly; take 1 ml of the mixed sample into a 2 ml polystyrene centrifuge tube and vortex for 30 s for testing.

[0073] 2. Detection steps

[0074] After diluting the fresh egg liquid to be tested, draw 200µl into the freeze-dried gold microwell, react at room temperature for 5 minutes, start timing when the test strip is inserted into the microwell, and after 5 minutes of reaction, the result can be determined according to the color depth of the test line (T line) and the quality control line (C line). If the T line and the C line show purple-red strips at the same time and the color of the T line is stronger than that of the C line, the result is negative; if the color of the T line is lighter than that of the C line or the C line is colored but the T line is not colored, the result is positive; if both the C line and the T line are not colored, the detection reagent has expired.

[0075] 3. Sample detection limit test

[0076] When the method described in 1 was used for sample pretreatment, metronidazole was added to blank egg samples to a mass fraction of 0, 0.1, 0.2, 0.5, and 1 µg / kg, and the test strips were used for detection. Three replicates were set for each concentration, and the test results were recorded to determine the detection limit of metronidazole in eggs by naked eye using the test strips. Figure 4 As shown in the figure, with the increase of metronidazole concentration, the color of T line gradually faded. When the mass fraction was 0.2 µg / kg, the color of T line was significantly weaker than that of C line, indicating a positive result. Therefore, it was determined that the detection limit of metronidazole in eggs by the naked eye of this test strip was 0.2 µg / kg.

[0077] 4. Accuracy test

[0078] Take 20 blank egg samples, pre-treat them according to the method described in 1, and then test them with test strips, count the positive results, and calculate the false positive rate; take 20 blank egg samples, add metronidazole to a mass fraction of 0.2 µg / kg, pre-treat them according to the method described in 1, and then test them with test strips, count the negative results, and calculate the false negative rate. One positive sample was detected in 20 blank egg samples, and the false positive rate was 5%, indicating that the test strip has a good negative resolution. The test results of positive egg samples were all positive, and the false negative rate was 0%, indicating that the test strip has a stable detection ability.

[0079] 5. Specificity test

[0080] Take ornidazole, secnidazole, tinidazole, dimetridazole, hydroxymethylnidazole, ronidazole, hydroxyisopropranidazole, isopropranidazole standard products and prepare them into standard solutions with different gradients for testing to verify the specificity of the test strips. The specificity test results are shown in Figure 5 The test strips were used to test fresh eggs containing 500 µg / kg of ornidazole, secnidazole, tinidazole, dimenoic acid, hydroxymethylnidazole, lonidazole, hydroxyisopropranidazole, and isopropranidazole. The test results were all negative, indicating that the test strips have good specificity for detecting metronidazole residues.

[0081] 6. Product stability test

[0082] The metronidazole colloidal gold product was placed at 4°C and 37°C for accelerated testing, and the metronidazole standard 0.2 µg / kg was detected at 0d, 7d, 14d, and 30d, respectively, to evaluate the stability performance of the product. When the metronidazole colloidal gold product was aged to 30 days, the metronidazole colloidal gold product at 4°C and 37°C still had good sensitivity in detecting the 0.2μg / kg standard, with no significant difference from the test results on the first day, and the CV was <10%, indicating that the test strip provided by the present invention has good stability, and the results are shown in Table 1.

[0083] Table 1 Stability of metronidazole colloidal gold test strips

[0084]

[0085] In summary, the colloidal gold test strip of the present invention has the characteristics of low detection limit, high accuracy, good specificity and good stability, and can be applied to the colloidal gold immunochromatography method for rapid detection of metronidazole residues in eggs.

Claims

1. A metronidazole hapten, characterized in that: The structure of the metronidazole hapten is shown in Formula I, Formula I.

2. A method for preparing the metronidazole hapten according to claim 1, characterized in that: S1. Accurately weigh metronidazole, dissolve it in tetrahydrofuran, slowly add glycolic acid, and stir for 10 minutes; S2, add trifluoroacetic acid, react at 25°C for 5h; S3, LCMS monitoring, spin-drying, adding purified water to dissolve, adjusting the pH of the solution, extracting with ethyl acetate, and combining and collecting the organic phase; S4. Wash the organic phase obtained in step S3 twice with purified water and dry it to obtain the hapten.

3. A complete metronidazole antigen, characterized in that: The metronidazole hapten according to claim 1 is coupled with a carrier protein, wherein the carrier protein is any one of bovine serum albumin and chicken ovalbumin.

4. A method for preparing the complete metronidazole antigen according to claim 3, characterized in that: When the carrier protein is bovine serum albumin, the method comprises: The metronidazole hapten and EDC / NHS described in claim 1 are dissolved in N,N-dimethylformamide solution, cooled to 0°C, and stirred for 5-8 hours to obtain a metronidazole hapten activation solution; bovine serum albumin is weighed and dissolved in a CB buffer solution, and then the metronidazole hapten activation solution is slowly added dropwise, stirred at room temperature for 12 hours, and the obtained reaction solution is dialyzed with a CB buffer solution, the buffer solution is replaced every two hours, and dialyzed at 4°C for 48 hours to obtain a complete metronidazole antigen with bovine serum albumin as the carrier protein.

5. A method for preparing the complete metronidazole antigen according to claim 3, characterized in that: When the carrier protein is chicken ovalbumin, the method comprises: The above metronidazole hapten was dissolved in N,N-dimethylformamide solution, triethylamine was slowly added dropwise, and the mixture was stirred for 30 min, and then isobutyl chloroformate was added and stirred for 2 h. Chicken ovalbumin was weighed and added to the above reaction solution, and the mixture was reacted for 4 h. The reaction solution was dialyzed with PBS buffer solution, and the buffer solution was replaced every two hours. The mixture was dialyzed at 4°C for 48 h to obtain the complete metronidazole antigen with chicken ovalbumin as the carrier protein.

6. Provide a metronidazole colloidal gold detection product, characterized in that The complete metronidazole antigen according to claim 3 is coated on the nitrocellulose membrane.

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