Rifampicin hapten, artificial antigen and preparation method and application thereof

Abstract

The invention discloses a rifampicin hapten, a rifampicin artificial antigen and a preparation method and application thereof, and relates to a rifampicin hapten, a rifampicin artificial antigen, a preparation method thereof and application in detection of rifampicin drug residues in agricultural products. An active arm is introduced from the carbonyl terminal of rifampicin, the prepared rifampicin hapten reserves the characteristic structures of all hydroxyl groups and piperazine rings on a rifampicin naphthalene ring, not only has a relatively good space structure, but also is basically consistent with the electron cloud density of rifampicin, and the immunogenicity of the rifampicin artificial antigen is improved; the rifampicin artificial antigen and the monoclonal antibody prepared by the invention have strong ELISA detection specificity, and the IC50 value is 0.37 [mu] g / L; the detection sensitivity of the rifampicin detection device prepared by the invention to rifampicin in a standard solution and a sample is 1mu g / kg.

Description

Technical Field

[0001] The present invention relates to the technical field of agricultural product safety detection. More specifically, the present invention relates to a rifampicin hapten, an artificial antigen, and a preparation method and application thereof. Background Art

[0002] Rifampicin is a semi-synthetic broad-spectrum antibacterial drug of the rifamycin class. It has obvious antibacterial effects on a variety of pathogenic microorganisms and is widely used in the treatment of tuberculosis, leprosy, and Mycobacterium avium complex (MAC) infections. In particular, it is widely used in aquaculture. Long-term consumption of such aquatic products will accumulate in the human body, which can cause damage to liver and kidney functions and various skin rashes and other diseases, seriously endangering people's physical health.

[0003] In related detection technologies, the detection method of rifampicin mainly relies on instrumental methods. However, due to the high cost of the required equipment and instruments, long detection time, and the need for professional personnel to operate, on-site detection and rapid point-of-care testing cannot be truly achieved, bringing great inconvenience to daily detection work. The performance of antigens and antibodies determines the key of immunoassay detection technology, and the key of antigens and antibodies is the hapten. Therefore, to obtain antigens and antibodies with excellent performance, the structural design of the hapten is particularly important. The artificial antigens directly prepared based on rifampicin itself in related technologies have the defects of poor sensitivity and low cross-reactivity rate, and cannot meet the actual use requirements of the existing market. Therefore, the development of highly specific rifampicin haptens or artificial antigens is of crucial significance for rapid, highly sensitive, and low-cost detection methods of rifampicin. Summary of the Invention

[0004] The purpose of the present invention is to provide a rifampicin hapten, an antigen, an antibody, a detection device, and their preparation and application for detecting the residue of rifampicin in agricultural products.

[0005] According to one aspect of the present invention, a rifampicin hapten is provided, and its structure is shown in formula (Ⅰ):

[0006]

[0007] According to another aspect of the present invention, a method for preparing a rifampicin hapten is provided, including the following steps:

[0008] S1. Rifampicin reacts with 2-(aminooxy)acetic acid to undergo an addition and dehydration reaction to obtain the rifampicin hapten as claimed in claim 1. The reaction formula of this step is shown in formula (Ⅱ):

[0009]

[0010] According to another aspect of the present invention, there is provided a rifampicin artificial antigen, which is a conjugate of a rifampicin hapten and a carrier protein, and its structural formula is shown in formula (III):

[0011]

[0012] In some embodiments, the carrier protein is any one of bovine serum albumin, lactoferrin, ovalbumin, human serum albumin or hemocyanin.

[0013] According to the fourth aspect of the present invention, there is provided a rifampicin antibody, which is prepared by immunizing an animal with the rifampicin artificial antigen, and the rifampicin antibody is a rifampicin monoclonal antibody.

[0014] According to the fifth aspect of the present invention, there is provided the application of a rifampicin hapten and a rifampicin artificial antigen in immunological detection for non-disease diagnosis purposes of rifampicin.

[0015] According to the sixth aspect of the present invention, there is provided the application of a rifampicin antibody in immunological detection for non-disease diagnosis purposes of rifampicin.

[0016] According to the seventh aspect of the present invention, there is provided a detection device for rifampicin, which is prepared by using the rifampicin artificial antigen and the rifampicin antibody.

[0017] According to the eighth aspect of the present invention, there is provided a detection method for rifampicin for non-disease diagnosis purposes, which is to detect the rifampicin drug residue in agricultural products by using a detection device for rifampicin.

[0018] Advantages of the present invention: (1) The present invention introduces a reactive arm from the carbonyl end of rifampicin, retains all the characteristic structures of the hydroxyl groups on the naphthalene ring and the piperazine ring of the rifampicin drug. The prepared rifampicin hapten not only has a good spatial structure, but also has basically the same electron cloud density as the original drug rifampicin, improving the immunogenicity of the rifampicin artificial antigen;

[0019] (2) The prepared rifampicin artificial antigen and monoclonal antibody in the present invention are highly specific for ELISA detection of rifampicin, and the IC 50 value is 0.37 μg / L;

[0020] (3) The rifampicin artificial antigen and monoclonal antibody of the present invention are used in the colloidal gold immunochromatography technique, and can quickly and conveniently achieve the qualitative detection of rifampicin. The detection sensitivity of the colloidal gold immunochromatography test strip prepared in the present invention for rifampicin in the standard solution is 1 μg / kg, and the detection sensitivity in the sample is 1 μg / kg. Description of the Drawings

[0021] Figure 1Mass spectrum of rifampicin hapten of an embodiment of the present invention.

[0022] Figure 2 ELISA standard curve graph established based on rifampicin monoclonal antibody of an embodiment of the present invention.

[0023] Figure 3 Result determination standard graph of a rifampicin colloidal gold immunochromatographic test strip of an embodiment of the present invention. Detailed implementation manners

[0024] The present invention is further described in detail through specific implementation cases. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the protection scope of the present invention. After reading the present invention, various equivalent forms of modification of the present invention fall within the scope defined by the appended claims of this application. Unless otherwise specified, all raw materials and reagents of the present invention are raw materials and reagents that can be purchased in the conventional market.

[0025] Example 1 Preparation of rifampicin hapten

[0026] The method for preparing rifampicin hapten includes the following steps:

[0027] Take 500 mg (0.61 mmol, CAS: 13292-46-1) of rifampicin in a 50 mL round-bottom flask, then successively add 5 mL of pyridine and 83 mg (0.91 mmol, CAS: 645-88-5) of 2-(aminooxy)acetic acid. After stirring and mixing well, heat to 50-60 °C and react for 18-24 h. Evaporate the solvent, wash the residue three times with dichloromethane, and dry to obtain 283 mg of rifampicin hapten.

[0028] The prepared rifampicin hapten was identified by mass spectrometry, and the obtained mass spectrum is shown in Figure 1 . From Figure 1 it can be seen that the molecular negative ion peak of this rifampicin hapten is 894 and it is the highest peak, which is consistent with the molecular weight of rifampicin hapten, 895, indicating that the rifampicin hapten shown in formula (I) was successfully synthesized.

[0029] Example 2 Preparation of rifampicin immunizing antigen and coating antigen

[0030] 2.1 Preparation of rifampicin immunizing antigen

[0031] (1) Take 5 mg of the rifampicin hapten prepared in Example 1, dissolve it in 0.1 mL of dimethylformamide (DMF), and after stirring well, add 5 mg of EDC and 5 mg of N-hydroxysuccinimide (NHS), and stir at room temperature for 4 h to obtain the activated ester of the hapten;

[0032] (2) Weigh 30 mg of lactoferrin (LF), and fully dissolve it in 5 mL of PBS solution with a concentration of 0.01 mol / L to form a lactoferrin carrier solution. While stirring, slowly add the above-mentioned hapten activated ester drop by drop to the lactoferrin carrier solution, and stir at room temperature for 16 - 24 h;

[0033] (3) Dialyze the solution prepared in step (2) with 0.01 mol / L PBS at room temperature for 3 days, changing the dialysis fluid 3 times a day to remove unreacted small molecules, obtaining a rifampicin hapten - LF conjugate, that is, an antigen for rifampicin immunization. Aliquot and store at 4 °C for later use.

[0034] 2.2 Preparation of antigen for rifampicin coating

[0035] (1) Take 10 mg of the rifampicin hapten prepared in Example 1, dissolve it in 0.2 mL of dimethylformamide (DMF), and after stirring well, add 5 mg of EDC and 5 mg of N - hydroxysuccinimide (NHS), and stir at room temperature for 4 h to obtain the hapten activated ester;

[0036] (2) Weigh 50 mg of bovine serum albumin (BSA), and fully dissolve it in 4 mL of PBS solution with a concentration of 0.01 mol / L to form a bovine serum albumin carrier solution. While stirring, slowly add the hapten activated ester from step (1) drop by drop to the bovine serum albumin carrier solution, and stir at room temperature for 16 - 24 h;

[0037] (3) Dialyze the solution prepared in step (2) with 0.01 mol / L PBS at room temperature for 3 days, changing the dialysis fluid 3 times a day to remove unreacted small molecules, obtaining a rifampicin hapten - BSA conjugate, that is, an antigen for rifampicin coating. Aliquot and store at 4 °C for later use.

[0038] Example 3 Preparation of monoclonal antibody against rifampicin

[0039] After emulsifying the rifampicin immunization antigen prepared in Example 2 with an equal volume of Freund's adjuvant, BALB / C mice were immunized. The immunization dose for each mouse was 50-100 μg, and the immunization interval was 2 weeks. After immunization 3 times, the serum titer was detected by collecting the tail venous blood of the mouse. If the antibody titer does not meet the requirements, it is necessary to strengthen the immunization. After the antibody titer no longer increases, 100 μg of full antigen is used for subcutaneous strengthening immunization. After 5 days, the mouse spleen cells are taken and fused with SP20 cells. The fused cells are screened in HAT culture medium, and the complete culture medium is replaced with HAT culture medium for culture after 5 days. The cell supernatant is detected by ELISA, and the cells in the wells with strong positive test results are cloned and cultured by limiting dilution method. After 3 clone culture tests, the cells in the wells that are positive are hybridoma cells that secrete monoclonal antibodies. After the hybridoma cells are amplified and cultured, they are inoculated into the mouse peritoneal cavity to produce ascites containing antibodies. Purify the ascites by caprylic acid-ammonium sulfate precipitation method to obtain high-purity and high-specificity rifampicin monoclonal antibodies.

[0040] Example 4 ELISA performance evaluation of rifampicin monoclonal antibody

[0041] The rifampicin coating antigen prepared in Example 2 was diluted to 0.08 μg / mL using carbonate buffer at pH = 9.6 as the coating diluent, and 100 μL / well was added to a polystyrene microplate, coated overnight at 4°C, spun dry, 1% BSA solution was added at 280 μL / well, blocked in phosphate buffer at 37°C for 1 h, spun dry, and vacuum packed for storage after drying.

[0042] The rifampicin monoclonal antibody prepared in Example 3 was diluted to 0.05 μg / mL using a phosphate buffer solution containing 0.05% sodium azide and pH=7.4, and stored at 4°C for later use.

[0043] Rifampicin standard solution was added to the microwell ELISA plate coated with rifampicin coating antigen at 100 μL / well, and then rifampicin monoclonal antibody solution was added at 20 μL / well, and the reaction was carried out at 37°C for 0.5 h; after drying, washing solution was added at 280 μL / well, and the plate was washed 3 times and patted dry; enzyme-labeled secondary antibody was added at 100 μL / well, and the reaction was carried out at 37°C for 0.5 h; the plate was washed 3 times and patted dry, and 50 μL / well of color developing solution A and color developing solution B were added respectively, and the reaction was carried out at 37°C for 15 min; 1 M sulfuric acid solution was added at 50 μL / well to terminate the reaction, and the OD value of each well was measured by setting the ELISA reader at a wavelength of 450 nm. The results are shown in Table 1.

[0044] Table 1 OD values ​​of rifampicin standard solutions at different concentrations

[0045]

[0046] Using the data in Table 1, a four-parameter Logistic curve fitting was performed with ELISA Calc software to plot the standard curve, as shown in the appendix Figure 2 , and the linear equation of this standard curve is:

[0047] y = (A - D) / [1 + (x / C)^B] + D, r 2 = 0.998, A = 1.112, B = 0.878, C = 0.290, D = 0.104, where x represents the concentration of the analyte to be measured and y represents the OD value. The IC 50 value of rifampicin antibody was calculated to be 0.37 μg / L, showing a linear relationship within the rifampicin concentration range of 0.1 - 8.1 μg / L.

[0048] Example 5 A detection device for rifampicin

[0049] A detection device for rifampicin prepared in this example is a rifampicin colloidal gold immunochromatographic test strip, and the specific preparation steps are as follows:

[0050] 5.1 Preparation of a reaction membrane coated with rifampicin-coated antigen and mouse IgG:

[0051] Using a nitrocellulose membrane (NC membrane) as the reaction membrane, the concentration of the rifampicin-coated antigen prepared in Example 2 was adjusted to 0.05 - 0.2 mg / mL with coating buffer, and the concentration of mouse IgG was adjusted to 0.05 - 0.2 mg / mL with coating buffer. According to the membrane liquid volume of 0.8 - 1.2 μL / cm, the rifampicin-coated antigen and mouse IgG were sprayed onto the corresponding test area (T line) and control area (C line) of the reaction membrane. The interval between the test area and the control area was 2.5 mm, and it was placed in an oven at 45°C for 12 - 16 h, and then placed in a constant temperature and humidity storage box for standby. The coating buffer used was 0.01 M PBS buffer containing 1% sucrose, 0.05% sodium azide, and pH = 7.6;

[0052] 5.2 Preparation of a microporous membrane containing nanogold-labeled rifampicin monoclonal antibody:

[0053] 5.2.1 Preparation of nanogold solution:

[0054] Take 1 g of chloroauric acid, dissolve it by ultrasonic in pure water and make up the volume to 100 mL, and store it in the dark at 4°C for standby. Take 4 mL of the above solution into 100 mL of pure water, heat it to boiling, add 1.2 mL of 0.06% sodium citrate solution, continue heating for 10 minutes, cool it to room temperature, and restore the volume to the original volume with pure water, and store it in the dark at room temperature for standby. All glassware used needs to be soaked overnight in a mixed solution of potassium permanganate and sulfuric acid, and washed and dried before use;

[0055] 5.2.2 Labeling of rifampicin monoclonal antibody:

[0056] Dispense 1 mL / bottle of nano-gold solution, and adjust the pH value of the colloidal gold solution with 0.1 mol / L K2CO3 solution. Add 3.5 μg of rifampicin monoclonal antibody to the colloidal gold solution with different pH values, and react at room temperature for 5 minutes. Observe the change in the color of the solution, and record the pH value that keeps the solution red. Add 10 μL of 10% bovine serum albumin solution for blocking, centrifuge at 12,000 rpm for ten minutes, and discard all the supernatant;

[0057] 5.2.3 Preparation of micro-wells:

[0058] Add 1 mL of gold dilution solution containing 2% Tris, 2% bovine serum albumin, 0.05% thimerosal, and 5% sucrose to thaw, dispense 14 μL / well into the micro-wells, and store for use after drying at 37 °C for 16 hours;

[0059] 5.3 Preparation of sample pads:

[0060] Soak the cut blank sample pad of 30*30 cm in the sample pad treatment solution, take it out after soaking for 5 min, dry it at 37 °C for 16 h, and store it in a constant temperature and humidity storage box for later use. The sample pad treatment solution used is 0.05 M PBS buffer containing 0.05% Tween 20, 1% sucrose, 0.5% PVP40, and 0.05% sodium azide;

[0061] 5.4 Assembly of colloidal gold immunochromatographic test cards:

[0062] Stack the reaction membrane prepared in 5.1 in the middle of the PVC board backing, stack the absorbent pad and the sample pad prepared in 5.3 at both ends respectively. The reaction membrane is connected to the absorbent pad and the sample pad respectively. The detection area is close to the sample pad, and the control area is close to the absorbent pad to obtain a test paper board. Cut the test paper board into 3 mm test strips, and load the test strips into the card shell to obtain a rifampicin colloidal gold immunochromatographic test card.

[0063] Example 6 Determination of the sensitivity of rifampicin colloidal gold immunochromatographic test cards

[0064] Prepare a series of rifampicin standard solutions with different concentrations using 0.01 M PBS buffer, then take 100 μL of rifampicin standard solutions with different concentrations and add them to the gold-labeled micro-wells respectively, and repeatedly pipette to dissolve the solution evenly. After standing for 3 min, transfer the solution in the gold-labeled micro-wells to the sample addition hole of the rifampicin colloidal gold immunochromatographic test card prepared in Example 5. Start timing after adding the sample, and the results can be observed in 5 - 8 min. The reading is invalid after 8 min.

[0065] The result judgment criteria are shown in the appendix Figure 3, specifically: when the color development of the T line is stronger than that of the C line or there is no obvious difference from the color development of the C line, it indicates a negative test result (-); when the color development of the T line is significantly weaker than that of the C line or the T line does not show color, it indicates a positive test result (+); invalid: if the C line does not appear, it indicates an incorrect operation process or the test strip has expired. Three sets of replicates were set for the detection test, and the rapid qualitative detection of the rifampicin colloidal gold immunochromatographic test strip can be achieved. The specific results are shown in Table 2 below.

[0066] Table 2 Determination results of rifampicin standard solutions at different concentrations

[0067]

[0068] As shown in Table 2, the rifampicin colloidal gold immunochromatographic test strip prepared in the present invention has a high sensitivity for the detection of rifampicin, up to 1 μg / L.

[0069] Example 7 Stability test of the rifampicin colloidal gold immunochromatographic test strip

[0070] The storage condition of the colloidal gold qualitative immunochromatographic test strip is at room temperature. To ensure the stability of the test strip, an accelerated destructive experiment was carried out on the test strip. It was continuously placed at room temperature and 45 °C for 60 days, and the color development changes of the negative and rifampicin standard solutions were detected on the 0th day, 5th day, 10th day, 20th day, 30th day, 40th day, 50th day and 60th day respectively. Three sets of replicates were set for the experiment, and the results are shown in Table 3 below: ("+" represents positive, "-" represents negative)

[0071] Table 3 Stability results of the rifampicin colloidal gold immunochromatographic test strip

[0072]

[0073] As can be seen from Table 3, after the colloidal gold immunochromatographic test strip is sealed and stored at room temperature and 45 °C for 60 days, there is no obvious change in the T / C color development depth reading result of the test strip, indicating that the colloidal gold immunochromatographic test strip can be stably stored for at least 60 days at 45 °C in the accelerated test. Therefore, the rifampicin colloidal gold immunochromatographic test strip prepared in the present invention can be stably stored at room temperature for more than one year, fully meeting the requirements of the market during storage and transportation.

[0074] Example 8 Detection of samples by the rifampicin colloidal gold immunochromatographic test strip

[0075] Take 3 g of the homogenized sample and place it in a 15 mL centrifuge tube. Add 3 mL of chloroform, 0.1 mL of triethylamine, and 2 g of sodium sulfate respectively. After vigorously shaking and mixing for 2 min, centrifuge at 4000 r / min for 5 min at room temperature. Take all the supernatant and add it to a 10 mL centrifuge tube. Dry it under nitrogen or air at 50 - 60 °C. Add 0.5 mL of 0.01 M PB buffer and n-hexane to the dried centrifuge tube, and vigorously shake and mix for 2 min. The lower layer is the test solution.

[0076] Take 100 μL of the test solution and add it to the gold-labeled micro well. Aspirate up and down 5 - 10 times until the reagents in the gold-labeled micro well are evenly mixed. React at room temperature for 3 min, and add all the reaction solution to the sample application hole of the rifampicin colloidal gold immunochromatographic test strip prepared in Example 5. Start timing after adding the sample. After 5 - 8 min, observe the results according to the Figure 3 judgment criteria attached.

[0077] Naked-eye interpretation method: If the color development of the T line is stronger than that of the C line or there is no obvious difference from the color development of the C line, it indicates that the sample is negative (-); if the color development of the T line is significantly weaker than that of the C line or the T line does not develop color, it indicates that the sample is positive (+); invalid: If the C line does not appear, it indicates an incorrect operation process or the test strip has expired.

[0078] Minimum detection limit: Use the rifampicin standard solution to perform gradient spike detection on 10 blank samples. The spike gradients are 0, 0.3, 1, 2, 4 μg / kg respectively. The results are shown in Table 4.

[0079] Table 4 Spike detection limits for agricultural product samples

[0080]

[0081] As can be seen from Table 4, the rifampicin colloidal gold immunochromatographic test strip prepared by the present invention has good repeatability in the detection results of 10 kinds of agricultural product samples. When the rifampicin content in the sample is less than 1 μg / kg, all are negative; when it is higher than 1 μg / kg, all are positive. Therefore, the detection limit of the rifampicin colloidal gold immunochromatographic test strip prepared by the present invention for rifampicin in the sample is 1 μg / kg.

[0082] The above are only some embodiments of the present invention. For those of ordinary skill in the art, without departing from the inventive concept of the present invention, several deformations and improvements can still be made, and these all belong to the protection scope of the present invention.

Claims

1. Rifampicin hapten, characterized in that, Its structure is as shown in formula (Ⅰ):

2. A method for preparing the rifampicin hapten according to claim 1, characterized in that, It includes the following steps: S1. Rifampicin reacts with 2-(aminooxy)acetic acid through addition and dehydration reactions to obtain the rifampicin hapten described in claim 1. The reaction formula for this step is as shown in formula (Ⅱ):

3. Rifampicin artificial antigen, characterized in that, The rifampicin artificial antigen is a conjugate of the rifampicin hapten described in claim 1 and a carrier protein, and its structural formula is as shown in formula (Ⅲ):

4. The rifampicin artificial antigen according to claim 3, wherein The carrier protein is any one of bovine serum albumin, lactoferrin, ovalbumin, human serum albumin or hemocyanin.

5. Rifampicin antibody, characterized in that, The rifampicin antibody is prepared by immunizing an animal with the rifampicin artificial antigen described in claim 3, and the rifampicin antibody is a rifampicin monoclonal antibody.

6. Use of the rifampicin hapten described in claim 1 and the rifampicin artificial antigen described in claim 3 in immunological detection for non-disease diagnosis purposes of rifampicin.

7. Use of the rifampicin antibody described in claim 5 in immunological detection for non-disease diagnosis purposes of rifampicin.

8. A detection device for rifampicin, characterized in that, The detection device is prepared using the rifampicin artificial antigen described in any one of claims 3 or 4 and the rifampicin antibody described in claim 5.

9. A detection method for rifampicin for non-disease diagnosis purposes, characterized in that, The method is to detect rifampicin drug residues in agricultural products using a detection device for rifampicin described in claim 8.

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