Rifampicin hapten, rifampicin antigen, rifampicin antibody, rifampicin detection device and preparation and application thereof

Abstract

The invention discloses a rifampicin hapten, an antigen, an antibody, a detection device as well as preparation and application thereof, and relates to the rifampicin hapten, the antigen, the antibody, the detection device as well as preparation and application of the rifampicin hapten, the antigen, the antibody and the detection device in detection of rifampicin drug residues in agricultural products. The rifampicin hapten prepared by the invention retains the characteristic structures of all hydroxyl groups on the naphthalene ring of rifampicin, not only has a relatively good space structure, but also is consistent with the electron cloud density of rifampicin, so that the immunogenicity of the rifampicin antigen is improved; the rifampicin antigen and the monoclonal antibody prepared by the invention have strong ELISA detection specificity, and the IC50 value is 0.34 [mu] g / L; the detection sensitivity of the colloidal gold immunochromatography detection card prepared by the invention to rifampicin in a standard solution and a sample is 3 mu g / kg.

Description

Technical Field

[0001] The present invention relates to the technical field of agricultural product safety detection, and more specifically, the present invention relates to rifampicin hapten, antigen, antibody, detection device and their preparation and application. Background Art

[0002] Rifampicin is a semi-synthetic broad-spectrum antibacterial drug of the rifamycin class, which 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.

[0003] In related detection technologies, the detection methods of rifampicin mainly rely on instrumental methods. However, due to the high cost of the required equipment, long detection time, and the need for professional operation, on-site detection and rapid point-of-care testing cannot be truly achieved, which brings 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 hapten. Therefore, to obtain antigens and antibodies with excellent performance, the structural design of hapten is particularly important. In related technologies, the artificial antigens directly prepared based on rifampicin itself have the defects of poor sensitivity and low cross-reaction rate, and cannot meet the actual use requirements of the existing market. Therefore, the development of highly specific rifampicin hapten or artificial antigen 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 rifampicin hapten, antigen, antibody, detection device and their preparation and application for detecting the residual rifampicin in agricultural products.

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

[0006]

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

[0008] S1. Rifampicin reacts with 4-bromobutyric acid to carry out quaternization reaction to obtain the rifampicin hapten as claimed in claim 1, and the reaction formula of this step is shown in formula (Ⅱ):

[0009]

[0010] According to still another aspect of the present invention, there is provided a rifampicin antigen, and the rifampicin antigen is a conjugate of rifampicin hapten and 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 a rifampicin antigen, and the rifampicin antibody is a rifampicin monoclonal antibody.

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

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

[0016] According to the seventh aspect of the present invention, there is provided a rifampicin colloidal gold immunochromatographic test strip, the reaction membrane of which is coated with a rifampicin antigen, and the gold-labeled micropore thereof contains a rifampicin antibody labeled with nano gold.

[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 rifampicin colloidal gold immunochromatographic test strip.

[0018] Advantages of the present invention: (1) The rifampicin hapten is prepared in the form of quaternary ammonium salt in the present invention, and the characteristic structure of all hydroxyl groups on the naphthalene ring of rifampicin is retained. The prepared rifampicin hapten not only has a good spatial structure, but also has the same electron cloud density as the detection object rifampicin, improving the immunogenicity of the rifampicin antigen;

[0019] (2) The rifampicin antigen and monoclonal antibody prepared in the present invention are highly specific for ELISA detection of rifampicin, and the IC 50 value is 0.34 μg / L;

[0020] (3) The rifampicin antigen and monoclonal antibody of the present invention are used in the colloidal gold immunochromatographic technique, and the qualitative detection of rifampicin can be quickly and conveniently realized. The detection sensitivity of the colloidal gold immunochromatographic test strip prepared in the present invention for rifampicin in the standard solution is 3 μg / kg, and the detection sensitivity in the sample is 3 μg / kg. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 It is the mass spectrum of the rifampicin hapten of an embodiment of the present invention.

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

[0023] Figure 3 It is a result determination standard diagram of a rifampicin colloidal gold immunochromatographic test strip for 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 modifications of the present invention by those skilled in the art all 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 0.50 g (0.61 mmol, CAS: 13292-46-1) of rifampicin in a 50 mL round-bottom flask, then successively add 10 mL of toluene, 10 mL of pure water and 0.15 g (0.91 mmol, CAS: 2623-87-2) of 4-bromobutyric acid. After stirring and mixing well, heat to 50-60 °C and react for 45-48 h. Cool to room temperature, separate the organic phase, extract the aqueous phase with ethyl acetate 3 times, and evaporate the aqueous phase under reduced pressure to obtain 0.14 g 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 negative ion peak of the rifampicin hapten molecule is 908 and it is the highest peak, which is consistent with the molecular weight 909 of the rifampicin hapten, 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), 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;

[0032] (2) Weigh 20 mg of lactoferrin (LF) and dissolve it fully 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 future use.

[0034] 2.2 Preparation of the antigen for rifampicin coating

[0035] (1) Take 10 mg of the rifampicin hapten prepared in Example 1 and dissolve it in 0.2 mL of dimethylformamide (DMF). 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 30 mg of bovine serum albumin (BSA) and dissolve it fully 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 future use.

[0038] Example 3 Preparation of rifampicin monoclonal antibody

[0039] The antigen for rifampicin immunization prepared in Example 2 was emulsified with an equal volume of Freund's adjuvant and used to immunize BALB / C mice. The immunization dose for each mouse was 50 - 100 μg, with an immunization interval of 2 weeks. After 3 immunizations, the tail vein blood of the mice was collected to detect the serum titer. If the antibody titer did not meet the requirements, booster immunization was needed. After the antibody titer no longer increased, subcutaneous booster immunization was carried out with 100 μg of the whole antigen. Five days later, the spleen cells of the mice were fused with SP20 cells. The fused cells were screened in HAT medium and cultured in complete medium after replacing HAT medium 5 days later. The cell supernatant was detected by ELISA, and the cells in the wells with strongly positive test results were cloned and cultured by the limiting dilution method. After 3 detections of cloned culture, the cells in the wells that were all positive were hybridoma cells secreting monoclonal antibodies. After amplifying the culture of the hybridoma cells, they were inoculated into the abdominal cavity of mice to produce ascites containing antibodies. The ascites was purified by the caprylic acid-ammonium sulfate precipitation method to obtain a rifampicin monoclonal antibody with high purity and high specificity.

[0040] Example 4 ELISA Performance Evaluation of Rifampicin Monoclonal Antibody

[0041] Using carbonate buffer with pH = 9.6 as the coating dilution solution, the rifampicin coating antigen prepared in Example 2 was diluted to 0.2 μg / mL and added to a polystyrene microplate at 100 μL / well. It was coated overnight at 4°C, then drained. 1% BSA solution was added at 280 μL / well and blocked at 37°C for 1 h in phosphate buffer, then drained and vacuum-packed for storage after drying.

[0042] Using phosphate buffer containing 0.05% sodium azide and pH = 7.4, the rifampicin monoclonal antibody prepared in Example 3 was diluted to 0.1 μg / mL and stored at 4°C for later use.

[0043] To the microplate enzyme-labeled plate coated with rifampicin coating antigen, rifampicin standard solution was added at 100 μL / well, and then rifampicin monoclonal antibody solution was added at 20 μL / well. After thorough mixing and standing for 5 min, the reaction was carried out at 37°C for 10 min; after draining, washing solution was added at 280 μL / well, washed 3 times and patted dry; then enzyme-labeled secondary antibody was added at 100 μL / well and the reaction was carried out at 37°C for 0.5 h; after washing 3 times again and patting dry, chromogenic solution A and chromogenic solution B were added at 50 μL / well respectively and the reaction was carried out at 37°C for 0.5 h; 1M sulfuric acid solution was added at 50 μL / well to terminate the reaction, and the OD value of each well was measured at a wavelength of 450 nm using an enzyme-labeled instrument. The results are shown in Table 1 below.

[0044] Table 1 OD Value Table of Rifampicin Standard Solution with 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, see attachment Figure 2 , and the linear equation of this standard curve is:

[0047] y = (A - D) / [1 + (x / C)^B] + D, r 2 = 0.99998, A = 1.04000, B = 0.96914, C = 0.41359, D = -0.10319, where x represents the concentration of the analyte to be measured and y represents the OD value. By calculation, the IC 50 value of the rifampicin antibody was 0.34 μg / L, showing a linear relationship within the rifampicin concentration range of 0.1 - 1.6 μg / L.

[0048] Example 5 A rifampicin colloidal gold immunochromatographic test strip

[0049] 5.1 Preparation of a reaction membrane coated with rifampicin-coated antigen and mouse IgG:

[0050] Using a nitrocellulose membrane (NC membrane) as the reaction membrane, the concentration of the rifampicin-coated antigen prepared in Example 2 was adjusted to 0.1 - 0.4 mg / mL with a coating buffer, and the concentration of mouse IgG was adjusted to 0.05 - 0.2 mg / mL with the coating buffer. According to a 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. 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;

[0051] 5.2 Preparation of a micropore containing a nanogold-labeled rifampicin monoclonal antibody:

[0052] 5.2.1 Preparation of the nanogold solution:

[0053] Take 1 g of chloroauric acid, dissolve it ultrasonically with 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. Place 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 then washed and dried before use;

[0054] 5.2.2 Labeling of the rifampicin monoclonal antibody:

[0055] 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 5 μg of rifampicin monoclonal antibody into the colloidal gold solution with different pH values, and react at room temperature for 5 minutes. Observe the color change 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;

[0056] 5.2.3 Preparation of micropores:

[0057] Add 1 mL of gold dilution solution containing 2% Tris, 2% bovine serum albumin, 0.05% thimerosal, and 5% sucrose for reconstitution, dispense 12 μL / well into the micropores, and store for later use after drying at 37°C for 16 hours;

[0058] 5.3 Preparation of sample pad:

[0059] Soak the cut 30*30 cm blank sample pad in the sample pad treatment solution for 5 minutes, then take it out and dry it at 37°C for 16 hours, and place it in a constant temperature and humidity storage box for later use. The sample pad treatment solution used is 0.05M PBS buffer containing 0.05% Tween 20, 1% sucrose, 0.5% PVP40, and 0.05% sodium azide;

[0060] 5.4 Assembly of colloidal gold immunochromatographic test card:

[0061] 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 strip board. Cut the test strip board into 3 mm test strips, and load the test strips into the card shell to obtain a rifampicin colloidal gold immunochromatographic test card.

[0062] Example 6 Determination of the sensitivity of rifampicin colloidal gold immunochromatographic test card

[0063] Prepare a series of rifampicin standard solutions with different concentrations using 0.01M PBS buffer, then take 100 μL of rifampicin standard solutions with different concentrations and add them into the gold-labeled micropores respectively, and blow and beat repeatedly to make the solution dissolve evenly. After standing for 3 minutes, transfer the solution in the gold-labeled micropores to the sample adding hole of the rifampicin colloidal gold immunochromatographic test card prepared in Example 5. Start timing after adding the sample, and the result can be observed in 5 - 8 minutes. It is judged invalid after 8 minutes.

[0064] The result judgment standard is shown in the appendix Figure 3, specifically as follows: the color development of the T line is stronger than that of the C line or shows no obvious difference from that of the C line, indicating a negative test result (-); the color development of the T line is significantly weaker than that of the C line or the T line does not show color, indicating a positive test result (+); invalid: the C line does not appear, indicating an incorrect operation process or the test strip has expired. Three groups 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.

[0065] Table 2 Determination results of rifampicin standard solutions with different concentrations

[0066]

[0067] As shown in Table 2, the rifampicin colloidal gold immunochromatographic test strip prepared by the present invention has high sensitivity for the detection of rifampicin, up to 3 μg / L.

[0068] Example 7 Stability test of the rifampicin colloidal gold immunochromatographic test strip

[0069] The storage condition of the colloidal gold qualitative immunochromatographic test strip is 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 change of the negative and rifampicin standard solutions was detected on the 0th day, 5th day, 10th day, 20th day, 30th day, 40th day, 50th day and 60th day respectively. Three groups of replicates were set for the experiment, and the results are shown in Table 3 below: ("+" represents positive, "-" represents negative)

[0070] Table 3 Stability results of the rifampicin colloidal gold immunochromatographic test strip

[0071]

[0072] 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 by 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.

[0073] Example 8 Detection of samples by the rifampicin colloidal gold immunochromatographic test strip

[0074] Take 5 g of the homogenized sample and place it in a 15 mL centrifuge tube. Add 5 mL of acetonitrile, 0.3 mL of triethylamine, and 2 g of sodium chloride 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 65 - 70 °C. Add 0.3 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.

[0075] 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 then add all the reaction solution to the sample addition 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.

[0076] 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.

[0077] Minimum detection limit: Use the rifampicin standard solution to perform gradient spiking detection on 10 blank samples. The spiking gradients are 0, 0.5, 3, 6, 10 μg / kg respectively. The results are shown in Table 4.

[0078] Table 4 Detection limit of spiked samples for agricultural product samples

[0079]

[0080] 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 lower than 3 μg / kg, all are negative; when it is higher than 3 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 3 μg / kg.

[0081] 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 modifications 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 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 undergoes a quaternization reaction with 4-bromobutyric acid to obtain the rifampicin hapten described in claim 1. The reaction formula for this step is shown in formula (Ⅱ):

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

4. The rifampicin 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 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 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 rifampicin colloidal gold immunochromatographic test strip, characterized in that, The reaction membrane of the rifampicin colloidal gold immunochromatographic test strip is coated with the rifampicin antigen described in claim 3 or 4, and the gold-labeled micropore of the rifampicin colloidal gold immunochromatographic test strip contains the rifampicin antibody described in claim 5 labeled with nanogold.

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 the rifampicin colloidal gold immunochromatographic test strip described in claim 8.

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