Enrofloxacin artificial hapten, and preparation method and application thereof

Abstract

The application provides an enrofloxacin artificial hapten, a preparation method and application thereof. The hapten increases the rigidity and stability of the molecule by introducing a biphenyl group as a cyclic connecting arm structure, and maximally retains the characteristics of the measured molecule, improves the immunogenicity of the hapten, and after coupling the hapten with a carrier protein, the enrofloxacin artificial antigen is used to de-immunize animals, which is more conducive to stably stimulating the enhancement of the immune response effect of the animals in the immune process, so as to prepare an antibody with stronger specificity and higher sensitivity. The antibody titer is determined by an indirect ELISA method, and it is found that the titer of the enrofloxacin monoclonal antibody of the application is ≥32000, the inhibition rate is 82%; the minimum detection limit (IC 10 ) of the enrofloxacin monoclonal antibody is 0.02 ng / mL, the IC 50 is 0.17 ng / mL, and the linear range (IC 20 -IC 80 ) is 0.05-0.59 ng / mL.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of food safety detection technology, in particular to an enrofloxacin artificial hapten, a preparation method and application thereof. BACKGROUND

[0002] 1-cyclopropyl-7-(4-ethyl-1-piperazinyl)-6-fluoro-1,4-dihydro-4-oxo-3-quinoline carboxylic acid (Enrofloxacin, for short, Enrofloxacin), its molecular formula is C 19 H 22 FN3O3, and the molecular weight is 359.40. Enrofloxacin belongs to fluoroquinolone antibacterial drugs, and has become one of the most important antibacterial drugs in the field of livestock and aquaculture due to its wide antibacterial spectrum, strong antibacterial activity, good oral absorption rate and wide distribution in vivo. Enrofloxacin can be widely used in the treatment, prevention of diseases and the promotion of animal growth as a veterinary drug. As a broad-spectrum bactericide, Enrofloxacin has a significant therapeutic effect on mycoplasma, and also shows bactericidal effect on a variety of bacteria, including Escherichia coli, Klebsiella, Salmonella, Proteus, Pseudomonas aeruginosa, Haemophilus, Pasteurella multocida, Pasteurella haemolytica, Staphylococcus aureus and Streptococcus, and has inhibitory effect on gram-positive bacteria, gram-negative bacteria and mycoplasma. At the same time, Enrofloxacin also belongs to broad-spectrum antibacterial agents due to its long in vivo half-life and good tissue distribution characteristics. However, Enrofloxacin has been listed as a key point for veterinary drug residue monitoring due to its drug resistance and potential carcinogenic risk, and the maximum residue limit (100 μg / kg) in the tissue has been stipulated by the European Union and China.

[0003] At present, the chemical methods for detecting Enrofloxacin residue mainly include gas chromatography (GC), liquid chromatography (HPLC), gas chromatography-mass spectrometry (GC / MS), liquid chromatography-mass spectrometry (HPLC / MS) and the like. Due to high instrument price and complicated pretreatment, these methods are not suitable for on-site monitoring and large sample screening. Immunoassay technology has a wide application prospect in the analysis of agricultural and veterinary drug residues due to its high specificity of antigen-antibody reaction, simple determination method, high sensitivity, low cost and suitability for on-site screening of a large number of samples. Patent CN 101962359A discloses an Enrofloxacin hapten, artificial antigen and antibody, and a preparation method and application thereof. In the patent, Enrofloxacin is used as raw material to react with aminobutyric acid or aminocaproic acid to generate a hapten containing 4 or 6 carbon arms, and the antibody titer is determined by an indirect ELISA method. It is found that the detection limit (IC 10 ) of Enrofloxacin is 0.05 ng / mL, the half inhibitory concentration (IC 50 ) is 3.43 ng / mL, and the linear range (IC 20 ~ IC 80 ) is 0.05 ng / mL~3.43 ng / mL.) is 0.1-8.1 ng / mL. However, the enrofloxacin hapten in the prior art has poor rigidity and stability in design, and the small molecule hapten is easily wrapped in the three-dimensional structure of the carrier protein, so that the small molecule hapten cannot be fully exposed to antigen presenting cells in the immune process, and the body cannot produce high-sensitivity and specific antibodies against the small molecule hapten. Therefore, there is still a need for a hapten for detecting enrofloxacin, which has good rigidity and stability, high sensitivity and specificity, so as to realize rapid detection of the content of enrofloxacin in food. SUMMARY

[0004] The purpose of the present application is to overcome the above-mentioned defects and deficiencies in the prior art, and to provide an enrofloxacin artificial hapten.

[0005] The second purpose of the present application is to provide a preparation method of the above-mentioned enrofloxacin artificial hapten.

[0006] The third purpose of the present application is to provide an enrofloxacin artificial antigen and a preparation method thereof.

[0007] The fourth purpose of the present application is to provide an enrofloxacin antibody.

[0008] The fifth purpose of the present application is to provide the application of the above-mentioned enrofloxacin artificial hapten, antigen and antibody in detecting enrofloxacin.

[0009] The above-mentioned purposes of the present application are realized by the following technical solutions:

[0010] The present application provides an enrofloxacin artificial hapten, and the structure formula of the hapten is shown as formula (I):

[0011]

[0012] In view of the problems of the enrofloxacin hapten in the prior art, such as poor rigidity and stability in design, and low immune response, the present application provides a hapten which can maximize the retention of the characteristic structure of enrofloxacin and has good rigidity and stability. The present application introduces a biphenyl group as a cyclic connecting arm structure to increase the rigidity and stability of the molecule, and maximizes the retention of the characteristic structure of the test substance, improves the immunogenicity of the hapten, and ensures that the body can be stably stimulated to produce antibodies with high sensitivity and strong specificity in the immune process.

[0013] Further, the preparation method of the enrofloxacin artificial hapten is to mix enrofloxacin and 4'-amino-4-biphenyl carboxylic acid and perform condensation reaction to obtain the enrofloxacin artificial hapten.

[0014]

[0015] Further, the molar ratio of enrofloxacin and 4'-amino-4-biphenyl carboxylic acid is 1:0.8-1.2.

[0016] Preferably, the molar ratio of enrofloxacin and 4'-amino-4-biphenyl carboxylic acid is 1:1.

[0017] Further, the catalyst of the condensation reaction is 1,8-diazabicycloundec-7-ene.

[0018] The present application provides an enrofloxacin artificial antigen, which is obtained by coupling the above-mentioned hapten with a carrier protein; the structure of the antigen is shown in formula (II):

[0019]

[0020] In the formula, BSA is bovine serum albumin; OVA is ovalbumin.

[0021] Further, the preparation method of the enrofloxacin artificial antigen is to couple the carrier protein with the enrofloxacin artificial hapten by using the active ester method.

[0022] Further, the preparation method is to couple the carrier protein with the carboxyl group of the above-mentioned enrofloxacin hapten by using the active ester method.

[0023] Further, the bovine serum albumin is used for preparing an immunogen; and the ovalbumin is used for preparing a coating antigen.

[0024] Specifically, the above-mentioned enrofloxacin artificial hapten, N-hydroxysuccinimide and 1,3-dicyclohexyl carbodiimide are dissolved in DMSO, and then added into a solution containing the carrier protein to react, so as to obtain the enrofloxacin artificial antigen.

[0025] The present application provides an enrofloxacin antibody, which is prepared by immunizing animals with the above-mentioned antigen. The enrofloxacin antibody can specifically react with enrofloxacin; and the enrofloxacin antibody is a monoclonal antibody, a polyclonal antibody or a genetically engineered antibody.

[0026] Further, the animals include but are not limited to mice or rabbits.

[0027] The present application introduces a cyclic connecting arm structure of biphenyl group to increase the rigidity and stability of the molecule, and to maximize the retention of the characteristics of the test substance molecule, so as to improve the immunogenicity of the hapten. After coupling the hapten with the carrier protein, the enrofloxacin artificial antigen is used to immunize animals, which is more conducive to stimulating the enhancement of the immune response effect of the animals, so as to facilitate the preparation of an antibody with stronger specificity and higher sensitivity. The antibody titer is determined by the indirect ELISA method, the titer of the enrofloxacin monoclonal antibody of the present application is ≥32000, and the inhibition rate is 82%; the minimum detection limit (IC50) of enrofloxacin is 0.1 ng / mL.10 ) is 0.02 ng / mL, IC 50 ) is 0.17 ng / mL, the linear range (IC 20- IC 80 ) is 0.05-0.59 ng / mL.

[0028] Therefore, the application further provides the application of the above enrofloxacin artificial hapten, enrofloxacin artificial antigen or enrofloxacin antibody in detecting enrofloxacin.

[0029] The application further provides the application of the above enrofloxacin artificial hapten, enrofloxacin artificial antigen or enrofloxacin antibody in preparing products for detecting enrofloxacin.

[0030] Further, the products include but are not limited to enrofloxacin ELISA kit, enrofloxacin colloidal gold test strip and enrofloxacin time-resolved fluorescence test strip.

[0031] Compared with the prior art, the application has the following beneficial effects:

[0032] The application provides an enrofloxacin artificial hapten, which introduces a biphenyl group as a cyclic connecting arm structure to increase the rigidity and stability of the molecule, and the molecule characteristics of the to-be-detected substance are retained to the maximum extent, the immunogenicity of the hapten is improved, and after the hapten is coupled with a carrier protein to obtain an enrofloxacin artificial antigen, the enrofloxacin artificial antigen is used to immunize animals, which is more conducive to stably stimulating the enhancement of the immune response effect of the animals in the immunization process, so that an antibody with stronger specificity and higher sensitivity is prepared. The antibody titer is determined by an indirect ELISA method, it is found that the titer of the enrofloxacin monoclonal antibody of the application is greater than or equal to 32,000, the inhibition rate is 82%, the minimum detection limit (IC 10 ) of enrofloxacin is 0.02 ng / mL, IC 50 ) is 0.17 ng / mL, the linear range (IC 20 -IC 80 ) is 0.05-0.59 ng / mL. The antigen-antibody synthesis process of the application is simple, lays a foundation for developing various immunological analysis rapid detection methods with low cost, high detection efficiency and simple operation, and has good application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0033] Figure 1 The antibody titer of enrofloxacin is determined by an indirect ELISA method.

[0034] Figure 2 The standard curve of an indirect competitive ELISA based on enrofloxacin monoclonal antibody is established. DETAILED DESCRIPTION

[0035] The present application will be further described in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present application in any form. Unless otherwise specified, the reagents, methods and equipment used in the present application are conventional reagents, methods and equipment in the art.

[0036] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0037] Example 1: Preparation of an artificial enrofloxacin hapten

[0038] Take 0.36 g (1 mM) enrofloxacin and 0.21 g (1 mM) 4'-amino-4-biphenyl carboxylic acid dissolved in 10 mL of dimethyl sulfoxide (DMSO), drop 0.29 mL (2 mM) 1,8-diazabicycloundec-7-ene (DBU) as catalyst, stir at room temperature for 3 h. After the reaction is completed, use 2M hydrochloric acid to adjust the pH value of the reaction to 2, add 100 mL ethyl acetate to extract three times, dry the organic phase with anhydrous sodium sulfate, concentrate under reduced pressure to obtain a yellowish crude product hapten, the crude product is separated and purified by column chromatography, and vacuum dried to obtain the final enrofloxacin artificial hapten.

[0039] The enrofloxacin artificial hapten prepared above is identified by nuclear magnetic resonance, and the results are as follows: 1 HNMR (300 MHz, DMSO-d6, ppm) δ: 12.65 (s, 1H, -COOH), 11.09 (s, 1H, -NH-CO), 8.98 (s, 1H, -N-CH=), 6.08-8.10 (m, 10H, Ph-H), 4.15 (m, 1H, N-CH-), 3.46 (m, 8H, Pr-CH2-), 2.40 (m, 2H, N-CH2-), 1.10-1.32 (m, 4H, -CH2-CH2-), 1.03 (m, 3H, -CH3). 13 C NMR (75 MHz, DMSO-d6, ppm) δ: 163.2-177.4 (3C, -CO), 152.8 (1C, Ph-C-F), 147.6 (1C, Ph-C-N), 144.2 (1C, Py-C-N), 102.6-146.2 (16C, Ph-C), 111.5 (1C, Py-C=), 46.3-49.5 (5C, Pr-CH2-), 35.6 (1C, N-CH-), 13.5 (1C, -CH3), 7.8 (2C, CH2-CH2).

[0040] The structure of the synthesized enrofloxacin artificial hapten is shown in the following formula (I):

[0041]

[0042] Example 2 A method for preparing an enrofloxacin artificial antigen

[0043] 1. A method for preparing an immunogen

[0044] (1) Take 13.86 mg (0.025 mM) of the enrofloxacin hapten described in Example 1, 10.84 mg of N-hydroxysuccinimide (NHS), and 10.30 mg of 1,3-dicyclohexyl carbodiimide (DCC), and dissolve them in 1.5 mL of DMSO. Stir the mixture magnetically at 25°C for 4 hours. After centrifugation, take the supernatant as solution A.

[0045] (2) Take 20 mg of the carrier protein BSA and dissolve it in 2 mL of PBS buffer (0.01 M, pH = 7.4). Stir the solution to prepare solution B.

[0046] (3) Under magnetic stirring, add solution A dropwise to solution B, and react at 4°C for 12 hours. Purify the product by dialysis against PBS for 3 days, changing the solution 3 times per day, to obtain an enrofloxacin artificial antigen coupled to bovine serum albumin. Store the product in aliquots at -20°C for later use.

[0047] 2. A method for preparing a coating antigen

[0048] The synthesis steps are the same as those for the immunogen, except that the carrier protein used is changed to ovalbumin OVA. This gives an enrofloxacin artificial antigen coupled to ovalbumin, which is stored in aliquots at -20°C for later use.

[0049] Example 3 A method for preparing an enrofloxacin antibody

[0050] 1. An enrofloxacin antibody, prepared by the following method:

[0051] (1) Animal immunization

[0052] Mix the immunogen prepared in Example 2 with an equal amount of adjuvant (complete Freund's adjuvant for the first time, and incomplete Freund's adjuvant thereafter), and use the mixture to immunize healthy 6-8 week old female Balb / c mice at intervals. Detect the effect of the mouse serum by ELISA, and take the spleen of the mouse with the best effect for cell fusion.

[0053] (2) Cell fusion and screening

[0054] Fuse the spleen cells of the Balb / c mouse producing specific antibodies with myeloma cells SP20, and use indirect competitive enzyme-linked immunoassay to determine the cell supernatant and screen for positive cell wells. Use limited dilution or microcloning to clone the positive cell wells, to obtain a hybridoma cell strain that can stably secrete an enrofloxacin monoclonal antibody.

[0055] (3) Ascites preparation and antibody purification

[0056] Ascites was prepared by intraperitoneal injection of 8-week-old Balb / c mice with sterile paraffin oil, and then hybridoma cells were injected intraperitoneally 7-14 days later. Ascites was collected 7-10 days later. The monoclonal antibody was purified from ascites by the caprylic acid-ammonium sulfate method, and was stored in a vial at -20°C.

[0057] (4) Antibody testing

[0058] The antibody titer was determined by indirect ELISA, and the absorbance value between 1.0 and 1.5 was used as the standard.

[0059] The experimental results are shown in Table 1. Figure 1 The titer of the enrofloxacin monoclonal antibody was ≥32000, and the inhibition rate was 82%.

[0060] 1. Determination of coating antigen concentration and antibody dilution factor

[0061] The enrofloxacin artificial antigen prepared in Example 2 was used as the coating antigen, and the appropriate coating antigen concentration and antibody dilution factor were determined by chess titration. The coating antigen was diluted to different concentrations, and the antibody was gradiently diluted. In the coated enzyme-labeled plate, 50 μL of different concentrations of enrofloxacin standard solution and antibody were added to each well, respectively, and incubated at 37°C for 30 min. PBST was washed five times, the liquid in the well was patted dry, and 1:5000 diluted enzyme-labeled secondary antibody (goat anti-mouse IgG-HRP) was added, and incubated at 37°C for 30 min. PBST was washed five times, the liquid in the well was patted dry, 100 μL of TMB substrate solution was added, and color development was performed at 37°C for 10 min in the dark; 50 μL of stop solution (2M H2SO4) was added to terminate the reaction, and the absorbance value at 450 nm was read by an enzyme-labeled instrument. The coating antigen concentration and antibody dilution factor were selected to be between 1.0 and 1.5. 450nm The coating antigen concentration and antibody dilution factor were selected to be between 1.0 and 1.5.

[0062] 2. Establishment of standard curve

[0063] Add 50 μL (1 μg / mL) of anti-enrofloxacin monoclonal antibody and a series of 50 μL enrofloxacin standards of different concentrations to each well of an ELISA plate with an original coating concentration of 250 ng / mL. Incubate at 37°C for 30 min. Wash five times with PBST, blot dry the liquid in the wells, add 1:5000 diluted enzyme-labeled secondary antibody (goat anti-mouse IgG-HRP), incubate at 37°C for 40 min, wash five times with PBST, blot dry the liquid in the wells, add 100 μL of TMB substrate solution, and develop color at 37°C in the dark for 10 min. Stop the reaction by adding 50 μL of stop solution (2M H2SO4). Read the absorbance at 450 nm using an ELISA reader. Plot the enrofloxacin standard concentration against the x-axis, B / B0 (OD value of the wells containing enrofloxacin). 450 / OD of the pores without enrofloxacin 450 Using y as the ordinate, establish an indirect competition standard curve.

[0064] The standard curve of indirect competitive ELISA based on monoclonal antibodies is shown below. Figure 2 As shown, the standard curve exhibits an S-shape, indicating good linear correlation and a low limit of detection (IC50) for enrofloxacin. 10 The concentration was 0.02 ng / mL, and the IC50 concentration was [missing information]. 50 The concentration was 0.17 ng / mL, with a linear range (IC50). 20 ~IC 80 The concentration ranges from 0.05 to 0.59 ng / mL, indicating high detection sensitivity and a wide linear range.

[0065] Example 5: Specificity detection of enrofloxacin monoclonal antibody

[0066] The enrofloxacin monoclonal antibody prepared in Example 3 was used to detect the cross-reactivity rate of fluoroquinolone veterinary drugs that may coexist, such as ciprofloxacin, norfloxacin, moxifloxacin, enoxacin, and ofloxacin. The specific method was the same as in Example 4.

[0067] The experimental results are shown in Table 1 below. The results show that the enrofloxacin monoclonal antibody prepared in Example 3 has no significant cross-contamination (less than 0.5%) with ciprofloxacin, norfloxacin, moxifloxacin, enoxacin, and ofloxacin, indicating that the enrofloxacin monoclonal antibody prepared in Example 3 has good specificity and is not prone to false positives.

[0068] Table 1. Cross-reactivity results of enrofloxacin structural and functional analogues

[0069]

[0070]

[0071] In summary, the enrofloxacin artificial hapten, antigen, and antibody provided by this invention have higher sensitivity and specificity.

Claims

1. An enrofloxacin artificial hapten, characterized in that, The structure of the hapten is shown in formula (I):

2. The process for the preparation of enoxacin artificial hapten as claimed in claim 1 wherein, The preparation method is to mix enrofloxacin and 4'-amino-4-biphenyl carboxylic acid and then to conduct condensation reaction to obtain the enrofloxacin artificial hapten.

3. The preparation method according to claim 2, characterized in that, The molar ratio of the enrofloxacin and 4'-amino-4-biphenyl carboxylic acid is 1:0.8-1.

2.

4. The preparation method according to claim 2, characterized in that, The catalyst of the condensation reaction is 1,8-diazabicycloundec-7-ene.

5. An enrofloxacin artificial antigen, characterized in that, The antigen is obtained by coupling the hapten of claim 1 with carrier protein; the structure of the antigen is shown in formula (II): In the formula, BSA is bovine serum albumin; OVA is ovalbumin.

6. The method for preparing enoxacin artificial antigen of claim 5, characterized in that, The carrier protein is coupled with the enrofloxacin artificial hapten by using active ester method.

7. An enrofloxacin antibody, characterized in that, The antibody is obtained by immunizing animals with the antigen of claim 5.

8. The enrofloxacin artificial hapten of claim 1, the enrofloxacin artificial antigen of claim 5 or the enrofloxacin antibody of claim 7 is used for detecting enrofloxacin.

9. The enrofloxacin artificial hapten of claim 1, the enrofloxacin artificial antigen of claim 5 or the enrofloxacin antibody of claim 7 is used for preparing products for detecting enrofloxacin.

10. Use according to claim 9, characterized in that, The products are enrofloxacin ELISA kit, enrofloxacin colloidal gold test strip or enrofloxacin time-resolved fluorescence test strip.

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