Enrofloxacin artificial hapten as well as preparation method and application thereof

Abstract

The invention provides an enrofloxacin artificial hapten and a preparation method and application thereof.According to the hapten, the rigidity and stability of molecules are improved by introducing a biphenyl group which is an annular connecting arm structure, the molecular characteristics of a to-be-detected object are reserved to the maximum extent, and the immunogenicity of the hapten is improved; the enrofloxacin artificial antigen obtained by coupling the hapten and a carrier protein is used for immunizing animals, so that the enhancement of the immune response effect of the animals can be stably stimulated in the immunization process, and an antibody with higher specificity and higher sensitivity can be prepared. An indirect ELISA method is used for determining the titer of the antibody, the titer of the enrofloxacin monoclonal antibody is more than or equal to 32000, and the inhibition rate is 82%; the lowest detection limit (IC10 of the enrofloxacin monoclonal antibody is 0.02 ng / mL, the IC50 of the enrofloxacin monoclonal antibody is 0.17 ng / mL, and the linear range (IC20-IC80) of the enrofloxacin monoclonal antibody is 0.05 ng / mL to 0.59 ng / mL.

Description

Technical Field

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

[0002] 1-cyclopropyl-7-(4-ethyl-1-piperazinyl)-6-fluoro-1,4-dihydro-4-oxo-3-quinolinecarboxylic acid (Enrofloxacin, abbreviated as enrofloxacin), whose molecular formula is C 19 H 22 FN 3 O 3 , molecular weight is 359.40. Enrofloxacin, a fluoroquinolone antibacterial drug, has become one of the most important antibacterial drugs in livestock and aquaculture due to its broad antibacterial spectrum, strong antibacterial activity, good oral absorption rate and wide distribution in the body. It can be used as a veterinary drug and is widely used in the treatment and prevention of diseases and the promotion of animal growth. As a broad-spectrum bactericide, enrofloxacin has a significant effect on mycoplasma. In addition, it has a bactericidal effect on a variety of bacteria, including Escherichia coli, Klebsiella, Salmonella, Proteus, Pseudomonas aeruginosa, Haemophilus, Pasteurella multocida, Pasteurella hemolytica, Staphylococcus aureus and Streptococcus, and has an inhibitory effect on Gram-positive bacteria, Gram-negative bacteria and mycoplasma; at the same time, because of its long half-life in the body and good tissue distribution characteristics, it is also a broad-effect antibacterial agent. However, in view of its drug resistance and potential carcinogenic risk, enrofloxacin has been listed as a priority for veterinary drug residue monitoring, and both the EU and China have set maximum residue limits in tissues (100 μg / kg).

[0003] At present, the chemical methods for detecting enrofloxacin residues mainly include gas chromatography (GC), liquid chromatography (HPLC), gas chromatography-mass spectrometry (GC / MS), liquid chromatography-mass spectrometry (HPLC / MS), etc. Due to the high price of the instruments and the complicated pre-treatment, they are not suitable for on-site monitoring and screening of a large number of samples. Immunoassay technology has shown broad application prospects in the analysis of pesticide and veterinary drug residues due to its high specificity of antigen-antibody reaction, as well as its advantages of simple, rapid, high sensitivity, low cost and suitability for on-site large-scale sample screening. Patent CN 101962359A discloses an enrofloxacin hapten, artificial antigen and antibody, and a preparation method and application thereof. In this patent, enrofloxacin is used as a raw material and reacted with aminobutyric acid or aminocaproic acid to generate a hapten containing 4 or 6 carbon atom arms. The antibody titer is determined by the indirect ELISA method, and the detection limit (IC 10 ) is 0.05 ng / mL, and the half inhibitory concentration (IC 50 ) was 3.43 ng / mL, and the linear range (IC 20 ~IC80 ) is 0.1~8.1ng / mL. However, the rigidity and stability of the enrofloxacin hapten design are poor. Due to the complex three-dimensional structure of the carrier protein, it is easy to wrap the small molecule hapten inside, resulting in it being unable to be fully exposed to the antigen presenting cells during the immunization process, and the body will not be able to produce highly sensitive and specific antibodies against the small molecule hapten. Therefore, there is still a need for a hapten that has good rigidity and stability, higher sensitivity and specificity, and can be used to detect enrofloxacin, so as to achieve rapid detection of the enrofloxacin content in food. Summary of the invention

[0004] The purpose of the present invention is to overcome the above defects and shortcomings in the prior art and provide an artificial hapten of enrofloxacin.

[0005] The second object of the present invention is to provide a method for preparing the above-mentioned enrofloxacin artificial hapten.

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

[0007] The fourth object of the present invention is to provide an enrofloxacin antibody.

[0008] The fifth object of the present invention is to provide the use of the above-mentioned enrofloxacin artificial hapten, antigen and antibody in the detection of enrofloxacin.

[0009] The above-mentioned object of the present invention is achieved through the following technical solutions:

[0010] The present invention provides an artificial hapten of enrofloxacin, wherein the structural formula of the hapten is as shown in formula (I):

[0011]

[0012] In view of the problems of poor rigidity and stability, low immune response, etc. in the design of enrofloxacin hapten in the prior art, the present invention provides a hapten that can retain the characteristic structure of enrofloxacin to the greatest extent and has good rigidity and stability. The present invention increases the rigidity and stability of the molecule by introducing a biphenyl group, a cyclic connecting arm structure, and retains the molecular characteristics of the analyte to the greatest extent, thereby improving the immunogenicity of the hapten, so as to ensure that the body can be stably stimulated to produce antibodies with high sensitivity and strong specificity during the immunization process.

[0013] Furthermore, the method for preparing the artificial hapten of enrofloxacin is to mix enrofloxacin and 4'-amino-4-biphenylcarboxylic acid for condensation reaction to obtain the artificial hapten of enrofloxacin.

[0014]

[0015] Furthermore, the molar ratio of enrofloxacin to 4'-amino-4-biphenylcarboxylic acid is 1:0.8-1.2.

[0016] Preferably, the molar ratio of enrofloxacin to 4'-amino-4-biphenylcarboxylic acid is 1::1.

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

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

[0019]

[0020] Among them, BSA is bovine serum albumin; OVA is ovalbumin.

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

[0022] Furthermore, the preparation method is to couple the carrier protein to the carboxyl group of the enrofloxacin hapten using an active ester method.

[0023] Furthermore, the bovine serum albumin is used to prepare the immunogen; and the ovalbumin is used to prepare the coating agent.

[0024] Specifically, the above-mentioned enrofloxacin artificial hapten, N-hydroxysuccinimide and 1,3-dicyclohexylcarbodiimide are dissolved in DMSO, and added into a solution containing a carrier protein for reaction, thereby obtaining the enrofloxacin artificial antigen.

[0025] The present invention provides an enrofloxacin antibody, which is prepared by immunizing an animal with the above antigen. The enrofloxacin antibody can generate a specific immune reaction with enrofloxacin; the enrofloxacin antibody is a monoclonal antibody, a polyclonal antibody or a genetically engineered antibody.

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

[0027] The present invention increases the rigidity and stability of the molecule by introducing a biphenyl group, a cyclic connecting arm structure, and retains the molecular characteristics of the test object to the greatest extent, thereby improving the immunogenicity of the hapten. The artificial enrofloxacin antigen obtained by coupling the hapten with a carrier protein is used to immunize animals, which is more conducive to stimulating the animal immune response effect, so as to prepare antibodies with stronger specificity and higher sensitivity. The indirect ELISA method is used to determine the antibody titer. The titer of the enrofloxacin monoclonal antibody of the present invention is ≥32,000, and the inhibition rate is 82%; the minimum detection limit (IC10 ) is 0.02ng / mL, IC 50 The linear range (IC 20- IC 80 ) is 0.05~0.59ng / mL.

[0028] Therefore, the present invention also provides the use of the above-mentioned enrofloxacin artificial hapten, enrofloxacin artificial antigen or enrofloxacin antibody in detecting enrofloxacin.

[0029] The present invention also provides the use of the above-mentioned enrofloxacin artificial hapten, enrofloxacin artificial antigen or enrofloxacin antibody in the preparation of a product for detecting enrofloxacin.

[0030] Furthermore, the products include but are not limited to enrofloxacin ELISA kits, enrofloxacin colloidal gold test strips, and enrofloxacin time-resolved fluorescence test strips.

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

[0032] The present invention provides an artificial hapten of enrofloxacin, which increases the rigidity and stability of the molecule by introducing a biphenyl group, a cyclic connecting arm structure, and retains the molecular characteristics of the test object to the greatest extent, thereby improving the immunogenicity of the hapten. The artificial hapten of enrofloxacin obtained by coupling the hapten with a carrier protein is used to immunize animals, which is more conducive to stably stimulating the enhancement of the immune response effect of the animal during the immunization process, so as to prepare antibodies with stronger specificity and higher sensitivity. The indirect ELISA method is used to determine the antibody titer, and it is found that the titer of the enrofloxacin monoclonal antibody of the present invention is ≥32000, and the inhibition rate is 82%; the minimum detection limit (IC 10 ) is 0.02ng / mL, IC 50 The linear range (IC 20 -IC 80 ) is 0.05-0.59 ng / mL. The antigen-antibody synthesis process of the present invention is simple, and lays a foundation for developing various immunoassay rapid detection methods with low cost, high detection efficiency and simple operation, and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 The enrofloxacin antibody titer was determined by indirect ELISA method.

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

[0035] The present invention is further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention 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 A method for preparing an artificial hapten of enrofloxacin

[0038] 0.36g (1mM) enrofloxacin original drug and 0.21g (1mM) 4'-amino-4-biphenylcarboxylic acid were dissolved in 10mL dimethyl sulfoxide (DMSO), 0.29mL (2mM) 1,8-diazabicycloundec-7-ene (DBU) was added dropwise as a catalyst, and stirred at room temperature for 3h. After the reaction was completed, the pH value of the reactant was adjusted to 2 using 2M hydrochloric acid, 100mL ethyl acetate was added for extraction three times, the organic phase was dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain a pale yellow crude product hapten, the crude product was separated and purified by chromatography column, and finally the enrofloxacin artificial hapten was obtained after vacuum drying.

[0039] The above-prepared enrofloxacin artificial hapten was subjected to nuclear magnetic resonance identification, and the results are as follows: 1 HNMR (300 MHz, DMSO-d 6 ,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-CH 2 -), 2.40(m,2H,N-CH 2 -), 1.10~1.32(m,4H,-CH 2 -CH 2 -), 1.03(m,3H,-CH 3 ). 13 C NMR (75 MHz, DMSO-d 6 ,ppm)δ: 163.2~177.4(3C,-CO), 152.8(1C,Ph-CF), 147.6(1C,Ph-CN), 144.2( 1C,Py-CN), 102.6~146.2(16C,Ph-C), 111.5(1C,Py-C=), 46.3~49.5(5C,Pr-CH 2 -), 35.6(1C,N-CH-), 13.5(1C,-CH 3 ), 7.8(2C,CH2 -CH 2 ).

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

[0041]

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

[0043] 1. Preparation method of immunogen

[0044] (1) 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-dicyclohexylcarbodiimide (DCC) were dissolved in 1.5 mL of DMSO and reacted at 25° C. with magnetic stirring for 4 h. After centrifugation, the supernatant was collected as solution A.

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

[0046] (3) Under magnetic stirring, add dropwise solution A to solution B and react at 4°C for 12 h. Purify by dialyzing with PBS for 3 days, changing the solution 3 times a day, to obtain the artificial enrofloxacin antigen coupled to bovine serum albumin, which is packaged and stored at -20°C for later use.

[0047] 2. Preparation method of coating

[0048] The synthesis steps are consistent with those of the immunogen, except that the carrier protein used is replaced with ovalbumin OVA, thereby obtaining an artificial enrofloxacin antigen coupled with ovalbumin, which is packaged and stored at -20°C for future use.

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

[0050] 1. An enrofloxacin antibody, the preparation method of which is:

[0051] (1) Animal immunization

[0052] The immunogen prepared in Example 2 was mixed with an equal amount of adjuvant (complete Freund's adjuvant was used for the first time, and incomplete Freund's adjuvant was used for subsequent immunization) and emulsified. Healthy 6-8 week old female Balb / c mice were immunized at intervals, and the mouse serum effect was detected by ELISA. The spleen of the mouse with the best effect was used for cell fusion.

[0053] (2) Cell fusion and screening

[0054] The spleen cells of Balb / c mice producing specific antibodies were fused with myeloma cells SP20, and the cell supernatant was determined by indirect competitive enzyme-linked immunosorbent assay to screen positive cell wells. The positive cell wells were cloned by limiting dilution method or microcloning method to obtain hybridoma cell lines that can stably secrete enrofloxacin monoclonal antibodies.

[0055] (3) Ascites preparation and antibody purification

[0056] Using the in vivo induction method, sterile paraffin oil was injected into the peritoneal cavity of 8-week-old Balb / c mice, hybridoma cells were injected into the peritoneal cavity 7-14 days later, and ascites was collected 7-10 days later. The ascites was purified using the caprylic acid-ammonium sulfate method to obtain monoclonal antibodies, which were packaged in vials and stored at -20°C.

[0057] (4) Antibody testing

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

[0059] The experimental results are as follows Figure 1 As shown, the titer of the enrofloxacin monoclonal antibody is ≥32,000, and the inhibition rate is 82%. Example 4 Establishment of the indirect competitive ELISA standard curve based on the enrofloxacin monoclonal antibody

[0060] 1. Determine the coating original concentration and antibody dilution multiple

[0061] The enrofloxacin artificial antigen prepared in Example 2 was used as the coating source, and the appropriate coating source concentration and antibody dilution multiple were determined by the chessboard titration method. The coating source was diluted to different concentrations for coating plates, and the antibody was diluted in a gradient manner. In the coated ELISA plate, 50 μL of different concentrations of enrofloxacin standard solution and antibody were added to each well, incubated at 37°C for 30 minutes, washed five times with PBST, the liquid in the well was patted dry, and a 1:5000 diluted enzyme-labeled secondary antibody (sheep anti-mouse IgG-HRP) was added, incubated at 37°C for 30 minutes, washed five times with PBST, the liquid in the well was patted dry, 100 μL of TMB substrate solution was added, and color was developed at 37°C in the dark for 10 minutes; 50 μL of stop solution (2M H 2 SO 4 ) to terminate the reaction and read the absorbance at 450nm using an ELISA reader. 450nm The coating original concentration and antibody dilution multiple between 1.0 and 1.5 were combined to draw the inhibition curve in the next step. The coating original concentration was finally determined to be 250 ng / mL and the antibody concentration was 1 μg / mL.

[0062] 2. Establishment of standard curve

[0063] To the ELISA plate with a coating original concentration of 250 ng / mL, 50 μL (1 μg / mL) anti-enrofloxacin monoclonal antibody and a series of 50 μL of enrofloxacin standards of different concentrations were added to each well, incubated at 37°C for 30 min, washed five times with PBST, patted the liquid in the wells, added 1:5000 diluted enzyme-labeled secondary antibody (sheep anti-mouse IgG-HRP), incubated at 37°C for 40 min, washed five times with PBST, patted the liquid in the wells, added 100 μL TMB substrate solution, and developed color at 37°C in the dark for 10 min; added 50 μL stop solution (2M H 2 SO 4 ) to terminate the reaction; read the absorbance at 450nm using an ELISA reader. The concentration of enrofloxacin standard is plotted against the horizontal axis, B / B 0 (OD of the wells to which enrofloxacin was added 450 / OD of the well without enrofloxacin 450 ) as the vertical axis, and establish an indirect competition standard curve.

[0064] The standard curve of indirect competitive ELISA established based on monoclonal antibodies is shown in the figure below. Figure 2 As shown in the figure, it can be seen that the standard curve is S-shaped, with good linear correlation, and the minimum detection limit (IC 10 ) is 0.02ng / mL, IC 50 The linear range (IC 20 ~IC 80 ) is 0.05~0.59ng / mL, with high detection sensitivity and 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-reaction rate of fluoroquinolone veterinary drugs that may remain at the same time, such as ciprofloxacin, norfloxacin, moxifloxacin, enoxacin and ofloxacin, and the specific method was the same as that 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 obvious cross-reactivity (less than 0.5%) to 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-reaction results of enrofloxacin structural and functional analogs

[0069]

[0070]

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

Claims

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

2. The method for preparing the artificial hapten of enrofloxacin according to claim 1, characterized in that: The preparation method comprises the steps of mixing enrofloxacin and 4'-amino-4-biphenylcarboxylic acid to carry out a condensation reaction, thereby obtaining an artificial hapten of enrofloxacin.

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

2.

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

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

6. The method for preparing the artificial enrofloxacin antigen according to claim 5, characterized in that: The carrier protein was coupled to the artificial hapten of enrofloxacin using the active ester method.

7. An enrofloxacin antibody, characterized in that: The antibody is prepared by immunizing an animal with the antigen according to claim 5.

8. Use of the artificial hapten of enrofloxacin according to claim 1, the artificial antigen of enrofloxacin according to claim 5 or the antibody of enrofloxacin according to claim 7 in detecting enrofloxacin.

9. Use of the artificial hapten of enrofloxacin according to claim 1, the artificial antigen of enrofloxacin according to claim 5 or the antibody of enrofloxacin according to claim 7 in the preparation of a product for detecting enrofloxacin.

10. The use according to claim 9, characterized in that: The product is an enrofloxacin ELISA kit, an enrofloxacin colloidal gold test strip or an enrofloxacin time-resolved fluorescence test strip.

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