CN114316027B - Fluoronii Xin Rengong antigen and preparation method and application thereof - Google Patents
Fluoronii Xin Rengong antigen and preparation method and application thereof Download PDFInfo
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- CN114316027B CN114316027B CN202011077631.5A CN202011077631A CN114316027B CN 114316027 B CN114316027 B CN 114316027B CN 202011077631 A CN202011077631 A CN 202011077631A CN 114316027 B CN114316027 B CN 114316027B
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Abstract
The invention relates to a flunixin Xin Rengong antigen and a preparation method and application thereof. The invention provides a flunixin Xin Rengong antigen which is a compound shown in a formula I, wherein K represents carrier protein. The artificial antigen provided by the invention can be used for immunizing animals to prepare the specific antibody with high sensitivity, and the method is simple, convenient and easy to implement. The artificial antigen and the antibody prepared by the artificial antigen provided by the invention are applied to veterinary drug residue detection, and can realize rapid high-flux detection of flunixin at trace level.
Description
Technical Field
The invention relates to the technical field of biochemical engineering, in particular to a flunixin Xin Rengong antigen and a preparation method and application thereof.
Background
Flunixin (FLU) is a non-steroidal anti-inflammatory drug specific for animals, and is often mixed with Meglumine (Meglumine) as a stabilizer in equal proportions to form stable Flunixin Meglumine salt (Flunixin Meglumine, FM). The pharmacological action mechanism is mainly to reduce the generation of inflammatory mediators such as prostaglandin and the like by inhibiting cyclooxygenase so as to play roles of relieving fever, easing pain and resisting inflammation. Clinically, the traditional Chinese medicine composition is often used for acute inflammation of large animals such as pigs, cows, horses and the like caused by various disease infections; in addition, the compound preparation is combined with antibiotics, has remarkable drug synergistic effect, and can effectively improve clinical symptoms of diseases. Since the success of development in the 90 s of the 20 th century, flunixin meglumine has been widely used in a number of countries including china and has become the largest non-steroidal anti-inflammatory drug in clinical use by veterinarians both at home and abroad.
In order to strengthen the residual supervision of the flunixin in animal foods and ensure the quality safety of animal products, the European Union committee prescribes that the maximum residual limits of the flunixin in the tissues of cow muscle, fat, liver and kidney are respectively 20, 30, 300 and 100 mug/kg; the US FDA specifies that the maximum residual limits of flunixin in porcine muscle and liver tissue are 25 and 30 μg/kg, respectively.
At present, liquid chromatography-mass spectrometry/mass spectrometry is mainly adopted for detecting the residue of flunixin in animal-derived foods in the national industry standard. The method has strong specificity and high sensitivity, but the sample pretreatment is complex and complicated, the detection time is long, the cost is high, and the popularization and the use are limited. The immunoassay method is a qualitative and quantitative analysis method based on the specific reaction of antigen and antibody, and the method has the advantages of strong specificity, high sensitivity, quick reaction, simple operation and suitability for real-time detection of a large number of samples on site.
In a limited study involving the preparation of flunixin Xin Rengong antigens and antibodies, lin et al used this mixture of flunixin meglumine as a hapten to carrier protein for the preparation of artificial antigens of flunixin. The artificial antigen prepared by the method cannot be accurately characterized in the preparation and application processes, is difficult to control quality, and is not beneficial to popularization and application of the method. Lin L, jiang W, xu L, et al development of IC-ELISA and immunochromatographic strip assay for the detection of flunixin meglumine in milk [ J ]. Food & Agricultural Immunology,2017:1-11.
Preparation of Fluoronixin Xin Rengong antigen by Chen et al using the metabolite 5-hydroxyflunixin of Fluoronixin coupled with a carrier proteinThe antibody prepared by the method can better recognize 5-hydroxyflunixin, but has poorer sensitivity to flunixin and IC 50 1.43ng/mL. Chen et al also attempted to prepare fluni Xin Rengong antigen using flunixin as hapten and carrier protein coupling, but was less immune than using 5-hydroxyflunixin, thus finally selecting 5-hydroxyflunixin coupled to carrier protein as artificial antigen of flunixin. Chen X, peng S, liu C, et al development of an indirect competitive enzyme-linked immunosorbent assay for detecting flunixin and 5-hydroxyflunixin residues in bovine muscle and milk [ J].Food&Agricultural Immunology,2019,30(1):320-332。
Disclosure of Invention
Aiming at solving the defects of the prior art, the invention aims to provide a flunixin Xin Rengong antigen with a strong immune effect, and a preparation method and application thereof.
To achieve the object of the present invention, in a first aspect, the present invention provides a flunixin Xin Rengong antigen, the structure of which is shown in formula i.
In the formula I, the carrier protein is bovine serum albumin, human serum albumin, ovalbumin or hemocyanin, preferably bovine serum albumin and hemocyanin. Wherein, K in the formula I is hemocyanin and is used as an immunogen; k is bovine serum albumin as coating source.
In a second aspect, the present invention provides a method for preparing the flunixin Xin Rengong antigen, using flunixin as hapten to chemically couple with carrier protein, the method comprising the steps of:
(1) Flunixin (FLU), 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC. HCL) and N-hydroxysuccinimide (NHS) were dissolved in N, N-Dimethylformamide (DMF) and reacted for 4-48 hours to give solution A.
(2) And dissolving the carrier protein in phosphate buffer solution to obtain solution B.
(3) Mixing the solution A and the solution B, and reacting for 6-48 hours at room temperature to obtain the flunixin Xin Rengong antigen.
Specifically, the method comprises the following steps:
(1) 10-30mg of FLU, 10-40mg of EDC. HCL and 10-40mg of NHS are dissolved in 0.2-2mL of DMF and stirred for reaction for 4-48 hours to obtain solution A.
(2) 10-70mg of carrier protein was dissolved in 10mL of PBS with a concentration of 0.01M and a pH of 7.4 to give solution B.
(3) The solution A is added into the solution B dropwise, stirred at room temperature and reacted overnight.
(4) Dialyzing the reaction solution in PBS for 1-3 days to obtain the compound shown in the formula I.
In order to obtain the flunixin Xin Rengong antigen with excellent immune effect and controllable quality, the invention selects high-purity flunixin as hapten and carrier protein to couple so as to prepare the artificial antigen of the flunixin with quantitatively-characterized coupling ratio.
In the third aspect, in order to improve the immune effect of the flunixin Xin Rengong antigen prepared by the invention, the invention also provides a matched novel immune program. The present invention provides novel immunization methods for preparing a fluni Xin Teyi antibody from the flunixin Xin Rengong antigen. The specific method is as follows:
female Balb/c mice with the age of 6-8 weeks and the weight of 8-20g are immunized after being mixed and emulsified by using Freund's complete adjuvant for primary immunization and Freund's incomplete adjuvant for booster immunization. The immunization mode is subcutaneous multipoint injection at the back of the neck, the immunization dose is 250 mug/dose, the immunization interval is 60 days, and the total immunization is 3 times. On day 7 after each immunization, tail vein blood is collected, centrifugation is carried out at 4000rpm for 10min, supernatant is collected and is taken as antiserum, and the antiserum is preserved at-20 ℃.
The immunization program provided by the invention increases the immunization dosage, prolongs the immunization period, allows the antibody to perform more sufficient affinity maturation in vivo, and obtains the flunixin antiserum with higher affinity.
According to the understanding of the person skilled in the art, the preparation of anti-flunixin artificial antigen or the flunixin Xin Rengong antigen prepared by the preparation method of the invention as an immunogen, and the preparation of anti-flunixin Xin Teyi antibodies, including polyclonal antibodies, monoclonal antibodies and recombinant antibodies, also belongs to the protection scope of the invention. The invention also provides any one of the following applications of the specific antibody:
(1) Use in the detection of flunixin;
(2) The application in preparing a flunixin detection kit;
(3) The application of the flunixin in preparing immunochromatography test strips.
The beneficial effects of the invention are as follows: the invention discloses a novel flunixin Xin Rengong antigen and a preparation method thereof for the first time, and the artificial antigen is used for immunizing animals to obtain a specific antibody with high titer and high sensitivity, the preparation process is simple and economical, and the detection sensitivity of the antibody can reach 0.40ng/mL. The method has strong practicability and great value for detecting the residue of the flunixin.
Drawings
FIG. 1 is a matrix assisted laser desorption tandem time of flight mass spectrometry (MALDI-TOF-MS) spectrum of the Fluoronii Xin Rengong antigen of example 1 of the present invention.
FIG. 2 is a graph showing the inhibition of the indirect competition ELISA standard of the cloned Fluoronii Xin Duo antibody in example 3 of the present invention.
Detailed Description
The following examples facilitate a better understanding of the present invention, but are not intended to limit the same. The experimental methods in the examples described below, unless otherwise indicated, are conventional methods well known to those skilled in the art. The test materials used in the examples described below, unless otherwise specified, were purchased from conventional biochemical reagent manufacturers. The quantitative tests in the following examples were all set up in triplicate and the results averaged. The phosphate buffers (abbreviated as PBS) used in the examples below were phosphate buffers of pH 7.4 and 0.01M, and the carbonate buffers (abbreviated as CB) used were sodium carbonate buffers of pH 9.6 and 0.05M. Bovine serum albumin is abbreviated as BSA, hemocyanin is abbreviated as KLH, 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride is abbreviated as EDC.HCl, N-hydroxysuccinimide is abbreviated as NHS, and N, N-dimethylformamide is abbreviated as DMF.
Example 1 preparation and characterization of the Flunii Xin Rengong antigen
1. Preparation of Fluoronii Xin Mianyi precursor
(1) 15mg of FLU was dissolved in 0.5mL of DMF, 15mg of EDC. HCl and 8mg of NHS were added thereto, and the reaction was magnetically stirred at room temperature for 10 hours to give a solution A.
(2) 10mg of KLH was dissolved in 10mL of PBS buffer to give solution B.
(3) And (3) dropwise adding the solution A into the solution B, stirring at room temperature, and reacting overnight to obtain a reaction product, namely the fluorous Xin Mianyi stock. The reaction product was dialyzed in PBS for 72 hours with 6 water changes in between. After dialysis, the reaction product was dispensed into 2mL centrifuge tubes and stored at-20℃for further use.
2. Preparation of Fluoronix Xin Bao quilt
(1) 15mg of FLU was dissolved in 0.5mL of DMF, 15mg of EDC. HCl and 8mg of NHS were added thereto, and the reaction was magnetically stirred at room temperature for 10 hours to give a solution A.
(2) 20mg of BSA was dissolved in 10mL of PBS buffer to obtain solution B.
(3) The solution A is added into the solution B dropwise, stirred at room temperature and reacted overnight to obtain a reaction product, namely flunixin Xin Bao quilt (FLU-BSA). The reaction product was dialyzed in PBS for 72 hours with 6 water changes in between. After dialysis, the reaction product was dispensed into 2mL centrifuge tubes and stored at-20℃for further use.
3. Determination of the ratio of the original coupling of Fluoronix Xin Bao
The synthesized product obtained in the step 3 is identified by MALDI-TOF-MS, and the coupling ratio is calculated. As a result, as shown in FIG. 1, the molecular weight of BSA was 66994.740, and the molecular weight of the synthesized product was 73573.701, indicating that the synthesis of the coating antigen was successful, and the coupling ratio was (73573.740-66994.701)/296=22, i.e., 22 flunixin haptens were coupled per BSA molecule on average.
Example 2 preparation of flunixin antiserum
Balb/c mice were immunized after emulsifying the source of Fluoronii Xin Mianyi obtained in example 1 with an equivalent amount of Freund's adjuvant, freund's complete adjuvant was used for the primary immunization, and Freund's incomplete adjuvant was used for the booster immunization. The immunization mode is subcutaneous multipoint injection of the cervical back, the immunization dose is 250 mug/mouse, the immunization interval is 8 weeks, and the immunization is performed three times. On day 7 after each immunization, tail vein blood is collected, centrifugation is carried out at 4000rpm for 10min, supernatant is collected and is taken as antiserum, and the antiserum is preserved at-20 ℃.
Example 3 identification of flunixin antisera
The antisera obtained in example 2 were identified using an indirect competition ELISA method. The specific operation steps are as follows:
(1) Coating: fluonig Xin Bao was originally diluted to 1. Mu.g/mL with CB solution, 100. Mu.L per well was added to the ELISA plate and incubated for 2 hours at 37 ℃;
(2) Washing: pouring out the liquid in the holes, washing 3 times with washing liquid for 1min each time, and drying on the absorbent paper;
(3) Closing: 150 μl of blocking solution was added to each well, incubated at 37deg.C for 1 hr, and washed;
(4) Sample adding: each well was added with 50. Mu.L of a series of flunixin Xin Biaozhun diluted in PBS and 50. Mu.L of working concentration of antiserum, incubated at 37℃for 30min, washed;
(5) Adding a secondary antibody: adding 100 mu L of HRP-goat anti-mouse IgG into each hole, incubating for 30min at 37 ℃, and washing;
(6) Color development: 100 mu L of freshly prepared TMB solution is added into each hole, and color development is carried out at 37 ℃ in a dark place for 15min;
(7) And (3) terminating: 50 mu L of H with the concentration of 2mol/L is added into each hole 2 SO 4 Stopping the reaction by the solution;
(8) Reading: reading OD of each well with an enzyme-labeled instrument 450nm A value;
(9) Establishing a standard curve: drawing a standard inhibition curve by taking the logarithmic value of the FLU standard substance concentration as an abscissa and the OD value corresponding to each concentration as an ordinate, and calculating IC 50 Values.
The result of the antiserum identification after three-phase is shown in figure 2, and the IC of the flunixin antiserum is obtained by calculation of a standard curve 50 0.16ng/mL.
While the invention has been described in detail in the foregoing general description and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.
Claims (1)
1. A method of preparing flunixin-specific antisera comprising the steps of:
(1) Mixing the fluorous Xin Mianyi antigen and equivalent Freund's adjuvant to emulsify immune mice;
(2) Primary immunization uses Freund's complete adjuvant, booster immunization uses Freund's incomplete adjuvant, and the immunization dose is 250 mug/dose, and the immunization interval is 8 weeks; the number of immunization times is 3;
the preparation method of the fluorous Xin Mianyi antigen comprises the following steps:
1) Dissolving 10-30mg flunixin, 10-40mg EDC.HCL and 10-40mg NHS in 0.2-2mL DMF, stirring and reacting for 4-48 hours to obtain solution A;
2) Dissolving 10-70mg hemocyanin in 10mL M phosphate buffer with pH of 7.4 to obtain solution B;
3) Dropwise adding the solution A into the solution B, stirring at room temperature, and reacting overnight;
4) Dialyzing the reaction solution in PBS for 1-3 days to obtain a fluorous-Xin Mianyi antigen;
the pro-Fluoronii Xin Mianyi is shown in a formula I, wherein K is hemocyanin;
a formula I;
(3) On day 7 after each immunization, tail vein blood is collected, centrifugation is carried out at 4000rpm for 10min, and supernatant fluid is collected, namely flunixin specific antiserum.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101955468A (en) * | 2010-08-19 | 2011-01-26 | 中国人民解放军军事医学科学院卫生学环境医学研究所 | Melamine hapten, melamine complete antigen and preparation method thereof |
| CN103833845A (en) * | 2013-12-05 | 2014-06-04 | 江南大学 | Method for synthesis of chlorothalonil artificial antigen |
| CN106867971A (en) * | 2017-04-27 | 2017-06-20 | 江南大学 | One plant of flunixin meglumine monoclonal antibody hybridoma cell strain YY and its application |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101955468A (en) * | 2010-08-19 | 2011-01-26 | 中国人民解放军军事医学科学院卫生学环境医学研究所 | Melamine hapten, melamine complete antigen and preparation method thereof |
| CN103833845A (en) * | 2013-12-05 | 2014-06-04 | 江南大学 | Method for synthesis of chlorothalonil artificial antigen |
| CN106867971A (en) * | 2017-04-27 | 2017-06-20 | 江南大学 | One plant of flunixin meglumine monoclonal antibody hybridoma cell strain YY and its application |
Non-Patent Citations (1)
| Title |
|---|
| Xiaona Chen,等.Development of an indirect competitive enzyme-linked immunosorbent assay for detecting flunixin and 5-hydroxyflunixin residues in bovine muscle and milk..Food and Agricultural Immunology.2019,第30卷(第30期),320-332. * |
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