CN114316027A

CN114316027A - Flunixin artificial antigen and preparation method and application thereof

Info

Publication number: CN114316027A
Application number: CN202011077631.5A
Authority: CN
Inventors: 温凯; 王战辉; 沈建忠; 于雪芝; 余文博; 米佳飞; 张素霞; 史为民
Original assignee: China Agricultural University
Current assignee: China Agricultural University
Priority date: 2020-10-10
Filing date: 2020-10-10
Publication date: 2022-04-12
Anticipated expiration: 2040-10-10
Also published as: CN114316027B

Abstract

The invention relates to a flunixin artificial antigen and a preparation method and application thereof. The artificial flunixin antigen provided by the invention is a compound shown in a formula I, wherein K represents a carrier protein. The artificial antigen provided by the invention can be used for immunizing animals to prepare high-sensitivity specific antibodies, and the method is simple, convenient and easy to implement. The artificial antigen and the antibody prepared by the artificial antigen are applied to veterinary drug residue detection, so that rapid high-throughput detection of flunixin on a trace level can be realized。

Description

Flunixin artificial antigen and preparation method and application thereof

Technical Field

The invention relates to the technical field of biochemical engineering, and particularly relates to a flunixin artificial antigen and a preparation method and application thereof.

Background

Flunixin (Flunixin, abbreviated as FLU) is a special non-steroidal anti-inflammatory drug for animals, and is usually presented with a stabilizer Meglumine (Meglumine) in an equal proportion to form stable Flunixin Meglumine salt (Flunixin Meglumine, abbreviated as FM). The pharmacological action mechanism of the compound mainly reduces 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 is usually used for treating acute inflammation of large animals such as pigs, cows, horses and the like caused by various disease infections; in addition, the compound is combined with antibiotics, has obvious drug synergism, and can effectively improve clinical symptoms of diseases. Since the successful development in the 90 s of the 20 th century, flunixin meglumine has been widely used in various countries including China, and has become the most widely used non-steroidal anti-inflammatory drug for veterinary clinical use at home and abroad.

In order to strengthen the supervision of the residual amount of the flunixin in the animal food and ensure the quality safety of the animal food, the European Committee stipulates that the maximum residual limit amount of the flunixin in the muscle, fat, liver and kidney tissues of the cattle is 20, 30, 300 and 100 mu g/kg respectively; the FDA in the united states prescribes maximum residual limits of flunixin in porcine muscle and liver tissue of 25 and 30 μ g/kg, respectively.

At present, the liquid chromatography-mass spectrometry/mass spectrometry method is mainly adopted for detecting the residue of flunixin in animal-derived food in national industrial standards. Although the method has strong specificity and high sensitivity, the sample pretreatment is 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 an antigen and an antibody, has strong specificity, high sensitivity, quick reaction and simple operation, and is suitable for the real-time detection of a large number of samples on site.

In limited research involving the preparation of artificial antigens and antibodies to flunixin, Lin et al used a mixture of flunixin meglumine as a hapten coupled to a carrier protein to prepare artificial antigens to flunixin. The artificial antigen prepared by the method cannot be accurately characterized in the preparation and application processes, is difficult to control the quality, and is not beneficial to the popularization and application of the method. Lin L, Jiang W, Xu L, et al.development of IC-ELISA and immunochemical strip assay for the detection of a thin peptide in milk [ J ]. Food & Agricultural Immunology,2017: 1-11.

Chen et al use the metabolite of flunixin 5-hydroxyflunixin coupled with carrier protein to prepare artificial antigen of flunixin, the antibody prepared by the method can better identify 5-hydroxyflunixin, but the sensitivity to flunixin is poor, IC₅₀It was 1.43 ng/mL. Chen et al also tried to prepare artificial antigen of flunixin by coupling flunixin as hapten with carrier protein, but the immune effect was worse than that of 5-hydroxyflunixin, so 5-hydroxyflunixin coupled with carrier protein was finally selected as artificial antigen of flunixin. Chen X, Peng S, Liu C, et al, development of an index complementary enzyme-linked immunological assay for detecting a fluent and 5-hydroxyfluent reactants in a bone muscle and mill [ J].Food&Agricultural Immunology,2019,30(1):320-332。

Disclosure of Invention

In order to solve the defects of the prior art, the invention aims to provide a flunixin artificial antigen with strong immune effect, and a preparation method and application thereof.

In order to achieve the purpose, the invention provides a flunixin artificial antigen with a structure shown in a formula I in a first aspect.

In 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 immunogen; when K is bovine serum albumin, it is used as a coating antigen.

In a second aspect, the present invention provides a method for preparing the artificial antigen of flunixin, wherein flunixin is used as a hapten to be chemically coupled with a carrier protein, and the preparation method comprises the following steps:

(1) flunixin (FLU), 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC. HCL) and N-hydroxysuccinimide (NHS) are dissolved in N, N-Dimethylformamide (DMF) and reacted for 4-48 hours to obtain solution A.

(2) And dissolving the carrier protein in a phosphate buffer solution to obtain a solution B.

(3) Mixing the solution A and the solution B, and reacting at room temperature for 6-48 hours to obtain the flunixin artificial antigen.

Specifically, the method comprises the following steps:

(1) dissolving FLU 10-30mg, EDC.HCL 10-40mg and NHS 10-40mg in DMF 0.2-2mL, and reacting under stirring for 4-48 hr to obtain solution A.

(2) 10-70mg of carrier protein was dissolved in 10mL of PBS (0.01M, pH 7.4) to obtain solution B.

(3) And dropwise adding the solution A into the solution B, stirring at room temperature, and reacting overnight.

(4) And 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 artificial antigen with excellent immune effect and controllable quality, the invention selects high-purity flunixin as hapten to be coupled with carrier protein to prepare the flunixin artificial antigen with quantitatively characterized coupling ratio.

In order to improve the immune effect of the artificial flunixin antigen prepared by the invention, the invention also provides a matched novel immune program. The invention provides a novel immunization method for preparing a flunixin specific antibody by the flunixin artificial antigen. The specific method comprises the following steps:

mixing and emulsifying the flunixin immunogen and equivalent Freund's adjuvant, and then immunizing female Balb/c mice with age of 6-8 weeks and weight of 8-20g, wherein Freund's complete adjuvant is used for primary immunization, and Freund's incomplete adjuvant is used for boosting immunization. The immunization mode is subcutaneous multipoint injection of neck and back, the immunization dose is 250 mug/mouse, the immunization interval is 60 days, and 3 times of immunization are carried out totally. On the 7 th day after each immunization, tail vein blood was collected, centrifuged at 4000rpm for 10min, and the supernatant was collected as antiserum, which was stored at-20 ℃.

The immunization program provided by the invention increases the immunization dose, prolongs the immune cycle, 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 skilled person, the preparation of anti-flunixin specific antibodies, including polyclonal antibodies, monoclonal antibodies and recombinant antibodies, by using the flunixin artificial antigen of the invention or the flunixin artificial antigen prepared by the preparation method of the invention as an immunogen also belongs to the protection scope of the invention. The invention also provides any one of the following applications of the specific antibody:

(1) the application of the flunixin in detecting flunixin;

(2) the application in the preparation of a detection kit of flunixin;

(3) the application in the preparation of an immunochromatographic test strip of flunixin.

The invention has the beneficial effects that: the invention discloses a novel flunixin artificial antigen and a preparation method thereof for the first time, 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 economic, and the detection sensitivity of the antibody can reach 0.40 ng/mL. The method is high in practicability and has 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 an artificial antigen of flunixin in example 1 of the present invention.

FIG. 2 is a graph showing the standard inhibition of indirect competitive ELISA with a polyclonal antibody against flunixin in example 3 of the present invention.

Detailed Description

The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are, unless otherwise specified, conventional procedures well known to those skilled in the art. The test materials used in the following examples were purchased from conventional biochemical manufacturers unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged. Phosphate buffer solutions (abbreviated as PBS) used in the following examples were all phosphate buffer solutions of pH 7.4 and 0.01M, and carbonate buffer solutions (abbreviated as CB) were all sodium carbonate buffer solutions 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 Flunixin Artificial antigen

1. Preparation of flunixin immunogens

(1) 15mg of FLU was dissolved in 0.5mL of DMF, and 15mg of EDC.HCL and 8mg of NHS were added, and the reaction was magnetically stirred at room temperature for 10 hours to obtain solution A.

(2) 10mg of KLH was dissolved in 10mL of PBS buffer to obtain solution B.

(3) And dropwise adding the solution A into the solution B, stirring at room temperature, and reacting overnight to obtain a reaction product, namely the flunixin immunogen. The reaction product was dialyzed against 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 flunixin coating antigen

(2) 20mg BSA was dissolved in 10mL PBS buffer to obtain solution B.

(3) And dropwise adding the solution A into the solution B, stirring at room temperature, and reacting overnight to obtain a reaction product, namely flunixin coated antigen (FLU-BSA). The reaction product was dialyzed against 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 coupling ratio of flunixin coating source

Identifying the synthesized product obtained in the step 3 by using MALDI-TOF-MS, and calculating the coupling ratio. The results are shown in figure 1, where BSA has a molecular weight of 66994.740 and the synthesized product has a molecular weight of 73573.701, indicating successful synthesis of the coating source and a coupling ratio of (73573.740-66994.701)/296 ═ 22, i.e. 22 flunixin haptens are coupled per BSA molecule on average.

EXAMPLE 2 preparation of Flunixin antiserum

The flunixin immunogen obtained in example 1 was mixed and emulsified with an equal amount of Freund's adjuvant and then used to immunize Balb/c mice with Freund's complete adjuvant for the primary immunization and Freund's incomplete adjuvant for the booster immunization. The immunization mode is subcutaneous multipoint injection of neck and back, the immunization dose is 250 mu g/mouse, the immunization interval is 8 weeks, and three times of immunization are carried out. On the 7 th day after each immunization, tail vein blood was collected, centrifuged at 4000rpm for 10min, and the supernatant was collected as antiserum, which was stored at-20 ℃.

Example 3 identification of Flunixin antisera

The antiserum obtained in example 2 was identified by indirect competition ELISA. The specific operation steps are as follows:

(1) coating: diluting the flunixin coating source to 1 mu g/mL by using CB solution, adding 100 mu L of flunixin coating source to an ELISA plate in each hole, and incubating for 2 hours at 37 ℃;

(2) washing: pouring out liquid in the holes, washing for 3 times with washing liquid for 1min each time, and patting dry on absorbent paper;

(3) and (3) sealing: adding 150 mu L of confining liquid into each hole, incubating for 1 hour at 37 ℃, and washing;

(4) sample adding: adding a serial concentration of flunixin standard substance diluted by 50 muL PBS and 50 muL of antiserum with working concentration into each hole, incubating for 30min at 37 ℃, and washing;

(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: adding 100 μ L of freshly prepared TMB solution into each well, and developing at 37 deg.C in dark for 15 min;

(7) and (4) terminating: 50 μ L of 2mol/L H was added to each well₂SO₄Terminating the reaction by the solution;

(8) reading: read OD of each well with microplate reader_450nmA value;

(9) establishing a standard curve: taking the logarithm value of the concentration of the FLU standard substance as an abscissa, taking the OD value corresponding to each concentration as an ordinate, drawing a standard inhibition curve, and calculating IC₅₀The value is obtained.

The antiserum after the three-immunization is identified as shown in figure 2, and the IC of the flunixin antiserum is calculated by a standard curve₅₀It was 0.16 ng/mL.

Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Claims

1. The flunixin artificial antigen is characterized by having a structural formula shown as a formula I:

2. the artificial antigen of flunixin according to claim 1, wherein K in formula i is bovine serum albumin or human serum albumin or ovalbumin or hemocyanin.

3. The artificial antigen of flunixin according to claim 1 or 2, wherein K in formula i is hemocyanin, as an immunogen; when K is bovine serum albumin, it is used as a coating antigen.

4. A process for the preparation of a flunixin artificial antigen as claimed in any one of claims 1 to 3, characterized in that flunixin is used as hapten for chemical coupling with a carrier protein.

5. The method of claim 4, comprising the steps of:

(1) dissolving flunixin, 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide in N, N-dimethylformamide, and reacting for 4-48 hours to obtain a solution A;

(2) dissolving carrier protein in phosphate buffer solution to obtain solution B;

6. The method of claim 5, comprising the steps of:

(1) dissolving 10-30mg of flunixin, 10-40mg of EDC, HCL and 10-40mg of NHS in 0.2-2mL of DMF, and reacting for 4-48 hours under stirring to obtain solution A;

(2) dissolving 10-70mg of carrier protein in 10mL of phosphate buffer solution with the concentration of 0.01M and the pH value 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 the artificial antigen of flunixin.

7. A novel immunization method for preparing a flunixin-specific antibody from the flunixin artificial antigen of any one of claims 1 to 3 or the flunixin artificial antigen prepared by the preparation method of any one of claims 4 to 6, comprising the following steps:

(1) mixing flunixin immunogen and equivalent Freund's adjuvant to emulsify and immunize mouse;

(2) the Freund complete adjuvant is used for primary immunization, the Freund incomplete adjuvant is used for boosting immunization, the immunization dose is 250 mu g/mouse, and the immunization interval is 60 days; the number of immunizations was 3.

8. Use of the artificial antigen of flunixin according to any one of claims 1 to 3 or the artificial antigen of flunixin prepared by the preparation method according to any one of claims 4 to 6 for preparing an anti-flunixin antibody.

9. Antibodies produced by using the artificial antigen of flunixin according to any one of claims 1 to 3 or the artificial antigen of flunixin produced by the production method according to any one of claims 4 to 6 as an immunogen, including polyclonal antibodies, monoclonal antibodies and recombinant antibodies.

10. Use of the antibody of claim 9 for any one of:

(1) the application of the flunixin in detecting flunixin;

(2) the application in the preparation of a detection kit of flunixin;