CN113527390A - Tulathromycin hapten, artificial antigen and antibody, and preparation method and application thereof - Google Patents
Tulathromycin hapten, artificial antigen and antibody, and preparation method and application thereof Download PDFInfo
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- CN113527390A CN113527390A CN202110749563.0A CN202110749563A CN113527390A CN 113527390 A CN113527390 A CN 113527390A CN 202110749563 A CN202110749563 A CN 202110749563A CN 113527390 A CN113527390 A CN 113527390A
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- tulathromycin
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H17/00—Compounds containing heterocyclic radicals directly attached to hetero atoms of saccharide radicals
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H1/00—Processes for the preparation of sugar derivatives
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/76—Albumins
- C07K14/765—Serum albumin, e.g. HSA
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/76—Albumins
- C07K14/77—Ovalbumin
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/795—Porphyrin- or corrin-ring-containing peptides
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/44—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material not provided for elsewhere, e.g. haptens, metals, DNA, RNA, amino acids
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/94—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving narcotics or drugs or pharmaceuticals, neurotransmitters or associated receptors
- G01N33/9446—Antibacterials
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Abstract
The invention discloses a tulathromycin hapten, an artificial antigen and an antibody, a preparation method and application thereof, the tulathromycin hapten provided by the invention furthest reserves the characteristic structure of tulathromycin, and introduces maleimide group,the immunogenicity of the tulathromycin hapten is obviously enhanced; the tulathromycin artificial antigen obtained by coupling tulathromycin hapten and carrier protein is used for immunizing animals, so that the tulathromycin artificial antigen is more favorable for stimulating the immune response of the animals to generate antibodies with stronger specificity and higher sensitivity, and the IC of the tulathromycin antibodies to the tulathromycin is detected by an indirect competitive enzyme-linked immunosorbent assay50Is 0.3 mu g/L, has low cross reaction rate with other macrolide antibiotics, and provides a foundation for the subsequent establishment of various immunoassay methods of the tulathromycin.
Description
Technical Field
The invention belongs to the field of food safety detection. More specifically, the invention relates to a tulathromycin hapten, an artificial antigen and an antibody, and a preparation method and application thereof.
Background
Tulathromycin (Tulathromycin), also known as Tulathromycin, is a novel macrolide antibiotic which gradually replaces other macrolide drugs which are widely used in the market but have different degrees of drug resistance due to high drug effect, and is mainly used for treating livestock respiratory diseases caused by actinobacillus pleuropneumoniae, pasteurella and mycoplasma. The tulathromycin has the advantages of rapid drug absorption, long half-life period, high bioavailability and the like after administration, and is allowed to be used in animal production for the first time in No. 957 bulletin of China Ministry of agriculture. However, researches show that with the prolonging of the administration time and the increasing of the dosage, the tulathromycin can generate drug residues of different procedures in animal tissues, so that the detection of the drug residues is enhanced, and the tulathromycin has important significance for guaranteeing the safety of animal-derived foods.
At present, the methods for analyzing and detecting the residue of macrolide drugs such as tulathromycin and the like at home and abroad mainly comprise high performance liquid chromatography, high performance liquid chromatography-tandem mass spectrometry and the like, but have the defects of complicated sample pretreatment, long detection time, expensive instruments and the like, can not be widely applied in China, and do not meet the requirements of on-site detection on accurate detection and screening of a large number of samples at low cost in a short time. The immunological detection and analysis technology has the advantages of high sensitivity, high specificity, rapidness, simple and convenient operation and the like, is widely applied to the field of drug residue detection, and has many advantages compared with detection methods such as instruments and the like. Therefore, the immunoassay provides a new analysis and detection method for the residual study of the tulathromycin.
When an immunological detection method is established and the detection method is applied to detecting the residual quantity of the tulathromycin, the key technology is that an antibody with strong specificity and high sensitivity can be obtained, and the aim is to realize the key technology, and the precondition is to synthesize and prepare a proper tulathromycin hapten. Patent CN104710488B discloses a method for synthesizing hapten and complete antigen of tulathromycin, and the antigen is used for immunizing mice, and the serum titer reaches 1:8000, IC5098.5 mug/L; the patent CN107312754B discloses a hybridoma cell strain 1B3 secreting tulathromycin monoclonal antibody and application thereof, wherein the monoclonal antibody secreted by the cell strain is determined by an indirect competitive enzyme-linked immunosorbent assay to IC of tulathromycin50It was 0.79. mu.g/L. The method is characterized in that hydroxyl of the tulathromycin is derived by a succinic anhydride method, and free carboxyl is introduced to synthesize tulathromycin hapten. However, the tulathromycin contains a plurality of hydroxyl groups which can be used as sites for introducing the connecting arms, so when the tulathromycin reacts with succinic anhydride, a plurality of products are generated, the yield of the hapten is reduced, and certain difficulty is brought to the subsequent coupling of the hapten and the carrier protein.
Disclosure of Invention
Aiming at the defects in the prior art, the invention provides a hapten which can reserve the characteristic structure of tulathromycin to the maximum extent and is provided with a connecting arm with a certain length and a preparation method of the hapten; the artificial antigen prepared by the hapten and the antibody with high detection sensitivity and strong specificity; and the use of such haptens.
In order to achieve the object of the present invention, in a first aspect, the present invention provides a tulathromycin hapten which has the following structural formula:
the tulathromycin hapten provided by the invention introduces a maleimide group on the original structure of tulathromycin, and can be coupled with carrier protein by an N-succinyl-3- (2-pyridine-disulfide) propionate method (SPDP method) to obtain an artificial antigen for immunization; the tulathromycin hapten reserves all characteristic groups of tulathromycin, changes the original structural characteristics of tulathromycin to the minimum, introduces a spacer arm with a benzene ring, enhances the immunogenicity of the tulathromycin after being coupled with carrier protein, and lays a foundation for generating an antibody with stronger specificity and higher sensitivity for subsequent stimulation of animal immune response.
In a second aspect, the invention provides a preparation method of the tulathromycin hapten, which is obtained by reacting tulathromycin with N- (p-maleimide phenyl) isocyanate.
Further, the preparation method comprises the following steps: adding acetonitrile into the tulathromycin, stirring and dissolving, adding triethylamine, fully stirring, adding N- (p-maleimide phenyl) isocyanate, stirring and reacting at room temperature, and then separating and purifying to obtain tulathromycin hapten; wherein the molar ratio of the tulathromycin to the N- (p-maleimidophenyl) isocyanate is 1: 1.
Further, the preparation method comprises the following specific steps: 0.806g of tulathromycin is taken, 50mL of acetonitrile is added, stirring and dissolving are carried out, 0.6mL of triethylamine is added, after full stirring, 0.214g of N- (p-maleimide phenyl) isocyanate is added, stirring and reacting are carried out for 2h at room temperature, reduced pressure and evaporation are carried out to obtain yellow oily matter, 10mL of ethanol is added, and recrystallization is carried out to obtain tulathromycin hapten.
The preparation method of the hapten mainly comprises the following steps: (1) generating corresponding functional groups by using the existing structure or intermediate of an object to be detected through oxidation, reduction, substitution, hydrolysis and other reactions; (2) transforming the metabolite or structural analogue of the hapten into a required hapten by using the metabolite or structural analogue as a primer; (3) raw materials and reagents are used for re-synthesis, and small molecules with functional groups are introduced at proper positions. The method synthesizes the tulathromycin hapten with the maleimide group by reacting the tulathromycin with N- (p-maleimide phenyl) isocyanate, compared with the latter two methods, the method has the advantages of easily obtained used raw materials, simpler reaction operation, easily controlled reaction conditions and higher purity and yield of the prepared tulathromycin hapten.
In a third aspect, the invention provides a tulathromycin artificial antigen which is a conjugate obtained by coupling a carrier protein and the tulathromycin hapten. The tulathromycin artificial antigen can be used as immunogen and also can be used as coating antigen.
Further, the carrier protein is bovine serum albumin, ovalbumin, human serum albumin or hemocyanin; bovine serum albumin and egg albumin are preferred.
More specifically, the following are: an immunogen formed by tulathromycin hapten-Bovine Serum Albumin (BSA); tulathromycin hapten-Ovalbumin (OVA).
The tulathromycin hapten molecule is only immunoreactive and not immunogenic. Therefore, in order to confer immunogenicity on the tulathromycin hapten molecule, it is also necessary to couple and bind the tulathromycin hapten molecule to a suitable carrier protein molecule, thereby producing a tulathromycin artificial antigen that is both immunoreactive and immunogenic.
In a fourth aspect, the invention provides a method for preparing the artificial antigen of tulathromycin, which comprises coupling a carrier protein to a maleimide group of the tulathromycin hapten by adopting an N-succinyl-3- (2-pyridine-disulfide) propionate method.
In a fifth aspect, the invention provides a tulathromycin antibody, which is obtained by immunizing animals with the tulathromycin artificial antigen and can generate specific immunoreaction with tulathromycin.
Further, the tulathromycin antibody is a monoclonal antibody or a polyclonal antibody. In addition, the tulathromycin antibody can be prepared by a method conventional in the art.
In a specific embodiment, the tulathromycin antibody is a murine monoclonal antibody specific for a tulathromycin artificial antigen to the tulathromycin hapten described above.
The tylosin antibody obtained by using the tylosin artificial antigen has better titer, specificity and affinity, and low cross reaction rate with other macrolide antibiotics.
In a sixth aspect, the invention provides an application of the tulathromycin antibody in detection of tulathromycin residues.
The invention induces immune animals to generate antibodies through the tulathromycin artificial antigen, thereby being used in tulathromycin immunoassay analysis.
The tulathromycin immunoassay comprises but is not limited to tulathromycin ELISA kit, tulathromycin colloidal gold test strip and tulathromycin time-resolved fluorescence test strip.
By the technical scheme, the invention at least has the following advantages and beneficial effects:
the tulathromycin hapten provided by the invention retains the characteristic structure of tulathromycin to the maximum extent, and introduces a maleimide group, so that the immunogenicity of the tulathromycin hapten is obviously enhanced; the tulathromycin artificial antigen obtained by coupling the tulathromycin hapten and the carrier protein is used for immunizing animals, so that the tulathromycin artificial antigen is more beneficial to stimulating the immune response of the animals to generate antibodies with stronger specificity and higher sensitivity, and a foundation is provided for the subsequent establishment of various immunoassay methods of tulathromycin.
The preparation method of the tulathromycin hapten has the advantages of easily available raw materials, simpler reaction operation, easily controlled reaction conditions and higher purity and yield of the prepared tulathromycin hapten.
The tylosin antibody obtained by adopting the tylosin artificial antigen has better titer, specificity and affinity, and has IC (integrated Circuit) on the tylosin detected by an indirect competitive ELISA (enzyme-linked immunosorbent assay)500.3. mu.g/L, and has a low cross-reactivity with other macrolide antibiotics.
Drawings
FIG. 1 is a scheme for the synthesis of the tulathromycin hapten of the invention
Detailed Description
The present invention will be described in further detail with reference to specific examples, which are only preferred embodiments of the present invention and are not intended to limit the present invention.
Example 1
A preparation method of tulathromycin hapten comprises the following steps:
0.806g of tulathromycin is taken, 50mL of acetonitrile is added, stirring and dissolving are carried out, 0.6mL of triethylamine is added, after full stirring, 0.214g of N- (p-maleimide phenyl) isocyanate is added, stirring and reacting are carried out for 2h at room temperature, reduced pressure and evaporation to dryness are carried out, yellow oily matter is obtained, 10mL of ethanol is added, and recrystallization is carried out, thus obtaining 0.76g of tulathromycin hapten.
Example 2
A preparation method of tulathromycin artificial antigen comprises the following steps:
dissolving 100mg of BSA in 4mL of 0.05mol/L sodium bicarbonate buffer solution, adding 0.5mL of N, N-dimethylformamide solution containing 13.91mg of N-succinyl-3- (2-pyridine-disulfide) propionate (SPDP), and reacting at room temperature for 4h to obtain solution A; dripping 3mL of 0.5mol/L PBS buffer solution containing 12.83mg of tris (2-carboxyethyl) phosphine hydrochloride (TCEP-HCl) into the solution A, reacting for 30min, passing through a gel column, eluting and collecting to obtain a thiolated BSA protein solution, and recording as solution B; 30mg of the tulathromycin hapten prepared in example 1 is added with 1mL of N, N-dimethylformamide solution for dissolving, added into the solution B, reacted overnight at 4 ℃, dialyzed and purified by 0.02mol/L PBS to obtain tulathromycin artificial antigen coupled with bovine serum albumin, and subpackaged and stored at-20 ℃.
Example 3
A preparation method of tulathromycin artificial antigen comprises the following steps:
dissolving OVA 70mg in 0.05mol/L sodium bicarbonate buffer solution 3mL, adding N, N-dimethylformamide solution 0.5mL containing SPDP 9.71mg, and reacting at room temperature for 4h to obtain solution A; dripping 3mL of 0.5mol/L PBS buffer solution containing 12.83mg of TCEP-HC1 into the solution A, reacting for 30min, passing through a gel column, eluting and collecting to obtain a sulfhydrylation OVA protein solution, and recording as solution B; 22.1mg of the tulathromycin hapten prepared in example 1 is dissolved in 1mL of N, N-dimethylformamide solution, added into the solution B, reacted overnight at 4 ℃, dialyzed and purified by 0.02mol/L PBS to obtain tulathromycin artificial antigen coupled with ovalbumin, and subpackaged and stored at-20 ℃.
Example 4
A tulathromycin antibody is prepared by the following steps:
1. animal immunization
Taking 10 healthy female Balb/c mice (divided into two groups of A and B, and 5 mice in each group) in 6-8 weeks, emulsifying the mice by Freund complete adjuvant for primary immunization, and injecting the mice at subcutaneous multiple points on the back of the neck, wherein the immunization dose of each mouse is 200 mu g of tulathromycin artificial antigen coupled with bovine serum albumin; then boosting immunity and injecting subcutaneous multi-point on the back of the neck once every two weeks, and emulsifying by Freund incomplete adjuvant; the last immunization uses normal saline to replace Freund's incomplete adjuvant, and is injected in the abdominal cavity, and the injection dosage is the same as the previous times. The specific immunization procedure is shown in table 1.
Table 1 mouse immunization procedure
And (3) for the third time, the fourth time and 7d after the boosting immunization, the tail of the mouse is cut off, blood is taken, and the serum titer of the mouse is measured by an ELISA method, wherein the specific steps are as follows:
(1) diluting tulathromycin artificial antigen coupled with ovalbumin with 0.05mol/L of carbonate buffer solution with pH9.6 by a ratio of 1:1000, coating an enzyme label plate by 100 mu L of each hole, incubating for 2h at 37 ℃, throwing off coating solution, washing for 1 time by PBST, and patting to dry;
(2) adding 150 mu L of sealing liquid into each hole, reacting at 37 ℃ for 2h, then pouring off the sealing liquid, and patting to dry;
(3) adding 50 mu L of antiserum diluted by PBS in each hole, reacting at 25 ℃ for 30min, then removing the reaction liquid, washing 3-5 times by PBST, and patting dry at intervals of 30s each time;
(4) adding 100 mu L/hole of horseradish peroxidase-labeled goat anti-mouse anti-antibody (1:1000) diluted by PBS, reacting for 30min at 25 ℃, washing for 3-5 times by PBST (PBST), and patting dry at intervals of 30s each time;
(5) adding 50 μ L of substrate developing solution A and B into each well, reacting at 25 deg.C in dark for 15min, adding 50 μ L of 2mol/L H into each well2SO4Terminating the reaction by the solution;
(6) measuring OD value with wavelength of 450nm by enzyme-labeling instrument, and determining OD of sample hole450The titer of positive sera was determined as a dilution factor close to 1.
2. Cell fusion
(1) Preparing feeder cells: the Balb/c mice of 8-10 weeks old are killed after neck breakage, soaked in 75% alcohol for 5min, immediately placed in an ultra-clean bench with the abdomen facing upwards in a plate or fixed on a dissecting plate. The skin of the abdomen of the mouse is clamped by an ophthalmic forceps, a small opening is cut by scissors, and the peritoneum is not cut to avoid the outflow and pollution of the abdominal cavity fluid. Then blunt dissection was performed up and down with scissors to fully expose the peritoneum. Wiping peritoneum with alcohol cotton ball for sterilization. 5mL of RPMI-1640 basic culture solution was aspirated by a syringe, injected into the abdominal cavity of the mouse, the syringe was gently withdrawn, and the leg and tail of the mouse were shaken several times. The liquid in the abdominal cavity is pumped back by the original syringe and is injected into the centrifuge tube. The operation is repeated for 3-4 times. Centrifuging at 1000r/min for 10min, and discarding the supernatant. Resuspending the cells with 20-50 mL of complete culture medium, adding 100 μ L/well dropwise to the culture plate, and placing in an incubator for later use.
(2) Preparation of splenocytes: 3d after enhancing the immunity, taking an immune Balb/c mouse, collecting blood from an orbit, dislocating and killing the mouse, disinfecting the mouse in 75% alcohol, taking the spleen, removing connective tissues, preparing a spleen cell suspension, transferring the spleen cell suspension into a 50mL centrifuge tube, adding RPMI-1640 to 30mL, centrifuging the spleen cell suspension at 1500-2000 r/min for 5min, removing a supernatant, adding RPMI-1640 to 30mL, and counting the number for later use.
(3) Myeloma cell preparation: taking 3 bottles of myeloma cells with good growth state (the number of living cells is more than 95 percent), completely blowing down the myeloma cells, transferring the myeloma cells into a 50mL centrifuge tube, adding RPMI-1640 to 30mL, centrifuging at 1500-2000 r/min for 5min, discarding the supernatant, adding RPMI-1640 to 30mL, and counting for later use.
(4) Cell mixing: spleen cells and myeloma cells are mixed and centrifuged at 1500-2000 r/min for 5min, wherein the ratio of the spleen cells to the myeloma cells is 8: 1.
(5) Cell fusion: centrifuging the mixed cells, pouring out the supernatant, making the precipitated cell mass into paste, placing the paste in a water bath at 37 ℃, adding 1mL of fusion agent which is polyethylene glycol (PEG)4000 within 1min, acting for 2min, slightly stirring the cells, adding 20mL of serum-free PEG nutrient solution within 4min, centrifuging at 1000r/min for 10min, and discarding the supernatant. The cells were resuspended in 20-50 mL of complete medium, plated on 96-well feeder cells-containing cell culture plates at 100. mu.L per well, and placed in an incubator.
3. Cell line selection
And (3) when the cells grow to 1/3-1/2 of the bottom of the hole, carrying out antibody detection. Screening culture wells with hybridoma cell growth by adopting an ELISA method, wherein the screening comprises two steps: in the first step, positive cell holes are screened by indirect ELISA, in the second step, tulathromycin is selected as a standard substance, and the inhibition effect of positive cells is measured by indirect competitive ELISA. And selecting a well with better inhibition to the tulathromycin standard, carrying out subcloning by adopting a limiting dilution method, and carrying out detection by using the same method. Repeating the steps for three times to obtain the cell strain capable of stably secreting the tulathromycin monoclonal antibody.
4. Preparation of ascites
Liquid paraffin was injected into Balb/c mice for 6-8 weeks at 500. mu.L/mouse. 10 days later, hybridoma cells in logarithmic growth phase were collected in RPMI-1640 basic medium, counted in a hemocytometer at a cell concentration of 1.0X 10 and a microscope6～1.5×106In the size per mL range. Each mouse was injected with 0.5mL hybridoma cells into the abdominal cavity. Note that after one week the abdomen of the mouse was enlarged, ascites was collected in the abdomen of the mouse with a sterile syringe once every one to two days, and this was repeated until the mouse died naturally. Centrifuging at 4 deg.C for 5min at 5000r/min, collecting supernatant, and removing fat and protein membrane floating on the upper layer of abdominal water.
5. Antibody purification
The monoclonal antibody is purified by an octanoic acid-ammonium sulfate method.
6. Antibody titer determination
And (3) measuring the antibody titer by adopting an indirect ELISA method, and referring to the step 1, measuring the serum titer of animal immunity. The result shows that the titer of the tulathromycin monoclonal antibody is more than or equal to 32000.
7. Antibody cross-reactivity assay
The indirect competitive ELISA method is adopted for determination, and the result shows that the cross-reaction rate of the tulathromycin monoclonal antibody to tulathromycin and other macrolide antibiotics is as follows: the content of the tulathromycin is 100 percent, and the content of the erythromycin, the roxithromycin, the clarithromycin, the azithromycin, the spiramycin, the tylosin, the tilmicosin and the kitasamycin is less than 1 percent. Therefore, the prepared antibody has better specificity.
The above-mentioned embodiments only express the embodiments of the present invention, and the description is more specific and detailed, but not understood as the limitation of the patent scope of the present invention, but all the technical solutions obtained by using the equivalent substitution or the equivalent transformation should fall within the protection scope of the present invention.
Claims (9)
1. A tulathromycin hapten, which has the following structural formula:
2. the method of preparing a tulathromycin hapten as claimed in claim 1, which is obtained by reacting tulathromycin with N- (p-maleimidophenyl) isocyanate.
3. The method of preparing a tulathromycin hapten of claim 2, wherein the method comprises: adding acetonitrile into the tulathromycin, stirring and dissolving, adding triethylamine, fully stirring, adding N- (p-maleimide phenyl) isocyanate, stirring and reacting at room temperature, and then separating and purifying to obtain tulathromycin hapten; wherein the molar ratio of the tulathromycin to the N- (p-maleimidophenyl) isocyanate is 1: 1.
4. The method for preparing the tulathromycin hapten as claimed in claim 2, which is characterized in that the method comprises the following steps: 0.806g of tulathromycin is taken, 50mL of acetonitrile is added, stirring and dissolving are carried out, 0.6mL of triethylamine is added, after full stirring, 0.214g of N- (p-maleimide phenyl) isocyanate is added, stirring and reacting are carried out for 2h at room temperature, reduced pressure and evaporation are carried out to obtain yellow oily matter, 10mL of ethanol is added, and recrystallization is carried out to obtain tulathromycin hapten.
5. An artificial antigen of tulathromycin which is a conjugate of a carrier protein and a tulathromycin hapten as defined in claim 1.
6. The tulathromycin artificial antigen of claim 5, wherein the carrier protein is bovine serum albumin, ovalbumin, human serum albumin, or hemocyanin.
7. The method for preparing the artificial antigen of tulathromycin as claimed in claim 5 or 6, wherein the carrier protein is coupled to the maleimide group of the tulathromycin hapten as claimed in claim 1 by N-succinyl-3- (2-pyridyldithio) propionate method.
8. A tulathromycin antibody which is obtained by immunizing an animal with the tulathromycin artificial antigen of claim 5 and which specifically immunoreacts with tulathromycin.
9. Use of the tulathromycin antibody of claim 8 for detecting tulathromycin residues.
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