CN108178777B - Tylosin hapten, artificial antigen and antibody as well as preparation methods and applications thereof - Google Patents

Tylosin hapten, artificial antigen and antibody as well as preparation methods and applications thereof Download PDF

Info

Publication number
CN108178777B
CN108178777B CN201711464700.6A CN201711464700A CN108178777B CN 108178777 B CN108178777 B CN 108178777B CN 201711464700 A CN201711464700 A CN 201711464700A CN 108178777 B CN108178777 B CN 108178777B
Authority
CN
China
Prior art keywords
tylosin
hapten
antibody
artificial antigen
enzyme
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
CN201711464700.6A
Other languages
Chinese (zh)
Other versions
CN108178777A (en
Inventor
沈玉栋
姚婵媛
杨金易
王弘
徐振林
孙远明
韦晓群
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
South China Agricultural University
Original Assignee
South China Agricultural University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by South China Agricultural University filed Critical South China Agricultural University
Priority to CN201711464700.6A priority Critical patent/CN108178777B/en
Publication of CN108178777A publication Critical patent/CN108178777A/en
Application granted granted Critical
Publication of CN108178777B publication Critical patent/CN108178777B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Classifications

    • 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
    • C07H17/04—Heterocyclic radicals containing only oxygen as ring hetero atoms
    • C07H17/08—Hetero rings containing eight or more ring members, e.g. erythromycins
    • 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
    • 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
    • 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
    • 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
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/581—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with enzyme label (including co-enzymes, co-factors, enzyme inhibitors or substrates)

Landscapes

  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Molecular Biology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Biophysics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Immunology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Toxicology (AREA)
  • Urology & Nephrology (AREA)
  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • Zoology (AREA)
  • Hematology (AREA)
  • Cell Biology (AREA)
  • Microbiology (AREA)
  • Food Science & Technology (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Peptides Or Proteins (AREA)

Abstract

The invention discloses a tylosin hapten, an artificial antigen and an antibody, and preparation methods and applications thereof. Specifically, the glyoxal functional group of the large-ring side chain of the tylosin is used as a derivative coupling site, and the glyoxal group is converted into carboxyl by a one-step oxidation method, so that the tylosin hapten of carboxymethylation coupling arms is obtained. The obtained hapten has high overlapping degree with a framework structure, and lays a foundation for the exposure of a tylosin characteristic structure and the generation of a high-specificity antibody. The constructed enzyme-linked immunosorbent assay method is high in accuracy and sensitivity, the detection limit reaches 0.051ng/mL, the half inhibition concentration is 1.39ng/mL, the operation is simple, the cost is low, the enzyme-linked immunosorbent assay method is very suitable for field detection, can be used for quickly and effectively detecting tylosin in environment and food samples, and has wide application prospect and economic value.

Description

Tylosin hapten, artificial antigen and antibody as well as preparation methods and applications thereof
Technical Field
The invention belongs to the technical field of drug residue detection. More particularly, relates to a tylosin hapten, an artificial antigen and an antibody, and a preparation method and application thereof.
Background
Tylosin (Tylosin, TYL), also known as tylon, is a macrolide antibiotic obtained from the culture broth of streptomyces fradiae in Hamill et al 1959. TYL has obvious inhibition effect on gram-positive bacteria, partial gram-negative bacteria and good treatment effect on livestock and poultry mycoplasma diseases, and is a preferred medicament for treating the mycoplasma diseases. Has certain inhibition and killing effects on most spirochaete, viruses and the like, and also has the effects of promoting the growth of animals and improving the efficiency of feed, so the feed additive is widely applied to the livestock breeding industry. However, the residues of these drugs can cause anaphylaxis (rash, anaphylactic shock, etc.) of human bodies, inhibit the growth of normal sensitive flora in intestinal tracts, cause pathogenic bacteria and candida to multiply greatly to cause systemic or local infection, and find that the tylosin-resistant enterococcus has resistance to erythromycin, and the cross-resistance with other antibiotics can cause the human bodies to generate resistance to the antibiotics, thereby bringing great harm to clinical treatment.
The use of tylosin as a growth promoter was banned by the european union committee in 1999. However, tylosin is also used in some countries as a growth promoter for poultry, pigs and cattle. For long-term interest in human health, the food code board, the european union board and some countries have established the maximum residual limit of tylosin in livestock food. The 235 th file published in 2002 by Ministry of agriculture in China, the highest residual quantity of veterinary drugs in animal food, also makes the highest residual quantity limit standard for tylosin. The limit values vary for different species and different tissues, e.g. 50 μ g/kg in milk and 200 μ g/kg in eggs and pork, entrails etc. Therefore, the research and the preparation of the method for simply, quickly, sensitively and reliably detecting the tylosin residue in the food have important significance for improving the food safety monitoring system in China.
At present, a plurality of methods for detecting the residual quantity of tylosin in animal-derived foods at home and abroad mainly comprise a microbiological method, a high performance liquid chromatography, a chromatography-mass spectrometry combined method, an immunoassay method (comprising an enzyme-linked immunosorbent assay and a colloidal gold immunochromatography) and the like. The microbiological method is gradually replaced by other methods due to the fatal defects of long analysis period, complicated experimental steps, low sensitivity and the like. The detection method of instruments such as the high performance liquid chromatography and the like is accurate in detection, high in sensitivity and good in repeatability, but the problems of high cost, long time, incapability of meeting high flux, field detection and the like still exist. The immunoassay method based on antigen-antibody specific reaction is mature at present, has high sensitivity, low cost and short detection time compared with other instrument methods, is also suitable for detecting high-throughput samples, and gradually becomes one of the main methods for rapidly screening and monitoring toxic and harmful residues.
At present, most of technologies for detecting tylosin by using an enzyme-linked immunosorbent assay at home and abroad utilize organic acid with amino to react with aldehyde groups on large rings of the tylosin to obtain derivative haptens with carboxyl structures. The haptens have complex structures, long coupling arms, easy folding, difficult exposure to the surface of carrier protein, poor repeatability of immune effect and high requirements on reaction raw materials, and cause the change of skeleton conformation.
Disclosure of Invention
The technical problem to be solved by the invention is to overcome the defects and shortcomings of the existing tylosin residue detection technology and provide a novel tylosin hapten structure, and particularly, the glyoxal group of a macrolide side chain of a tylosin molecule is directly oxidized into carboxyl as a hapten in one step, and an acetic acid arm after oxidation is moderate in length, is not suitable for folding and has high overlapping degree with a skeleton structure, so that a tylosin three-dimensional structure is maintained to the maximum extent, stable immune induction of an antibody is facilitated, and further, the hapten is coupled with a carrier protein, the specific structure of the hapten protrudes out of the surface of the carrier protein, an epitope as a carrier is exposed to an animal immune system, a specific antibody can be obtained, and a foundation is laid for establishing an immune detection method.
The invention aims to provide a tylosin hapten and a preparation method thereof.
The invention also aims to provide a tylosin artificial antigen and a preparation method thereof.
Still another object of the present invention is to provide a tylosin antibody and uses thereof.
The above purpose of the invention is realized by the following technical scheme:
a tylosin hapten, the molecular structural formula of which is shown in formula (I):
Figure BDA0001530909450000021
the application of the tylosin hapten in preparing the tylosin artificial antigen is also in the protection scope of the invention.
The tylosin artificial antigen is obtained by coupling the tylosin hapten and carrier protein, and the molecular structural formula of the tylosin artificial antigen is shown as a formula (II):
Figure BDA0001530909450000031
wherein the carrier protein is Bovine Serum Albumin (BSA), Ovalbumin (OVA) or Keyhole Limpet Hemocyanin (KLH).
The application of the tylosin hapten or the tylosin artificial antigen in preparing tylosin antibodies (including polyclonal antibodies) also falls within the protection scope of the present invention.
The immunogen and the coating antigen combination of the enzyme-linked immunoassay tylosin are obtained by coupling the hapten and BSA (bovine serum albumin), and the coating antigen is obtained by coupling the hapten and OVA.
The application of the immunogen and the coating antigen combination in the detection of tylosin or the construction of a tylosin enzyme-linked immunoassay method also belongs to the protection scope of the invention.
A tylosin antibody (including polyclonal antibody) prepared by using the above tylosin artificial antigen as immunogen is also within the protection scope of the present invention.
In addition, the preparation method of the tylosin hapten provided by the invention is to oxidize the special aldehyde group of the macrolide side chain in the tylosin molecule into carboxyl in one step to obtain the tylosin hapten.
Specifically, the preparation method of the tylosin hapten comprises the following steps:
(1) dissolving tylosin tartrate in a mixed solution of tert-butyl alcohol and water, and sequentially adding NaH at 0 DEG C2PO4Dimethyl sulfoxide, NaClO2Carrying out aldehyde group oxidation reaction;
(2) adding ethyl acetate and water into the reaction solution for extraction, and collecting an organic phase; extracting the water phase with ethyl acetate, and collecting to obtain an organic phase; the combined organic phases were washed with water and brine, then anhydrous MgSO4Drying and filtering, evaporating the filtrate under reduced pressure to remove the solvent, and purifying the residue by silica gel column chromatography to obtain white solid, namely the tylosin hapten.
Preferably, the volume ratio of the tert-butyl alcohol to the water in the step (1) is 4-6: 3.
more preferably, the volume ratio of the tert-butanol to the water in the step (1) is 5: 3.
preferably, the tylosin tartrate and NaH of step (1)2PO4、NaClO2In a molar ratio of 1: 3-5: 1.5 to 2.5.
More preferably, the tylosin tartrate and NaH of step (1)2PO4、NaClO2In a molar ratio of 1: 4: 2.
in addition, the preparation method of the tylosin artificial antigen is that the tylosin hapten and carrier protein are coupled by an active ester method to prepare the tylosin artificial antigen; the method specifically comprises the following steps:
(1) dissolving tylosin hapten as defined in claim 1 in DMF, adding DCC and NHS, and reacting under stirring at 4 ℃;
(2) weighing carrier protein, dissolving the carrier protein in a phosphate buffer solution, centrifuging the reaction solution obtained in the step (1), taking out supernatant, dropwise adding the supernatant into the phosphate buffer solution of the carrier protein, and continuing to react at 4 ℃;
(3) dialyzing the reaction solution at room temperature for 3 days, and changing the dialyzate for 2 times a day to obtain the tylosin artificial antigen.
Preferably, the dosage ratio of the tylosin hapten to DCC and NHS in the step (1) is 1: 1.5-2.5: 1.5 to 2.5.
More preferably, the dosage ratio of tylosin hapten to DCC and NHS in step (1) is 1: 2: 2.
preferably, the molar ratio of carrier protein to tylosin hapten is 60: 1.
preferably, the volume ratio of DMF to phosphate buffer solution is 1: 10.
preferably, the reaction time in the step (1) is 8-12 h.
Preferably, the reaction time in the step (2) is 8-12 h.
The invention has the following beneficial effects:
according to the invention, the glyoxal functional group of the large-ring side chain of theiletin is used as a derivative coupling site, and aldehyde group is converted into carboxyl by a one-step oxidation method, so that the tyliletin hapten of carboxymethylation coupling arm is obtained; the length of the oxidized acetic acid arm is moderate, the acetic acid arm is not suitable for folding, the hapten is highly consistent with the tylosin molecule, the overlapping degree with a skeleton structure is high, the stable conformation of the acetic acid arm is completely overlapped with the stable conformation of the tylosin molecule, the three-dimensional structure characteristic of the tylosin molecule is kept to the maximum extent, the stable immune induction of an antibody is facilitated, the hapten is coupled with carrier protein, the specific structure of the hapten protrudes out of the surface of the carrier protein, the hapten is used as an antigen epitope of a carrier and is exposed to an animal immune system, a specific antibody can be obtained, and a foundation is laid for establishing an immune detection method.
The method further adopts an active lipid method to couple the hapten with carrier protein to obtain an artificial antigen, obtains a tylosin high-specificity antibody through immunizing animals with the artificial antigen, has no cross with other structural and functional analogues, further optimizes and establishes a tylosin enzyme-linked immunosorbent assay method by using the antigen-antibody, has the detection limit of 0.051ng/mL, the half inhibition concentration of 1.39ng/mL, good accuracy and sensitivity, can be used for quickly and effectively detecting tylosin in environmental and food samples, and has wide application prospect.
Moreover, the method for synthesizing the hapten is simple and easy to master, and the established enzyme-linked immunosorbent assay has high sensitivity and accuracy, simple operation and low cost and is very suitable for field detection; the method lays a good foundation for efficient, cheap and rapid immunoassay products, and has good application prospect and economic benefit.
Drawings
FIG. 1 is a structural formula of tylosin hapten.
FIG. 2 is a mass spectrum of tylosin hapten.
FIG. 3 is a tylosin standard curve.
Detailed Description
The invention is further described with reference to the drawings and the following detailed description, which are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise specified; the reagents and materials used in the following examples are all commercially available.
EXAMPLE 1 preparation of tylosin hapten
(1) Dissolving 1g of tylosin tartrate in a mixture of 50mL of tert-butanol and 30mL of water, and sequentially adding 0.62g of NaH at 0 DEG C2PO41mL of dimethyl sulfoxide, 0.2g of NaClO2Reacting for 30 min;
(2) ethyl acetate (50mL) and water (10mL) were added to the reaction mixture and extracted 3 times, and the organic phase was collected. The aqueous phase was extracted three more times with ethyl acetate (50 mL). The combined organic phases were washed with water (20mL) and brine (20mL), anhydrous MgSO4Dried and filtered. The filtrate was rotary evaporated under reduced pressure and purified by silica gel column chromatography (methanol: chloroform: 1: 10) to obtain a white solid, which was the hapten.
The mass spectrum of the tylosin hapten is shown in FIG. 2, and an ion peak with m/z of 932.5 appears, which indicates that the molecular weight of the product after deprotonation is 931.5, and is consistent with the theoretical molecular weight of the target product of 931.5. It was confirmed to have the structure shown by the formula (I) (see FIG. 1):
Figure BDA0001530909450000051
EXAMPLE 2 preparation of tylosin artificial antigen
(1) 93.2mg (0.1mmoL) of the hapten prepared in example 1 was dissolved in 0.5mL of DMF, 51.2mg (0.2mmoL) of DCC and 23mg (0.2mmoL) of NHS were added with stirring, and the mixture was reacted overnight under magnetic stirring at 4 ℃ and centrifuged to obtain a supernatant A.
(2) Carrier protein (BSA, OVA)15mg was weighed out and dissolved in 5mL of 0.1mol/L phosphate buffer solution (pH 8.0) to prepare solution B. Gradually dropping the solution A into the solution B under magnetic stirring, and reacting at 4 deg.C for 12 h.
(3) And (3) filling the reaction solution into a dialysis bag, dialyzing for 3 days at 4 ℃ by using a phosphate buffer solution with the pH value of 8.0, and replacing the dialysate for 2-3 times every day to obtain the tylosin artificial antigen.
The structural formula of the artificial antigen of tylosin is shown as formula (II):
Figure BDA0001530909450000061
EXAMPLE 3 preparation of tylosin antibody
The antibody preparation methods described in this example refer to methods conventional in the art.
Mixing tylosin artificial antigen with equal dose of immunologic adjuvant (Freund's complete adjuvant is used for immunization 1, and Freund's incomplete adjuvant is used for boosting immunization later), completely emulsifying with a stirrer, and performing subcutaneous immunization on healthy New Zealand white rabbits at multiple points on the back, and boosting immunization once every three weeks later. The serum titer and inhibition rate are measured by indirect ELISA, and the immunization is strengthened once after the titer is stable. Blood is collected from the heart one week after the last immunization, and serum is retained by centrifugation. And purifying the serum by adopting an octanoic acid-ammonium sulfate salting-out method to obtain the antibody with high specificity.
EXAMPLE 4 establishment of enzyme-linked immunoassay method for tylosin
The working concentration of the antigen and the antibody is determined by a chessboard method, the working concentration of the coating antigen is 0.16 mu g/mL, and the concentration of the tylosin antibody is 0.5 mu g/mL.
The test solutions were prepared from tylosin standard solutions of different concentrations, and competition was performed under gradient dilution using 9 parallel experiments (n-3).
Indirect competitive ELISA method: the coating stock was diluted to the above concentration with 0.1M carbonate buffer (PBS) pH 7.4 and coated overnight at 37 ℃. Washing with PBS buffer solution containing 0.05% Tween for 2 times, adding blocking solution (PBS buffer solution containing 2% skimmed milk powder and 0.05% Tween), shaking, mixing, and incubating at 37 deg.C for 3 hr; and (5) drying the liquid in the holes, and drying at 37 ℃ for later use. Adding the tylosin standard solution into a plate hole, adding the diluted antibody, setting a blank hole and a negative control hole at the same time, incubating for 40min at 37 ℃, washing the plate for 5 times, and adding the diluted enzyme-labeled secondary antibody solution; incubating at 37 ℃ for 30 min; washing the plate for 5 times, adding a substrate chromogenic solution, carrying out chromogenic reaction for 10min at 37 ℃, adding a stop solution to stop the reaction, measuring the light absorption value of each hole at the wavelength of 450nm by using an enzyme labeling instrument, drawing a semilogarithmic standard curve graph by using the light absorption value as a vertical coordinate and using the log10 value of the concentration of the tylosin standard solution as a horizontal coordinate, and referring to a tylosin ELISA competition standard curve shown in the attached figure 3.
The result shows that the standard curve has a complete reverse S shape and is provided with an upper platform and a lower platform, the parallel determination times of the standard curve are 3 times, the experimental repeatability is good, and the relative standard deviation is within 15 percent.
The 10% inhibition (LOD) and half-Inhibition (IC) were obtained from the standard curve50) And detecting the antibody performance.
The results showed that the tylosin polyclonal antibody had half the inhibitory amount (IC) against tylosin50) 1.39ng/mL, and a minimum detection Limit (LOD) of 0.051 ng/mL.
A standard curve is drawn by the same method, and the half Inhibition (IC) of other structural or functional analogs is calculated50) And calculating the cross-reactivity. The detection results in the table 1 show that the method has good accuracy and sensitivity, can be used for detecting tylosin in environmental and food samples, and is very convenient and quick in practical use.
TABLE 1 semi-inhibitory concentration and crossover rate of the antigen and antibody detection tylosin analogues of the present invention
Figure BDA0001530909450000071
Figure BDA0001530909450000081
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Claims (3)

1. The application of the molecule shown in the formula (I) as a tylosin hapten in preparing a tylosin artificial antigen;
Figure FDA0002740242090000011
2. an immunogen and coating antigen combination for enzyme-linked immunoassay of tylosin, wherein the immunogen is a molecule shown in formula (II) obtained by coupling tylosin hapten and carrier protein according to claim 1 through an active ester method, and the carrier protein is bovine serum albumin;
the coating antigen is a molecule shown in a formula (II) obtained by coupling tylosin hapten and carrier protein according to claim 1 through an active ester method, and the carrier protein is ovalbumin;
Figure FDA0002740242090000012
3. the use of the immunogen and coating antigen combination of claim 2 in the preparation of a tylosin enzyme-linked immunoassay reagent.
CN201711464700.6A 2017-12-28 2017-12-28 Tylosin hapten, artificial antigen and antibody as well as preparation methods and applications thereof Active CN108178777B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201711464700.6A CN108178777B (en) 2017-12-28 2017-12-28 Tylosin hapten, artificial antigen and antibody as well as preparation methods and applications thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201711464700.6A CN108178777B (en) 2017-12-28 2017-12-28 Tylosin hapten, artificial antigen and antibody as well as preparation methods and applications thereof

Publications (2)

Publication Number Publication Date
CN108178777A CN108178777A (en) 2018-06-19
CN108178777B true CN108178777B (en) 2020-12-11

Family

ID=62548663

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201711464700.6A Active CN108178777B (en) 2017-12-28 2017-12-28 Tylosin hapten, artificial antigen and antibody as well as preparation methods and applications thereof

Country Status (1)

Country Link
CN (1) CN108178777B (en)

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112129942A (en) * 2020-08-21 2020-12-25 华南农业大学 Tylosin and tilmicosin latex microsphere immunochromatographic test strip as well as preparation and application thereof
CN112961073B (en) * 2021-02-08 2022-02-22 华南农业大学 Tiamulin hapten TMLH, artificial antigen, antibody and preparation method and application thereof
CN113717255B (en) * 2021-08-13 2023-04-28 中国农业大学 Polymyxin B and colistin hapten, artificial antigen, and preparation methods and applications thereof

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102827222A (en) * 2012-08-25 2012-12-19 河北农业大学 Hapten, artificial antigen and monoclonal antibody of tylosin, and preparation method and application thereof

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN100533148C (en) * 2006-02-17 2009-08-26 中国农业大学 Method for detecting tylosin and special enzyme-linked immune reagent kit thereof

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102827222A (en) * 2012-08-25 2012-12-19 河北农业大学 Hapten, artificial antigen and monoclonal antibody of tylosin, and preparation method and application thereof

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
D. Kim et al..NEW MACROCYCLES WITH POTENT ANTITUBERCULOSIS ACTIVITY ACCESSED BY ONE-POT MULTICOMPONENT REACTIONS.《Chemistry of Heterocyclic Compounds》.2013,第49卷(第6期),第849-859页. *
NEW MACROCYCLES WITH POTENT ANTITUBERCULOSIS ACTIVITY ACCESSED BY ONE-POT MULTICOMPONENT REACTIONS;D. Kim et al.;《Chemistry of Heterocyclic Compounds》;20130930;第49卷(第6期);849-859 *

Also Published As

Publication number Publication date
CN108178777A (en) 2018-06-19

Similar Documents

Publication Publication Date Title
CN108178777B (en) Tylosin hapten, artificial antigen and antibody as well as preparation methods and applications thereof
CN101962358B (en) Ciprofloxacin hapten, artificial antigen and antibody and preparation method and application thereof
CN102827076A (en) Universal hapten, artificial antigen and broad-spectrum monoclonal antibody for fluoroquinolone medicines as well as preparation method and application of universal hapten
CN106866568A (en) Furaxone metabolite AOZ derivatizations haptens, the preparation method and applications of artificial antigen
CN111269262B (en) Profenofos hapten, artificial antigen and antibody, and preparation method and application thereof
CN106588805A (en) Furaltadone metabolite (AMOZ) derivatization hapten, preparation method of artificial antigen and application of artificial antigen
CN105566493B (en) Monoclonal antibody, enzyme-linked immunosorbent assay method and kit for detecting florfenicol
CN109824645B (en) Warfarin hapten and artificial antigen as well as preparation method and application thereof
CN102816196A (en) Tildipirosin hapten and artificial antigen as well as preparation methods thereof
CN105273021B (en) Erythromycin haptens, artificial antigen and antibody and its preparation method and application
CN1677107A (en) Immune antibody for testing residual of polyether-like antibiotic and use thereof
CN106831498A (en) Furacilin metabolite SEM derivatizations haptens, the preparation method and applications of artificial antigen
CN109824599A (en) A kind of albendazole haptens and its preparation method and application
CN109438424B (en) Ribavirin hapten and artificial antigen as well as preparation method and application thereof
CN110683965B (en) Nifurtida hapten and artificial antigen as well as preparation methods and application thereof
CN105646536A (en) Ceftiofur hapten as well as colloidal gold detection device and preparation method of ceftiofur hapten
CN101135683B (en) Bifenthrin antigen, antibody and uses thereof
WO2003006507A1 (en) Method of preparing antinarcotic shellfish toxin antibody, novel antibody, elisa kit with the use of the antibody and labelled toxin sample prepared by the method
CN105524174B (en) Monoclonal antibody, enzyme linked immunosorbent assay method and kit for detecting thiamphenicol and florfenicol
CN104031886A (en) Method of detecting monensin by immunomagnetic bead purification-enzyme-linked immunoassay and special monoclonal antibody used therein
CN1904613A (en) Artificial antigen used for immune analysis of sulfanilamide multi kind residue and antibody and its preparation
CN113896758B (en) Solanum nigrum hapten, artificial antigen and antibody as well as preparation methods and application thereof
CN103509068B (en) Amikacin haptens and its preparation method and application
CN111875652B (en) Spinosad hapten, artificial antigen and antibody as well as preparation methods and applications thereof
CN108107223A (en) Detect method and its enzyme linked immunological kit used of estradiol and/or oestradiol benzoate

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant