CN108178777A - A kind of tylosin haptens, artificial antigen and antibody and its preparation method and application - Google Patents
A kind of tylosin haptens, artificial antigen and antibody and its preparation method and application Download PDFInfo
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- CN108178777A CN108178777A CN201711464700.6A CN201711464700A CN108178777A CN 108178777 A CN108178777 A CN 108178777A CN 201711464700 A CN201711464700 A CN 201711464700A CN 108178777 A CN108178777 A CN 108178777A
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- tylosin
- haptens
- artificial antigen
- antibody
- antigen
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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
- C07H17/04—Heterocyclic radicals containing only oxygen as ring hetero atoms
- C07H17/08—Hetero rings containing eight or more ring members, e.g. erythromycins
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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
- C07K14/435—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)
Abstract
The invention discloses a kind of tylosin haptens, artificial antigen and antibody and its preparation method and application.Specifically using the aldehyde-base functional group of the big ring side chain of Taylor's rhzomorph as derivative conjugation sites, by one-step oxidation process by convert aldehyde groups for carboxyl to get being coupled Taylor's rhzomorph haptens of arm to carboxy methylation.Gained haptens is high with skeleton structure degree of overlapping, and basis has been carried out for the exposure of tylosin feature structure and the generation of antibody with high specificity.Haptens and carrier protein couplet are further obtained into artificial antigen, and immune animal obtains Taylor's rhzomorph antibody with high specificity, the enzyme-linked immunoassay method accuracy established is high, and sensitivity is good, and detection limit reaches 0.051 ng/mL, 503nhibiting concentration is 1.39 ng/mL, and it is easy to operate, it is at low cost, it is very suitable for Site Detection, available for the fast and effective detection of tylosin in environment and food samples, have broad application prospects and economic value.
Description
Technical field
The invention belongs to medicament residue detection technique fields.More particularly, to a kind of tylosin haptens, artificial anti-
Former and antibody and its preparation method and application.
Background technology
Tylosin (Tylosin, TYL), also known as safe agriculture are that Hamill is equal to culture solution of the nineteen fifty-nine from streptomyces fradiae
A kind of macrolide antibiotics of middle acquisition.TYL has gram-positive bacteria apparent inhibition, to part Gram-negative
Bacterium also has inhibiting effect, has good therapeutic effect to livestock and poultry mycoplasma disease, is the choice drug for treating mycoplasmal diseases.It is right
Most of conveyor screw, virus etc. have effects that certain inhibition and kill also have and promote growth of animal, improve feed effect
The effect of rate, is therefore widely used in livestock breeding industry.But the residual of these drugs can cause the allergic reaction of human body
(fash, anaphylactic shock etc.) inhibits the growth of normal sensitive flora in enteron aisle, make pathogenic bacteria, candida albicans be largely proliferated and
Lead to whole body or local infection, and it was found that the enterococcus of resistance to tylosin is resistant to erythromycin, other anti-with some
The cross resistance of raw element can cause human body to develop immunity to drugs antibiotic, and very big harm is brought to clinical treatment.
EU Committee forbade tylosin to be used as growth accelerator in 1999.It is however, safe happy in some countries
Still as poultry, the growth promoter of pig and ox is used rhzomorph.Consider for the long-term interest of human health, Codex Alimentarius
The committee, EU Committee and some countries all establish maximum residue limit of the tylosin in poultry group food.China
The Ministry of Agriculture is in No. 235 file of publication in 2002《Animal food herbal medicine highest residual quantity》Tylosin is also made that
Maximum residue limit standard.Different for different species and its Limited Doses of different tissues, limitation is 50 μ g/ such as in milk
Kg, the limitation in egg and pork, internal organ etc. are 200 μ g/kg.Therefore, study and make can it is simple, quick, sensitive,
It reliably detects out the remaining method of tylosin in food and is of great significance to improving China's food safety monitoring system.
There is the method for tylosin residual quantity in many detection animal derived foods both at home and abroad at present, mainly including microorganism
Method, high performance liquid chromatography, chromatograph-mass spectrometer coupling method, immunoassay (including enzyme-linked immunization and colloidal gold immunochromatographimethod)
Deng.Wherein, microbial method because its analytical cycle it is long, the fatefulue shortcoming such as experimental procedure is cumbersome, and sensitivity is low is gradually by other
Method is substituted.The detection of the instruments such as high performance liquid chromatography detection method is accurate, high sensitivity, reproducible, but still deposits
It is of long duration of high cost, it is impossible to meet it is high-throughput, the problems such as Site Detection.Immune inspection based on antigen and antibody specific reaction
Survey method is now more mature, and compared with other instruments method, immunoassay method high sensitivity is at low cost, during detection
Between it is short, apply also for the detection of high-throughput sample, be increasingly becoming the main method that noxious residual chemicals quickly screen monitoring
One of.
At present, the technology for using enzyme-linked immunoassay method detection tylosin both at home and abroad is utilized with the organic of amino
The derivative haptens with carboxyl structure is obtained by the reaction with the aldehyde radical on the big ring of tylosin in acid.These haptens structures are more multiple
Miscellaneous, coupled arm is longer, easily folds, bone framework image is caused to change, is unfavorable for being exposed to carrier protein surface, immune effect weight
Renaturation is bad, and more demanding to reaction raw materials.
Invention content
The technical problem to be solved by the present invention is to overcome the defects of above-mentioned existing tylosin residue detection technology and deficiency,
A kind of new tylosin haptens structure is provided, specifically by the aldehyde-base direct one of tylosin molecule macrolide side chain
Step is oxidized to carboxyl as haptens, and the acetic acid arm length after aoxidizing is moderate, should not fold, with skeleton structure degree of overlapping
Height maintains Taylor's rhzomorph stereochemical structure to greatest extent, is conducive to the stabilization immune induction of antibody, and then the haptens is with carrying
Body protein is coupled, and specificity structure protrudes from the surface of carrier protein, and an epitope as carrier is exposed to animal
Immune system can obtain specific antibody, lay the foundation to establish immunologic detection method.
The object of the present invention is to provide a kind of tylosin haptens and preparation method thereof.
Another object of the present invention is to provide a kind of tylosin artificial antigen and preparation method thereof.
Still a further object of the present invention is to provide a kind of tylosin antibody and its application.
Above-mentioned purpose of the present invention is achieved through the following technical solutions：
A kind of tylosin haptens, shown in molecular structural formula such as formula (I)：
Application of the tylosin haptens in tylosin artificial antigen is prepared, also in protection scope of the present invention
Within.
A kind of tylosin artificial antigen is to obtain above-mentioned tylosin haptens with carrier protein couplet, molecule
Shown in structural formula such as formula (II)：
Wherein, the carrier protein is bovine serum albumin (BSA), ovalbumin (OVA) or keyhole limpet hemocyanin (KLH).
The tylosin haptens or tylosin artificial antigen are preparing tylosin antibody (including Anti-TNF-α
Body) in application, also should be within protection scope of the present invention.
Immunogene and the coating antigen combination of a kind of enzyme linked immunosorbent detection tylosin, immunogene are the haptens and BSA
Coupling obtains, and coating antigen is coupled to obtain for the haptens with OVA.
The immunogene and coating antigen combination are in detection tylosin or structure tylosin enzyme-linked immune detection method
Application, also should be within protection scope of the present invention.
More than one are stated tylosin artificial antigen and tylosin antibody are prepared (including Anti-TNF-α for immunogene
Body), it also should be within protection scope of the present invention.
In addition, the preparation method of tylosin haptens of the present invention, is by macrolide side in tylosin molecule
Peculiar one step of aldehyde radical of chain is oxidized to carboxyl and obtains tylosin haptens.
Specifically, the preparation method of the tylosin haptens specifically comprises the following steps：
(1) tartaric acid Taylor's rhzomorph is dissolved in the mixed liquor of the tert-butyl alcohol and water, NaH is sequentially added at 0 DEG C2PO4, two
Methyl sulfoxide, NaClO2, carry out aldehyde radical oxidation reaction；
(2) ethyl acetate and water extraction are added in reaction solution, collects organic phase；Water phase is extracted with ethyl acetate again, receives
Collect to obtain organic phase；Merge organic phase water and salt water washing, then with anhydrous MgSO4It dries and filters, solvent is evaporated off in filtrate decompression
Afterwards, residue purifies through silica gel column chromatography and obtains white solid, as described tylosin haptens.
Wherein it is preferred to the volume ratio of step (1) tert-butyl alcohol and water is 4~6：3.
It is highly preferred that the volume ratio of step (1) tert-butyl alcohol and water is 5：3.
Preferably, step (1) the tartaric acid Taylor rhzomorph and NaH2PO4、NaClO2Molar ratio be 1：3~5：1.5~
2.5。
It is highly preferred that step (1) the tartaric acid Taylor rhzomorph and NaH2PO4、NaClO2Molar ratio be 1：4：2.
In addition, the preparation method of the tylosin artificial antigen, is using active ester method that the tylosin half is anti-
Former and carrier protein is coupled, and prepares tylosin artificial antigen；Specifically comprise the following steps：
(1) tylosin haptens described in claim 1 is dissolved in DMF, then adds in DCC and NHS, stirred at 4 DEG C
It mixes and is reacted；
(2) it weighs carrier protein to be dissolved in phosphate buffer solution, the reaction solution of above-mentioned steps (1) is centrifuged, in taking-up
Clear liquid is added dropwise in the phosphate buffer solution of carrier protein, and the reaction was continued at 4 DEG C；
(3) reaction solution is dialysed at room temperature 3 days, changes 2 dialyzates daily, obtain tylosin artificial antigen.
Wherein it is preferred to the rate of charge of step (1) the tylosin haptens and DCC and NHS are 1：1.5~2.5：
1.5~2.5.
It is highly preferred that the rate of charge of step (1) the tylosin haptens and DCC and NHS are 1：2：2.
Preferably, the molar ratio of carrier protein and tylosin haptens is 60：1.
Preferably, the volume ratio of the DMF and phosphate buffer solution is 1：10.
Preferably, the time of step (1) described reaction is 8~12h.
Preferably, the time of step (2) described reaction is 8~12h.
The invention has the advantages that：
The present invention passes through one-step oxidation process using the aldehyde-base functional group of the big ring side chain of Taylor's rhzomorph as derivative conjugation sites
It is carboxyl by convert aldehyde groups, obtains Taylor's rhzomorph haptens of carboxy methylation coupling arm；And the acetic acid arm length after aoxidizing
It is moderate, it should not fold, haptens is consistent with Taylor's rhzomorph numberator height, with skeleton structure degree of overlapping height, Stable conformation and Thailand
It is completely overlapped to strangle rhzomorph Stable molecular conformation, maintains Taylor's rhzomorph molecule stereo structure feature to greatest extent, is conducive to antibody
Stabilization immune induction, and then the haptens and carrier protein couplet, specificity structure protrude from the surface of carrier protein,
An epitope as carrier is exposed to animal immune system, specific antibody can be obtained, to establish immunologic detection method
It lays the foundation.
Haptens and carrier protein couplet are further obtained, and pass through artificial antigen and exempt from by artificial antigen using active fat method
Epidemic disease animal obtains Taylor's rhzomorph antibody with high specificity, with other structures and functional analogue without intersecting, further with described anti-
Original antibody optimization establishes Taylor's rhzomorph ELISA adsorption analysis method, and detection limit reaches 0.051ng/mL, 503nhibiting concentration
For 1.39ng/mL, accuracy and sensitivity are fine, available for the fast and effective detection of tylosin in environment and food samples,
It has broad application prospects.
Moreover, the present invention synthesis haptens method it is simple, be easy to grasp, the enzyme-linked immunoassay method sensitivity established and
Accuracy is high, easy to operate, at low cost, is very suitable for Site Detection；To be efficient, cheap, quick immune detection product is beaten
Lower excellent basis, has a good application prospect and economic benefit.
Description of the drawings
Fig. 1 is the structural formula of tylosin haptens.
Fig. 2 is the mass spectrogram of tylosin haptens.
Fig. 3 is tylosin standard curve.
Specific embodiment
It is further illustrated the present invention below in conjunction with Figure of description and specific embodiment, but embodiment is not to the present invention
It limits in any form.Unless stated otherwise, the reagent of the invention used, method and apparatus are routinely tried for the art
Agent, method and apparatus；Following embodiment agents useful for same and material are purchased in market.
The preparation of 1 tylosin haptens of embodiment
(1) 1g Tylan Tartrates are dissolved in the mixed liquor of the 50mL tert-butyl alcohols and 30mL water, are sequentially added at 0 DEG C
0.62g NaH2PO4, 1mL dimethyl sulfoxide (DMSO)s, 0.2g NaClO2, react 30min；
(2) ethyl acetate (50mL) is added in reaction solution and water (10mL) extracts 3 times, collects organic phase.Water phase is used again
Ethyl acetate (50mL) extracts three times.The organic phase of merging is washed with water (20mL) and brine (20mL), with anhydrous MgSO4It is dry
And it filters.After filtrate decompression revolving (methanol is purified through silica gel column chromatography：Chloroform=1：10) white solid is obtained, as half is anti-
It is former.
The mass spectrogram of tylosin haptens illustrates that the product is gone as shown in Fig. 2, there is the quasi-molecular ions that m/z is 932.5
Molecular weight after proton is 931.5, consistent with the theoretical molecular weight 931.5 of target product.Prove its knot shown in formula (I)
Structure (such as Fig. 1)：
The preparation of 2 tylosin artificial antigen of embodiment
(1) it weighs haptens 93.2mg (0.1mmoL) made from embodiment 1 to be dissolved in 0.5mL DMF, stirring adds in
51.2mg (0.2mmol) DCC and 23mg (0.2mmoL) NHS, overnight, centrifuged supernatant is A liquid for magnetic agitation reaction at 4 DEG C.
(2) phosphate buffer solution (pH that carrier protein (BSA, OVA) 15mg is dissolved in a concentration of 0.1mol/L of 5mL is weighed
=8.0) in, stirring and dissolving prepares B liquid.Under magnetic agitation, A liquid is gradually dropped in B liquid, reacts 12h at 4 DEG C.
(3) reaction solution is packed into bag filter, is dialysed 3 days with the phosphate buffer solution of pH=8.0 at 4 DEG C, replace 2 daily
~3 dialyzates, obtain tylosin artificial antigen.
Shown in the artificial antigen structural formula such as formula (II) of tylosin：
The preparation of 3 tylosin antibody of embodiment
Preparation method for antibody described in the present embodiment is with reference to this field conventional method.
Tylosin artificial antigen is mixed with isodose immunologic adjuvant (the 1st immune Freund's complete adjuvant, after
Booster immunization uses incomplete Freund's adjuvant), after being emulsified completely with blender, back multiple spot subcutaneous inoculation health New Zealand great Bai
Rabbit, booster immunization is primary every three weeks later.Serum titer and inhibiting rate are measured using indirect ELISA, after titer plateaus, then
Booster immunization is primary.The immune latter all Culling heart bloods of last time, centrifugation retain serum.Using octanoic acid-ammonium sulfate salting-out process pair
Serum is purified to obtain the antibody of high specific.
The foundation of 4 tylosin enzyme-linked immune detection method of embodiment
The working concentration of antigen and antibody is determined by chessboard method, the working concentration of coating antigen is 0.16 μ g/mL, safe happy bacterium
Plain antibody concentration is 0.5 μ g/mL.
Experimental solutions are done with the tylosin standard solution of various concentration, is at war under gradient dilution, is put down using 9 groups
Row experiment (n=3).
Indirect Competitive ELISA method：Coating antigen is diluted to 7.4 carbonate buffer solutions of 0.1M pH (PBS)
Concentration is stated, 37 DEG C of coatings are overnight.It is washed 2 times with the PBS buffer solutions containing 0.05% tween, adds in confining liquid and (contain 2% defatted milk
The PBS buffer solutions of 0.05% tween of powder), vibrate mixing, 37 DEG C of incubation 3h；Liquid in hole is dried, 37 DEG C are dried for standby.It will be safe
Happy rhzomorph standard solution adds in plate hole, adds the antibody after dilution, while set blank well and negative control hole, 37 DEG C incubate
40min is educated, board-washing 5 times adds in the ELIAS secondary antibody solution diluted；37 DEG C of incubation 30min；It is molten to add in substrate colour developing for board-washing 5 times
Liquid, 37 DEG C of colour developing 10min, adds in terminate liquid and terminates reaction, and light absorption value of each hole at wavelength 450nm is measured with microplate reader, with
Light absorption value is ordinate, using the log10 values of tylosin standard solution concentration as abscissa, draws semilog standard curve
Figure, is shown in tylosin ELISA competition test curves shown in attached drawing 3.
The result shows that standard curve has complete reverse-s shape shape, and have upper mounting plate and lower platform, standard curve it is parallel
Measure number 3 times, experimental repeatability is good, and relative standard deviation is within 15%.
10% amount of suppression (LOD) and half amount of suppression (IC are obtained according to standard curve50), detect antibody performance.
The result shows that half amount of suppression (IC of the tylosin polyclonal antibody to tylosin50) it is 1.39ng/mL, most
Low detection limit (LOD) is 0.051ng/mL.
Standard curve is drawn by same method, calculates the half amount of suppression (IC of other structures or functional analogue50), and
Calculate cross reacting rate.It can illustrate that the method for the present invention accuracy and sensitivity are fine from the testing result of table 1, available for environment
It is very convenient quick in actual use with the detection of tylosin in food samples.
The 503nhibiting concentration and crossing-over rate of 1 antigen of the present invention of table, antibody test tylosin analog
Above-described embodiment is the preferable embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment
Limitation, other any Spirit Essences without departing from the present invention with made under principle change, modification, replacement, combine, simplification,
Equivalent substitute mode is should be, is included within protection scope of the present invention.
Claims (10)
- A kind of 1. tylosin haptens, which is characterized in that its molecular structural formula such as formula（Ⅰ）It is shown：。
- 2. application of the tylosin haptens in tylosin artificial antigen is prepared described in claim 1.
- 3. a kind of tylosin artificial antigen, which is characterized in that be by tylosin haptens described in claim 1 and carrier egg White coupling obtains, molecular structural formula such as formula（Ⅱ）It is shown：。
- 4. tylosin artificial antigen according to claim 3, which is characterized in that the carrier protein is cow's serum egg In vain, ovalbumin or keyhole limpet hemocyanin.
- 5. immunogene and the coating antigen combination of a kind of enzyme linked immunosorbent detection tylosin, which is characterized in that immunogene will for right 1 haptens is asked to be coupled to obtain with bovine serum albumin, coating antigen is coupled for haptens described in claim 1 with ovalbumin It arrives.
- 6. tylosin artificial antigen described in tylosin haptens described in claim 1 or claim 3 is preparing safe happy bacterium Application in plain antibody.
- 7. immunogene described in claim 5 and coating antigen combination are in detection tylosin or structure tylosin enzyme linked immunosorbent detection Application in method.
- 8. a kind of tylosin antibody, which is characterized in that be using by tylosin artificial antigen described in claim 3 as immunogene It is prepared.
- 9. the preparation method of tylosin haptens described in claim 1, which is characterized in that will be in ring big in tylosin molecule Peculiar one step of aldehyde radical of ester side chain is oxidized to carboxyl and obtains tylosin haptens.
- 10. the preparation method of tylosin artificial antigen described in claim 3, which is characterized in that will be weighed using active ester method Profit requires the 1 tylosin haptens and carrier protein to be coupled, and prepares tylosin artificial antigen；It specifically includes Following steps：（1）Tylosin haptens described in claim 1 is dissolved in DMF, then add in DCC and NHS, stirred at 4 DEG C into Row reaction；（2）It weighs carrier protein to be dissolved in phosphate buffer solution, by above-mentioned steps（1）Reaction solution centrifugation, take out supernatant It is added dropwise in the phosphate buffer solution of carrier protein, the reaction was continued at 4 DEG C；（3）Reaction solution is dialysed at room temperature 3 days, change 2 dialyzates daily, obtain tylosin artificial antigen.
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| 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 |
| CN112961073A (en) * | 2021-02-08 | 2021-06-15 | 华南农业大学 | Tiamulin hapten TMLH, artificial antigen, antibody and preparation method and application thereof |
| CN113717255A (en) * | 2021-08-13 | 2021-11-30 | 中国农业大学 | Polymyxin B, colistin hapten and artificial antigen as well as preparation method and application thereof |
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| 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 |
| CN112961073A (en) * | 2021-02-08 | 2021-06-15 | 华南农业大学 | Tiamulin hapten TMLH, artificial antigen, antibody and preparation method and application thereof |
| CN113717255A (en) * | 2021-08-13 | 2021-11-30 | 中国农业大学 | Polymyxin B, colistin hapten and artificial antigen as well as 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 |
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