CN101962359B - Hapten, artifical antigen and antibody of enrofloxacin and preparation method as well as application thereof - Google Patents
Hapten, artifical antigen and antibody of enrofloxacin and preparation method as well as application thereof Download PDFInfo
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Abstract
The invention provides a hapten, an artifical antigen and an antibody of enrofloxacin and a preparation method as well as application thereof. The preparation method comprises the following steps of: taking enrofloxacin as raw materials to react with aminobutyric acid or aminocaproic acid to produce the hapten containing four or six carbon atom arms; coupling the hapten with protein to prepare the artifical antigen through an active lipid method; mithridatizing a mice with the artifical antigen, and adopting the cell fusion technology to prepare a monoclonal antibody with high specificity to the enrofloxacin. Immunodetection established by adopting the monoclonal antibody can be used for on-site rapid detection of the enrofloxacin, and has practical significance for rapid detection on residual enrofloxacin in aquatic animals.
Description
Technical field
The invention belongs to the food safety technical field.Be specifically related to preparation method and the application of Enrofloxacin haptens, artificial antigen and antibody.
Background technology
Enrofloxacin (Enrofloxacin, EF), its molecular formula are C
19H
22FN
3O
3, molecular weight is 359.40, little yellow or greenish orange yellow crystalline powder, odorless, mildly bitter flavor; Meet and photochromicly fade to orange redly, its structural formula is shown in the formula (1):
Enrofloxacin is one of Typical Representative of third generation quinolones (FQs), have has a broad antifungal spectrum, toxic side effect is little, Plasma Concentration is high, long half time, various, the drug effect cost ratio high of dosage form, various Gram-negatives and positive bacteria all had powerful bacteriostatic action, with penicillin, cynnematin and amino medicine all without characteristics such as cross resistances, have specific action for treatment anthrax illness.
The FQs medicine because of its can anti-bacteria DNA helicase, has a broad antifungal spectrum, efficient, low toxicity, tissue penetration strong, anti-microbial effect is nearly thousand times of sulfonamides, it is chemical synthetic drug in addition, cheap, so very cache gets widespread use in medical science and veterinary science, especially in aquaculture, become one of most important anti-infectives.The FQs medicine has in various degree untoward reaction to most Organ and tissues of animal body and human body: because the FQs medicine is fat-soluble, can enter cerebral tissue by hemato encephalic barrier, suppress γ-aminobutyric acid (GABA) and produce the neural system reaction with its receptors bind.So the most common with neural damage, secondly be the damage of skin and annex thereof.Most untoward reaction symptoms are lighter, and reversible, but also have heavier even life-threatening.Experimentation on animals shows that the FQs medicine can cause cub sacroiliitis, scorching with key, also can cause anaphylactic shock, feel sick, vomit, the symptom such as phlebitis and the carcinogenic character that some is potential, because pathogenic bacterium produce resistance, the residue problem of enrofloxacin has caused extensive concern simultaneously.The maximum residue limit(MRL) that the U.S. and European Union are defined in side in the fowl edible tissue is that Enrofloxacin and Enrofloxacin are 30 μ g/Kg; It is 50 μ g/Kg that the standard NY5070-2001 of China Ministry of Agriculture " fishing medicine residue limits in the pollution-free food fishery products " regulation enrofloxacin residual is limited the quantity of.
At present, the detection of enrofloxacin residual in the aquatic animal is mainly adopted the instrument detection methods such as high performance liquid chromatography (HPLC), gas phase.Yet when using instrumental method, its sensitivity is subjected to the impact of the purification of sample, the step such as concentrated very large, the measuring method existence is complicated, loaded down with trivial details, the detection flux is little, need professional and the defectives such as professional skill, testing cost costliness, can not realize the rapid detection analysis of batch samples.Therefore, be necessary to develop more easily detection method of simple and fast.
Immunologic detection method is one of important method of present food safety rapid detection, and testing cost is very low, uses simply, and the screening that is fit to a large amount of samples detects, and has brought into play vital role in food safety detection.Artificial antigen synthetic be haptens material immune analysis method set up key, for Enrofloxacin, because itself contains carboxyl, do not need to derive, all be directly to utilize this carboxyl to be connected with protein at present therefore.For example, Cai Qinren etc. have delivered paper " preparation of enrofloxacin monoclonal antibody and evaluation " (Scientia Agricultura Sinica, 2004), adopt carbodlimide method, and directly the amino of coupling carboxyl and protein has synthesized Enrofloxacin Artificial Antigens; Tang Hong etc. disclose a kind of patent " enrofloxacin monoclonal antibody and application (application number 200910027793.5) ", adopt carbodlimide method with Enrofloxacin and carrier proteins BSA, HSA and OVA coupling.
But specificity and the insight of the Enrofloxacin antibody that prior art obtains are inadequate.
Summary of the invention
An object of the present invention is to fill up the deficiency of the existing detection technique of Enrofloxacin, a kind of Enrofloxacin artificial semiantigen, artificial antigen and monoclonal antibody specific are provided.
Another object of the present invention provides the preparation method of described Enrofloxacin artificial semiantigen, artificial antigen and monoclonal antibody specific.
A further object of the invention provides the application of described Enrofloxacin artificial semiantigen, artificial antigen and monoclonal antibody specific.
Purpose of the present invention is achieved by the following technical programs:
A kind of Enrofloxacin artificial semiantigen, artificial antigen and monoclonal antibody specific are provided, and described Enrofloxacin artificial semiantigen has molecular structure shown in the formula (II):
Wherein, n is-CH
2The group number, n=4 or 6, preferred n=4.
Described Enrofloxacin Artificial Antigens has molecular structure shown in the formula (III):
Wherein, n is-CH
2The group number, n=4 or 6, preferred n=4.
The invention provides the haptenic preparation method of described Enrofloxacin, is with Enrofloxacin and aminobutyric acid (H
2NCH
2CH
2CH
2COOH) or hexosamine (H
2NCH
2(CH
2)
4COOH) reaction generates the haptens that contains 4 or 6 carbon atom arms under NHS, DDC and DMF existence condition.
Enrofloxacin haptens synthetic route (in the generating structure formula n value as 4 as example):
The haptenic preparation of Enrofloxacin of the present invention may further comprise the steps:
(1) be Enrofloxacin, nitrogen N-Hydroxysuccinimide (NHS) and fat-soluble carbodiimide (DDC) to be placed beaker in 1: 1: 1 by molar ratio, and with said mixture with a small amount of DMF dissolving, lucifuge stirs and spends the night under the room temperature;
(2) will get A liquid through the reactant centrifuging and taking supernatant that step (1) obtains; The centrifugal 10min of the preferred 10000r/min of described centrifugally operated;
Get aminobutyric acid or hexosamine with the Enrofloxacin equimolar amount, be dissolved in a small amount of distilled water, get B liquid;
(3) B liquid slowly is added in the A liquid, stirred overnight at room temperature, the centrifuging and taking supernatant liquor is transferred supernatant liquor pH value, with the sedimentation and filtration of separating out, collects supernatant liquor;
The centrifugal 10min of the preferred 10000r/min of the described centrifugally operated of step (3);
Step (3) preferably adopts saturated NaHCO
3Regulate the pH value of supernatant liquor, preferred adjust pH is 8.0～8.5;
(4) the supernatant liquor adjust pH that step (3) is made, collecting precipitation obtains Enrofloxacin haptens (CPFX-ABA).
It is 5.0～6.0 with the supernatant liquor adjust pH that step (4) preferably adopts the dilute hydrochloric acid of 1mol/L.
The preparation of artificial antigen of the present invention can be adopted active fat method, according to this area design philosophy, also can adopt other ordinary method preparations of this area.Adopt the concrete steps of the standby antigen of the present invention of active fat legal system as follows:
(1) equimolar Enrofloxacin haptens claimed in claim 1, DDC, NHS are dissolved among the DMF, the reaction of room temperature lucifuge is spent the night;
(2) with step (1) gained reactant centrifuging and taking supernatant liquor, supernatant liquor is joined in the borate buffer that contains carrier proteins, mixture is spent the night 4 ℃ of lower reactions;
(3) after step (2) reaction is finished, the mixed solution dialysis tubing of packing into after will react, dialysing obtains described artificial antigen.
Enrofloxacin monoclonal antibody specific preparation of the present invention, its concrete steps are as follows:
1) experiment selects the Healthy female Balb/c mouse of 8 weeks about ages as immune object, fundamental immunity dosage be 0.1～0.2mg/ only, booster immunization dosage be 03～0.5mg/ only.Artificial antigen and the abundant mixing of equivalent adjuvant, adopt the subcutaneous multi-point injection in back, every the 15d booster immunization once, after the 3rd immunity in 7～10 days, antiserum titre and specificity are measured in afterbody blood sampling, after its antiserum titre and specificity are qualified, the booster immunization that does not once contain adjuvant, cytogamy was carried out in immunity in rear the 3rd day; Merge with mouse boosting cell and SP2/0 myeloma cell, obtain hybridoma, hybridoma is placed female Balb/c mouse culturing in vivo, obtain to contain the ascites of high-concentration monoclonal antibody, and the ascites purifying is obtained the anti-enrofloxacin monoclonal antibody of high specific.
2) antibody purification adopts caprylic acid-ammonium or Protein G/A affinitive layer purification.
The Enrofloxacin monoclonal antibody specific can be applicable to the field quick detection aspect of enrofloxacin residual in immunodetection aspect, the especially aquatic animal of enrofloxacin residual.
The invention has the beneficial effects as follows:
The present invention is take Enrofloxacin as raw material, and Enrofloxacin and aminobutyric acid or hexosamine reaction generate the haptens that contains 4 or 6 carbon atom arms, and haptens of the present invention is exposed to animal immune system more fully, strengthens specificity and the insight of antibody; Haptens of the present invention and protein coupling are prepared artificial antigen, adopt this area routine techniques with the artificial antigen immune mouse of the present invention, the employing cell-fusion techniques makes the monoclonal antibody to the Enrofloxacin high specific, use described monoclonal antibody to set up immunodetection and can be applicable to the Enrofloxacin field quick detection, and sensitivity is higher, detectability has reached 0.05ng/mL, and the rapid detection that realizes enrofloxacin residual in the aquatic animal is had important practical significance.
Description of drawings
Fig. 1 Enrofloxacin typical curve
Embodiment
Further describe the present invention below in conjunction with the drawings and specific embodiments.The medicament such as Enrofloxacin all is conventional commercial use for laboratory reagent unless stated otherwise among the embodiment.
Embodiment 1: haptens is synthetic
Take by weighing Enrofloxacin 38.6mg (about 0.1mmoL), NHS 11.5mg (about 0.1mmoL) and DDC 20.3mg (about 0.1mmoL) place round-bottomed flask, and with said mixture with a small amount of DMF dissolving, lucifuge stirs the reactant that spends the night to get under the room temperature;
With the centrifugal 10min of reactant 10000r/min, get supernatant, get A liquid; Get aminobutyric acid 10.3mg (0.1mmoL), be dissolved in the 1mL distilled water, be B liquid; B liquid slowly is added in the A liquid, stirred overnight at room temperature, the centrifugal 10min of 10000r/min gets supernatant liquor, uses saturated NaHCO
3There is Precipitation adjust pH to 8.0～8.5, with sedimentation and filtration, collect supernatant liquor.With dilute hydrochloric acid (1mol/L) supernatant liquor is transferred to acidity (pH value 5.0～6.0), collecting precipitation is Enrofloxacin-aminobutyric acid haptens.
Embodiment 2: haptens is synthetic
Take by weighing Enrofloxacin 38.6mg (about 0.1mmoL), NHS 11.5mg (about 0.1mmoL) and DDC 20.3mg (about 0.1mmoL) place round-bottomed flask, and with said mixture with a small amount of DMF dissolving, lucifuge stirs the reactant that spends the night to get under the room temperature;
With the centrifugal 10min of reactant 10000r/min, get supernatant, get A liquid; Get aminobutyric acid 10.3mg (0.1mmoL), be dissolved in the 1mL distilled water, be B liquid; B liquid slowly is added in the A liquid, stirred overnight at room temperature, the centrifugal 10min of 10000r/min gets supernatant liquor, uses saturated NaHCO
3Adjust pH to 8.0 has Precipitation, with sedimentation and filtration, collects supernatant liquor.With dilute hydrochloric acid (1mol/L) supernatant liquor is transferred to acidity (pH value 6.0), collecting precipitation is Enrofloxacin-aminobutyric acid haptens.
Embodiment 3: haptens is synthetic
Take by weighing Enrofloxacin 38.6mg (about 0.1mmoL), NHS 11.5mg (about 0.1mmoL) and DDC 20.3mg (about 0.1mmoL) place round-bottomed flask, and with said mixture with a small amount of DMF dissolving, lucifuge stirs the reactant that spends the night to get under the room temperature;
With the centrifugal 10min of reactant 10000r/min, get supernatant, get A liquid; Get hexosamine 0.1mmoL, be dissolved in the 1mL distilled water, be B liquid; B liquid slowly is added in the A liquid, stirred overnight at room temperature, the centrifugal 10min of 10000r/min gets supernatant liquor, uses saturated NaHCO
3Adjust pH to 8.5 has Precipitation, with sedimentation and filtration, collects supernatant liquor.With dilute hydrochloric acid (1ol/L) supernatant liquor is transferred to acidity (pH value 6.0), collecting precipitation is Enrofloxacin-aminobutyric acid haptens.
Embodiment 4: artificial antigen synthetic
Optional taking by weighing among Enrofloxacin haptens that embodiment 1～3 prepares, the DMF (DMF) that each 0.1mmoL of DDC, NHS is dissolved in 2mL, the reaction of room temperature lucifuge is spent the night.With the centrifugal 10min of reactant 10000r/min, get supernatant, and it is slowly joined in the borate buffer of 5mL0.1moL/L pH9.0, its damping fluid contains carrier proteins BSA or OVA, and wherein the concentration of carrier proteins is at 20～30mg/mL, mixture is spent the night 4 ℃ of lower reactions, after question response was finished, then the dialysis tubing of packing into used 0.9% normal saline dialysis 3d, change dialyzate every day 3～5 times, the solution that obtains dialysing is artificial antigen (EF-ABA-BSA or EF-ABA-OVA).
The preparation method is the same for Enrofloxacin-aminobutyric acid envelope antigen, only with the OVA of the quality such as BSA changes into.
The same Enrofloxacin of the synthetic method of Enrofloxacin-hexosamine artificial antigen-aminobutyric acid artificial antigen method only changes Enrofloxacin-aminobutyric acid into equimolar Enrofloxacin-hexosamine.
Embodiment 5: the evaluation of artificial antigen:
Take Enrofloxacin-aminobutyric acid artificial semiantigen as example, according to the obtained the maximum absorption of spectrophotometry haptens, carrier proteins and conjugate, calculate the coupling ratio of conjugate.Between 200～400nm, respectively raw material, BSA, OVA and conjugate ultra-violet absorption spectrum are scanned, identify whether haptens and carrier proteins coupling occurs.Estimate simultaneously haptens and carrier protein couplet ratio:
The result is as follows as calculated:
EF-ABA-BSA 22∶1 EF-ABA-OVA 19∶1
Embodiment 6: immune animal and polyclonal antibody preparation
6.1 immune animal prepares antiserum(antisera)
Experiment selects the Healthy female clean level Balb/c experiment mice of laboratory about conventional 8 ages in week of using as immune object, fundamental immunity dosage be 0.2mg/ only, booster immunization dosage be 0.4mg/ only.With artificial antigen and the abundant mixing of equivalent freund adjuvant, adopt the subcutaneous multi-point injection in back, every the 15d booster immunization once, after the 3rd immunity in 7～10 days, antiserum titre and specificity are measured in afterbody blood sampling, after its antiserum titre and specificity are qualified, the booster immunization that does not once contain adjuvant, cytogamy was carried out in immunity in rear the 3rd day; Merge with mouse boosting cell and SP2/0 myeloma cell, obtain hybridoma, hybridoma is placed female Balb/c mouse culturing in vivo, obtain to contain the ascites of high-concentration monoclonal antibody, and the ascites purifying is obtained the anti-enrofloxacin monoclonal antibody of high specific.
6.2 the purifying of antibody
Antibody purification can adopt sad-ammonium sulfate salting-out process also can adopt Protein G/A affinitive layer purification.
Embodiment 7: the Enrofloxacin enzyme-linked immunoassay method is set up
7.1 coated: adopt the EF-ABA-OVA of packing to be coated with, coated concentration is 1 μ g/mL envelope antigen.Every hole adds the coating buffer that 100 μ L contain envelope antigen on 96 hole enzyme plates, and 4 ℃ are spent the night.
7.2 sealing: take out coated good enzyme plate, use washing lotion to wash plate twice, every hole adds 120 μ L confining liquids, with incubation 3h in 37 ℃ of incubators.After confining liquid is dried, put baking oven 1h.
7.3 some plate: every hole adds each the concentration standard liquid 50 μ L of Enrofloxacin through the serial dilution preparation in half good bar of sealing, adds the enzyme labelled antibody 50 μ L of 20000 times of antibody diluent dilutions again.In 37 ℃ of thermostat containers, react 30min.Use washing lotion to wash plate 6 times.
Add the sheep anti mouse horseradish peroxidase diluent 100 μ L that diluted through 1: 5000 7.4 add the every hole of ELIAS secondary antibody, be placed in 37 ℃ of thermostat containers and react 20min.Use washing lotion to wash plate 6 times.
7.5 colour developing: every hole adds substrate TMB-superoxol 100 μ L, behind 37 ℃ of colour developing 10min with 10% the H of 50 μ L
2SO
4Termination reaction.Light absorption value under microplate reader mensuration 450nm wavelength.Mapping namely obtains typical curve according to the relation of the semilog between inhibiting rate and the Enrofloxacin concentration, sees shown in the accompanying drawing 1.
The calculating formula of described inhibiting rate is:
Inhibiting rate (%)=1-B/B
0
Wherein, B is the average light absorption value of standardized solution, B
0It is the mean light absorbency value of 0 concentration standard solution.Analysis obtains the detectability (IC of Enrofloxacin
10) be 0.05ng/mL, linearity range (IC
20～IC
80) be 0.1～8.1ng/mL.
Add rate of recovery experiment:
4 portions of chicken of weighing, every part of 2g places the 15mL centrifuge tube, and adding 0,20,100,600 μ L concentration in 4 centrifuge tubes respectively is the EF standard substance of 100ng/mL, and then chicken contains that EF concentration is respectively 0,1,5,30ng/g in 4 centrifuge tubes, vibration 2min.Add again a small amount of PBS in the centrifuge tube, vortex vibration 5min, lucifuge leaves standstill 15min, adds the 4mL PBS 5min that fully vibrates, and boils 10min in the boiling water, and the centrifugal 10min of 6000r/min collects supernatant liquor.Add an amount of normal hexane in the supernatant liquor that extracts, subnatant is collected in extraction degreasing 3 times; Be settled to 6mL.Be settled to 6mL.Detect with Enrofloxacin Artificial Antigens of the present invention and antibody.
Experimental result see Table 1 and table 2 shown in:
Table 1 Enrofloxacin ELISA basic parameter
Table 2 direct competitive ELISA detects actual sample and adds the rate of recovery
By table 1 and table 2 as can be known, the Enrofloxacin artificial semiantigen that the inventive method prepares, antigen and antibody sensitivity are higher, sensing range 0.1～8.1ng/mL, detectability has reached 0.05ng/mL, the rate of recovery reaches 103～122%, and the variation coefficient has produced good effect 3.8～13.2.
Claims (6)
1. Enrofloxacin artificial semiantigen it is characterized in that reacting under NHS, DDC and DMF existence condition with Enrofloxacin and aminobutyric acid and prepares, and described haptens contains 4 carbon atom arms, has molecular structure shown in the formula (II):
Wherein, n is-CH
2The group number, n=3;
The preparation method of described Enrofloxacin artificial semiantigen may further comprise the steps:
(1) be Enrofloxacin, N-hydroxy-succinamide and fat-soluble carbodiimide to be placed reaction vessel in 1: 1: 1 by molar ratio, and with said mixture with a small amount of DMF dissolving, lucifuge stirs and spends the night under the room temperature;
(2) will get A liquid through the reactant centrifuging and taking supernatant that step (1) obtains;
The aminobutyric acid of getting with the Enrofloxacin equimolar amount is dissolved in a small amount of distilled water, gets B liquid;
(3) B liquid slowly is added in the A liquid, stirred overnight at room temperature, the centrifuging and taking supernatant liquor, transferring supernatant liquor is alkalescence, with the sedimentation and filtration of separating out, collects supernatant liquor;
(4) supernatant liquor that step (3) is made transfers to acidity, and collecting precipitation obtains the Enrofloxacin haptens.
2. Enrofloxacin artificial semiantigen according to claim 1, the described accent supernatant liquor of step (3) that it is characterized in that the preparation method is that alkalescence is that supernatant liquor pH value is adjusted to 8.0～8.5; The described accent supernatant liquor of step (4) is that acidity is that supernatant liquor pH value is adjusted to 5.0～6.0.
3. an Enrofloxacin Artificial Antigens it is characterized in that the described Enrofloxacin haptens of claim 1 and protein OVA coupling are prepared; Have molecular structure shown in the formula (III):
Wherein, n is-CH
2The group number, n=3;
The preparation method of described Enrofloxacin Artificial Antigens may further comprise the steps:
(1) will wait the described Enrofloxacin haptens of mole, DDC, NHS to be dissolved among the DMF, the reaction of room temperature lucifuge is spent the night;
(2) with step (1) gained reactant centrifuging and taking supernatant liquor, supernatant liquor is joined in the borate buffer that contains carrier proteins, mixture reaction spends the night;
(3) after step (2) reaction is finished, the mixed solution dialysis tubing of packing into after will react, dialysing obtains described artificial antigen.
4. Enrofloxacin Artificial Antigens according to claim 3 is characterized in that described preparation method's the described carrier proteins of step (2) is OVA, and the concentration of carrier proteins is at 20～30mg/mL; Described temperature of reaction is 4 ℃.
5. Enrofloxacin Artificial Antigens according to claim 3, the described dialysis of step (3) that it is characterized in that described preparation method adopted 0.9% normal saline dialysis 3 days, changed dialyzate every day 3～5 times.
6. the application of the described antigen of claim 3 is characterized in that being applied to prepare Enrofloxacin antibody or being applied to Enrofloxacin immunodetection aspect.
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| 宓晓黎等.恩诺沙星免疫抗原的制备与鉴定.《中国动物检疫》.2010,第27卷(第6期),43-45. |
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