CN102353769A - Fluoroquinolones labeling method with HRP - Google Patents
Fluoroquinolones labeling method with HRP Download PDFInfo
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- CN102353769A CN102353769A CN2011101700479A CN201110170047A CN102353769A CN 102353769 A CN102353769 A CN 102353769A CN 2011101700479 A CN2011101700479 A CN 2011101700479A CN 201110170047 A CN201110170047 A CN 201110170047A CN 102353769 A CN102353769 A CN 102353769A
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- fluoroquinolones
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- hrp
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Abstract
The invention relates to a fluoroquinolones labeling method with HRP and is characterized by coupling fluoroquinolones with a carrier protein to obtain a pre-conjugate at first and then coupling the pre-conjugate with horseradish peroxidase to obtain a required enzyme-labelled antigen. The carrier protein is used in the invention as an intermediate conjugate to accomplish fluoroquinolones labeling with HRP. The method provided by the invention is simple to operate and provides necessary enzyme-labelled antigen for direct competitive ELISA detection of fluoroquinolones residues in animal food.
Description
[technical field]
The present invention relates to technological field of biochemistry, relate in particular to the method for a kind of fluoroquinolones mark HRP.
[background technology]
Fluoquinolone (fl μ oroq μ inolones, FQNS or FQS) type medicine, such medicine comprise the husky star of single promise, Difloxacin, Enrofloxacin and sarafloxacin etc., belong to the chemosynthesis antimicrobial, and such medicine is widely used in the breed of fowl poultry, aquatic products.But owing to interests are ordered about, to this type of veterinary drug overdose, ultra scope use, of common occurrence in accordance with abominable phenomenons such as off-drug periods.FQNS class medicament residue not only has direct harm to human body, and even more serious is that pathogenic bacteria strengthen its drug resistance just gradually, and has found that some FQNS has potential carcinogenicity, and its residue problem has caused widely to be paid close attention to.At present, to fluoroquinolones class medicine strict regulation is arranged all in detection of veterinary drugs in food both at home and abroad, like residual the limiting the quantity of of Japan's amended " positive list system ", domestic also have relevant detection GB and a rower.
Detect fluoroquinolones residual in animal foodstuff; Should select high specificity, highly sensitive, efficient detection method fast; Enzyme linked immunosorbent detection (ELISA) method has highly sensitive and the good characteristics of specificity; And do not need expensive instrument and equipment; Be applicable to the fast detecting of a large amount of samples outside the laboratory; Direct competitive ELISA has the advantages that with respect to indirect competitive ELISA the reaction time is short, operation steps is few, highly sensitive, and exploitation direct competitive ELISA then needs corresponding enzyme-labelled antigen (marker enzyme in antigen).The present invention provides essential enzyme-labelled antigen for using direct competitive ELISA to detect fluoroquinolones residual in animal foodstuff.In fact, detect residual all the be to use indirect competitive ELISA of fluoroquinolones in animal foodstuff at present, be because at present in fluoroquinolones (antigen) marker enzyme be difficult to the realization.
[summary of the invention]
The technical matters that the present invention will solve provides the method for a kind of fluoroquinolones mark HRP (horseradish peroxidase); Simple to operate, for using direct competitive ELISA to detect fluoroquinolones residual in animal foodstuff essential enzyme-labelled antigen is provided.
Above-mentioned technical matters realizes through following technical scheme:
The method of a kind of fluoroquinolones mark HRP is characterized in that, earlier fluoroquinolones and carrier protein couplet is got preparatory conjugate, then preparatory conjugate and horseradish peroxidase coupling is got required enzyme-labelled antigen.
Said carrier protein is a kind of in bovine serum albumin(BSA) (BSA), human serum albumins (HSA), oralbumin (OVA), the poly-D-lysine (PLL).
The method of fluoroquinolones and carrier protein couplet adopts mixed anhydride method or carbodiimide method.
The method of conjugate and horseradish peroxidase coupling adopts glutaraldehyde two step method or sodium periodate method in advance.
Said fluoroquinolones comprises Enrofloxacin, Ofloxacin, sarafloxacin, Ciprofloxacin, flumequine.
Visible by such scheme; The present invention uses carrier protein to realize horseradish peroxidase is marked at fluoroquinolones as middle conjugate; Simple to operate, for using direct competitive ELISA to detect fluoroquinolones residual in animal foodstuff essential enzyme-labelled antigen is provided.
[embodiment]
Embodiment one Ciprofloxacin (CIF) mark HRP
One, CIF-BSA's is synthetic---adopt carbodiimide method
101) take by weighing Ciprofloxacin 0.35g in the 50ml beaker, add the dissolving of 15ml hydrochloric acid (1mol) magnetic agitation, get CPFX solution;
102) take by weighing N-hydroxy-succinamide (NHS) 0.1g (30 * 3 * 10
-5Mol), 1-ethyl-3-(3-dimethyl aminopropyl)-carbodiimides (EDC) 0.17g (30 * 3 * 10
-5Mol), add in the above-mentioned CIF solution stirring reaction 2h, the CIF active ester that must activate; Take by weighing BSA (bovine serum albumin(BSA)) 2.04g (3 * 10
-5Mol) put into the 50ml beaker, add 20ml pure water and stirrer in beaker, be placed on to stir on the magnetic stirring apparatus and make it dissolving, BSA solution;
103) the CIF active ester is slowly splashed in the BSA solution, keep stirring, the process of splashing into is approximately 20min, afterwards stirring reaction 2h;
104) stop to stir, reacted CIF-BSA solution 5000 commentariess on classics/min are centrifugal, get supernatant and pack in the bag filter, put 4 ℃ of dialysis of refrigerator with physiological saline or conventional PBS, changed dislysate twice in one day, dialysed continuously three days, must required preparatory conjugate CIF-BSA; Subsequent use or the freeze-drying of tubule packing, every pipe 1ml.
Two, mark HRP (horseradish peroxidase)---adopt the sodium periodate method
201) take by weighing HRP 4mg and be dissolved in the 1ml deionized water, add the NaIO that 0.2ml newly joins
4Solution (21.4mg/ml=0.1mol/L), room temperature lucifuge stirring reaction 20min;
202) use acetate buffer (1mM pH=4.4) dialysed overnight;
203) using 0.01M carbonate buffer solution adjust pH is 9.0～9.5;
204) add the preparatory conjugate CIF-BSA of 5mg, room temperature lucifuge stirring reaction 2h;
205) add freshly prepared 0.1mlNaBH
4Solution (4mg/ml), 4 ℃ of lucifuge stirring reaction 2h;
206) reaction product solution 5000 commentaries on classics/min are centrifugal, get supernatant and pack in the bag filter, put 4 ℃ of dialysis of refrigerator with physiological saline or conventional PBS, changed dislysate twice in one day, dialysed continuously three days.
Embodiment two Enrofloxacins (ENF) mark HRP
One, ENF-HAS is synthetic---adopt carbodiimide method
101) take by weighing ENF 0.38g in the 50ml beaker, add the dissolving of 15ml hydrochloric acid (1mol) magnetic agitation, get ENF solution;
102) take by weighing NHS 0.1g (30 * 3 * 10
-5Mol), EDC 0.17g (30 * 3 * 10
-5Mol), add in the above-mentioned ENF solution stirring reaction 2h, the ENF active ester that must activate; Take by weighing HAS (human serum albumins) 2.10g and put into the 50ml beaker, add 20ml pure water and stirrer in beaker, be placed on to stir on the magnetic stirring apparatus and make it dissolving, HAS solution;
103) the ENF active ester is slowly splashed in the HAS solution, keep stirring, the process of splashing into is approximately 20min, afterwards stirring reaction 2h;
104) stop to stir, reacted ENF-HAS solution 5000 commentariess on classics/min are centrifugal, get supernatant and pack in the bag filter, put 4 ℃ of dialysis of refrigerator with physiological saline or conventional PBS, changed dislysate twice in one day, dialysed continuously three days, must required preparatory conjugate ENF-HAS; Subsequent use or the freeze-drying of tubule packing, every pipe 1ml.
Two, mark HRP (horseradish peroxidase)---adopt the glutaraldehyde two step method
201) take by weighing HRP25mg and be dissolved in 1.25% glutaraldehyde solution, in the room temperature standing over night;
202) reacted enzyme solutions is used the physiological saline wash-out through Sephadex G-25 chromatographic column, and flow speed control was collected brown effluent at 1ml/1 minute, greater than 5ml, then was concentrated into 5ml with PEG like volume, places in the 25ml small beaker, slowly stirs;
203) ENF-HAS 12.5mg to be marked is diluted to 5ml with physiological saline, dropwise adds in the enzyme solutions under stirring;
204), continue to stir 3 with 1M PH9.5 carbonic acid buffer 0.25ml? Hour;
205) add 0.2M lysine 0.25ml, behind the mixing, put room temperature 2 hours;
206) under agitation dropwise add the equal-volume saturated ammonium sulfate, put 4 ℃ 1 hour;
207) 3000rpm centrifugal half an hour, abandon supernatant; Sediment is washed secondary with semi-saturation ammonium sulfate, and last sediment is dissolved among the PBS of a small amount of 0.15M PH7.4;
208) above-mentioned solution is packed in the bag filter, to the PB buffer saline dialysis of 0.15M PH7.4, remove (detecting with Nai Shi reagent) behind the ammonium ion, 10000rpm removed and precipitates in centrifugal 30 minutes, and supernatant is enzyme conjugates, after the packing, and stored frozen.
Embodiment three sarafloxacins (OLA) mark HRP
One, OLA-OVA is synthetic---adopt carbodiimide method
101) take by weighing OLA 0.43g in the 50ml beaker, add the dissolving of 15ml hydrochloric acid (1mol) magnetic agitation, get OLA solution;
102) take by weighing NHS 0.1g (30 * 3 * 10
-5Mol), EDC0.17g (30 * 3 * 10
-5Mol), add in the above-mentioned OLA solution stirring reaction 2h, the OLA active ester that must activate; Take by weighing OVA (oralbumin) 1.88g and put into the 50ml beaker, add 20ml pure water and stirrer in beaker, be placed on to stir on the magnetic stirring apparatus and make it dissolving, OVA solution;
103) the OLA active ester is slowly splashed in the OVA solution, keep stirring, the process of splashing into is approximately 20min, afterwards stirring reaction 2h;
104) stop to stir, reacted OLA-OVA solution 5000 commentariess on classics/min are centrifugal, get supernatant and pack in the bag filter, put 4 ℃ of dialysis of refrigerator with physiological saline or conventional PBS, changed dislysate twice in one day, dialysed continuously three days, must required preparatory conjugate OLA-OVA; Subsequent use or the freeze-drying of tubule packing, every pipe 1ml.
Two, mark HRP (horseradish peroxidase)---adopt the glutaraldehyde two step method
201) taking by weighing HRP25mg is dissolved in the 1.25% concentration glutaraldehyde solution, in the room temperature standing over night;
202) reacted enzyme solutions is used the physiological saline wash-out through Sephadex G-25 chromatographic column, and flow speed control was collected brown effluent at 1ml/1 minute, greater than 5ml, then was concentrated into 5ml with PEG like volume, places in the 25ml small beaker, slowly stirs;
203) OLA-OVA 12.5mg to be marked is diluted to 5ml with physiological saline, dropwise adds in the enzyme solutions under stirring;
204), continue to stir 3 hours with 1M PH9.5 carbonic acid buffer 0.25ml;
205) add 0.2M lysine 0.25ml, behind the mixing, put room temperature 2 hours;
206) under agitation dropwise add the equal-volume saturated ammonium sulfate, put 4 ℃ 1 hour;
207) 3000rpm centrifugal half an hour, abandon supernatant; Sediment is washed secondary with semi-saturation ammonium sulfate, and last sediment is dissolved among the PBS of a small amount of 0.15M PH7.4;
208) above-mentioned solution is packed in the bag filter, to the PB buffer saline dialysis of 0.15M PH7.4, remove (detecting with Nai Shi reagent) behind the ammonium ion, 10000rpm removed and precipitates in centrifugal 30 minutes, and supernatant is enzyme conjugates, after the packing, and stored frozen.
Embodiment four Ofloxacins (OFL) mark HRP
One, OFL-PLL is synthetic---adopt mixed anhydride method
101) take by weighing OFL 0.55g, add 4mL and contain in the pyridine solution of 22.8mg (about 0.2mmol) glutaric anhydride, stirring at room reaction 22h;
102) after reaction was accomplished, nitrogen dried up pyridine; With 8mL solvent (DMF and 1, the 4-dioxane mixes at 1: 1) dissolving OFL-glutaric anhydride semialdehyde, add the positive tri-n-butylamine of 52.4 μ L (about 0.2mmol), stir 10 min in the ice;
103) add isobutyl chlorocarbonate 28.8 μ L (about 0.2mmol), stirring at room reaction 1h dropwise adds the OFL solution that activates in 10mL, the pH 8.5 ice-cold dobell's solutions (containing 0.1mol/L poly-D-lysine PLL), adds in the 1h, and the stirring at room reaction is spent the night;
104) the conjugate OFL-PLL that will react gained crosses Sephadex G-25M chromatographic column and carries out purifying, (ultraviolet absorption method is measured carrier concn as conjugate concentration), required conjugate OFL-PLL ,-20 ℃ of preservations.
Two, mark HRP (horseradish peroxidase)---adopt the sodium periodate method
201) take by weighing HRP 4mg and be dissolved in the 1ml deionized water, add the NaIO that 0.2ml newly joins
4Solution (21.4mg/ml=0.1mol/L), room temperature lucifuge stirring reaction 20min;
202) use acetate buffer (1mM pH=4.4) dialysed overnight;
203) using 0.01M carbonate buffer solution adjust pH is 9.0～9.5;
204) add the preparatory conjugate OFL-PLL of 5mg, room temperature lucifuge stirring reaction 2h;
205) add freshly prepared 0.1mlNaBH
4Solution (4mg/ml), 4 ℃ of lucifuge stirring reaction 2h;
206) reaction product solution 5000 commentaries on classics/min are centrifugal, get supernatant and pack in the bag filter, put 4 ℃ of dialysis of refrigerator with physiological saline or conventional PBS, changed dislysate twice in one day, dialysed continuously three days.
The present invention is not limited to the foregoing description; For example, fluoroquinolones is not limited to include only Enrofloxacin, Ofloxacin, sarafloxacin, Ciprofloxacin, flumequine; Based on simple replacement the foregoing description, that do not make creative work, should belong to the scope that the present invention discloses.
Claims (5)
1. the method for a fluoroquinolones mark HRP is characterized in that, earlier fluoroquinolones and carrier protein couplet is got preparatory conjugate, then preparatory conjugate and horseradish peroxidase coupling is got required enzyme-labelled antigen.
2. method according to claim 1 is characterized in that, said carrier protein is a kind of in bovine serum albumin(BSA), human serum albumins, oralbumin, the poly-D-lysine.
3. method according to claim 1 is characterized in that, the method for fluoroquinolones and carrier protein couplet adopts mixed anhydride method or carbodiimide method.
4. method according to claim 1 is characterized in that, the method for conjugate and horseradish peroxidase coupling adopts glutaraldehyde two step method or sodium periodate method in advance.
5. method according to claim 1 is characterized in that said fluoroquinolones comprises Enrofloxacin, Ofloxacin, sarafloxacin, Ciprofloxacin, flumequine.
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| CN2011101700479A CN102353769A (en) | 2011-06-22 | 2011-06-22 | Fluoroquinolones labeling method with HRP |
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| CN2011101700479A CN102353769A (en) | 2011-06-22 | 2011-06-22 | Fluoroquinolones labeling method with HRP |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102680677A (en) * | 2012-05-08 | 2012-09-19 | 嘉兴博泰生物科技发展有限公司 | Enzyme linked immunosorbent assay kit and detection method for detecting ofloxacin |
| CN102707045A (en) * | 2012-05-04 | 2012-10-03 | 嘉兴博泰生物科技发展有限公司 | Enzyme linked immunosorbent assay kit and method for detecting ciprofloxacin |
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| WO2002093162A2 (en) * | 2001-05-17 | 2002-11-21 | Mcgill University | Individualization of therapy with antibiotic agents |
| CN1707265A (en) * | 2004-06-11 | 2005-12-14 | 中国兽医药品监察所 | Enzyme-linked immunological kit for detecting tetracycline drug |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102707045A (en) * | 2012-05-04 | 2012-10-03 | 嘉兴博泰生物科技发展有限公司 | Enzyme linked immunosorbent assay kit and method for detecting ciprofloxacin |
| CN102680677A (en) * | 2012-05-08 | 2012-09-19 | 嘉兴博泰生物科技发展有限公司 | Enzyme linked immunosorbent assay kit and detection method for detecting ofloxacin |
| CN102680677B (en) * | 2012-05-08 | 2015-04-01 | 嘉兴博泰生物科技发展有限公司 | Enzyme linked immunosorbent assay kit and detection method for detecting ofloxacin |
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Application publication date: 20120215 |