CN102749451A - Rapid gentamicin detection strip as well as preparation method and application thereof - Google Patents
Rapid gentamicin detection strip as well as preparation method and application thereof Download PDFInfo
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- CN102749451A CN102749451A CN201210238317XA CN201210238317A CN102749451A CN 102749451 A CN102749451 A CN 102749451A CN 201210238317X A CN201210238317X A CN 201210238317XA CN 201210238317 A CN201210238317 A CN 201210238317A CN 102749451 A CN102749451 A CN 102749451A
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Abstract
The invention discloses a rapid gentamicin detection strip as well as a preparation method and an application thereof, belonging to the field of immunology. According to the rapid gentamicin detection strip, a gentamicin antibody and antigen are subjected to collaurum immunochromatography, so that the rapid detection of gentamicin residue in dairy products can be realized. The invention mainly provides the rapid gentamicin detection strip which consists of a sample gasket, a gold-labelled antibody combining gasket, a nitrocellulose film, a water absorbing gasket and an MAX line, wherein the gold-labelled antibody gasket is a gentamicin monoclonal antibody labeled by colloidal gold, a detection line and a control line are enveloped at the middle part of the nitrocellulose film, the detection line is a gentamicin-protein conjugate being subjected to specific binding with gentamicin monoclonal antibody labeled by colloidal gold, and the control line is Goat anti Mouse IgG monoclonal antibody. According to the rapid gentamicin detection strip, a cutting mode is adopted, the operation is simple, the carrying is convenient, the result can be judged rapidly and accurately, the whole detection process only needs 10 minutes and the rapid gentamicin detection strip can be used for on-site supervision and is suitable for qualitative screening of a large number of samples.
Description
Technical field
The invention belongs to field of immunology, relate to the kit and the method for aminoglycoside antibiotics gentamicin residue in the fast detecting food.
Background technology
Since two thousand eight, the safety problem of dairy products in the whole nation so that the whole world caused tremendous influence, not only consumers in general's rights and interests have received infringement, whole dairy products industry comprises that numerous dairy farmers' interests have also received huge infringement.The food-safety problem of dairy products industry seriously has been related to the national economy of China, is badly in need of solving.
The safety of dairy products both need begin from the source, through each item rules and regulations quality was controlled layer by layer, also needed various detection techniques to set up successive check.Owing to raw material enormous amount, source that dairy products production relates to are numerous, therefore how to realize a large amount of raw material milk samples are screened quickly and accurately, be that present detection technique is badly in need of one of difficult point that breaks through.
Aminoglycoside antibiotics is one of milk cow common drug; Comprise streptomysin, dihydrostreptomycin, gentamicin and kanamycins etc., this type of medicine all has ototoxicity and Toxicity of Kidney, can damage cranial nerve, cochlea nerve; The lighter's auditory dysesthesia; Weight person's permanent deafness can have infringement to the near-end renal tubules,convoluted, causes blood urine, renal failure etc.Detection method commonly used at present is liquid phase chromatography, TTC method and ELISA, and these three kinds of methods all can't be implemented in on-the-spot fast detecting, is difficult to satisfy the detection sample size that increases gradually at present, and the demand of control is detected at the scene in milk source.
The immune chromatograph testing technology is a new technology that grows up the early 1990s; Because of its easy (without any need for equipment), quick, directly perceived, sensitivity, special; Be used widely at numerous areas such as clinical diagnosises, be used for the antigenic component of fast detecting serum or urine sample; Also can be used for pathogenic bacteria and other objectionable constituent etc. in the test sample.Highly sensitive and the high specificity of this method, reaction fast, and is easy and simple to handle, do not need any special instruments and equipment, is applicable to that rapid screening detects with on-the-spot.
Summary of the invention
The technical matters that the present invention solves provides gentamicin fast detecting bar and detection method and application in the food; To be to overcome traditional detection method loaded down with trivial details for the invention fundamental purpose, detect consuming time, defective such as application limitation is big in real work, and a kind of quicker, simple and direct, safe detection kit and detection method thereof are provided.
Gentamicin gold mark fast detecting bar in the food, said detector bar is overlapped in order and is pasted on end liner by sample pad, golden labeling antibody pad, nitrocellulose filter, adsorptive pads, sticks the MAX line at the sample pad place.Encapsulate the gentamicin monoclonal antibody of colloid gold particle mark on the gold labeling antibody pad; Be coated with detection line and control line on the nitrocellulose filter; Contain gentamicin-protein conjugate in the said detection line, be placed with the sheep anti mouse monoclonal antibody in the said control line.
Said protein conjugate is a bovine serum albumin(BSA);
The colloid gold particle diameter that encapsulates on the pad is 25nm, and the amount of 1mL colloid gold particle mark gentamicin monoclonal antibody is 3ng, forms collaurum-antibody complex, and the package amount of colloid gold particle is 2mL/30cm on the binding site pad.
The package amount of gentamicin-protein conjugate is 1 μ L/cm on the said detection line;
The sheep anti-mouse antibody package amount is 1 μ L/cm on the control line;
Sheep anti-mouse antibody concentration is 1mg/mL;
A kind of preparation method of gentamicin gold mark fast detecting bar sets forth as follows:
1) preparation of golden labeling antibody pad
A. the preparation of blank colloidal gold solution:
Adopt the citrate reducing process to prepare colloid gold particle; In Erlenmeyer flask, add the 97.5ml distilled water and be heated to boiling back adding 1.5ml trisodium citrate; Continue to add 1ml 1% gold chloride behind the heating 5min, continue heated and boiled to liquid and present salmon pink, keep behind the fluidized state 10min flask being placed room temperature; After fully cooling off, stored refrigerated;
B. the preparation of colloid gold label antibody:
Get blank colloidal gold solution 100ml, add the pH value that 0.1M sal tartari 150～200ul comes regulator solution.Under magnetic agitation, dropwise adding concentration is the gentamicin monoclonal antibody of 0.2mg/ml; Making final concentration is 3ng/ml, continue to stir 30min, and 12000 rev/mins after centrifugal 30 minutes; Absorb supernatant, with the resuspended liquid of golden labeling antibody that the precipitation of gold labeling antibody is fully resuspended;
The preparation of c. golden labeling antibody pad:
Resuspended good golden labeling antibody encapsulated in well cutting in advance be of a size of on the plain film of spun glass of 1cmX30cm, package amount is 2ml/30cm, and 37 ℃ of dried overnight are taken out the dry place of valve bag sealing lucifuge and preserved;
2) the encapsulating of T, C line on the nitrocellulose filter
A. the preparation of gentamicin-protein conjugate
Take by weighing 40mg sulmycin and 10mg bovine serum albumin(BSA) (BSA), be dissolved in respectively in the distilled water of 1ml, BSA solution is dropwise joined in the gentamycin solution, under the room temperature in stirring on the magnetic stirring apparatus more than the 2h.Get 120mg EDC.HCl and be dissolved in the 1ml distilled water, dropwise join again in the above-mentioned solution, at room temperature continue to stir more than the 1h, spend the night in 4 ℃ of placements then.After PBS dialysis 3 days, packing ,-20 ℃ of storages.
Encapsulating of b.T, C line
The good two-dimentional specking platform of debugging; The gentamicin protein conjugate of the suitable concn mode through line is coated on the nitrocellulose filter as detection line, carries out encapsulating of sheep anti mouse monoclonal antibody simultaneously, 37 ℃ of dry 2h as control line; Sealing, the dry place of lucifuge preserves;
3) assembling of test strips
By the requirement of power in 1 sample pad, golden labeling antibody pad, nitrocellulose filter and adsorptive pads are overlapped according to the order of sequence and to paste on the PVC base plate; And be covered with the MAX line on the surface of sample pad and golden labeling antibody pad, at last the base plate that posts is cut into the wide test strips of 4mm that is on cutting cutter.
Encapsulating the colloid gold particle diameter on the pad is 25nm, and the amount of 1mL colloid gold particle mark gentamicin monoclonal antibody is 3ng, forms collaurum-antibody complex, and the package amount of colloid gold particle is 2mL/30cm on the binding site pad.
Detection line encapsulates gentamicin-protein conjugate on the nitrocellulose filter, and concentration is 0.13mg/mL, and package amount is 1 μ L/cm; Encapsulating sheep anti-mouse antibody concentration on the control line is 1mg/mL, and package amount is 1 μ L/cm.
The resuspended formula of liquid of gold labeling antibody is formed as follows
Use kit of the present invention and detect gentamicin residue in the food, may further comprise the steps:
One, detects sample
1. raw material milk
-get the ELISA Plate micropore of requirement, place on the microwell plate;
-in each micropore, add 100 μ L sample diluting liquids and 100 μ L milk appearance to be measured, mixing successively.
-in every hole, insert gold mark detector bar, wait for 5 minutes observationss, the result is invalid after 30 minutes.
2. milk powder
-take by weighing the 1.2g powdered milk sample, add the 8.8mL dissolved in distilled water;
-get the micropore of requirement, place on the microwell plate;
-in each micropore, add 100 μ L sample diluting liquids and 100 μ L milk appearance to be measured, mixing successively.
-in every hole, insert gold mark detector bar, wait for 5 minutes observationss, the result is invalid after 30 minutes.
3. non-lactic acid fluid-like state finished milk (white milk, high-end milk, pattern milk, children's milk etc.)
-get the micropore of requirement, place on the microwell plate;
-in each micropore, add 140 μ L sample diluting liquids and 70 μ L milk appearance to be measured, mixing successively.
-in every hole, insert gold mark detector bar, wait for 5 minutes observationss, the result is invalid after 30 minutes.
4. lactic acid fluid-like state finished milk (yoghurt, milk beverage etc.)
-get volume required sample diluting liquid, adding adjuvant to concentration is 2%, vibration dissolving (with 48 detections is example, and desirable 7.5mL sample diluting liquid adds the 0.15g adjuvant);
-get the micropore of requirement, place on the microwell plate;
-get milk to be measured appearance, the centrifugal 10min of 3000g;
-get the 70uL supernatant to micropore, add the sample diluting liquid that 140uL has added 2% adjuvant again, mixing;
-in every hole, insert gold mark detector bar, wait for 5 minutes observationss, the result is invalid after 10 minutes.
Annotate:
1) the adjuvant dissolving is very slow, needs the long period, and in the vibration course of dissolution, like the adjuvant caking, available rifle head is chosen it broken, with accelerate dissolution; If any constent temperature heater, also can about 35 ℃, heat and help its dissolving.
2) milk beverage centrifugal after, the upper strata possibly still be muddy state, can directly draw the liquid of the superiors, is used for detecting getting final product.
Two, the result judges:
When reading as a result, detector bar should place observer's front by disposing way as shown in the figure.Please don't or be inverted detector bar from the detector bar side and carry out observation as a result.
● feminine gender: T line (detection line is near an end of sample solution) and C line (control line) all develop the color, and are judged as feminine gender, and gentamicin concentration is lower than 100ppb or does not contain gentamicin residue in the expression milk sample.
● the positive: only C line colour developing, and the T line does not have colour developing, is judged as the positive, gentamicin concentration is higher than 100ppb in the expression milk sample;
● invalid: the C line does not develop the color, and shows that incorrect operating process or detector bar lost efficacy.In the case, should read over instructions once more, and test again with new detector bar.Still exist like problem, should stop using this lot number product, and get in touch with local supplier.
Three, specificity:
Other aminoglycoside antibiotics cross reacting rates such as this product and streptomysin, kanamycins 0.1%, with other kind medicine cross reacting rates < 0.1% such as beta-lactam, Tetracyclines, chloromycetin, sulfamido and quinolones.
The know-why of kit of the present invention is following:
According to the antigen-antibody reaction principle; Carry out the significant notation of antibody colloidal gold; On nitrocellulose filter, draw system gentamicin-protein conjugate simultaneously; Gentamicin residue thing in this conjugate and the sample is competed golden mark gentamicin monoclonal antibody jointly and is formed effective competition resistance reaction as detection line, and sheep anti-mouse antibody is as control line.Gold mark compound combines on object in the chromatography process in the positive sample and the gold-marking binding pad; When chromatography arrives detection line; Free golden labeling antibody with detection line on protein conjugate combine; Simultaneously because the reaction of antigen-antibody is reversible reaction, thus protein conjugate also compete the seized survey line of golden labeling antibody that has combined with object and catch colour developing, the many more detection lines colour developings of object are just shallow more in the sample; When the object in the sample reached finite concentration, detection line no longer developed the color owing to catching less than golden labeling antibody; Unnecessary gold mark compound continues chromatography, catches colour developing with the control line that is coated with sheep anti-mouse antibody when reaching control line.
Beneficial effect:
This kit is that modal gentamicin in the micromolecule veterinary drug aminoglycoside medicaments is realized the fast detecting to gentamicin residue thing in the dairy products through the colloidal gold immunochromatographimethod technology; Through to the simple pre-treatment of sample, in 10-20min, can realize residue detection to gentamicin.This kit has following characteristics:
1. pre-treatment is simple: only need just can realize the detection to dairy products through centrifugal or interpolation;
2. detection time is short: in 10-20min, can realize gentamicin residue is detected;
3. it is low to detect cost: detect with respect to instrument and ELISA, the required testing cost of single sample is lower;
4. applied widely: as, to can be used for laboratory and open-air and on-the-spot detection because kit does not have an expensive instrument requirement simultaneously to operating environment is less demanding;
5. personnel require low: owing in whole testing process, need the instrument and the reagent of usefulness less, and professional less demanding to the experimenter, the whole operation process is simple, and the testing result naked eyes just can carry out interpretation;
6. security is higher: no any poisonous and harmful substance in the kit.
The gentamicin gold labeled quick detection reagent box that the present invention studied has been realized gentamicin residue in the dairy products is detected, and its detection time is short, and it is low to detect cost, and sensing range is wide; Detection sensitivity meets country and European Union's standard.Therefore; It meets the demand for development of country to detection technique in food security and the inspection and quarantining for import/export; Help improving the efficient and the accuracy rate of food security and inspection and quarantining for import/export work, help guaranteeing food security and foreign trade, thereby play a significant role at public safety field.
Description of drawings
Fig. 1: detector bar testing result decision method;
Fig. 2: the detector bar result judges; 0,12.5,25,50 and 100 for adding concentration shown in the figure, and unit is ng/ml, and preceding 4 interpolation concentration testing results are negative, last positive result;
Fig. 3: detector bar structural representation: 1PVC base plate wherein, 2 sample pad, 3 gold-marking binding pads, 4NC nitrocellulose filter, 5 detection line T, 6 nature controlling line C, 7 adsorptive pads.
Embodiment
Below the present invention is done further practical implementation explanation,
The sensitivity experiment of embodiment 1 kit
Get 5 parts and do not contain the milk sample of aminoglycoside antibiotics, carry out the interpolation (table 1) of variable concentrations standard items through instrument detecting (No. 1163 bulletin-7-2009 methods of the GB/T 22969-2008 national standard method and the Ministry of Agriculture).Every duplicate samples replication 5 times the detection sensitivity of the least concentration of positive findings as kit occur with 95%.
The sensitivity determination result of 5 parts of milk appearance variable concentrations standard items interpolation sees table 1.The result shows, gentamicin gold mark fast detecting bar, 100ng/mL adds the milk appearance of concentration, only have in detecting for 75 times 1 part negative, positive rate is 98.7%, therefore, the sensitivity of this kit can reach 100ng/mL.
Table 1 gentamicin sensitivity experiment result
Annotate: "-" expression is negative; "+" expression is positive; The quantity that occurs positive or negative during numeral repeats for 5 times.
The specificity of so-called detector bar is meant that this detector bar and other drug have no cross reaction, and this experiment is chosen several types of common veterinary drug standard items and added test experience (seeing table 2), and the interpolation final concentration of all medicines is 0.1 and 1ug/ml.Testing result shows no cross reaction between gentamicin gold mark fast detecting bar and other drug, has the specificity of height.。
The testing result of table 2 gentamicin gold mark fast detecting bar
Annotate: "-" expression testing result is negative, and "+" expression testing result is positive.
Claims (9)
1. the gentamicin gold is marked the fast detecting bar, and said detector bar is overlapped in order and pasted on end liner by sample pad, golden labeling antibody pad, nitrocellulose filter, adsorptive pads, pastes the MAX line at the sample pad place; Encapsulate the gentamicin monoclonal antibody of colloid gold particle mark on the gold labeling antibody pad; Be coated with detection line and control line on the nitrocellulose filter; Contain gentamicin-protein conjugate in the said detection line, be placed with the sheep anti mouse monoclonal antibody in the said control line.
2. according to the said gentamicin gold of claim 1 mark fast detecting bar, it is characterized in that said gentamicin-protein conjugate is a bovine serum albumin(BSA).
3. according to the said gentamicin gold of claim 1 mark fast detecting bar, it is characterized in that the colloid gold particle diameter that encapsulates on the said golden labeling antibody pad is 25nm, the amount of 1mL colloid gold particle mark gentamicin monoclonal antibody is 3ng.
4. according to the said gentamicin gold of claim 1 mark fast detecting bar, it is characterized in that the package amount of colloid gold particle is 2mL/30cm on the said binding site pad.
5. according to the said gentamicin gold of claim 1 mark fast detecting bar, it is characterized in that the package amount of gentamicin-protein conjugate is 1 μ L/cm on the said detection line.
6. according to the said gentamicin gold of claim 1 mark fast detecting bar, it is characterized in that the sheep anti-mouse antibody package amount is 1 μ L/cm on the said control line, sheep anti-mouse antibody concentration is 1mg/mL.
7. the preparation method like the said gentamicin gold of power 1-6 mark fast detecting bar comprises the steps:
1) preparation of golden labeling antibody pad
A. the preparation of blank colloidal gold solution:
Adopt the citrate reducing process to prepare colloid gold particle; In Erlenmeyer flask, add the 97.5ml distilled water and be heated to boiling back adding 1.5ml trisodium citrate; Continue to add 1ml 1% gold chloride behind the heating 5min, continue heated and boiled to liquid and present salmon pink, keep behind the fluidized state 10min flask being placed room temperature; After fully cooling off, stored refrigerated;
B. the preparation of colloid gold label antibody:
Get blank colloidal gold solution 100ml, add the pH value that 0.1M sal tartari 150～200ul comes regulator solution.Under magnetic agitation, dropwise adding concentration is the gentamicin monoclonal antibody of 0.2mg/ml, and final concentration is 3ng/ml; Continue to stir 30min, 12000 rev/mins after centrifugal 30 minutes, absorb supernatant, with the resuspended liquid of golden labeling antibody that the precipitation of gold labeling antibody is fully resuspended;
The preparation of c. golden labeling antibody pad:
Resuspended good golden labeling antibody encapsulated in well cutting in advance be of a size of on the plain film of spun glass of 1cmX30cm, package amount is 2ml/30cm, and 37 ℃ of dried overnight are taken out the dry place of valve bag sealing lucifuge and preserved;
2) the encapsulating of T, C line on the nitrocellulose filter
A. the preparation of gentamicin-protein conjugate
Take by weighing 40mg sulmycin and 10mg bovine serum albumin(BSA), be dissolved in respectively in the distilled water of 1ml, BSA solution is dropwise joined in the gentamycin solution, under the room temperature in stirring on the magnetic stirring apparatus more than the 2h.Get 120mg EDC.HCl and be dissolved in the 1ml distilled water, dropwise join again in the above-mentioned solution, at room temperature continue to stir more than the 1h, spend the night in 4 ℃ of placements then.After PBS dialysis 3 days, packing ,-20 ℃ of storages.
Encapsulating of b.T, C line
The good two-dimentional specking platform of debugging is coated on the nitrocellulose filter mode of gentamicin protein conjugate through line as detection line, carries out encapsulating as control line of sheep anti mouse monoclonal antibody simultaneously, 37 ℃ of dry 2h, and the dry place of sealing lucifuge preserves;
3) assembling of test strips
By the requirement of power in 1 sample pad, golden labeling antibody pad, nitrocellulose filter and adsorptive pads are overlapped according to the order of sequence and to paste on the PVC base plate; And be covered with the MAX line on the surface of sample pad and golden labeling antibody pad, the base plate that posts is cut into the wide test strips of 4mm that is on cutting cutter.
8. preparation method like the said gentamicin of claim 7 gold mark fast detecting bar, it is characterized in that encapsulating on the said control line sheep anti-mouse antibody concentration is 1mg/mL, package amount is 1 μ L/cm.
9. one kind like the application of the said gentamicin of power 1-6 gold mark fast detecting bar in dairy produce detects.
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Application publication date: 20121024 |