CN102980980A - Multi-residue colloidal-gold rapid detection kit, and detection method and application thereof - Google Patents
Multi-residue colloidal-gold rapid detection kit, and detection method and application thereof Download PDFInfo
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Abstract
The invention discloses a multi-residue colloidal-gold rapid detection kit and a detection method and application thereof, belonging to the field of immunology. The kit provided by the invention mainly comprises a multi-residue colloidal-gold rapid detection card; the detection card comprises a detector bar and a plastic card shell, and the detector bar is composed of a sample pad, a colloidal-gold antibody binding pad, a nitrocellulose membrane and a water absorbing pad. The colloidal-gold antibody binding pad comprises a colloidal gold labeled gentamycin monoclonal antibody, a colloidal gold labeled kanamycin monoclonal antibody and a colloidal gold labeled fluoroquinolone monoclonal antibody. The central part of the nitrocellulose membrane is coated with a detection line and a control line, wherein the detection line is arranged to be a protein conjugate and the control line is a goat anti-mouse IgG monoclonal antibody. The rapid detection kit provided by the invention can realize simultaneous detection of a plurality of residues only through simple pre-treatment, the whole operation process is simple, the kit is convenient to carry, result determination is rapid and accurate, and the kit is applicable to on-site monitoring and to qualitative screening of considerable samples.
Description
Technical field
The invention belongs to field of immunology, relate to 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 been subject to infringement, whole Dairy Industry comprises that numerous dairy farmers' interests also have been subject to huge infringement.The food-safety problem of Dairy Industry seriously has been related to the national economy of China, is badly in need of solving.The safety of dairy products had both needed by every rules and regulations quality to be controlled layer by layer from the source, 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.
On the other hand, because of increasing and the enhancing of bacterial drug resistance of Antibiotics, the medicament categories that is used at present cow disease prevention and treatment also increases greatly, so that the medicament residue in the raw material milk is except common beta-lactam antibiotic, the residual ratio of fluoroquinolone antibiotics, aminoglycoside antibiotics also increases greatly.These medicament residues exist potential safety hazard equally to human body, easily cause the nerve system of human body bad reaction such as residual in human body of fluoroquinolones, also can bring out epileptics when serious, and with the potential abnormal effect of urging, aminoglycoside medicaments then all has ototoxicity and Toxicity of Kidney, can damage cranial nerve, cochlea nerve, the lighter's Hearing, the severe one permanent deafness, can infringement be arranged to the near-end renal tubules,convoluted, cause blood urine, renal failure etc., in addition, the long-term taking of these medicines also easily causes bacterial drug resistance.Therefore, in former milk purchase link, is badly in need of a kind of method that can the fast detecting multiple antibiotic residues and saves time, reduce cost, raise the efficiency with help inspection body and enterprise.
At present, mostly the detection method for fluoquinolone and aminoglycoside antibiotics is the methods such as Enzyme-linked Immunosorbent Assay reaction (ELISA), high performance liquid chromatography (HPLC) or LC-MS (LC-MS/MS).These methods have the characteristics such as accurate, highly sensitive, good reproducibility as a kind of laboratory conventional method, yet, owing to exist requirements such as equipment, environment, operative skills, sense cycle is long, expensive, thereby its detection mainly concentrates in large-scale scientific research testing agency or the large enterprise laboratory, and the extensive screening sample that is not suitable for medium-sized and small enterprises and numerous milking stations uses.Aspect fast detecting, existing method for quick mainly concentrates beta-lactam antibiotic to detect, such as UNISENSOR, β-STAR, ROSA, SNAP etc., fast detecting product for fluoquinolone and aminoglycoside antibiotics does not then almost have, and these methods all are only to detect for a kind of microbiotic, because it is more that the milk microbiotic detects index, the method of single detection index means that then a former milk sample uses a plurality of testing products to carry out repeated detection with regard to needs, both expend the time, also wasted manpower, financial resources.Therefore be badly in need of the exploitation multiple index quick detecting method to satisfy the detection demand that day by day increases.
Summary of the invention
The technical matters that the present invention solves provides and a kind ofly can detect simultaneously multiple antibiotic residues detection kit and detection method and application in the food; It is loaded down with trivial details that the invention fundamental purpose is to overcome traditional detection method, detect length consuming time, the defective such as application limitation is large in real work, changed simultaneously that unitem detects and multiple test item simply merges the situation of detection, thereby a kind of quicker, simple and direct, detection kit and detection method thereof that cost performance is high, safe are provided.
Many residue golds labeled quick detection reagent box in the food, described kit comprise that detector bar and plastics get stuck, and described detector bar is comprised of sample pad, golden labeling antibody pad, nitrocellulose filter, adsorptive pads.Described golden labeling antibody pad is comprised of the gentamicin monoclonal antibody of colloid gold label, the kanamycins monoclonal antibody of colloid gold label and the fluoroquinolones monoclonal antibody of colloid gold label; Described nitrocellulose filter middle part is coated with detection line and control line, detection line is provided with the protein conjugate that can carry out with three kinds of monoclonal antibodies on the golden labeling antibody pad specific binding, protein conjugate is respectively Ciprofloxacin-BSA conjugate, gentamicin-BSA conjugate, kanamycins-BSA conjugate, and control line is provided with the sheep anti-mouse igg monoclonal antibody.
Colloid gold label gentamicin monoclonal antibody on the described golden labeling antibody pad is combined by the colloid gold particle of gentamicin monoclonal antibody and 25nm;
Colloid gold label kanamycins monoclonal antibody is combined by the colloid gold particle of kanamycins monoclonal antibody and 25nm;
Colloid gold label fluoroquinolones monoclonal antibody is combined by the colloid gold particle of fluoroquinolones monoclonal antibody and 60nm.
The coated concentration of the Ciprofloxacin of detection line-BSA conjugate, gentamicin-BSA conjugate, kanamycins-BSA conjugate is respectively 0.08,0.13 and 0.15mg/mL on the described nitrocellulose filter, and package amount is 1 μ L/cm;
Coated sheep anti-mouse igg monoclonal antibody on the control line, concentration is 1mg/mL, package amount is 1 μ L/cm.
A kind of preparation method of many residue golds labeled quick detection reagent box is described below:
1) preparation of golden labeling antibody pad
A. the preparation of blank collaurum:
Adopt the citrate reducing process to prepare colloid gold particle, prepare the big or small colloidal gold solution for 25nm and 60nm of colloid gold particle by regulating gold chloride and trisodium citrate addition.The color of the solution can change in the refining process, keeps behind the fluidized state 10min flask being placed room temperature when the color of the solution no longer changes, and fully after the cooling, takes out the rotor of reaction usefulness, 4 ℃ of stored refrigerated of colloidal gold solution, and the shelf-life is 1 month;
B. the preparation of the resuspended liquid of golden labeling antibody
The first step takes by weighing Tris 0.605g with analytical balance, adds in the 1L beaker.
Second step takes by weighing sucrose 25.000g with analytical balance, adds in the above beaker.
The 3rd step 500mL graduated cylinder is measured pure water 500mL, adds in the above-mentioned beaker, and it is fully dissolved.
The 4th goes on foot gentle agitation on magnetic stirring apparatus, makes that each component fully dissolves, mixing.
The 5th step was regulated pH value to 8.2 ± 0.1 with aforesaid liquid with 4mol HCl.
The 6th step took by weighing bovine serum albumin(BSA) (BSA) 5.000g with analytical balance, added in the above beaker
The 7th step gentle agitation on magnetic stirring apparatus again makes that BSA fully dissolves, mixing.
The 8th step, with 0.22 μ m miillpore filter aseptic filtration, the liquid after the filtration was packed in the 500mL reagent bottle with aforesaid liquid, labeling, and 4 ℃ are for subsequent use, the preservation term of validity 2 months.
C. the preparation of colloidal gold labeled monoclonal antibody:
Get respectively the gold grain size and be the colloidal gold solution 100mL of 25nm or 60nm, under stirring, add 0.1M sal tartari 150～200 μ L, keep after 7～10 minutes, slowly add the golden mark that corresponding monoclonal antibody carries out antibody, continue to stir and dropwise to add 25% BSA solution behind the 30min and make it final concentration and reach 0.4%, continue to stir 30min, centrifugal at last, absorb supernatant, with the resuspended liquid of golden labeling antibody that the precipitation of gold labeling antibody is fully resuspended; Whole temperature of reaction control room temperature.
The gentamicin monoclonal antibody uses the colloid gold particle solution of 25nm to carry out mark, and labelled amount is 3 μ g/mL; The kanamycins monoclonal antibody uses the colloid gold particle solution of 25nm to carry out, and labelled amount is 3 μ g/mL; The fluoroquinolones monoclonal antibody uses the colloid gold particle solution of 60nm to carry out mark, and labelled amount is 5 μ g/mL.
D. the preparation of golden labeling antibody pad:
Resuspended good golden labeling antibody is coated on the glass fibre element film that well cutting in advance is of a size of 1cm*30cm, and package amount is 2mL/30cm, 37 ℃ of dried overnight, and the dry place of sealing lucifuge preserves;
2) T, C line coated on the nitrocellulose filter
A. the preparation of gentamicin-protein conjugate
Take by weighing 40mg sulmycin and 10mgBSA, 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 120mgEDCHCl and be dissolved in the 1mL distilled water, dropwise join again in the mentioned solution, at room temperature continue to stir more than the 1h, then spend the night in 4 ℃ of placements.After PBS dialysis 3 days, packing ,-20 ℃ of storages.
B. kanamycins-protein conjugate preparation
Take by weighing Kanamycin Sulfate 28mg and BSA14mg and be dissolved in respectively in the 500 μ L distilled water, on magnetic stirring apparatus, stir after mixing.Get 76mgEDC.HCl and be dissolved in the 1mL distilled water, dropwise join again in the above-mentioned mixed solution, at room temperature stir and spend the night.Dialyse more than 3 times centrifugal packing ,-20 ℃ of storages with 0.01MPBS.
C. the preparation of Ciprofloxacin-protein conjugate
Take by weighing 20mg ciprofloxacin hydrochloride, 10mgNHS and 12.5mg EDCHCl and fully be dissolved in the 1mL dimethyl formamide (DMF), under room temperature, stir 24h, obtain Ciprofloxacin (CPLX) reactant liquor.Take by weighing again BSA 50mg, make it fully to be dissolved among the 3mL PBS (pH value 7.4), the CPLX reactant liquor dropwise slowly is added drop-wise in this BSA solution, and under room temperature, stir 3h, then with the PBS 3d that dialyses, change dislysate every day 2 times, to remove unreacted small-molecule substance.With the centrifugal 20min of 3000r/min, collect supernatant, and packing-20 ℃ storage.
Being coated with of d.T, C line
The good two-dimentional specking platform of debugging, three kinds of protein conjugates that prepare are coated on the nitrocellulose filter as detection line in order by the mode of ruling, carry out simultaneously the coated as control line of sheep anti mouse monoclonal antibody, 37 ° of dry 2h of C, the dry place of sealing lucifuge preserves;
Be coated with three detection lines and a control line on the nitrocellulose filter, article three, the T1 line is that Ciprofloxacin-BSA conjugate, T2 line are that gentamicin-BSA conjugate, T3 line are kanamycins-BSA conjugate in the detection line, its coated concentration is respectively 0.08,0.13 and 0.15mg/mL, and package amount is 1 μ L/cm; Coated sheep anti mouse monoclonal antibody on the control line, concentration is 1mg/mL, package amount is 1 μ L/cm.
3) assembling of test strips
Sample pad, golden labeling antibody pad, nitrocellulose filter and adsorptive pads overlapped according to the order of sequence paste on the PVC base plate, the base plate that posts is cut into the wide test strips of 4mm that is at cutting cutter, test strips is fixed in plastics get stuck.
Use kit of the present invention and detect gentamicin residue in the food, may further comprise the steps:
One, milk sample operations
The complete reading operation instructions of elder generation before detecting;
Degreasing: get the 1-2mL milk sample in centrifuge tube, 2000 * g (being equivalent to 5000rpm), centrifugal 10min removes the upper strata fats portion;
Draw degreasing milk sample 100 μ L in test tube, add again 100 μ L sample diluting liquids, mixing.
Draw solution to be measured with plastic dropper, vertically drip 4 in the well of test card, application of sample begins timing simultaneously;
Can carry out as a result interpretation in 10-15 minute, the result is invalid after 30 minutes.
Two, the result judges:
During reading result, test card as shown in the figure disposing way places observer's front.Please don't or be inverted from the test card side test card and carry out as a result observation.(the T1 line detects fluoroquinolones, the T2 line detects gentamicin, T3 line detection kanamycins)
Negative: T line (detection line is near well one end) and C line (control line, topmost) all colour developings are judged as feminine gender, and aminoglycoside medicaments and fluoroquinolones concentration are lower than the minimum residue detection limit of stipulating in the instructions in the expression milk sample.
Positive: the colour developing of C line, as long as the result that the T line does not develop the color occurs, judge that the residual quantity of corresponding detection of drugs surpasses the detectability of instructions regulation, is judged to be the positive;
Invalid: the C line does not develop the color, and shows that incorrect operating process or detector bar lost efficacy.In the case, should again read over instructions, and retest with new detector bar.Still exist such as problem, should stop using this lot number product, and contact with local supplier.
Three, specificity:
The cross reacting rate of the other drugs such as this product and streptomysin, neomycin, macrolides, beta-lactam, Tetracyclines, chloromycetin and sulfamido＜0.1%.
Four, storage and the term of validity:
This product should be at room temperature, the dry place of shady and cool lucifuge preserves, be sure not freezing, the term of validity 12 months.Lot number and date of manufacture are seen packing box.
The know-why of kit of the present invention is as follows:
According to the antigen-antibody reaction principle, carry out the significant notation of antibody colloidal gold, on nitrocellulose filter, draw successively simultaneously Ciprofloxacin, gentamicin and kanamycins-protein conjugate, fluoroquinolones in conjugate and the sample, kanamycins and gentamicin residue thing are jointly competed colloid gold label fluoroquinolones, gentamicin and kanamycins monoclonal antibody and are formed effective competition resistance reaction as detection line, and sheep anti-mouse antibody is as control line.Gold mark compound carries out combination on object in chromatography process in the positive sample and the gold-marking binding pad, when chromatography arrives detection line, free golden labeling antibody with combine with protein conjugate on the detection line, simultaneously because the reaction of antigen-antibody is reversible reaction, thereby protein conjugate is also competed the tested survey line of golden labeling antibody of being combined with object and is caught colour developing, the more detection line colour developings of object are just more shallow 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 by the fast detecting of colloidal gold immunochromatographimethod technology realization to multiple residuals in the dairy products with modal gentamicin, kanamycins and fluoroquinolones in the little molecule veterinary drug aminoglycoside medicaments, mainly by sample is carried out simple pre-treatment, in 10min, can realize the residue detection of multiple residuals.This kit has following characteristics:
1. pre-treatment is simple: only need two to go on foot the detection that just can realize dairy products, centrifugal, two doubling dilutions;
2. detection time is short: can realize the residue detection to fluoroquinolones, gentamicin and kanamycins in 10-20min;
3. testing cost is low: with respect to the independent operation detection of instrument, ELISA and each test item, the required testing cost of many residue detection is lower;
4. applied widely: since kit to the less demanding while of operating environment without expensive instrument requirement, can be used for laboratory and open-air and on-the-spot detection;
5. personnel require low: owing in whole testing process, need instrument and the reagent of usefulness less, and professional less demanding to the experimenter, whole operating process is simple, and the testing result naked eyes just can carry out interpretation;
6. security is higher: kit is interior without any poisonous and harmful substance.
Many residue golds labeled quick detection reagent box that the present invention studies has been realized the residue detection to Multiple Classes of Antibiotics in the dairy products, and its detection time is short, and testing cost is low, and sensing range is wide; Detection sensitivity meets country and European Union's standard.Therefore, it meets country to the demand for development of detection technique in food security and the inspection and quarantining for import/export, be conducive to improve efficient and the accuracy rate of food security and inspection and quarantining for import/export work, be conducive to guarantee food security and foreign trade, thereby play a significant role at public safety field.
Description of drawings
Fig. 1: kit testing process synoptic diagram;
Fig. 2: testing result decision method;
Fig. 3: the judgement of actual result;
Fig. 4: the judgement of actual result
Fig. 5: kit structural representation
Embodiment
Below the present invention is done the explanation of further implementation:
The sensitivity experiment of embodiment 1 kit
One, detects sample
Randomly draw 20 parts of warps " gentamicin residue detects high performance liquid chromatography in No. 1163 bulletin-7-2009 animal foods of the Ministry of Agriculture " method, " GB/T 22969-2008 milk powder and milk streptomycin, the mensuration Liquid Chromatography-Tandem Mass Spectrometry of dihydrostreptomycin and yapamicin relict amount " method and " 14 kinds of quinolone medicine method for detecting residue liquid chromatography-mass spectrography/mass spectroscopies in the GB/T 21312-2007 animal derived food " method detected the cloudy sample that does not contain this class material and carried out the sensitivity test experience.
Two, the preparation of contaminated sample
1. the preparation of standard solution
Prepare respectively gentamicin and kanamycins mixed standard solution, final concentration is gentamicin 10 μ g/mL, kanamycins 10 μ g/mL; Prepare respectively the standard solution of 8 kinds of fluoroquinolones, such as Enrofloxacin, Danofloxacin, Ciprofloxacin etc., the concentration of solution is 5 μ g/mL.
2. sample adds
Get 20 parts of negative milk samples, every part of milk sample is divided into 4 groups: one group as blank; One group is added according to 1mL milk sample+accurate solution of 5 μ L; One group is added according to 1mL milk sample+10 μ L standard solution; One group is added according to 1mL milk sample+15 μ L standard solution.It is 7mL that every group of every part of milk sample prepares volume.The milk sample that according to said method is mixed with, wherein each medicine of contained aminoglycoside is followed successively by gentamicin and kanamycins 0,0.050,0.100 and 0.150 μ g/mL, and the content of 8 kinds of fluoroquinolones is respectively 0,0.025,0.050 and 0.075 μ g/mL.
Three, sensitivity experiment
Get above-mentioned prepare 80 parts and pollute milk samples, use respectively many residue golds labeled quick detection reagent box of three different lot numbers to detect, every part of milk sample duplicate detection 3 times, detection method is according to the kit instructions.With 95% the least concentration of positive findings appears as the detection sensitivity of kit.
The testing result that 20 parts of milk sample variable concentrations add samples sees Table 1, table 2 and table 3.The result shows that the detection sensitivity of gentamicin and kanamycins is 0.100 μ g/mL, and the detection sensitivity of 8 kinds of fluoroquinolones is 0.050 μ g/mL.
Table 1 more than residue gold labeled quick detection reagent box detects the testing result of gentamicin residue
Annotate: every group every part milk sample is 20 parts, and every part of 3 Duplicate Samples are counted 60 and detected sample.The negative milk sample of "-" expression testing result, the positive milk sample of "+" expression testing result.
As shown in Table 1,3 batches of detector bars are 0% to the positive rate of negative milk sample; Gentamicin interpolation concentration is that the positive rate of 0.050 μ g/mL milk sample is 1.1%; Gentamicin interpolation concentration is that the positive rate of 0.100 μ g/mL milk sample is 100%; Gentamicin interpolation concentration is that the positive rate of 0.150 μ g/mL milk sample is 100%.
Table 2 more than residue gold labeled quick detection reagent box detects the testing result of yapamicin relict
Annotate: every group every part milk sample is 20 parts, and every part of 3 Duplicate Samples are counted 60 and detected sample.The negative milk sample of "-" expression testing result, the positive milk sample of "+" expression testing result.
As shown in Table 2,3 batches of detector bars are 0% to the positive rate of negative milk sample; Kanamycins interpolation concentration is that the positive rate of 0.050 μ g/mL milk sample is 1.1%; Kanamycins interpolation concentration is that the positive rate of 0.100 μ g/mL milk sample is 100%; Kanamycins interpolation concentration is that the positive rate of 0.150 μ g/mL milk sample is 100%.
Table 3 more than residue gold labeled quick detection reagent box detect 8 kinds of fluo quinolone drug residuals testing result
Annotate:
1.8 planting fluoroquinolones is followed successively by: Ciprofloxacin, Enrofloxacin, Norfloxacin, Ofloxacin, Danofloxacin, Enoxacin, marbofloxacin and Nadifloxacin.
2. every group every part milk sample is 20 parts, and every part of 3 Duplicate Samples are counted 60 and detected sample.The negative milk sample of "-" expression testing result, the positive milk sample of "+" expression testing result.
As shown in Table 3,3 batches of detector bars are 0% to the positive rate of negative milk sample; 8 kinds of medicines are when interpolation concentration is 0.025 μ g/mL, and the highest positive rate of milk sample is 5%; When interpolation concentration was 0.050 μ g/mL, the minimum positive rate of milk sample was respectively 100%; When interpolation concentration was 0.075 μ g/mL, the positive rate of milk sample was 100%.
Among Fig. 3 during positive sample detection the interpolation concentration of quinolones be that the interpolation concentration of 50 μ g/kg, gentamicin and kanamycins is 100 μ g/kg; Among Fig. 4 during positive sample detection the interpolation concentration of quinolones be that the interpolation concentration of 50 μ g/kg, gentamicin and kanamycins is 100 μ g/kg.Fig. 5 shows many residue golds labeled quick detection reagent box, and described kit comprises that detector bar and plastics get stuck, and described detector bar is comprised of sample pad, golden labeling antibody pad, nitrocellulose filter, adsorptive pads.
Claims (8)
1. the box of residue gold labeled quick detection reagent more than a kind, described kit comprises that detector bar and plastics get stuck, described detector bar is comprised of sample pad, golden labeling antibody pad, nitrocellulose filter, adsorptive pads; It is characterized in that described golden labeling antibody pad is comprised of the gentamicin monoclonal antibody of colloid gold label, the kanamycins monoclonal antibody of colloid gold label and the fluoroquinolones monoclonal antibody of colloid gold label; Described nitrocellulose filter middle part is coated with detection line and control line, detection line is provided with the protein conjugate that can carry out with three kinds of monoclonal antibodies on the golden labeling antibody pad specific binding, protein conjugate is respectively Ciprofloxacin-BSA conjugate, gentamicin-BSA conjugate, kanamycins-BSA conjugate, and control line is provided with the sheep anti-mouse igg monoclonal antibody.
2. a kind of many residue golds labeled quick detection reagent box according to claim 1, the gentamicin monoclonal antibody of the colloid gold label on the described golden labeling antibody pad is combined with the colloid gold particle of 25nm by the gentamicin monoclonal antibody and is formed.
3. a kind of many residue golds labeled quick detection reagent box according to claim 1, the kanamycins monoclonal antibody of described colloid gold label is combined with the colloid gold particle of 25nm by the kanamycins monoclonal antibody and is formed.
4. a kind of many residue golds labeled quick detection reagent box according to claim 1, the fluoroquinolones monoclonal antibody of described colloid gold label is combined with the colloid gold particle of 60nm by the fluoroquinolones monoclonal antibody and is formed.
5. a kind of many residue golds labeled quick detection reagent box according to claim 1, the coated concentration of detection line Ciprofloxacin-BSA conjugate, gentamicin-BSA conjugate, kanamycins-BSA conjugate is respectively 0.08,0.13 and 0.15mg/mL on the described nitrocellulose filter, and package amount is 1 μ L/cm.
6. a kind of many residue golds labeled quick detection reagent box according to claim 1 is coated with the sheep anti-mouse igg monoclonal antibody on the described control line, and concentration is 1mg/mL, and package amount is 1 μ L/cm.
7. the preparation method of a kind of many residue golds labeled quick detection reagent box according to claim 1 comprises the steps:
1) preparation of golden labeling antibody pad
A. the preparation of blank collaurum:
Adopt the citrate reducing process to prepare colloid gold particle, prepare the colloidal gold solution that grain size is 25nm and 60nm by regulating gold chloride and trisodium citrate addition;
B. the preparation of the resuspended liquid of golden labeling antibody;
C. the preparation of colloidal gold labeled monoclonal antibody:
Get respectively the gold grain size and be the colloidal gold solution 100mL of 25nm or 60nm, under stirring, add 0.1M sal tartari 150～200 μ L, keep after 7～10 minutes, slowly add the golden mark that corresponding monoclonal antibody carries out antibody, continue to stir and dropwise to add 25% BSA solution behind the 30min and make it final concentration and reach 0.4%, continue to stir 30min, centrifugal at last, absorb supernatant, with the resuspended liquid of golden labeling antibody that the precipitation of gold labeling antibody is fully resuspended; Whole temperature of reaction control room temperature;
The gentamicin monoclonal antibody uses the colloid gold particle of 25nm to carry out mark, and labelled amount is 3 μ g/mL; The kanamycins monoclonal antibody uses the colloid gold particle of 25nm to carry out, and labelled amount is 3 μ g/mL; The fluoroquinolones monoclonal antibody uses the colloid gold particle of 60nm to carry out mark, and labelled amount is 5 μ g/mL;
D. the preparation of golden labeling antibody pad:
Resuspended good golden labeling antibody is coated on the glass fibre element film that well cutting in advance is of a size of 1cm*30cm, and package amount is 2mL/30cm, 37 ℃ of dried overnight, and the dry place of sealing lucifuge preserves;
2) T, C line coated on the nitrocellulose filter
A. the preparation of gentamicin-protein conjugate
Take by weighing 40mg sulmycin and 10mgBSA, 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 EDCHCl and be dissolved in the 1mL distilled water, dropwise join again in the mentioned solution, at room temperature continue to stir more than the 1h, then spend the night in 4 ℃ of placements; After PBS dialysis 3 days, packing ,-20 ℃ of storages;
B. kanamycins-protein conjugate preparation
Take by weighing Kanamycin Sulfate 28mg and BSA14mg and be dissolved in respectively in the 500ul distilled water, on magnetic stirring apparatus, stir after mixing.Get 76mgEDCHCl and be dissolved in the 1mL distilled water, dropwise join again in the above-mentioned mixed solution, at room temperature stir and spend the night.Dialyse more than 3 times centrifugal packing ,-20 ℃ of storages with 0.01MPBS;
C. the preparation of Ciprofloxacin-protein conjugate
Take by weighing 20mg ciprofloxacin hydrochloride, 10mgNHS and 12.5mgEDCHCl and fully be dissolved among the 1mLDMF, under room temperature, stir 24h, obtain the CPLX reactant liquor.Take by weighing BSA 50mg again, make it fully to be dissolved in 3mL PBS, pH value 7.4 dropwise slowly is added drop-wise to the CPLX reactant liquor in this BSA solution, and stirs 3h under room temperature, then with the PBS 3d that dialyses, changes dislysate every day 2 times, to remove unreacted small-molecule substance.With the centrifugal 20min of 3000r/min, collect supernatant, and packing-20 ℃ storage;
Being coated with of d.T, C line
The good two-dimentional specking platform of debugging, three kinds of protein conjugates that prepare are coated on the nitrocellulose filter as detection line in order by the mode of ruling, carry out simultaneously the coated as control line of sheep anti mouse monoclonal antibody, 37 ℃ of dry 2h, the dry place of sealing lucifuge preserves;
Be coated with three detection lines and a control line on the nitrocellulose filter, article three, detection line is Ciprofloxacin-BSA conjugate, gentamicin-BSA conjugate, kanamycins-BSA conjugate, its coated concentration is respectively 0.08,0.13 and 0.15mg/mL, and package amount is 1 μ L/cm; Coated sheep anti mouse monoclonal antibody on the control line, concentration is 1mg/mL, package amount is 1 μ L/cm;
3) assembling of test strips
Sample pad, golden labeling antibody pad, nitrocellulose filter and adsorptive pads overlapped according to the order of sequence paste on the PVC base plate, the base plate that posts is cut into the wide test strips of 4mm that is at cutting cutter, test strips is fixed in plastics get stuck.
8. such as the application of the arbitrary described many residue golds labeled quick detection reagent box of claim 1-6 in food inspection.
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| CN103412124A (en) * | 2013-07-25 | 2013-11-27 | 北京陆桥技术有限责任公司 | Aflatoxin M1 gold label quick detectiontest card and preparation method and application thereof |
| CN104569326A (en) * | 2013-10-23 | 2015-04-29 | 上海捷门保林迈生物工程有限公司 | Method for detecting beta-lactamase in milk and test paper |
| CN104678095A (en) * | 2013-11-27 | 2015-06-03 | 北京维德维康生物技术有限公司 | Kanamycin and erythromycin integrated gold-labeled microporous test strip |
| CN104678100A (en) * | 2013-11-27 | 2015-06-03 | 北京维德维康生物技术有限公司 | Gentamycin and quinolones two-in-one gold-labeled microporous test paper strip |
| CN104678101A (en) * | 2013-11-27 | 2015-06-03 | 中国农业大学 | Melamine and quinolone two-in-one gold-labeled microporous test paper strip |
| CN104678069A (en) * | 2013-11-27 | 2015-06-03 | 中国农业大学 | Kanamycin, erythrocin and lincomycin three-in-one micro-hole gold-labeled test strip |
| CN105424924A (en) * | 2015-11-02 | 2016-03-23 | 广州璞雅医药生物科技有限公司 | Antibiotic test paper strip and preparation method and application thereof |
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| CN108181468A (en) * | 2018-02-02 | 2018-06-19 | 江苏维尔生物科技有限公司 | For detecting colloid gold test paper of syphilis helicoid antibody and preparation method thereof and application method in saliva |
| CN108196050A (en) * | 2018-02-02 | 2018-06-22 | 江苏维尔生物科技有限公司 | For detecting the colloid gold test paper and its preparation and application of human immune defect virus antibody and syphilis helicoid antibody in saliva |
| CN116643049A (en) * | 2023-07-27 | 2023-08-25 | 云南省农业科学院质量标准与检测技术研究所 | Profenofos pesticide colloidal gold marker based on modified nano gold material and application thereof |
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| CN103412124A (en) * | 2013-07-25 | 2013-11-27 | 北京陆桥技术有限责任公司 | Aflatoxin M1 gold label quick detectiontest card and preparation method and application thereof |
| CN104569326A (en) * | 2013-10-23 | 2015-04-29 | 上海捷门保林迈生物工程有限公司 | Method for detecting beta-lactamase in milk and test paper |
| CN104678101B (en) * | 2013-11-27 | 2017-01-11 | 中国农业大学 | Melamine and quinolone two-in-one gold-labeled microporous test paper strip |
| CN104678100A (en) * | 2013-11-27 | 2015-06-03 | 北京维德维康生物技术有限公司 | Gentamycin and quinolones two-in-one gold-labeled microporous test paper strip |
| CN104678101A (en) * | 2013-11-27 | 2015-06-03 | 中国农业大学 | Melamine and quinolone two-in-one gold-labeled microporous test paper strip |
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| CN104678100B (en) * | 2013-11-27 | 2017-01-11 | 北京维德维康生物技术有限公司 | Gentamycin and quinolones two-in-one gold-labeled microporous test paper strip |
| CN106248894A (en) * | 2015-05-30 | 2016-12-21 | 北京艾旗斯德科技有限公司 | A kind of paper chip detecting Residue of Antibiotics in Milk |
| CN105424924A (en) * | 2015-11-02 | 2016-03-23 | 广州璞雅医药生物科技有限公司 | Antibiotic test paper strip and preparation method and application thereof |
| CN108181468A (en) * | 2018-02-02 | 2018-06-19 | 江苏维尔生物科技有限公司 | For detecting colloid gold test paper of syphilis helicoid antibody and preparation method thereof and application method in saliva |
| CN108196050A (en) * | 2018-02-02 | 2018-06-22 | 江苏维尔生物科技有限公司 | For detecting the colloid gold test paper and its preparation and application of human immune defect virus antibody and syphilis helicoid antibody in saliva |
| CN116643049A (en) * | 2023-07-27 | 2023-08-25 | 云南省农业科学院质量标准与检测技术研究所 | Profenofos pesticide colloidal gold marker based on modified nano gold material and application thereof |
| CN116643049B (en) * | 2023-07-27 | 2023-09-29 | 云南省农业科学院质量标准与检测技术研究所 | Profenofos pesticide colloidal gold marker based on modified nano gold material and application thereof |
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