CN101907623A - Method for detecting gentamicin and/or micronomicin and special quantum dot fluorescence immunoassay kit thereof - Google Patents
Method for detecting gentamicin and/or micronomicin and special quantum dot fluorescence immunoassay kit thereof Download PDFInfo
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Abstract
The invention discloses a method for detecting gentamicin and/or micronomicin and a special quantum dot fluorescence immunoassay kit thereof. The quantum dot fluorescence immunoassay kit for detecting the gentamicin comprises gentamicin specific antibodies, a coating antigen and standard product solution, wherein the coating antigen is the conjugate of gentamicin hapten and carrier proteins. The experiment of the invention proves that the kit of the invention has the characteristics of simple sample pretreatment process, simple and convenient operation, low cost, high specificity, high sensitivity, high accuracy, field monitoring capability and the like and is suitable for the screening of a great number of samples.
Description
Technical field
The present invention relates to a kind of method and special quantum dot fluorescent immunoassay kit thereof that detects gentamicin and/or micronomicin.
Background technology
Its veterinary drug residue problem is one of main factor that influences animal food safety at present, because animal specimen complicated component, testing concentration is lower, and most of sampling amount seldom, and this just has higher requirement to the selectivity and the sensitivity of analytical approach.Quantum dot (Quantum dot, QD) owing to have unique optics and electrical properties, it is more and more used in fluoroimmunoassay, as fluorescence probe, the optical characteristics of QD has the exciting light spectrum width of obvious superiority QD than frequent traditional chromophore that adopts such as rhodamine 6G or other organic dye molecule in fluoroimmunoassay, and continuous distribution, and emission spectrum is symmetrical distribution and width is narrow, the fluorescent emission wavelength can be regulated by the size that changes quantum dot, thereby the semiconductor-quantum-point of different sizes can be sent the fluorescence of different colours by the optical excitation of single wavelength.Quantum dot is sustainable luminous, its fluorescence lifetime can reach more than 100 times of dye molecule, anti-background interference ability than conventional enzyme-linked immunosorbent assay method is strong, the automaticity height, improved the precision of analytical approach, will have more wide application prospect at aspects such as veterinary science, medical science, food analyses.(Gentamicin is a kind of aminoglycoside broad-spectrum antibiotic GEN) to gentamicin, can act on multiple Gram-positive and negative bacterium, is mainly used in veterinary clinic treatment and feed medicated premixs such as respiratory tract infection and intramammary infection.On veterinary clinic, gentamicin is mainly by injection administration, but the drainage of this medicine is slower, if improper use causes residual the accumulating in animal product easily.Medication meeting lack of standardization causes livestock products, especially in the milk of lactation animal medicament residue is arranged, and human beings'health is constituted potential hazard, causes ototoxicity and Toxicity of Kidney.Stipulated its maximum residue limit(MRL) in animal food in succession both at home and abroad, China Ministry of Agriculture maximum residue limit(MRL) of regulation gentamicin in milk is 200 μ g/L.The detection method of gentamicin residue mainly adopts liquid phase chromatography, liquid chromatography-Lian mass spectroscopy, euzymelinked immunosorbent assay (ELISA) etc. in the animal tissue.
Summary of the invention
An object of the present invention is to provide a kind of method and special quantum dot fluorescent immunoassay kit thereof that detects gentamicin and/or micronomicin.
The quantum dot fluorescence immune reagent kit of detection gentamicin provided by the invention and/or micronomicin comprises gentamicin specific antibody, coating antigen and standard solution; Described coating antigen is the conjugate of gentamicin haptens and carrier protein; Its structural formula is suc as formula shown in the I:
Described kit comprises that also concentrated cleaning solution, concentrated redissolution liquid, bag are cushioned liquid, confining liquid and quantum dot-labeled antiantibody;
Described kit is cushioned liquid, confining liquid and quantum dot-labeled antiantibody and is formed by gentamicin specific antibody, coating antigen, standard solution, concentrated cleaning solution, concentrated redissolution liquid, bag.
Described gentamicin specific antibody is gentamicin monoclonal antibody or gentamicin polyclonal antibody, and described gentamicin monoclonal antibody is to be the antibody to the monoclonal hybridoma strain GEN of gentamicin secretion of CGMCC No.3778 by preserving number.
The concentration of standard items is 0 μ g/L, 1 μ g/L, 5 μ g/L, 10 μ g/L, 50 μ g/L or 100 μ g/L in the described standard solution, and described standard items are gentamicin;
Described concentrated cleaning solution is to be that 0.02M, pH are that 7.4 phosphate buffer mixes and obtains solution with 0.05g sodium azide and 100mL concentration;
Described concentrated redissolution liquid is to be that 0.05mol/L, pH are that 7.4 phosphate buffer mixes the solution that obtains with 0.1g bovine serum albumin(BSA) and 100mL concentration;
It is that the pH value is that 9.6 concentration is the carbonate buffer solution of 0.03mol/L that described bag is cushioned liquid;
Described confining liquid is to be that 0.03mol/L, pH value are that 7.4 phosphate solutions mix the solution obtain with 0.01g sodium azide, 10g bovine serum albumin(BSA) and 100mL concentration.
Described coating antigen is prepared as follows and obtains: with every 100mg gentamicin haptens, 20mg carrier protein and 2mL 50mM phosphate buffer mixing, obtain mixed liquor, note is made solution A; 150mg ethyl dimethylamino carbodiimides (EDC) is dissolved in the 1mL water, obtains solution B; Solution B is dropwise added in the solution A, 25 ℃ of stirring reaction 2h, the product dialysis with obtaining obtains described coating antigen.
The haptenic structural formula of described gentamicin is suc as formula shown in the II:
Described gentamicin haptens is a gentamicin.
Described quantum dot-labeled antiantibody is a quantum dot QD650 mark sheep anti mouse antiantibody;
Described carrier protein is mouse serum albumin, bovine serum albumin(BSA), ovalbumin or hemocyanin, is preferably ovalbumin.
Another object of the present invention provides a kind of method that detects gentamicin and/or micronomicin.
The method of detection gentamicin provided by the invention and/or micronomicin may further comprise the steps:
1) sample pre-treatments:
The trichloroacetic acid solution that in every 10mL milk, adds 200 μ l 50% (volumn concentration), mixing; With the centrifugal 5min of 3000g; Get supernatant, obtain sample solution after liquid dilutes 5 times with redissolving; Described redissolution liquid for will described concentrated redissolution liquid be that 7.4 phosphate buffer dilutes 10 times and obtains with 0.05mol/L, pH value;
2) utilize the quantum dot fluorescence immune reagent kit of described detection gentamicin to detect sample solution in the step 1).
By preserving number is that the gentamicin monoclonal antibody to the monoclonal hybridoma strain GEN of gentamicin secretion of CGMCC No.3778 also belongs to protection scope of the present invention.
Preserving number is that the monoclonal hybridoma strain GEN to gentamicin of CGMCC No.3778 also belongs to protection scope of the present invention.This cell line is preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on April 12nd, 2010 and (is called for short CGMCC, address: Datun Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, postcode 100101), preserving number is CGMCC No.3778.
The adopted name of gentamicin is a gentamicin, and chemical name is 9 α-fluorine-based-11 β, 17 alpha-dihydroxy-s-6 Alpha-Methyls-pregnane-1,4-diene-3,20-ketone.
Of the present invention experimental results show that, quantum dot fluorescence immune reagent kit of the present invention mainly adopts the residual quantity of the qualitative or detection by quantitative gentamicin of indirect competition method, the working fluid form that the main contents thing of this kit has adopted convenience to use, working fluid keeping quality and good stability; Utilize kit of the present invention to detect the method for the residual quantity of gentamicin, can be used for detecting the residual quantity of gentamicin in the milk sample, have that sample pretreatment process is simple, easy and simple to handle, expense is cheap, specificity is high, characteristics such as highly sensitive, degree of accuracy height, can on-site supervision and the examination of suitable great amount of samples.Therefore detection method of the present invention and dedicated kit thereof will be in animal derived food play a significant role in the residue detection of gentamicin.
Description of drawings
Fig. 1 is the gentamicin canonical plotting
Embodiment
Employed experimental technique is conventional method if no special instructions among the following embodiment.
Used material, reagent etc. if no special instructions, all can obtain from commercial channels among the following embodiment.
The detection principle of each kit is as follows among the following embodiment:
When on quantum dot fluorescence plate micropore, wrapping in advance by the conjugate of gentamicin haptens and carrier protein, after adding sample solution or standard solution, add gentamicin antibody solution again, the gentamicin coupled antigen competition gentamicin antibody of bag quilt on residual gentamicin or gentamicin standard items and the quantum dot fluorescence plate in the sample, add quantum dot-labeled antiantibody, use the fluorescence microplate reader fluorescence intensity, the content of gentamicin becomes negative correlation in sample fluorescence intensity level and the sample, relatively can draw the residual quantity of gentamicin in the sample with typical curve.
The preparation of embodiment 1, quantum dot fluorescence immune reagent kit and detection method thereof
One, the quantum dot fluorescence immune reagent kit comprises:
(1) coating antigen is dissolved in bag and is cushioned and obtains in the liquid, wherein the concentration of coating antigen in coating antigen solution is 0.08 μ g/mL; Coating antigen is the conjugate of gentamicin haptens and carrier protein couplet thing.
(2) quantum dot-labeled sheep anti mouse antiantibody working fluid: obtain with the quantum dot-labeled sheep anti mouse antiantibody of diluted, dilutability is 1: 500;
Dilution is that bovine serum albumin(BSA) and the mixing of 50mL phosphate buffer obtain; The concentration of described phosphate buffer is 0.02M, and the pH value is 7.4.
The sheep anti mouse antiantibody is available from Beijing Bo Aosen, and catalog number is bs-0295G.
(3) gentamicin standard solution: standard items are dissolved in obtain in the dilution, wherein standard items are at the concentration of gentamicin standard solution 0 μ g/L respectively, 1 μ g/L, 5 μ g/L, 10 μ g/L, 50 μ g/L, 100 μ g/L, dilution are the phosphate buffer of pH7.4,0.05M.
The gentamicin standard items are gentamicin, and available from China Veterinery Drug Inspection Office, catalog number is K0070604.
(4) gentamicin monoclonal antibody working fluid:
Monoclonal antibody is dissolved in obtains in the dilution, the proportioning of monoclonal antibody and dilution is 1: 1000; Monoclonal antibody is the monoclonal hybridoma strain GEN generation to gentamicin of CGMCC No.3778 by preserving number.
Dilution is that 25g casein, 0.03g sodium azide and the mixing of 1000mL phosphate buffer obtain.
(6) concentrated cleaning solution: with 0.05g nitrine sodium sodium and 100mL concentration is that 0.02M, pH are that 7.4 phosphate buffer mixes and obtains.
(7) concentrate to redissolve liquid: with 0.1g bovine serum albumin(BSA) and 100mL concentration is that 0.05mol/L, pH value are that 7.4 phosphate buffer mixes.The 400mL/ bottle, 1 bottle.
(8) bag is cushioned liquid: the pH value is that 9.6 concentration is the carbonate buffer solution of 0.03mol/L.
(9) confining liquid: with 0.01g sodium azide, 10g bovine serum albumin(BSA) and 100mL concentration is that 0.03mol/L, pH value are that 7.4 phosphate solution mixes.
Two, the preparation of kit
1, the preparation of quantum dot fluorescence plate:
(1) gentamicin is haptenic synthetic:
Contain amino in the gentamicin structure, can with the direct coupling of carrier protein, therefore need not carry out structure of modification, can be directly as haptens.
The haptenic structural formula of described gentamicin is suc as formula shown in the II:
(formula II).
(2) preparation of coating antigen: adopt carbodlimide method that gentamicin haptens and ovalbumin coupling are obtained coating antigen.
100mg gentamicin (GEN), 20mg ovalbumin OVA and 2mL 50mM PBS stirring and evenly mixing are obtained mixed liquor, and note is made solution A; Take by weighing 150mg EDC (ethyl dimethylamino carbodiimides) then in addition and be dissolved in the 1mL water, obtain solution B; Solution B is dropwise added in the solution A 25 ℃ of stirring reaction 2h; Reactant liquor is placed 0.01M PBS (pH 7.4), and behind 4 ℃ of stirring dialysis 72h, the centrifuging and taking supernatant obtains coating antigen.Described coating antigen is the conjugate of gentamicin haptens and carrier protein; Its structural formula is suc as formula shown in the I:
(3) preparation of quantum dot fluorescence plate:
Be cushioned the coating antigen (being gentamicin haptens and ovalbumin conjugate) that liquid obtains step (2) with bag and be diluted to 0.08 μ g/mL, every hole adds 100 μ l, 37 ℃ of incubation 2h, and coating buffer inclines, cleansing solution with 20 times of dilutions washs 2 times, each 30 seconds, pat dry, in every hole, add 150 μ l confining liquids then, 37 ℃ of incubation 1h, the liquid in the hole that inclines, dry back obtains to be coated with the quantum dot fluorescence plate of coating antigen, preserves with the vacuum seal of aluminium film.
2, gentamicin MONOCLONAL ANTIBODIES SPECIFIC FOR:
(1) immunogene is synthetic:
Gentamicin haptens and bovine serum albumin(BSA) are obtained immunogene by the carbodlimide method coupling.
Concrete preparation process is as follows: identical with the preparation method of coating antigen, different is that ovalbumin (OVA) is replaced with bovine serum albumin(BSA) BSA.
(2) animal immune and Fusion of Cells
Adopt the Balb/c mouse as immune animal, carry out immunity with the immunogene that (1) of step 2 obtains, immunizing dose is 100 μ g/, Freund's complete adjuvant with immunogene and equivalent when head exempts from is mixed and made into emulsifying agent, the subcutaneous multi-point injection of nape portion, At intervals of two to three weeks are got the same dose immunogene and are added equivalent incomplete Freund mixing and emulsifying, and booster immunization once, four exempt from the pneumoretroperitoneum booster immunization once, extracting spleen cell after 3 days.
Get immune BALB/c mouse splenocyte, in 5: 1 ratio (quantitative proportion) merge with SP2/0 myeloma cell.Adopt indirect competitive ELISA to measure cell conditioned medium liquid, screen positive hole.Utilize limiting dilution assay that cloning is carried out in positive hole, up to the hybridoma cell strain that obtains the stably excreting monoclonal antibody, with this cell line called after GEN.
Obtain monoclonal hybridoma strain GEN through screening to gentamicin, this cell line is preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on April 12nd, 2010 and (is called for short CGMCC, address: Datun Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, postcode 100101), preserving number is CGMCCNo.3778.
(3) cell cryopreservation and recovery: above-mentioned monoclonal hybridoma strain GEN CGMCC No.3778 is made 5 * 10 with cryopreserving liquid
6The cell suspension of individual/mL is sub-packed in frozen pipe, in the medium-term and long-term preservation of liquid nitrogen.Take out frozen pipe during recovery, put into 37 ℃ of water-bath middling speeds immediately and melt, behind the centrifugal removal cryopreserving liquid, move in the culture flask and cultivate.
(4) MONOCLONAL ANTIBODIES SPECIFIC FOR and purifying
The increment cultivation: the hybridoma of above-mentioned cultivation is placed cell culture medium, under 37 ℃ of conditions, cultivates, with following sad-the saturated ammonium sulfate method carries out purifying with the nutrient solution that obtains, obtains monoclonal antibody ,-20 ℃ of preservations.
Described cell culture medium is for adding the cell culture medium that calf serum and sodium bicarbonate obtain in the RPMI-1640 nutrient culture media, making the final concentration of calf serum in cell culture medium is 20% (volumn concentration), and making the final concentration of sodium bicarbonate in cell culture medium is 0.2% (quality percentage composition); The pH of described cell culture medium is 7.4.
Sad-saturated ammonium sulfate method 1) 50% saturation degree saltouts: get above-mentioned cell culture fluid 5mL, the PBS that adds equivalent 0.01mol/L, pH7.4 (contains potassium dihydrogen phosphate 0.27g in the 1L solution, 12 hypophosphite monohydrate disodium hydrogen 2.86g, potassium chloride 0.2g, sodium chloride 8.8g) mixing, drip isopyknic saturated ammonium sulfate (pH7.4) solution (making the saturation degree of ammonium sulfate reach 50%) then gradually, the limit edged stirs, room temperature is placed 30min, and the centrifugal 30min of 3000g abandons supernatant and stays precipitation.2) 33% saturation degree is saltoutd: add 5mL 0.01mol/LPBS respectively and (contain potassium dihydrogen phosphate 0.27g in the 1L solution in the precipitation that step 1) obtains, 12 hypophosphite monohydrate disodium hydrogen 2.86g, potassium chloride 0.2g, sodium chloride 8.8g) dissolution precipitation, add saturated ammonium sulfate solution again and reach 33% saturation degree, the limit edged stirs, and room temperature is placed 30min, abandons supernatant and stays precipitation.Repetitive operation 2 times.3) desalination: the PBS that gets 0.01mol/L, pH7.4 (contains potassium dihydrogen phosphate 0.27g in the 1L solution, 12 hypophosphite monohydrate disodium hydrogen 2.86g, potassium chloride 0.2g, sodium chloride 8.8g) dissolving step 2) precipitation that obtains, be loaded in the bag filter, be suspended from the PBS that fills 0.01mol/L, pH7.4 and (contain potassium dihydrogen phosphate 0.27g in the 1L solution, 12 hypophosphite monohydrate disodium hydrogen 2.86g, potassium chloride 0.2g, sodium chloride 8.8g) desalination in the beaker, be positioned over 4 ℃, change liquid 3-4 every day, 1%BaCl
2Detection is in dislysate till the sulfate radical-free ion.4) dialysis finishes, and the centrifugal 5min of 3000g gets the gentamicin monoclonal antibody that supernatant obtains purifying, and-20 ℃ of refrigerators are preserved.
3, the preparation of quantum dot and sheep anti-mouse antibody:
Surface amino groups (NH
2) the water-soluble quantum dot QD650 that modifies is available from thing source, BeiJing ZhongKe, catalog number is W-4007-650; Sheep anti-mouse antibody is available from Beijing Bo Aosen, and catalog number is bs-0259G;
(1) activation quantum dot: surface amino groups (NH
2) the water-soluble QD6502mL that modifies is through 20 μ l coupling agent SMCC (succinimide 4-[N-citraconic acid]-1-carboxylic cyclohexane) activation, forms active surface, the quantum dot that obtains activating.Gel column is removed excessive SMCC.
(2) reduction sheep anti-mouse antibody: add reductive agent DTT 25 μ l (dithiothreitol, dithiothreitol (DTT)) among the sheep anti-mouse antibody 2mL, disconnect the sulfydryl that disulfide bond forms.Gel column is removed DTT.
(3) quantum dot of activation and the sheep anti-mouse antibody of reduction are mixed, 37 ℃ were reacted 1 hour, and 10 μ l beta-mercaptoethanol cessation reactions form quantum dot-labeled sheep anti-mouse antibody.
Three, use the method for gentamicin residual in the described kit test sample of step 1
Method is as follows:
1, sample pre-treatments
Sample is the milk sample.
The trichloroacetic acid solution that in 10mL milk, adds 200 μ l 50% (volumn concentration), mixing; With the centrifugal 5min of 3000g; Get 200 μ l supernatants, obtain sample solution after liquid dilutes 5 times, carry out analysis of experiments with redissolving; Described redissolution liquid for will described concentrated redissolution liquid be that 7.4 phosphate buffer dilutes 10 times and obtains with 0.05mol/L, pH value.
2, detect
Obtain to be coated with adding gentamicin standard solution or sample solution 50 μ l in the quantum dot fluorescence plate micropore of coating antigen (gentamicin haptens and ovalbumin conjugate) to step 1, add gentamicin monoclonal antibody working fluid 50 μ l again, with cover plate film shrouding, react 30min in 37 ℃ of constant temperature ovens; Pour out liquid in the hole, every hole adds 250 μ l cleansing solutions, pours out liquid in the hole after 30 seconds, and so repetitive operation is washed plate 5 times altogether, pats dry with thieving paper; Every hole adds quantum dot-labeled sheep anti mouse antiantibody working fluid 100mL, reacts 30min in 37 ℃ of constant temperature ovens, pours out liquid in the hole, the repeated washing step; Use fluorescence microplate reader, measure every hole fluorescence intensity level.
3, interpretation of result
Multiply by 100% with the fluorescence intensity mean value (B) of the standard solution of each concentration that is obtained again divided by the fluorescence intensity level (B0) of first standard solution (0 standard), i.e. the percentage fluorescent value.Computing formula is:
Percentage fluorescent value (%)=(B/B0) * 100%
Semilog value with the concentration (μ g/L) of gentamicin standard solution is an X-axis, and the percentage fluorescent value is a Y-axis, drawing standard curve map (Fig. 1).With the percentage fluorescent value of same way calculation sample solution, the concentration of corresponding each sample then can be read the residual quantity of gentamicin the sample from typical curve.The analysis of testing result also can be adopted regression equation method among the present invention, calculates sample solution concentration.The analysis of testing result can also utilize computer professional software among the present invention, the be more convenient for express-analysis of a large amount of samples of this method, and whole testing process only needed to finish in 1.5 hours.
Obtain three batches of kits (01 batch, 02 batch, 03 batch) according to the method described above.
Embodiment 2, kit sensitivity, accuracy and storage life test
One, kit sensitivity experiment
Zero standard solution (being that dilution is the phosphate buffer of pH7.4,0.05M) is carried out 20 times detect, the mean value of measurement result adds the lowest detectable limit of 3 times of standard deviations as kit.
Table 1 zero standard measurement result statistical form μ g/L
As shown in Table 1, the lowest detection of kit is limited to 1.0 μ g/L.
Two, standard items precision test:
Every batch is extracted 10 kits from three batches of kits described in the embodiment 1 (01 batch, 02 batch, 03 batch), measures the luminous intensity values of 10 μ g/L standard solutions, calculates the coefficient of variation.Experiment three is described consistent among detection method and the embodiment 1.
3 repetitions are established in experiment, and the result is as shown in table 2, shows coefficient of variation scope between 4.2%～10.5%, meets precision and is less than or equal to 20% regulation.
The repeatable test of table 2 standard (CV%)
Three, sample precision and accuracy test
1, sample precision test:
After the milk that does not contain gentamicin carried out sample pre-treatments according to the method for embodiment 1, add the gentamicin standard items, making its final concentration is 20 μ g/L.Every batch is extracted 3 kits from three batches of kits described in the embodiment 1 (01 batch, 02 batch, 02 batch), experimentizes, and each experiment repeats 5 times, calculates the coefficient of variation respectively, result's (numerical value in the table is 5 mean values that repeat) as shown in table 3.The result shows the Variation Lines number average of milk sample less than 20%, met " Ministry of Agriculture's file " farming doctor and sent out [2005] No. 17 annex 2 kits and put on record with reference to the precision standard of the 4th precision in the judgment criteria and accuracy.
The repeatable test of table 3 milk sample
2, sample accuracy test
The milk that does not contain gentamicin is handled according to the sample-pretreating method described in the embodiment 1, in milk, added the gentamicin standard solution then, make its final concentration be respectively 50 μ g/L and 100 μ g/L; Detect gentamicin in the milk with the kit described in the embodiment 1 then, each concentration do 4 parallel, accuracy in computation (promptly adding the recovery) (accuracy=measured value/interpolation value) respectively.The result is as shown in table 4, shows that each sample adds the recovery all between 76.4%-95.2% with 50 μ g/L and 200 μ g/L gentamicins.
The accuracy of table 4 kit
Four, cross reacting rate test
Select to have 8 kinds of drug monitoring cross reacting rates of similar structures and similar functions with gentamicin.Typical curve by various medicines obtains its 50% inhibition concentration respectively.Calculate the cross reacting rate of kit with following formula to other medicines.Cross reaction is more little, and this kit is just good more to the detection specificity of gentamicin so.
Cross reacting rate (%)=(the gentamicin analog concentration that suppresses the concentration/inhibition 50% of 50% gentamicin standard items) * 100%
The specificity of table 5 kit
Experimental result shows that the kit that the present invention developed is good to the specificity of gentamicin and micronomicin.
Five, kit storage life test
The kit preservation condition is 2-8 ℃, preserves after 6 months, measures the actual interpolation of 50% inhibition concentration, gentamicin of kit and measures, and the result shows that 50% inhibition concentration of kit is all within normal range.Consideration has improper preservation condition and occurs in transportation and use, and kit was placed 6 days under the condition of 37 ℃ of preservations, carries out the accelerated deterioration experiment, and the result shows that the every index of this kit meets the requirements fully.Consider that the freezing situation of kit takes place, kit was put into-20 ℃ of refrigerators freezing 5 days, measurement result shows that also the every index of kit is normal fully.Can draw kit from above result can preserve more than 6 months at least at 2-8 ℃.
Claims (10)
1. a quantum dot fluorescence immune reagent kit that detects gentamicin and/or micronomicin comprises gentamicin specific antibody, coating antigen and standard solution; Described coating antigen is the conjugate of gentamicin haptens and carrier protein; Its structural formula is suc as formula shown in the I:
(formula I).
2. quantum dot fluorescence immune reagent kit according to claim 1 is characterized in that: described kit comprises that also concentrated cleaning solution, concentrated redissolution liquid, bag are cushioned liquid, confining liquid and quantum dot-labeled antiantibody;
Described kit is cushioned liquid, confining liquid and quantum dot-labeled antiantibody and is formed by gentamicin specific antibody, coating antigen, standard solution, concentrated cleaning solution, concentrated redissolution liquid, bag.
3. quantum dot fluorescence immune reagent kit according to claim 1 and 2, it is characterized in that: described gentamicin specific antibody is gentamicin monoclonal antibody or gentamicin polyclonal antibody, and described gentamicin monoclonal antibody is to be the antibody to the monoclonal hybridoma strain GEN of gentamicin secretion of CGMCC No.3778 by preserving number.
4. according to arbitrary described quantum dot fluorescence immune reagent kit among the claim 1-3, it is characterized in that:
The concentration of standard items is 0 μ g/L, 1 μ g/L, 5 μ g/L, 10 μ g/L, 50 μ g/L or 100 μ g/L in the described standard solution, and described standard items are gentamicin;
Described concentrated cleaning solution is to be that 0.02M, pH are that 7.4 phosphate buffer mixes and obtains solution with 0.05g sodium azide and 100mL concentration;
Described concentrated redissolution liquid is to be that 0.05mol/L, pH are that 7.4 phosphate buffer mixes the solution that obtains with 0.1g bovine serum albumin(BSA) and 100mL concentration;
It is that the pH value is that 9.6 concentration is the carbonate buffer solution of 0.03mol/L that described bag is cushioned liquid;
Described confining liquid is to be that 0.03mol/L, pH value are that 7.4 phosphate solutions mix the solution obtain with 0.01g sodium azide, 10g bovine serum albumin(BSA) and 100mL concentration.
5. according to arbitrary described quantum dot fluorescence immune reagent kit among the claim 1-4, it is characterized in that:
Described coating antigen is prepared as follows and obtains: with every 100mg gentamicin haptens, 20mg carrier protein and 2mL 50mM phosphate buffer mixing, obtain mixed liquor, note is made solution A; 150mg ethyl dimethylamino carbodiimides is dissolved in the 1mL water, obtains solution B; Solution B is dropwise added in the solution A, 25 ℃ of stirring reaction 2h, the product dialysis with obtaining obtains described coating antigen;
The haptenic structural formula of described gentamicin is suc as formula shown in the II:
6. according to arbitrary described quantum dot fluorescence immune reagent kit among the claim 1-5, it is characterized in that:
Described gentamicin haptens is a gentamicin.
7. according to arbitrary described quantum dot fluorescence immune reagent kit among the claim 1-6, it is characterized in that:
Described quantum dot-labeled antiantibody is a quantum dot QD650 mark sheep anti mouse antiantibody;
Described carrier protein is mouse serum albumin, bovine serum albumin(BSA), ovalbumin or hemocyanin, is preferably ovalbumin.
8. method that detects gentamicin and/or micronomicin may further comprise the steps:
1) sample pre-treatments:
The trichloroacetic acid solution that in every 10mL milk, adds 200 μ l 50% (volumn concentration), mixing; With the centrifugal 5min of 3000g; Get supernatant, obtain sample solution after liquid dilutes 5 times with redissolving; Described redissolution liquid for will described concentrated redissolution liquid be that 7.4 phosphate buffer dilutes 10 times and obtains with 0.05mol/L, pH value;
2) utilize the quantum dot fluorescence immune reagent kit of arbitrary described detection gentamicin among the claim 1-7 to detect sample solution in the step 1).
9. by preserving number the gentamicin monoclonal antibody of CGMCC No.3778 to the monoclonal hybridoma strain GEN of gentamicin secretion.
10. preserving number is the monoclonal hybridoma strain GEN to gentamicin of CGMCC No.3778.
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| CN102608319A (en) * | 2012-02-27 | 2012-07-25 | 华中农业大学 | Monoclonal antibody, enzyme linked immunosorbent assay method and kit for detecting gentamicin and sisomicin |
| CN105158477A (en) * | 2015-06-15 | 2015-12-16 | 中国科学院上海微系统与信息技术研究所 | Quantum dot fluorescent probe and application thereof |
| CN105628929A (en) * | 2014-11-25 | 2016-06-01 | 北京维德维康生物技术有限公司 | Quantum-dot immunofluorescence kit for simultaneously detecting gentamycin and quinolone medicines |
| CN109781976A (en) * | 2018-12-27 | 2019-05-21 | 中国农业大学 | Fluorescence immune analysis method based on carbon quantum dot |
| CN111876148A (en) * | 2020-09-08 | 2020-11-03 | 咸阳师范学院 | Imidazole 4, 5-dicarboxylic acid modified nitrogen-doped carbon quantum dot nanocomposite and preparation method and application thereof |
| CN112904008A (en) * | 2021-02-04 | 2021-06-04 | 浙江省食品药品检验研究院 | Enzyme linked immunosorbent assay kit for detecting protein A and other impurities in biological products and application thereof |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102608319A (en) * | 2012-02-27 | 2012-07-25 | 华中农业大学 | Monoclonal antibody, enzyme linked immunosorbent assay method and kit for detecting gentamicin and sisomicin |
| CN102608319B (en) * | 2012-02-27 | 2014-02-05 | 华中农业大学 | Monoclonal antibody, enzyme linked immunosorbent assay method and kit for detecting gentamicin and sisomicin |
| CN105628929A (en) * | 2014-11-25 | 2016-06-01 | 北京维德维康生物技术有限公司 | Quantum-dot immunofluorescence kit for simultaneously detecting gentamycin and quinolone medicines |
| CN105158477A (en) * | 2015-06-15 | 2015-12-16 | 中国科学院上海微系统与信息技术研究所 | Quantum dot fluorescent probe and application thereof |
| CN109781976A (en) * | 2018-12-27 | 2019-05-21 | 中国农业大学 | Fluorescence immune analysis method based on carbon quantum dot |
| CN111876148A (en) * | 2020-09-08 | 2020-11-03 | 咸阳师范学院 | Imidazole 4, 5-dicarboxylic acid modified nitrogen-doped carbon quantum dot nanocomposite and preparation method and application thereof |
| CN111876148B (en) * | 2020-09-08 | 2023-06-23 | 咸阳师范学院 | Imidazole 4, 5-dicarboxylic acid modified nitrogen-doped carbon quantum dot nanocomposite, and preparation method and application thereof |
| CN112904008A (en) * | 2021-02-04 | 2021-06-04 | 浙江省食品药品检验研究院 | Enzyme linked immunosorbent assay kit for detecting protein A and other impurities in biological products and application thereof |
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| CN101907623B (en) | 2013-05-08 |
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