CN103389377A - Liquid-phase chip method for detecting chloromycetin in aquatic product - Google Patents
Liquid-phase chip method for detecting chloromycetin in aquatic product Download PDFInfo
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- CN103389377A CN103389377A CN2012101396948A CN201210139694A CN103389377A CN 103389377 A CN103389377 A CN 103389377A CN 2012101396948 A CN2012101396948 A CN 2012101396948A CN 201210139694 A CN201210139694 A CN 201210139694A CN 103389377 A CN103389377 A CN 103389377A
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Abstract
The invention belongs to the technical field of biological engineering, and relates to a liquid-phase chip kit used for detecting chloromycetin in an aquatic product. The method comprises the steps that: (1) a chloromycetin artificial antigen is prepared; (2) chloromycetin monoclonal antibody is prepared and purified; (3) the artificial antigen obtained in the step (1) is used for coupling liquid-phase chip fluorescent microspheres; (4) the monoclonal antibody obtained in the step (2) is used for biotinylation; and (5) the coupled microspheres obtained in the step (3) and the biotinylated antibody obtained in the step (4) are used for detecting chloromycetin in an aquatic product through the liquid-phase chip method. The method provided by the invention can be used for detecting chloromycetin residue index with high speed, good specificity, and high specificity. A chloromycetin detection limit is lower than 0.1ng/L.
Description
Technical field
The invention belongs to technical field of bioengineering, relate to a kind of liquid-phase chip and detect the detection method of chloromycetin in aquatic products.
Background technology
Chloromycetin (chloramphenicol, CAP) be to extract a class broad-spectrum antibiotic that makes from the Venezuela Streptothrix, since listing in 1948, it is the choice drug for the treatment of typhoid fever, paratyphoid and salmonella always, and high inexpensive because of its effect, at home and abroad also widely apply in animal husbandry.But, along with the dark people's research to the chloromycetin clinical practice, find that long-term trace takes the photograph people's chloromycetin, not only make salmonella, Escherichia coli etc. produce drug resistance, also can cause the imbalance of body normal flora, suppress bone marrow function, cause the relevant diseases such as alpastic anemia and other malignant hematologic diseases, the mankind's health in serious harm.The U.S., European Union, Japan and other countries all provide against chloromycetin for edible animal in recent years, and must not detect chloromycetin in exported product.
China Ministry of Agriculture expressly provided that this medicine is the animal veterinary drug of forbidding for all food, can not detect in food in 2002 in " animal food herbal medicine maximum residue limit(MRL) " file.But part livestock and poultry cultivation family is blindly pursued the phenomenon of economic benefit, abuse CAP and is still existed.
Summary of the invention
The problem to be solved in the present invention is to provide a kind of method that detects CAP in aquatic products.Be intended to solve that detection of veterinary drugs in food is single, consuming time, the high in cost of production problem, also for further detecting simultaneously four residue of veterinary drug indexs, lay a good foundation.The foundation of the method can meet the detection requirement fast and accurately of aquatic products chloromycetin, simultaneously, and for the daily monitoring of China's aquatic products residue of veterinary drug provides a kind of new usability methods.
The invention provides a kind of method that detects chloromycetin in aquatic products, described method concrete steps comprise the following steps:
(1) prepare the artificial antigen of chloromycetin;
(2) prepare monoclonal antibody and the purifying of chloromycetin;
(3) the artificial antigen coupling liquid-phase chip fluorescent microsphere that utilizes step (1) to obtain;
(4) monoclonal antibody of utilizing step (2) to obtain is carried out biotinylation;
(5) use the microballoon of the coupling that step (3) obtains and the biotinylated antibody that step (4) obtains, by Luminex, detect Determination of Chloramphenicol Residues In Aquatic Products By Charm Ii.
In described step (2), the titer of ascites of the monoclonal antibody of chloromycetin is 1:2.56 * 10
6.
In step (5), the step of described detection Determination of Chloramphenicol Residues In Aquatic Products By Charm Ii is followed successively by: the microballoon that has been coated with adds the chloromycetin sample solution of handling well, adds respectively subsequently coupling that microballoon and the biotinylated chloramphenicol antibody of CAP antigen are arranged; Then add the SA-PE that has diluted, add finally stop buffer, fluorescence intensity.
In step (5), a microballoon part that has been coated with adds the chloromycetin sample solution of handling well, and another part adds serial chloromycetin standard solution; The concentration of standard solution comprise 25ng/mL, 12.5ng/mL, 6.25ng/mL,, 3.125ng/mL, 1.6ng/mL, 0.8ng/mL, 0.4ng/mL, 0.2ng/mL and 0.1ng/mL.
Described fluorescence intensity is by Luminex 200 instrument fluorescence intensity values, and with Luminex version2.1 software analysis data.
The antibody dilution of chlorine detection mycin is: 1:3200.
The dilutability of described SA-PE is 1:100.
Particularly, method of the present invention comprises the steps:
1, prepare chloromycetin artificial antigen;
2, the preparation of chloromycetin monoclonal antibody and purifying;
3, the biotinylation of chloromycetin monoclonal antibody;
4, preparation is coupled the liquid-phase chip fluorescent microsphere of chloromycetin antigen;
5, set up the Luminex that detects chloromycetin in aquatic products.
In step 1, the preparation method of described chloromycetin antigen is specially: chloromycetin and oralbumin are carried out coupling, form artificial antigen.
In step 2, preparation and the purification process of described chloromycetin monoclonal antibody are specially: adopt haptens Chloramphenicol Succinate and the carrier protein (OVA) that active ester method will contain the shuttle base to carry out coupling, haptens and the carrier protein that will contain fragrant amido with the diazotising method carry out coupling, antigen emulsification.Immune mouse, booster immunization, gather the chloromycetin antiserum, obtains purer chloromycetin monoclonal antibody.
Process immune and purifying is specially: get 4 of BALB/C mice, before immunity, eye socket is got blood as negative serum, and first immunisation is injected with CAP-OVA and equal-volume complete Freund's adjuvant emulsification pneumoretroperitoneum, and dosage is 100 μ g/; Immunity again after using CAP-OVA and incomplete Freunds adjuvant mixes after 2 weeks; , every 1 all booster immunizations 1 time, use incomplete Freund's adjuvant instead successively at the tail vein injection injection later.Before merging, the 3d reinforced immunological is 1 time, and mouse tail vein is slowly only directly injected CAP-OVA 250 μ g/.Carry out according to a conventional method Fusion of Cells: the splenocyte of immune mouse and SP2/0 bone marrow cell are merged, then, through 4 subclones, screen the hybridoma cell strain of energy stably excreting CAP monoclonal antibody.The hybridoma of building after strain is carried out amplification cultivation.The BALB/c female mice in ages in lumbar injection 7 week, with every 0.5mL lumbar injection, the 10d pneumoretroperitoneum inoculation hybridoma that is in exponential phase that full nutrient culture media dilutes that toos many or too much for use, inject respectively 2 * 10 for every with sterilizing norphytane
6Individual cell, treat that mouse web portion expands, and when One's spirits are drooping, gathers ascites, with ammonium sulfate precipitation method purifying ascites, and with after DEAE cellulose chromatography post purifying ascites, CAP monoclonal antibody that must be purer.
The titer of ascites of the monoclonal antibody of chloromycetin is 1:2.56 * 10
6.
In step 3, the biotinylation of described chloromycetin monoclonal antibody specific is specially: extracting chloromycetin monoclonal antibody 1mg, dissolve with 45 μ L 10mM Sulfo-NHS-Biotin.Under room temperature, stirring reaction 4-5h, make the abundant biotinylation of antibody.Then use 0.1mol/L, the PBS of pH7.4 crosses the YM-10 post, and the displacement damping fluid is also removed unreacted little molecule BNHS.The various biotinylated antibodies of a small amount of packing ,-20 ℃ of freezing preservations.
In step 4, described preparation is coupled the liquid-phase chip fluorescent microsphere of chloromycetin antigen, is with chloromycetin monoclonal antibody and the microballoon coupling of sending different fluorescence.Concrete operation step is: the microballoon of 4 ℃ of preservations is positioned over room temperature 20～30min; vortex microballoon (2～3min is advisable); each draw 100 μ L fluorescent microspheres join 2 the pipe 1.5ml centrifuge tube in; the centrifugal 4min of 13000r/min removes protection liquid; after the tri-distilled water washing, with the 400 μ L0.1M pH 6.2 resuspended microballoons of phosphate buffer.Add respectively 10 μ L 50mg/mL Sulfo-NHS and 10 μ L 50mg/ml EDC, mix rear 37 ℃ of effect 20min.The centrifugal supernatant that goes, with microballoon with 900 μ L 0.05M MES Eddy diffusions, the centrifugal supernatant that goes, twice of washing microballoon.With the 400 resuspended microballoons of μ L0.05M MES, add respectively the chloromycetin artificial antigen of purifying, during mixing rear 37 ℃ of effect 2～3h(, every the 15min vortex, mix once).The centrifugal supernatant that goes, add 900 μ L PBS-TBN(to contain the 0.01M PBS of 0.05%Tween-20,0.1%BSA, 0.05% Sodium azide) resuspended microballoon, mix the rear centrifugal supernatant that goes, twice of washing microballoon.Add the 200 resuspended microballoons of μ L PBS-TBN.Count with hemacytometer.
In step 5, the method that described Luminex detects chloromycetin in aquatic products comprises sample pre-treatments, microballoon and sample is hatched, added the SA-PE reaction and stop.
The step of described pre-treatment testing sample comprises: the tissue that homogenate is good adds distilled water and ethyl acetate; Centrifugal, pipette supernatant, nitrogen dries up; The residue n-hexane dissolution, then add PBS, centrifugal; Absorb the upper strata normal hexane, take off layer solution for detection of.
Concrete steps are:
1. sample pre-treatments.Accurately take homogenate good organize 3.0g, the distilled water of 3ml mixes, then adds 6mL ethyl acetate, maximal rate concussion 10min; Under room temperature condition, the centrifugal 10min of 6000rpm; Pipette the 4mL supernatant to another new test tube, 60 ℃ of water-bath nitrogen dry up, and residue is with the n-hexane dissolution of 1.0mL, then add the PBS of 0.5mL, vortex oscillation 1min; Under room temperature condition, the centrifugal 10min of 6000rpm, absorb the upper strata normal hexane, get 50 μ L lower floor solution for detection of.
2. the microballoon that coupling is good is placed room temperature 20～30min, vortex microballoon (2～3min is advisable), add respectively in 96 well culture plates the serial chloromycetin standard solution that 25 μ L/ holes prepare (25ng/mL, 12.5ng/mL, 6.25ng/mL,, 3.125ng/mL, 1.6ng/mL, 0.8ng/mL, 0.4ng/mL, 0.2ng/mL and 0.1ng/mL), add the sample solution 25 μ L that handle well in other micropore, add respectively subsequently coupling to have each 1 μ L/2000 of microballoon of CAP individual, and each 25 μ L of biotinylated chloramphenicol antibody; Also add 25 μ L dilutions to make blank in a hole, all the other steps are with the operation of sample solution.37 ℃ of oscillation action 60min.
3. add the SA-PE that has diluted, 37 ℃ of oscillation action 30min.
4. add 100 μ L stop buffers, by Luminex 200 instrument fluorescence intensity values (median fluorescence intensity, MFI), and with Luminex version 2.1 software analysis data.
The invention provides a kind of method that detects the liquid-phase chip of chloromycetin in aquatic products.At first the artificial antigen for preparing chloromycetin, simultaneously, prepared the chloromycetin monoclonal antibody also with it purifying.Respectively chloromycetin artificial antigen is coupled the liquid-phase chip fluorescent microsphere, respectively with the chloramphenicol antibody biotinylation.According to the principle of indirect competitive ELISA, set up the Luminex that detects simultaneously Determination of Chloramphenicol Residues In Aquatic Products By Charm Ii, and definite optimum test condition.The inventive method can detect the residual chloromycetin index, whole reaction can be completed in 2 hours, its characteristics quick, responsive, special and that synchronously detect a plurality of residues of veterinary drug can be applied to the daily monitoring of aquatic products residue of veterinary drug, and its detection sensitivity is that the minimum detectability of chloromycetin is lower than 0.1ng/mL.
The above-mentioned liquid-phase chip method that detects simultaneously chloromycetin in aquatic products has been used liquid-phase chip technology, this utilization specific fluorescent microsphere, these microballoons are take polystyrene as matrix, mixed red in the Microsphere manufacture process and orange two kinds of fluorescent dyes (these two kinds of dyestuffs respectively have 10 kinds of different differentiations), thereby but 100 kinds of different colors on mark form an array that contains 100 different microballoons with unique spectrum address.Utilize this 100 kinds of microballoons, can distinguish 100 kinds of different probe molecules on mark, need all types of target that detects molecule to carry out specific binding in probe molecule on different microballoons and sample, reporter molecules and target molecule specific binding, namely formed the liquid-phase chip system, can detect reaching 100 kinds of different target molecules in a sample simultaneously.
Compare with traditional detection diagnostic method, liquid-phase chip technology has following advantage:
1. highly sensitive, high specificity, good stability, good reproducibility.The sensitivity of liquid-phase chip is 10～100 times of the sensitivity of conventional ELISA method; The reaction of liquid-phase chip occurs in the liquid environment of accurate homogeneous phase, and liquid phase environment more is conducive to keep the native conformation of protein in reaction, not only is conducive to the reaction of probe and detected material, also more can guarantee specificity and the stability of reacting; In detection system two bundle laser detects respectively microballoon classification fluorescence (specificity) and report fluorescence (susceptibility) simultaneously, only has the report fluorescence signal that occurs simultaneously with classification fluorescence just to be detected record, has more improved the specificity of result.Liquid-phase chip technology in the detection to each index, has its 1000～5000 corresponding identical microballoons in same reaction system.During detection, extract 100～500 microballoon readings wherein, final data are to get the median of its all values, can reduce to minimum to error like this, thereby make the testing result good reproducibility.
2. the sample requirement is little, and cost is relatively low.The amount of serum of liquid-phase chip technology is wanted much less than ELISA method, chemoluminescence method, Electrochemiluminescince.In liquid-phase chip, microsphere surface is long-pending large, and single microballoon can be in conjunction with up to about (1～2) * 10
6Individual target molecule, therefore only need the minute quantity sample can carry out simultaneously Multiple components and detect, and is convenient to clinical sampling analysis; Reagent dosage is few simultaneously, and required cost is lower, and is cost-saved, reduces the manpower consumption.
3. reaction is fast, consuming time short.The reaction environment of liquid-phase chip technology is liquid phase, probe (or antibody) fixing on microballoon all reacts with sample to be checked in solution, collision probability and speed can increase more than 10 times with respect to reaction patterns such as solid phase chip or ELISA to each other for they, reaction time even can shorten to tens minutes, therefore can improve reaction velocity.And microsphere diameter very little (approximately 5～6 μ m), easily be suspended in liquid, for biomolecular reaction provides approximate physiological liquid phase environment, reaction fast, fully, take short.The result of the proteins interactions such as Ag-Ab can be recorded with the form of data message by computer in moment after the fluidic cell detection system is judged, more saved the time of course of reaction.
4. high flux, can carry out multiplexed analyses, and is applied widely.The commercialization microballoon adopts the Two Colour Fluorescence coding techniques at present, can detect 100 kinds of target molecules simultaneously.Once can detect simultaneously many indexes, this compares with the detection one by one of classic method is a qualitative leap; By the modification to microsphere surface, various antibody or the acceptor molecules such as microsphere surface can coated antibody, antigen, cell factor, protein or nucleic acid, part, acceptor, thus meet different the detection and the research needs of function, applicable to various analysis and research; Powerful software analysis system and high flux detection system, more convenient and quicker obtains more information intuitively.
Compared with prior art, the present invention can detect CAP and can carry out as required flexible combination, joint-detection, and simple to operate, the used time is short, and traditional high efficiency chromatography method and ELISA method can't be accomplished this point.Utilize this invention can be fast, cheap, detect residue of veterinary drug in aquatic products exactly.
Description of drawings
Fig. 1 is the typical curve that liquid-phase chip detects CAP.
Fig. 2 is that liquid-phase chip detects CAP specificity figure.
Embodiment
Below embodiments of the invention are elaborated: the present embodiment is to implement under prerequisite in technical solution of the present invention, provided detailed embodiment and concrete operating process, but the present embodiment only is used for explanation the present invention, has been not used in and limits the scope of the invention.Should be pointed out that for the person of ordinary skill of the art, without departing from the inventive concept of the premise, can also make some distortion and improvement, these all belong to protection scope of the present invention.Therefore, protection scope of the present invention should be as the criterion with claims.
Chloromycetin and oralbumin are carried out coupling, form artificial antigen.
Preparation and the purifying of embodiment 2 chloromycetin monoclonal antibodies
Haptens Chloramphenicol Succinate and the carrier protein (OVA) that will contain the shuttle base with active ester method carry out coupling, and haptens and the carrier protein that will contain fragrant amido with the diazotising method carry out coupling, antigen emulsification.Get 4 of BALB/C mice, before immunity, eye socket is got blood as negative serum, and first immunisation is with the injection of CAP-OVA and equal-volume complete Freund's adjuvant emulsification pneumoretroperitoneum, dosage be 100 μ/; Immunity again after using CAP-OVA and incomplete Freunds adjuvant mixes after 2 weeks; , every 1 all booster immunizations 1 time, use incomplete Freund's adjuvant instead successively at the tail vein injection injection later.Before merging, the 3d reinforced immunological is 1 time, and mouse tail vein is slowly only directly injected CAP-OVA 250 μ L/.Carry out according to a conventional method Fusion of Cells: the splenocyte of immune mouse and SP2/0 bone marrow cell are merged, then, through 4 subclones, screen the hybridoma cell strain of energy stably excreting CAP monoclonal antibody.The hybridoma of building after strain is carried out amplification cultivation.The BALB/c female mice in ages in lumbar injection 7 week, with every 0.5mL lumbar injection, the 10d pneumoretroperitoneum inoculation hybridoma that is in exponential phase that full nutrient culture media dilutes that toos many or too much for use, inject respectively 2 * 10 for every with sterilizing norphytane
6Individual cell, treat that mouse web portion expands, and when One's spirits are drooping, gathers ascites, with ammonium sulfate precipitation method purifying ascites, and with after DEAE cellulose chromatography post purifying ascites, CAP monoclonal antibody that must be purer.
The biotinylation of embodiment 3 chloromycetin monoclonal antibody specifics
Extracting chloromycetin monoclonal antibody 1mg, dissolve with 45 μ L 10mM Sulfo-NHS-Biotin respectively.Under room temperature, stirring reaction 4-5h, make the abundant biotinylation of antibody.Then use 0.1mol/L, the PBS of pH7.4 crosses the YM-10 post, and the displacement damping fluid is also removed unreacted little molecule BNHS.The various biotinylated antibodies of a small amount of packing ,-20 ℃ of freezing preservations.
Embodiment 4 preparations are coupled the liquid-phase chip microballoon of chloromycetin antigen
The microballoon of 4 ℃ of preservations is positioned over room temperature 20～30min; vortex microballoon (2～3min is advisable); each draw 100 μ L fluorescent microspheres join 2 the pipe 1.5mL centrifuge tube in; the centrifugal 4min of 13000r/min removes protection liquid; after the tri-distilled water washing, with the 400 μ L0.1M pH 6.2 resuspended microballoons of phosphate buffer.Add respectively 10 μ L50mg/mL Sulfo-NHS and 10 μ L 50mg/ml EDC, mix rear 37 ℃ of effect 20min.The centrifugal supernatant that goes, with microballoon with 900 μ L 0.05M MES Eddy diffusions, the centrifugal supernatant that goes, twice of washing microballoon.With the 400 resuspended microballoons of μ L 0.05M MES, add respectively the chloromycetin artificial antigen of purifying, during mixing rear 37 ℃ of effect 2～3h(, every the 15min vortex, mix once).The centrifugal supernatant that goes, add 900 μ L PBS-TBN(to contain the 0.01M PBS of 0.05%Tween-20,0.1%BSA, 0.05% Sodium azide) resuspended microballoon, mix the rear centrifugal supernatant that goes, twice of washing microballoon.Add the 200 resuspended microballoons of μ L PBS-TBN.Count with hemacytometer.
Embodiment 5 sample pre-treatments
Accurately take homogenate good organize 3.0g, the distilled water of 3ml mixes, then adds 6mL ethyl acetate, maximal rate concussion 10min; Under room temperature condition, the centrifugal 10min of 6000rpm; Pipette the 4mL supernatant to another new test tube, 60 ℃ of water-bath nitrogen dry up, and residue is with the n-hexane dissolution of 1.0mL, then add the PBS of 0.5mL, vortex oscillation 1min; Under room temperature condition, the centrifugal 10min of 6000rpm, absorb the upper strata normal hexane, get 50 μ L lower floor solution for detection of.
Embodiment 6 Luminexes detect chloromycetin in aquatic products simultaneously
Get the good microballoon of coupling and place room temperature 20～30min, vortex microballoon (2～3min is advisable), add respectively in 96 well culture plates the serial chloromycetin standard solution that 25 μ L/ holes prepare (25ng/mL, 12.5ng/mL, 6.25ng/mL,, 3.125ng/mL, 1.6ng/mL, 0.8ng/mL, 0.4ng/mL, 0.2ng/mL and 0.1ng/mL), add the sample solution 25 μ L that handle well in other micropore, add respectively subsequently coupling to have each 1 μ L/2000 of microballoon of CAP individual, and each 25 μ L of biotinylated chloramphenicol antibody; Also add 25 μ L dilutions to make blank in a hole, all the other steps are with the operation of sample solution.37 ℃ of oscillation action 60min.Add the SA-PE that has diluted, 37 ℃ of oscillation action 30min.Add 100 μ L stop buffers, by Luminex 200 instrument fluorescence intensity values (median fluorescence intensity, MFI), and with Luminex version 2.1 software analysis data.
Embodiment 7 testing results are judged
The average fluorescent strength (median fluorescence intensity, MFI) that first calculates standard model is ordinate, with the logarithm value of the concentration of CAP, does respectively horizontal ordinate, does typical curve.Just can read from typical curve the concentration of its corresponding sample according to the MFI of each test sample.Multiply by again corresponding extension rate.
1) typical curve and sensitivity
Take MFI as ordinate, the logarithm value of CAP concentration of standard solution is horizontal ordinate, draws out typical curve (Fig. 1).As shown in Figure 1, the Logistic that two kinds of residue of veterinary drug liquid-phase chips that the method is drawn out detect returns the typical curve equation, and curved line relation is good, coefficient of determination R
2＞0.99.The detection sensitivity of the method is lower than 0.1ng/mL.
2) degree of accuracy test
With batch in error and batch between error represent the degree of accuracy of the method.1. criticize interior error: each standard model concentration is done 5 times and is repeated, and with its variation within batch coefficient, represents batch interior error.2. criticize an error: the method operation that repeats to have set up, make continuously error between representing to criticize with its interassay coefficient of variation 5 times.Testing result is checked through SPSS, in batch (in Table 1) and batch between result there was no significant difference all, good reproducibility.
Table 1 is with a collection of standard items reproducible results
3) recovery is calculated
Be taken in the blank swamp eel meat of 3g sample and add respectively chloromycetin, addition is respectively 20ng/g, 8ng/g, 1ng/g, and each adds the concentration sample and does three repetitions, carries out sample pre-treatments, and extract is measured.Obtain concentration value from typical curve, with the recovery, determine its accuracy (seeing the following form 2).
Table 2 swamp eel meat sample chloromycetin adds the recovery test result
DMPA with gradient dilution, Medroxyprogesterone, megestrol acetate, diethylstilbestrol, leucobase of malachite green, coloured malachite green, medroxyprogesterone acetate, Thiamphenicol, Florfenicol is as mortifier, test, obtain the average fluorescent strength of sample, result as shown in Figure 2, Fig. 2 has shown that each drug concentration is the mean fluorecence value of 100ng/ml, wherein chloromycetin has strong inhibition to fluorescence intensity, the average fluorescent strength of other drug is close in the blank value, result shows, no cross reaction in this system, with megestrol acetate, Medroxyprogesterone, diethylstilbestrol, leucobase of malachite green, coloured malachite green, medroxyprogesterone acetate, Thiamphenicol, other veterinary drug thing no cross reactions such as Florfenicol.
Claims (7)
1. a liquid-phase chip detects the method for chloromycetin in aquatic products, it is characterized in that, it comprises step:
(1) prepare the artificial antigen of chloromycetin;
(2) prepare monoclonal antibody and the purifying of chloromycetin;
(3) the artificial antigen coupling liquid-phase chip fluorescent microsphere that utilizes step (1) to obtain;
(4) monoclonal antibody of utilizing step (2) to obtain is carried out biotinylation;
(5) use the microballoon of the coupling that step (3) obtains and the biotinylated antibody that step (4) obtains, by Luminex, detect Determination of Chloramphenicol Residues In Aquatic Products By Charm Ii.
2. liquid-phase chip according to claim 1 detects the method for chloromycetin in aquatic products, it is characterized in that, in described step (2), the titer of ascites of the monoclonal antibody of chloromycetin is 1:2.56 * 10
6.
3. liquid-phase chip according to claim 1 detects the method for chloromycetin in aquatic products, it is characterized in that, the step of the detection Determination of Chloramphenicol Residues In Aquatic Products By Charm Ii described in step (5) is followed successively by: coated microballoon adds the chloromycetin sample solution of handling well, adds respectively subsequently coupling that the microballoon of CAP antigen and the antibody of biotinylated chloromycetin are arranged; Then the SA-PE that adds dilution, add stop buffer, fluorescence intensity finally.
4. liquid-phase chip according to claim 3 detects the method for chloromycetin in aquatic products, it is characterized in that, a coated microballoon part adds the chloromycetin sample solution of processing, and another part adds serial chloromycetin standard solution; The concentration of standard solution comprises 25ng/mL, 12.5ng/mL, 6.25ng/mL, 3.125ng/mL, 1.6ng/mL, 0.8ng/mL, 0.4ng/mL, 0.2ng/ml and 0.1ng/ml.
5. the method for chloromycetin in detection aquatic products according to claim 3, is characterized in that, described fluorescence intensity is by Luminex 200 instrument fluorescence intensity values, and with Luminex version 2.1 software analysis data.
6. liquid-phase chip according to claim 3 detects the method for chloromycetin in aquatic products, it is characterized in that, the antibody dilution of chlorine detection mycin is: 1:3200.
7. liquid-phase chip according to claim 3 detects the method for chloromycetin in aquatic products, it is characterized in that, the dilutability of SA-PE is 1:100.
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| EP1178316A1 (en) * | 2000-08-04 | 2002-02-06 | The Jordanian Pharmaceutical Manufacturing and Medical Equipment Co.Ltd. | Medical Kit and method for the determination of a drug |
| CN101458253A (en) * | 2008-12-22 | 2009-06-17 | 中国海洋大学 | One-step immune detecting method for enrofloxacin residual in aquatic product |
| CN101788560A (en) * | 2010-03-17 | 2010-07-28 | 北京阿匹斯生物技术有限公司 | Enzyme-linked immunoassay kit for detecting residual chloramphenicol and detection method thereof |
| CN102288765A (en) * | 2011-07-07 | 2011-12-21 | 清华大学深圳研究生院 | Immunofluorescence chloramphenicol detecting method based on quantum dots and special kit |
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Application publication date: 20131113 |