CN103207272A - A method of simultaneous detection of medroxyprogesterone acetate and diethylstilbestrol in aquatic products - Google Patents
A method of simultaneous detection of medroxyprogesterone acetate and diethylstilbestrol in aquatic products Download PDFInfo
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Abstract
The invention belongs to the field of bio-engineering techniques, and relates to a suspension array method of simultaneous detection of medroxyprogesterone acetate and diethylstilbestrol in aquatic products. The method comprises the following steps: (1) preparing artificial antigens of medroxyprogesterone acetate and diethylstilbestrol individually; (2) preparing and purifying monoclonal antibodies against medroxyprogesterone acetate and diethylstilbestrol; (3) conjugating suspension array fluorescent microspheres to the artificial antigens obtained in step (1) separately; (4) biotinylating monoclonal antibodies obtained in step (2); and (5) simultaneously detecting medroxyprogesterone acetate and diethylstilbestrol residues in aquatic products by using conjugated fluorescent microspheres obtained in step (3) and biotinylated antibodies obtained in step (4) through the suspension array method. . The method provided by the invention can simultaneously detect two indicators of medroxyprogesterone acetate and diethylstilbestrol residues, and is fast, sensitive, and highly specific; wherein the minimum detection limits of both medroxyprogesterone acetate and diethylstilbestrol are 0.4ng/ml.
Description
Technical field
The invention belongs to technical field of bioengineering, relate to the detection method that a kind of liquid-phase chip method detects medroxyprogesterone acetate and diethylstilbestrol in the aquatic products simultaneously.
Background technology
Medroxyprogesterone acetate (medroxyprogesterone acetate, MPA) be a kind of progestogens medicine, in veterinary drug, be used as growth accelerator, be widely used in aspects such as the growth encourage of peace feelings and synchronization of estrus to promote growth of animal, improve food conversion ratio, save cost, to increase economic efficiency, it has toxic and side effect widely to the people, bad reactions such as mental depression, allergy be can cause, even breast cancer and infertility caused.EU member country's explicit order already forbids to use in food source property animal feeding management, China Ministry of Agriculture expressly provided that medroxyprogesterone acetate is the animal veterinary drug of forbidding for all food, can not detect in the food in 2002 in " animal food herbal medicine maximum residue limit(MRL) " file.Diethylstilbestrol (diethylstilbestrol, DES) be a kind of non-steroidal hormone, once be widely used in promoting growth of animal, increased lean meat percentage, its medicine original shape or metabolic product residue in the organ, tissue of food animal inevitably or enter external environment by excreta, become environmental estrogens, have serious toxic side effects such as causing monster, cancer knurl.(The US Food and Drug Administration FDA) just forbade that diethylstilbestrol was used for animal and fattens as far back as 1979 in Food and Drug Administration.1989, European Union did not enter animal and the animal food enforcement envelope pass that all contain somatropin.
China Ministry of Agriculture expressly provided that this medicine is the animal veterinary drug of forbidding for all food, can not detect in the food in 2002 in " animal food herbal medicine maximum residue limit(MRL) " file.But the part livestock and poultry cultivation family blindly phenomenon of economic pursuit benefit, abuse DES still exists.
Summary of the invention
The problem to be solved in the present invention provides a kind of method that can detect MPA and DES in the aquatic products simultaneously.Be intended to solve problems such as detection of veterinary drugs in food is single, consuming time, cost height, also lay a good foundation for further detecting four residue of veterinary drug indexs simultaneously.The foundation of this method can be satisfied medroxyprogesterone acetate and diethylstilbestrol detection requirement fast and accurately in the aquatic products, 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 medroxyprogesterone acetate and diethylstilbestrol in the aquatic products simultaneously, described method concrete steps may further comprise the steps:
(1) prepares the artificial antigen of medroxyprogesterone acetate and diethylstilbestrol respectively;
(2) prepare monoclonal antibody and the purifying of medroxyprogesterone acetate and diethylstilbestrol respectively;
(3) the artificial antigen difference 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, it is residual to detect in the aquatic products medroxyprogesterone acetate and diethylstilbestrol simultaneously by the liquid-phase chip method.
In the described step (2), it is 1: 2.56 * 10 that the ascites of the monoclonal antibody of medroxyprogesterone acetate is tired
6
In the described step (2), it is 1: 3.2 * 10 that the ascites of the monoclonal antibody 1F5 of diethylstilbestrol is tired
6
In the described step (2), the hybridoma 2D8 cell ascites of diethylstilbestrol is tired and is respectively 1: 1.6 * 10
6
In the step (5), the residual steps in sequence of medroxyprogesterone acetate and diethylstilbestrol is in the described detection aquatic products: coupling has the microballoon of MPA and DES to add medroxyprogesterone acetate and the diethylstilbestrol sample solution of handling well, adds biotinylated medroxyprogesterone acetate and diethylstilbestrol antibody subsequently respectively; Add the good SA-PE of dilution then, add stop buffer at last, fluorescence intensity.
In the step (5), bag is added medroxyprogesterone acetate and the diethylstilbestrol sample solution of handling well by a good microballoon part, and another part adds serial medroxyprogesterone acetate and diethylstilbestrol 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 and 0.4ng/mL.
Described fluorescence intensity is by Luminex 200 instrument fluorescence intensity values, and with Luminex version2.1 software analysis data.
The antibody dilution that detects medroxyprogesterone acetate is 1: 3000.
The antibody dilution that detects diethylstilbestrol is 1: 6400.
The dilutability of described SA-PE is 1: 100.
Particularly, method of the present invention comprises the steps:
1, preparation medroxyprogesterone acetate artificial antigen;
2, medroxyprogesterone acetate MONOCLONAL ANTIBODIES SPECIFIC FOR and purifying;
3, preparation diethylstilbestrol artificial antigen;
4, diethylstilbestrol MONOCLONAL ANTIBODIES SPECIFIC FOR and purifying;
5, the biotinylation of medroxyprogesterone acetate monoclonal antibody specific and diethylstilbestrol monoclonal antibody;
6, preparation is coupled the liquid-phase chip fluorescent microsphere of medroxyprogesterone acetate antigen and diethylstilbestrol antigen respectively;
7, set up the liquid-phase chip method detect medroxyprogesterone acetate and diethylstilbestrol in the aquatic products simultaneously.
In the step 1, the preparation method of described medroxyprogesterone acetate antigen is specially: medroxyprogesterone acetate and oralbumin are carried out coupling, form artificial antigen.
In the step 2, described medroxyprogesterone acetate MONOCLONAL ANTIBODIES SPECIFIC FOR and purification process are specially: adopt (MPS) haptens of the acetic acid synthesized medroxyprogesterone acetate-3-of O-(ethyloic) hydroxylamine assay (O-ethyloic).With the dicyclohexyl carbodiimide coupling method DMPA-3-(O-ethyloic) is synthesized artificial immunogene fully with the BSA coupling.Immune mouse, screening obtains hybridoma, collects the mouse ascites purifying, obtains purer medroxyprogesterone acetate monoclonal antibody.
The process of immunity and purifying is specially: get 4 of BALB/C mice, first immunisation is injected with MPA-BSA and equal-volume complete Freund's adjuvant emulsification metapedes pad, dosage is 100ug/, later on every 3 all booster immunizations 1 time, use incomplete Freund's adjuvant instead successively at subcutaneous, muscle, abdominal cavity multi-point injection, dosage is 150ug/.The 3d reinforced immunological is 1 time before merging, and mouse tail vein is slowly only directly injected MPA-BSA 250ug/.Carry out Fusion of Cells according to a conventional method: splenocyte and the SP2/0 bone marrow cell of immune mouse are merged, again through 4 subclones, screen the hybridoma cell strain of energy stably excreting MPA monoclonal antibody.The hybridoma of building after the strain is carried out amplification cultivation.The BALB/c female mice in ages in lumbar injection 7 week, with the sterilization norphytane with every 0.5mL lumbar injection, the 10d pneumoretroperitoneum is inoculated the hybridoma that is in exponential phase of full nutrient culture media dilution that toos many or too much for use, inject 2 * 106 cells respectively for every, treat that mouse web portion expands, when One's spirits are drooping, gather ascites, with ammonium sulfate precipitation method purifying ascites, and with after the DEAE cellulose chromatography post purifying ascites, MPA monoclonal antibody that must be purer.
It is 1: 2.56 * 10 that the ascites of the monoclonal antibody of medroxyprogesterone acetate is tired
6
In the step 3, the concrete grammar of described diethylstilbestrol artificial antigen preparation is: diethylstilbestrol and oralbumin are carried out coupling, form artificial antigen.
In the step 4, the concrete grammar of described diethylstilbestrol MONOCLONAL ANTIBODIES SPECIFIC FOR and purifying is: the diethylstilbestrol molecule is modified transformation, synthetic diethylstilbestrol-list-carboxyl propyl group-ether.Utilize active ester method to prepare immunogene (DES-BSA).Immune mouse, screening obtains hybridoma, collects the mouse ascites purifying, obtains purer diethylstilbestrol monoclonal antibody.
The process of immunity and purifying is specially: get 4 of 6-8 health Balb/c in age in week mouse, first immunisation is injected with DES-BSA and equal-volume complete Freund's adjuvant emulsification metapedes pad, dosage is 100ug/, later on every 3 all booster immunizations 1 time, use incomplete Freund's adjuvant instead successively at subcutaneous, muscle, lumbar injection, dosage is 150ug/.Adopt indirect elisa method and indirect competitive ELISA method to measure tiring and specificity of serum antibody.Splenocyte and the SP2/0 myeloma cell of immune mouse are merged, filter out the hybridoma cell strain of energy stably excreting diethylstilbestrol monoclonal antibody.The hybridoma of building after the strain is carried out amplification cultivation.Get the Balb/c female mice in 7 ages in week, with the sterilization norphytane with every 0.5mL lumbar injection, the inoculation of 7d pneumoretroperitoneum has the hybridoma that is in exponential phase of incomplete nutrient culture media dilution, observe the mouse ascites production every day, treat that mouse web portion expands, when One's spirits are drooping, gather ascites with No. 12 syringe needles, the centrifuging and taking supernatant after ammonium sulfate precipitation method purifying ascites, is used DEAE cellulose chromatography post purifying again.
It is 1: 3.2 * 10 that the ascites of the monoclonal antibody of diethylstilbestrol is tired
6It is 1: 1.6 * 10 that the hybridoma ascites of diethylstilbestrol is tired
6
In the step 5, the biotinylation of described medroxyprogesterone acetate monoclonal antibody specific and diethylstilbestrol monoclonal antibody, be specially: get medroxyprogesterone acetate and diethylstilbestrol monoclonal antibody 1mg respectively, dissolve with 45uL 10mM Sulfo-NHS-Biotin.Under the room temperature, stirring reaction 4-5h makes 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 the step 6, described preparation is coupled the liquid-phase chip fluorescent microsphere of medroxyprogesterone acetate antigen and diethylstilbestrol antigen, be with medroxyprogesterone acetate antigen and diethylstilbestrol antigen respectively with different fluorescence-encoded microballoon couplings.The concrete operations step is: the microballoon of 4 ℃ of preservations is positioned over room temperature 20～30min; vortex microballoon (2～3min is advisable); each is drawn 100 μ L fluorescent microspheres and joins in 1.5 milliliters the centrifuge tube of 2 pipes; the centrifugal 4min of 13000r/min removes protection liquid; after the tri-distilled water washing, with the resuspended microballoon of 400 μ L 0.1M pH 6.2 phosphate buffers.Add 10 μ L 50mg/mL Sulfo-NHS and 10 μ L50mg/ml EDC respectively, 37 ℃ of effect 20min behind the mixing.The centrifugal supernatant that goes suspends microballoon again with 900 μ L 0.05M MES, the centrifugal supernatant that goes, twice of washing microballoon.With the resuspended microballoon of 400 μ L 0.05M MES, add medroxyprogesterone acetate artificial antigen and the diethylstilbestrol artificial antigen of purifying respectively, 37 ℃ of effect 2～3h (during, every 15min vortex mixing once) behind the mixing.The centrifugal supernatant that goes adds the resuspended microballoon of 900 μ L PBS-TBN (0.01MPBS that contains 0.05%Tween-20,0.1%BSA, 0.05% Sodium azide), the centrifugal supernatant that goes behind the mixing, twice of washing microballoon.Add the resuspended microballoon of 200 μ L PBS-TBN.Count with hemacytometer.
In the step 7, described liquid-phase chip method detects simultaneously that the method for medroxyprogesterone acetate and diethylstilbestrol comprises sample pre-treatments in the aquatic products, the SA-PE reaction is hatched, added to microballoon and sample and stop.
The step of described pre-treatment testing sample comprises: the tissue that homogenate is good adds ethyl acetate and CaCl
2Centrifugal, pipette supernatant, nitrogen dries up; The residue n-hexane dissolution adds PBS-methyl alcohol again, and is centrifugal; Absorb the upper strata normal hexane, take off layer solution for detection of.
Concrete steps are:
1. sample pre-treatments.Accurately take by weighing homogenate good organize 3.0g, add 9mL ethyl acetate, add 0.3CaCl then
2, maximal rate concussion 3min; Under the room temperature condition, the centrifugal 5min of 6000rpm; Pipette the 6mL supernatant to another new test tube, 60-70 ℃ of water-bath nitrogen dries up, and residue is with the n-hexane dissolution of 2.0mL, and residue is with the n-hexane dissolution of 2.0mL, add again 1.0mL 1 * PBS-methyl alcohol (3: 2, v/v), vortex oscillation 2min; Under the room temperature condition, the centrifugal 10min of 6000rpm absorbs the upper strata normal hexane, get 50uL 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), in 96 well culture plates, add serial medroxyprogesterone acetate and diethylstilbestrol standard solution (25ng/mL that 25 μ L/ holes prepare respectively, 12.5ng/mL, 6.25ng/mL, 3.125ng/mL, 1.6ng/mL, 0.8ng/mL, 0.4ng/mL), add the sample solution 25 μ L that handle well in other micropore, adding coupling subsequently respectively has each 1 μ L/2000 of microballoon of MPA and DES individual, and biotinylated medroxyprogesterone acetate and each 25 μ L of diethylstilbestrol antibody; Also add 25 μ L cleansing solutions and make blank in a hole, all the other steps are with the operation of sample solution.37 ℃ of oscillation action 60min.
3. add the good SA-PE of dilution, 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 pre-treating method of sample can carry out according to conventional method, also can operate as follows:
Get the good tissue of homogenate, add ethyl acetate and CaCl
2Pipette supernatant to another new test tube, nitrogen dries up, the residue n-hexane dissolution, add again PBS-methyl alcohol (3: 2, v/v); Centrifugal, remove supernatant, take off layer solution for detection of.
The invention provides a kind of method that detects the liquid-phase chip of medroxyprogesterone acetate and diethylstilbestrol in the aquatic products simultaneously.The artificial antigen that at first prepares medroxyprogesterone acetate and diethylstilbestrol simultaneously, has prepared medroxyprogesterone acetate and diethylstilbestrol monoclonal antibody also with it purifying.Respectively medroxyprogesterone acetate and diethylstilbestrol artificial antigen are coupled the liquid-phase chip fluorescent microsphere, respectively with medroxyprogesterone acetate and diethylstilbestrol monoclonal antibody biotinylation.According to the principle of indirect competitive ELISA, set up and detect medroxyprogesterone acetate and the residual liquid-phase chip method of diethylstilbestrol in the aquatic products simultaneously, and definite optimum test condition.The inventive method can detect medroxyprogesterone acetate and two kinds of residual indexs of diethylstilbestrol simultaneously, entire reaction can be finished in 2 hours, its characteristics quick, responsive, special and that detect a plurality of residues of veterinary drug synchronously can be applied to the daily monitoring of aquatic products residue of veterinary drug, and its detection sensitivity is that the minimum detectability of medroxyprogesterone acetate and diethylstilbestrol is 0.4ng/mL.
The above-mentioned liquid-phase chip method of medroxyprogesterone acetate and diethylstilbestrol that detects simultaneously in the aquatic products has been used liquid-phase chip technology, this technology has been used specific fluorescent microsphere, these microballoons are matrix with the polystyrene, redness and orange two kinds of fluorescent dyes (these two kinds of dyestuffs respectively have 10 kinds of different differentiations) in the microballoon manufacture process, have been mixed, thereby but 100 kinds of different colors on the 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 the mark, the all types of target molecule that probe molecule on the different microballoons needs to detect in sample carries out specificity is combined, reporter molecules is combined with the target molecule specificity, namely constituted the liquid-phase chip system, can detect reaching 100 kinds of different target molecules in the 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 the detection system two bundle laser detects microballoon classification fluorescence (specificity) and report fluorescence (susceptibility) simultaneously respectively, has only the report fluorescence signal that occurs simultaneously with classification fluorescence just to be detected record, has improved result's specificity more.Liquid-phase chip technology has its 1000～5000 corresponding identical microballoons in same reaction system in the detection to each index.During detection, extract 100～500 microballoon readings wherein, final data is 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 serum consumption of liquid-phase chip technology is wanted much less than ELISA method, chemoluminescence method, electrochemiluminescence method.Microsphere surface is long-pending big in the liquid-phase chip, and single microballoon can be in conjunction with up to about (1～2) * 10
6Therefore individual target molecule only needs the minute quantity sample to carry out multiple composition detection simultaneously, 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 quick, weak point consuming time.The reaction environment of liquid-phase chip technology is liquid phase, probe (or antibody) fixing on the microballoon all reacts in solution with sample to be checked, 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 (about 5～6 μ m), be suspended in easily in the liquid, for biomolecular reaction provides approximate physiological liquid phase environment, react quick, abundant, take weak point.The result of proteins interactions such as Ag-Ab can be noted with the form of data message by computer after the fluidic cell detection system is judged in moment, had 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 multiple index simultaneously, this compares with the detection one by one of classic method is a qualitative leap; By the modification to microsphere surface, various antibody or acceptor molecules such as microsphere surface can coated antibody, antigen, cell factor, protein or nucleic acid, part, acceptor, thus satisfy 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 MPA and DES simultaneously and can carry out flexible combination as required, joint-detection, and simple to operate, the time spent 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 the aquatic products exactly.
Description of drawings
Fig. 1 is the typical curve that liquid-phase chip detects MPA.
Fig. 2 is the typical curve that liquid-phase chip detects DES.
Embodiment
Below embodiments of the invention are elaborated: present embodiment is to implement under the prerequisite in technical solution of the present invention, provided detailed embodiment and concrete operating process, be not used in but present embodiment only is used for explanation the present invention and limit 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.
Medroxyprogesterone acetate and oralbumin are carried out coupling, form artificial antigen.
Embodiment 2 medroxyprogesterone acetate MONOCLONAL ANTIBODIES SPECIFIC FOR and purifying
Adopt (MPS) haptens of the acetic acid synthesized medroxyprogesterone acetate-3-of O-(ethyloic) hydroxylamine assay (O-ethyloic).With the dicyclohexyl carbodiimide coupling method DMPA-3-(O-ethyloic) is synthesized artificial immunogene fully with the BSA coupling.Get 4 of BALB/C mice, first immunisation is 100ug/ with MPA-BSA and equal-volume complete Freund's adjuvant emulsification metapedes pad injection, dosage, later on every 3 all booster immunizations 1 time, use incomplete Freund's adjuvant instead successively at subcutaneous, muscle, abdominal cavity multi-point injection, dosage is 150ug/.The 3d reinforced immunological is 1 time before merging, and mouse tail vein is slowly only directly injected MPA-BSA 250ug/.Carry out Fusion of Cells according to a conventional method: splenocyte and the SP2/0 bone marrow cell of immune mouse are merged, again through 4 subclones, screen the hybridoma cell strain of energy stably excreting MPA monoclonal antibody.The hybridoma of building after the 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 of full nutrient culture media dilution that toos many or too much for use injects 2 * 10 respectively for every with the sterilization norphytane
6Individual cell treats that mouse web portion expands, and when One's spirits are drooping, gathers ascites, with ammonium sulfate precipitation method purifying ascites, and with after the DEAE cellulose chromatography post purifying ascites, MPA monoclonal antibody that must be purer.Double fastener heart ELISA Preliminary Determination antigen valence and serum dilution.
The preparation of embodiment 3 diethylstilbestrol artificial antigens
Diethylstilbestrol and oralbumin are carried out coupling, form artificial antigen.
Embodiment 4 diethylstilbestrol MONOCLONAL ANTIBODIES SPECIFIC FOR and purifying
The diethylstilbestrol molecule is modified transformation, synthetic diethylstilbestrol-list-carboxyl propyl group-ether.Utilize active ester method to prepare immunogene (DES-BSA).Get 4 of 6-8 health Balb/c in age in week mouse, first immunisation is 100ug/ with DES-BSA and equal-volume complete Freund's adjuvant emulsification metapedes pad injection, dosage, later on every 3 all booster immunizations 1 time, use incomplete Freund's adjuvant instead successively at subcutaneous, muscle, lumbar injection, dosage is 150ug/.Adopt indirect elisa method and indirect competitive ELISA method to measure tiring and specificity of serum antibody.Splenocyte and the SP2/0 myeloma cell of immune mouse are merged, filter out the hybridoma cell strain of energy stably excreting diethylstilbestrol monoclonal antibody.The hybridoma of building after the strain is carried out amplification cultivation.Get the Balb/c female mice in 7 ages in week, with the sterilization norphytane with every 0.5mL lumbar injection, the inoculation of 7d pneumoretroperitoneum has the hybridoma that is in exponential phase of incomplete nutrient culture media dilution, observe the mouse ascites production every day, treat that mouse web portion expands, when One's spirits are drooping, gather ascites with No. 12 syringe needles, the centrifuging and taking supernatant after ammonium sulfate precipitation method purifying ascites, is used DEAE cellulose chromatography post purifying again.
The biotinylation of embodiment 5 medroxyprogesterone acetate monoclonal antibody specifics and diethylstilbestrol monoclonal antibody
Get medroxyprogesterone acetate and diethylstilbestrol monoclonal antibody 1mg respectively, dissolve with 45uL 10mM Sulfo-NHS-Biotin.Under the room temperature, stirring reaction 4-5h makes 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 6 preparations are coupled the liquid-phase chip microballoon of medroxyprogesterone acetate antigen and diethylstilbestrol antigen
The microballoon of 4 ℃ of preservations is positioned over room temperature 20～30min; vortex microballoon (2～3min is advisable); each is drawn 100 μ L fluorescent microspheres and joins in 1.5 milliliters the centrifuge tube of 2 pipes; the centrifugal 4min of 13000r/min removes protection liquid; after the tri-distilled water washing, with the resuspended microballoon of 400 μ L 0.1M pH 6.2 phosphate buffers.Add 10 μ L 50mg/mL Sulfo-NHS and 10 μ L 50mg/ml EDC respectively, 37 ℃ of effect 20min behind the mixing.The centrifugal supernatant that goes suspends microballoon again with 900 μ L 0.05M MES, the centrifugal supernatant that goes, twice of washing microballoon.With the resuspended microballoon of 400 μ L 0.05M MES, add medroxyprogesterone acetate artificial antigen and the diethylstilbestrol artificial antigen of purifying respectively, 37 ℃ of effect 2～3h (during, every 15min vortex mixing once) behind the mixing.The centrifugal supernatant that goes adds the resuspended microballoon of 900 μ L PBS-TBN (the 0.01M PBS that contains 0.05%Tween-20,0.1%BSA, 0.05% Sodium azide), the centrifugal supernatant that goes behind the mixing, twice of washing microballoon.Add the resuspended microballoon of 200 μ L PBS-TBN.Count with hemacytometer.
Embodiment 7 sample pre-treatments
Accurately take by weighing homogenate good organize 3.0g, add 9mL ethyl acetate, add 0.3 CaCl2 then, maximal rate concussion 3min; Under the room temperature condition, the centrifugal 5min of 6000rpm; Pipette the 6mL supernatant to another new test tube, 60-70 ℃ of water-bath nitrogen dries up, and residue is with the n-hexane dissolution of 2.0mL, and residue is with the n-hexane dissolution of 2.0mL, add again 1.0mL 1 * PBS-methyl alcohol (3: 2, v/v), vortex oscillation 2min; Under the room temperature condition, the centrifugal 10min of 6000rpm absorbs the upper strata normal hexane, get 50uL lower floor solution for detection of.
Embodiment 8 liquid-phase chip methods detect medroxyprogesterone acetate and diethylstilbestrol in the aquatic products simultaneously
Get the good microballoon of coupling and place room temperature 20～30min, vortex microballoon (2～3min is advisable), in 96 well culture plates, add serial medroxyprogesterone acetate and diethylstilbestrol standard solution (25ng/mL that 25 μ L/ holes prepare respectively, 12.5ng/mL, 6.25ng/mL, 3.125ng/mL, 1.6ng/mL, 0.8ng/mL, 0.4ng/mL), in other micropore, add the sample solution 25 μ L that handle well, add coupling subsequently respectively respectively 1 μ L/2000 of MPA and DES microballoon arranged, and biotinylated medroxyprogesterone acetate and each 25 μ L of diethylstilbestrol antibody, also in a hole, adding 25 μ L cleansing solutions and make blank, all the other steps are with the operation of sample solution.37 ℃ of oscillation action 60min.Add the good SA-PE of dilution, 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 9 testing results are judged
(median fluorescence intensity MFI) is ordinate, does horizontal ordinate respectively with the logarithm value of the concentration of MPA and DES, does typical curve to calculate earlier the average fluorescent strength of two kinds of standard models.Just can read the concentration of its corresponding sample from typical curve according to the MFI of each test sample.Multiply by corresponding extension rate again.
1) typical curve and sensitivity
Be ordinate with MFI, the logarithm value of MPA and DES concentration of standard solution is horizontal ordinate, draws out typical curve (Fig. 1).From Fig. 1 and Fig. 2 as can be seen, the Logistic that two kinds of residue of veterinary drug liquid-phase chips that this method is drawn out detect returns the typical curve equation, and curved line relation is good, coefficient of determination R2＞0.99.The detection sensitivity of this method is MPA 0.4ng/mL, DES 0.4ng/mL.
2) degree of accuracy test
Criticize the degree of accuracy that an error is represented this method with batch interior sum of errors.1. criticize interior error: each standard model concentration is done 5 times and is repeated, and represents batch interior error with its variation within batch coefficient.2. criticize an error: the method operation that repeats to have set up, make error between representing to criticize with its interassay coefficient of variation continuously 5 times.Testing result is checked through SPSS, in batch (seeing Table 1) and batch between result's 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 the 3g sample and add medroxyprogesterone acetate and diethylstilbestrol respectively, addition is respectively 25ng/g, 10ng/g, 2ng/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, determine its accuracy (seeing the following form 2) with the recovery.
Table 2 swamp eel meat sample medroxyprogesterone acetate and diethylstilbestrol add the recovery test result
, experimentize as mortifier with the DMPA of gradient dilution, Medroxyprogesterone, megestrol acetate, diethylstilbestrol, leucomalachite green, coloured malachite green, chloromycetin, Thiamphenicol, Florfenicol, obtain the average fluorescent strength of sample.The crossing-over rate of megestrol acetate is 50%, and this is consistent with other experiment reports, shows that the influence of c=c Dichlorodiphenyl Acetate Medroxyprogesterone antibody specificity is big on the more female ring structure of aceticoceptor.
Table 2 liquid-phase chip specific detection result (MFI value)
Claims (10)
1. a method that detects medroxyprogesterone acetate and diethylstilbestrol in the aquatic products simultaneously is characterized in that, said method comprising the steps of:
(1) prepares the artificial antigen of medroxyprogesterone acetate and diethylstilbestrol respectively;
(2) prepare monoclonal antibody and the purifying of medroxyprogesterone acetate and diethylstilbestrol respectively;
(3) the artificial antigen difference 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, it is residual to detect in the aquatic products medroxyprogesterone acetate and diethylstilbestrol simultaneously by the liquid-phase chip method.
2. the method that detects medroxyprogesterone acetate and diethylstilbestrol in the aquatic products simultaneously according to claim 1 is characterized in that in the described step (2), it is 1: 2.56 * 10 that the ascites of the monoclonal antibody of medroxyprogesterone acetate is tired
6
3. the method that detects medroxyprogesterone acetate and diethylstilbestrol in the aquatic products simultaneously according to claim 1 is characterized in that in the described step (2), it is 1: 3.2 * 10 that the ascites of the monoclonal antibody of diethylstilbestrol is tired
6
4. the method that detects medroxyprogesterone acetate and diethylstilbestrol in the aquatic products simultaneously according to claim 1 is characterized in that in the described step (2), it is 1: 1.6 * 10 that the hybridoma ascites of diethylstilbestrol is tired
6
5. the method that detects medroxyprogesterone acetate and diethylstilbestrol in the aquatic products simultaneously according to claim 1, it is characterized in that, the residual steps in sequence of medroxyprogesterone acetate and diethylstilbestrol is in the detection aquatic products described in the step (5): coupling has the microballoon of MPA and DES to add medroxyprogesterone acetate and the diethylstilbestrol sample solution of handling well, adds the antibody of biotinylated medroxyprogesterone acetate and diethylstilbestrol subsequently; Add the good SA-PE of dilution then, add stop buffer at last, fluorescence intensity.
6. the method that detects medroxyprogesterone acetate and diethylstilbestrol in the aquatic products simultaneously according to claim 5, it is characterized in that, bag is added medroxyprogesterone acetate and the diethylstilbestrol sample solution of handling well by a good microballoon part, and another part adds serial medroxyprogesterone acetate and diethylstilbestrol 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 and 0.4ng/mL.
7. the method that detects medroxyprogesterone acetate and diethylstilbestrol in the aquatic products simultaneously according to claim 5, it is characterized in that, described fluorescence intensity is by Luminex 200 instrument fluorescence intensity values, and with Luminex version2.1 software analysis data.
8. the method that detects medroxyprogesterone acetate and diethylstilbestrol in the aquatic products simultaneously according to claim 5 is characterized in that, the antibody dilution that detects medroxyprogesterone acetate is 1: 3000.
9. the method that detects medroxyprogesterone acetate and diethylstilbestrol in the aquatic products simultaneously according to claim 5 is characterized in that, the antibody dilution that detects diethylstilbestrol is 1: 6400.
10. the method that detects medroxyprogesterone acetate and diethylstilbestrol in the aquatic products simultaneously according to claim 5 is characterized in that the dilutability of SA-PE is 1: 100.
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