CN102707073A - Enzyme linked immunosorbent assay kit for detecting metandienone and using method of enzyme linked immunosorbent assay kit - Google Patents
Enzyme linked immunosorbent assay kit for detecting metandienone and using method of enzyme linked immunosorbent assay kit Download PDFInfo
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Abstract
The invention discloses an enzyme linked immunosorbent assay kit for detecting metandienone and a using method of the enzyme linked immunosorbent assay kit, relating to the technical field of enzyme linked immunization and veterinary drug residue detection. The enzyme linked immunosorbent assay kit for detecting the metandienone comprises a metandienone specific antibody, a conjugate of metandienone and carrier protein and an enzyme-labeled secondary antibody. The enzyme linked immunosorbent assay kit for detecting metandienone has the advantages of sensitivity, rapidness and accuracy, and is mainly used for screening a large quantity of samples; main reagents in the kit are provided in working solution types, thus the enzyme linked immunosorbent assay kit is convenient for use, and has the characteristics of high specificity, high sensitivity, high accuracy, high veracity and the like, and is capable of rapidly detecting metandienone remained in forage and the animal by-products.
Description
Technical field
The present invention relates to enzyme linked immunological and detection of veterinary drugs in food technical field, particularly, the present invention relates to a kind of enzyme linked immunological kit and method of application that is used to detect protobolin.
Technical background
(1-dehydro-17-methyltestosterone DMT), has another name called metandienone, U.S. androsterone, molecular formula: C to protobolin
20H
28O
2, molecular weight 300.44.DMT generally is used for alpastic anemia, male sex's hypoevolutism, burn processing, operation back chronic wasting disease, senile osteoporosis and tumour at present and dislikes juice disease etc. on clinical medicine.But DMT belongs to the protein anabolic hormones, and taking for a long time or in a large number to have a lot of spinoffs to human body.As causing hepatosis, reproductive function is disorderly, increases to suffer from cardiovascular disease risk etc.DMT also has the effect that promotes the interior nutriment deposition of animal body and improve production performance, in animal husbandry, often is used as animal feed additive, to promote growth of animal, improves output.But when protein anabolic hormone is applied to herding production as feed addictive; Can be residual in animal body; When people are for a long time edible when containing the animal food of this medicament residue; Can cause people's reproductive system obstacle, dysplasia and some cancer such as mastocarcinoma, carcinoma of testis etc. equally, the serious harm human health.
At present, mostly the detection method that DMT is commonly used is the instrument detecting method, like high performance liquid chromatography (HPLC), LC-MS (LC-MS) and gas chromatography mass spectrometry (GC-MS) etc.These instrument detecting methods need complicated instrument, and process is loaded down with trivial details, the examination of incompatible on-the-spot a large amount of samples.For the analyzing and testing of micromolecule residue a new approach is provided based on the immunoreactive immunology detection technology of antigen-antibody.
Because DMT is a micromolecular compound, itself does not have immunogenicity, must itself and macromolecular carrier albumen coupling be obtained having immunogenic artificial antigen.DMT artificial antigen and specific antibody and set up immune analysis method on this basis and do not appear in the newspapers as yet.
Summary of the invention
The object of the present invention is to provide enzyme linked immunological kit and the method for application of a kind of fast detecting DMT.
The invention provides the enzyme linked immunological kit of a kind of fast detecting DMT, this kit comprises: DMT specific antibody, DMT-carrier protein couplet thing and ELIAS secondary antibody, three's volume proportion of composing is 1:2:2.
Wherein the DMT specific antibody is the monoclonal antibody of mouse-anti DMT, and available DMT and carrier protein couplet thing make through immune BALB/c mouse as immunogene.
Said carrier protein is bovine serum albumin(BSA) (BSA), and the conjugate of said DMT and carrier protein can obtain through DMT and carrier protein are carried out coupling with active ester method.
The enzyme linked immunological kit of a kind of fast detecting DMT of the present invention comprises further that also the carrier mass that is used for fixing DMT and carrier protein couplet thing is a lot, for example, and polystyrene, tygon, polypropylene.The form of carrier is the micro-reaction plate shrinkage pool.
Be convenient on-the-spot the detection and the great amount of samples examination, described kit can further include DMT standard solution, substrate developer, cleansing solution, stop buffer and concentrating sample dilution.
Described cleansing solution is 0.5% Tween-20 phosphate buffer.
Described developer is formed (for example, volume ratio is 1:19) by developer A liquid and developer B liquid, and developer A liquid is hydrogen peroxide, and developer B liquid is tetramethyl benzidine.
Described concentrating sample dilution is 0.01M, the phosphate buffer of PH7.4.
On the other hand, the present invention also provides the method for utilizing DMT content in the mentioned reagent box test sample, carries out according to following step:
(1) sample pre-treatments: the blank sample (meat and water equivalent weight stir) of getting the pork of 10 g (wherein meat 5 g are accurate to 0.01 g) rubbing places 50 mL centrifuge tubes; Add t-butyl methyl ether 20 mL after adding a certain amount of DMT titer, vortex vortex mixer mixing 1 min places ultrasonic generator Extraction by Ultrasound 10 min, takes out centrifugal 5 min of 8000 r/min, and organic phase is transferred in another 50 mL centrifuge tube; Lower floor divides 2 extractions with 20 mL t-butyl methyl ether again, and organic phase is transferred in the corresponding centrifuge tube; 40 ℃ of rotary evaporations add 2 % methyl alcohol-PBS and are settled to 10 mL; Carrying out ELISA measures.
(2) detect with kit; In the ELISA Plate hole that is coated with protobolin antigen, add standard items or sample solution, add Methandienone monoclonal antibody again, hatch the back washing and drain; It is anti-to add enzyme labeling two; Hatch the back washing and drain, colour developing, termination are measured absorbance with ELIASA.
(3) analyzing and testing result.
The detection principle of kit of the present invention is:
DMT antigen is adsorbed on the solid phase carrier; Add sample or DMT standard solution and add DMT antibody working fluid, the DMT antigenic competition DMT antibody that encapsulates on DMT and the solid phase carrier in the testing sample adds the amplification that the enzyme labeling antiantibody carries out enzymatic activity again; The colour developing back stops; The absorbance of working sample, the amount of DMT is negative correlation in this value and the sample, relatively can draw the DMT concentration range with typical curve.
Beneficial effect:
The kit that the present invention detects DMT mainly adopts DMT content in the qualitative or quantitative sample of indirect competitive enzyme-linked immunosorbent method; Pre-treatment requirement to sample is low, and sample pretreatment process is simple, simultaneously the fast detecting gross sample.
This kit of the present invention adopts the DMT monoclonal antibody of high specific; Main agents provides with the working fluid form; Can reduce the operation steps of kit; For the user saves time and reduces the error that causes because of operation steps is miscellaneous; That the present invention has is highly sensitive, high specificity, high precision, pin-point accuracy, low to instrument requirement, the reagent holding time is long, automaticity is high, "dead" isotopic contamination etc. advantage, can in feed and animal derived product detect, play a significant role.
Description of drawings
Fig. 1. DMT enzyme-linked immunologic detecting kit structural drawing, wherein 1 is the kit box body, 2 for having encapsulated the ELISA Plate of DMT-BSA antigen; 3 is series standard solution, and 4 is developer A, and 5 is developer B; 6 is antibody-solutions, and 7 is ELIAS secondary antibody solution, and 9 is concentrated cleaning solution; 10 is the concentrating sample dilution, and 11 is the reagent bottle stand.
Fig. 2. DMT suppresses curve.
Embodiment
The preparation method of DMT-carrier protein couplet thing is referring to patent 201010213218.7 (Dong Ying, Zhang Xun, Wang Yun, Chu Xiaogang).
The ELIAS secondary antibody that the present invention uses is commercial sheep anti-mouse igg antibody, available from Genescript company.
Embodiment below the present invention is only as the further specifying of content of the present invention, can not be as scope perhaps in the qualification of the present invention.Through embodiment the present invention is described further below.
1.1 the preparation of the conjugate of DMT and carrier protein
Adopt active ester method to carry out coupling DMT and bovine serum albumin(BSA) (BSA) and obtain conjugate.
1.2 DMT MONOCLONAL ANTIBODIES SPECIFIC FOR and purifying
(1) MONOCLONAL ANTIBODIES SPECIFIC FOR: get the healthy BALB/c mouse in 6～8 ages in week, every mouse peritoneal is injected 0.5 m through autoclaved whiteruss; After about 10 days, collect the hybridoma 1D6F10B4 (by laboratory previous work preparation, patent applied for) of logarithmic phase, centrifugal 10 min of 1 000 r/min abandon supernatant; With 1640 incomplete nutrient solutions cell is hanged, count every injected in mice cell 5.0 * 10 after the trypan blue dyeing that adds equal volume that takes a morsel
5Individual; The inoculation hybridoma extracted ascites with syringe after 7～10 days, and the ascites that obtains through centrifugal 15 min of 12 000 r/min, is abandoned deposition, collected supernatant.
(2) adopt caprylic acid-saturated ammonium sulfate method to carry out purifying, purification step is specific as follows: get in right amount through pretreated mouse ascites, dilute 2 times with the acetate buffer solution of 0.06 mol/L, pH 4.8; In the ratio of the original ascites of 33 μ L/mL, dropwise add caprylic acid in 30 min, stir while dripping; In 4 ℃ leave standstill 2 h after, centrifugal 20 min of 12 000 r/min get the supernatant filter paper filtering; Add 0.1 mol/L of 1/10 supernatant volume, the PBS of pH 7.4, and transfer pH to 7.4 with NaOH; Supernatant adds the ammonium sulfate powder in the ratio of 277 g/L under 4 ℃ of condition of ice bath, stir simultaneously, adds in 30 min, leave standstill 2 h above or 4 ℃ spend the night; At 4 ℃ of following centrifugal 30 min of 12 000 r/min, abandon supernatant, collecting precipitation is dissolved in the PBS of certain volume with deposition, and under 4 ℃, places 50 ~ 100 times PBS dialysis desalination 2 ~ 3 days, during change dislysate more than 3 times.Frozen afterwards subsequent use down in-20 ℃.
2.1 detect the structure of the enzyme linked immunological kit of DMT
Detect DMT enzyme linked immunological kit structure like Fig. 1. shown in: wherein 1 is the kit box body, and 2 for having encapsulated the ELISA Plate of DMT-BSA antigen, and 3 is series standard solution; 4 is developer A, and 5 is developer B, and 6 is antibody-solutions; 7 is ELIAS secondary antibody solution; 9 is concentrated cleaning solution, and 10 is the concentrating sample dilution, and 11 is the reagent bottle stand.Be shaped on shrinkage pool on the reagent bottle stand 11, mentioned reagent bottle 3-10 is placed in the shrinkage pool of reagent bottle stand, and reagent bottle stand 11 and ELISA Plate 2 are placed in the box body.Wherein ELISA Plate 2 is made up of plastic stent and detachable enzyme mark bar.
2.2 the preparation of agents useful for same
A.DMT standard solution: 6 bottles of DMT series standard solution, 1-3 mL/ bottle.
B. encapsulate the carbonate buffer solution of damping fluid: pH 9.6,0.05 mL.
C. confining liquid: 3 % polyglycol.
D. concentrated cleaning solution: contain the phosphate buffer (0.01 M, pH 7.4) of 1 % tween, be 20 times of normal working concentration, 30-50 mL, 1 bottle.
2.3 the preparation of ELISA Plate
Be diluted to 1 μ g/mL with encapsulating the conjugate of damping fluid with encapsulating damping fluid with DMT and carrier protein, every hole 100 μ L, 37 ℃ of temperature are bathed 2h; Inclining coating buffer, with cleansing solution washing 3 times, each 30 seconds; Clap and do, in every hole, add 200 μ L confining liquids then, 4 ℃ are spent the night; The liquid in the hole that inclines, preserve with the preservative film sealing dry back.
The foundation of embodiment 3 immunologic detection methods
3.1 the ELISA method confirms that the best encapsulates concentration and antibody working concentration
The bovine serum albumin(BSA) DMT-BSA conjugate of 4 μ g/mL, 2 μ g/mL, 1 μ g/mL, 0.5 μ g/mL, 0.25 μ g/mL, 0.125 μ g/mL series concentration is pressed every hole 100 μ L coated elisa plates; A kind of concentration of every row, 4 ℃ encapsulate and spend the night, and wash 3 times; Clap and do; By 37 ℃ of sealings of every hole 200 μ L confining liquids, 2 h, wash 3 times, clap and do.The antibody 100 μ L that add 1:1000,1:2000,1:4000,1:8000,1:12000,1:16000,1:24000,1:32000 dilution, a kind of concentration of every row, 37 ℃ of effect 50 min; Wash 3 times, add 100 μ L enzymes mark sheep anti-mouse antibody immediately, 37 ℃ of effect 1h; Wash 3 times, add 100 μ L substrate solutions, 37 ℃ of lucifuge effect 15 min; 50 μ L stop buffer cessation reactions, ELIASA detects A value (450nm).Parallel repeating hole is set simultaneously, get the OD value when being 1.0 left and right sides encapsulate concentration and the antibody working concentration is an optium concentration.Experimental data is listed in table 1.Can confirm that it is 1 μ g/mL that the best encapsulates concentration, the optimum antibody working concentration is 8000 times of dilutions.
Table 1. chessboard method confirms that the best encapsulates concentration and antibody working concentration
3.2 indirect ELISA detects antibody titer
With 1 μ g/mL concentration coated elisa plate, antibody is diluted 4000,8000,16000,32000,64000,128000,256000 times respectively, 5000 times of negative serum dilutions, ELISA procedure by 2.1.OD with antibody
450Value is more than or equal to the OD of negative serum contrast
4502.1 times of value are tired as sero-fast, and the sero-fast testing result of tiring is seen table 2.Tiring of the antibody of the present invention's preparation more than 64000.
The mensuration that table 2. is tired
| Dilutability | 4000 | 8000 | 16000 | 32000 | 64000 | 128000 | 256000 | Negative |
| The OD value | 1.289 | 1.045 | 0.712 | 0.446 | 0.271 | 0.157 | 0.126 | 0.092 |
| The P/N value | 14.01 | 11.36 | 7.74 | 4.85 | 2.95 | 1.71 | 1.40 | ? |
3.3 indirect competitive ELISA detects antibody specificity
The ELISA method of operating with 2.1. different be the DMT standard items that every hole adds 50 μ L variable concentrations, add antiserum subsequently, draw different OD values.Table is seen in putting in order of DMT series concentration and test hole, and repeating hole and blank hole are set simultaneously.OD value with 0 inhibition hole is maximal value B
0, other inhibition concentration hole OD values are B, B/B
0The DMT concentration of=50% o'clock correspondence is the IC of antibody for this reason
50Value.The specific detection result's of antibody data are listed in table.Then, draw out DMT with the gained data and suppress curve.
Can know DMT antiserum IC by diagram data
50Value is 3.20 ng/mL, shows that the antiserum that the present invention prepares is to have good specific anti-DMT monoclonal antibody.
Embodiment 4 detects the establishment of the enzyme linked immunological kit of DMT
Set up the enzyme linked immunological kit that detects DMT, make it comprise following component:
(1) encapsulates the ELISA Plate of DMT antigen;
(2) protein concentration be 3.46 mg/mL anti-DMT monoclonal antibody;
(3) with the sheep anti mouse antiantibody of horseradish peroxidase-labeled;
(4) the DMT standard solution is 6 bottles, and concentration is respectively 0.1,1,5,10,50 and 100 ng/mL;
(5) substrate developer A liquid is hydrogen peroxide, and substrate developer B liquid is tetramethyl benzidine;
(6) cleansing solution is the phosphate buffer that contains 0.5% polysorbas20;
(7) the concentrating sample dilution is 0.01M, the phosphate buffer of PH7.4;
(8) stop buffer is the sulfuric acid solution of 2 mol/L.
The residual detection of DMT in embodiment 5 samples
5.1 sample pre-treatments
Get the pork sample (meat and water equivalent weight stir) that 10g (wherein meat 5 g are accurate to 0.01 g) rubs, place 50 mL centrifuge tubes; Add t-butyl methyl ether 20 mL, vortex vortex mixer mixing 1 min places ultrasonic generator Extraction by Ultrasound 10 min, takes out centrifugal 5 min of 8000 r/min, and organic phase is transferred in another 50 mL centrifuge tube; Lower floor divides 2 extractions with 20 mL t-butyl methyl ether again, merges 40 ℃ of rotary evaporations of organic phase, adds 2 % methyl alcohol-PBS and is settled to 10 mL; Carrying out ELISA measures.
5.2 detect with kit
In the ELISA Plate micropore that the DMT-BSA conjugate encapsulates, add series standard article or sample solution 50 μ L, add antibody working fluid (the anti-DMT monoclonal antibody of 0.5 mg/L) 50 μ L again, hatch 50 min under 37 ℃.Throw away liquid in the hole, every hole adds 300 μ L through 10 times of cleansing solutions that diluted, and throws away liquid in the hole after 30 seconds, and so repetitive operation is washed plate 3 times altogether, claps with thieving paper and does.Every hole adds enzyme labeling antiantibody 100 μ L, hatches 60min under 37 ℃.Substrate colour developing liquid A liquid (hydrogen peroxide) 5 μ L; Add B liquid (tetramethyl benzidine) 95 μ L again, the mixing that vibrates gently, 37 ℃ of lucifuge effect 15 min; Every hole adds stop buffer (2 mol/L sulfuric acid) 50 μ L, detects every hole absorbance (450 nm) with ELIASA.
Interpretation of result:
Calculate percentage absorbance and drawing standard curve, the concentration of DMT can be read from typical curve in corresponding each sample.According to the comparison of the depth and the series concentration standard solution color of the sample of color on the ELISA Plate, can judge the concentration range of DMT in the sample.
Test Example one, kit sensitivity test:
Confirming of table 3. kit sensitivity
| Number of times | IC 10（ng/ml） | IC 50（ng/ml） |
| 1 | 0.056 | 3.212 |
| 2 | 0.048 | 2.953 |
| 3 | 0.063 | 3.123 |
| 4 | 0.027 | 2.706 |
| 5 | 0.069 | 3.167 |
| 6 | 0.060 | 3.157 |
| 7 | 0.050 | 2.986 |
| 8 | 0.066 | 3.206 |
| 9 | 0.061 | 3.191 |
| 10 | 0.048 | 2.888 |
Test Example two, the test of kit precision:
This Test Example is the repeatable test of standard.Concrete operations are:
From 3 batches of ELISA Plates, respectively extract 4 holes, measure the OD value of 1ng/mL DMT standard solution, calculate coefficient of variation CV%.
The result sees table 4, and coefficient of variation scope has met the regulation of the coefficient of variation less than 15 % between 3.16～4.64 %, explains that this kit standard items precision has reached requirement.
The repeatable experiment of table 4. standard
The recovery test of Test Example three, kit:
Get the DMT standard specimen of 6 concentration, sample is added recovery test, respectively calculate recovery rate.
The result sees table 5, and the recovery is at 87.41 % ~ 110.70 %, and the coefficient of variation is at 2.05 % ~ 10.81 %, and average coefficient of variation is 5.76 %, and the Variation Lines number average shows that less than 15 % kit has higher accuracy.
Table 5. pork sample recovery rate
Test Example four, the test of kit specificity:
Select analogue testosterone propionate ester, the Trenbolone of DMT to make mortifier, competitive ELISA is measured the IC of each mortifier
50, method is calculated the cross reacting rate with DMT with 2.3.Cross reacting rate is low more, and specificity is strong more.
Cross reacting rate=(the IC of DMT
50The IC of/competition thing
50) * 100%.
The result sees table 6, and the cross reacting rate of DMT and two kinds of analogues is all less than 0.1%, and DMT enzyme linked immunological quick detection kit and other analogue of illustrative experiment preparation almost do not have cross reaction, and specificity is good.
The mensuration of table 6. cross reacting rate
| Compound | IC 50（ng/mL） | Cross reacting rate (%) |
| DMT | 3.06 | 100 |
| The testosterone propionate ester | >;1000 | <0.5 |
| Trenbolone | >;1000 | <0.5 |
Test Example five, kit are preserved test:
Kit 37 ℃ of preservation condition held 6 days, is carried out accelerated aging test.In 6 days, detect its Amax, IC
50
The result sees table 7, and along with the prolongation of kit storage life, the Amax value descends to some extent, IC
50Value also changes to some extent, but Amax/IC
50Change not quite, explain that kit quality in 4 ℃ of 6 months storage lives keeps stable.
Table 7. is preserved experiment for 37 ℃
| Holding time (d) | Amax | IC 50（ng/mL） | Amax/ |
| 0 | 1.236 | 3.157 | 0.392 |
| 1 | 1.237 | 3.123 | 0.396 |
| 3 | 1.219 | 3.206 | 0.380 |
| 6 | 1.118 | 2.961 | 0.378 |
Being merely preferred embodiment of the present invention in sum, is not to be used for limiting practical range of the present invention.Be that all equivalences of doing according to the content of claim of the present invention change and modification, all should be technological category of the present invention.
Claims (6)
1. the enzyme linked immunological kit of a fast detecting DMT is characterized in that comprising: DMT specific antibody, DMT-carrier protein couplet thing and ELIAS secondary antibody.
2. according to the enzyme linked immunological kit of the said a kind of fast detecting DMT of claim 1, it is characterized in that DMT specific antibody, DMT-carrier protein couplet thing and ELIAS secondary antibody three volume proportion of composing are 1:2:2.
3. according to the enzyme linked immunological kit of the said a kind of fast detecting DMT of claim 1, it is characterized in that wherein the DMT specific antibody is the monoclonal antibody of mouse-anti DMT, make through immune BALB/c mouse as immunogene with DMT and carrier protein couplet thing; Said carrier protein is bovine serum albumin(BSA) (BSA), and the conjugate of said DMT and carrier protein obtains through DMT and carrier protein are carried out coupling with active ester method.
4. according to the enzyme linked immunological kit of the said a kind of fast detecting DMT of claim 1, it is characterized in that also further comprising the carrier mass that is used for fixing DMT and carrier protein couplet thing, like polystyrene, tygon, polypropylene; The form of carrier is the micro-reaction plate shrinkage pool.
5. according to the enzyme linked immunological kit of the said a kind of fast detecting DMT of claim 1, it is characterized in that described kit can further include DMT standard solution, substrate developer, cleansing solution, stop buffer and concentrating sample dilution;
Described cleansing solution is 0.5% Tween-20 phosphate buffer;
Described developer is that developer A liquid and the developer B liquid of 1:19 is formed by volume ratio, and developer A liquid is hydrogen peroxide, and developer B liquid is tetramethyl benzidine;
Described concentrating sample dilution is 0.01M, the phosphate buffer of PH7.4.
6. the method for DMT content in the enzyme linked immunological kit test sample of the said a kind of fast detecting DMT of claim 1 is characterized in that carrying out according to following step:
(1) sample pre-treatments: the blank sample of getting the pork of 10 g rubbing places 50 mL centrifuge tubes; Add t-butyl methyl ether 20 mL after adding a certain amount of DMT titer, vortex vortex mixer mixing 1 min places ultrasonic generator Extraction by Ultrasound 10 min, takes out centrifugal 5 min of 8000 r/min, and organic phase is transferred in another 50 mL centrifuge tube; Lower floor divides 2 extractions with 20 mL t-butyl methyl ether again, and organic phase is transferred in the corresponding centrifuge tube; 40 ℃ of rotary evaporations add 2 % methyl alcohol-PBS and are settled to 10 mL; Carrying out ELISA measures;
(2) detect with kit; In the ELISA Plate hole that is coated with protobolin antigen, add standard items or sample solution, add Methandienone monoclonal antibody again, hatch the back washing and drain; It is anti-to add enzyme labeling two; Hatch the back washing and drain, colour developing, termination are measured absorbance with ELIASA;
(3) analyzing and testing result.
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| CN104808005A (en) * | 2015-04-15 | 2015-07-29 | 中山大学 | Method for identifying male giant grouper |
| CN106771263A (en) * | 2016-12-01 | 2017-05-31 | 无锡艾科瑞思产品设计与研究有限公司 | Methyltestosterone detection method and kit in a kind of chicken |
| CN107015010A (en) * | 2017-06-09 | 2017-08-04 | 深圳大学 | It is a kind of for kit of testosterone residue detection and preparation method thereof and detection method |
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