CN103421072B - Dexamethasone hapten and its preparation method and application - Google Patents
Dexamethasone hapten and its preparation method and application Download PDFInfo
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Abstract
The invention discloses a kind of hapten and its preparation method and application, be specifically related to a kind of dexamethasone hapten.The invention also discloses described haptenic preparation method and applications.The quick detection kit product set up based on dexamethasone hapten, easy to use, testing cost is low, detection method is efficient, accurate, quick, large batch of sample can be detected simultaneously, be suitable to on-site supervision and the examination of great amount of samples of the residual of dexamethasone in animal derived food.
Description
Technical field
The present invention relates to a kind of hapten and its preparation method and application, be specifically related to dexamethasone hapten and its preparation method and application.
Background technology
Dexamethasone (dexamethasone, Dex) is the derivant of glucocorticoid cortisone class, is also dexamethasone, is also that clinical medicine uses widest potent 17-hydroxy-11-dehydrocorticosterone.Cortisone hormones is commonly used to treat anaphylaxis and serious infections, tool antiinflammatory and antianaphylactic effect.This parahormone still irritates appetite, increases hepatic glycogen synthesis, rises the effects such as hyperglycemia.But this hormone remains in food, having people potentially hazardous, therefore the states such as European Union all forbid dexamethasone, dexamethasone etc., as the growth promoter of domestic animal, uses in animal husbandry.
At animal derived food MRL, European Union specifies that the MRL of dexamethasone is muscle 0.75 μ g/kg, liver 2.0 μ g/kg, kidney 0.75 μ g/kg, milk 0.3 μ g/kg.The muscle 0.75 μ g/kg, liver 2.0 μ g/kg, kidney 0.75 μ g/kg, milk 0.3 μ g/kg that MRL is cattle/pig/horse of the Ministry of Agriculture of China 235 bulletin regulation dexamethasone.
The technology such as thin layer chromatography, immunoassay, liquid chromatograph, gas chromatogram, gas-matter, liquid-matter are had at present about the detection method that glucocorticoids is conventional.The mensuration Liquid Chromatography-Tandem Mass Spectrometry of dexamethasone residual quantity in GB/T20741-2006 livestock meat, this standard defines the liquid chromatography-tandem mass spectrometry method for determining of dexamethasone residual quantity in beef, Carnis Sus domestica, Carnis caprae seu ovis and Carnis Gallus domesticus, and method detection is limited to 0.2 μ g/kg.The mensuration Liquid Chromatography-Tandem Mass Spectrometry of dexamethasone residual quantity in GB/T22978-2008 milk and milk powder, this standard defines the liquid chromatography-tandem mass spectrometry method for determining of dexamethasone residual quantity in milk and milk powder, method detection is limited to milk 0.2 μ g/kg, milk powder 1.0 μ g/kg.Dexamethasone method for detecting residue high performance liquid chromatography in the Ministry of Agriculture 958 bulletin-6-2007 pig edible tissue, this standard defines sample preparation and the high-performance liquid chromatogram determination method of dexamethasone residue detection in pig muscle and liver, this method detects in Swine muscle and is limited to 0.75 μ g/kg, quantitatively it is limited to 1 μ g/kg, in pig liver tissue, detection is limited to 1 μ g/kg, is quantitatively limited to 2.0 μ g/kg.The response rate is 60% ~ 120%.Dexamethasone, betamethasone, Triamcinolone and double dexamethasone determination of residual amount method euzymelinked immunosorbent assay (ELISA) in SN/T1970-2007 Imported and exported animals derived food, this standard defines dexamethasone in animal derived food, betamethasone, Triamcinolone and the enzyme-linked immunoassay method of double dexamethasone residual quantity, mensuration lower bound is dexamethasone, betamethasone, Triamcinolone 0.3 μ g/kg, double dexamethasone 1.5 μ g/kg.Due to instrument costly, complex operation, costly, be not suitable for carrying out extensive Site Detection etc., ELISA method has the advantages such as highly sensitive, simple to operate, low cost, extensive detection, can quickly detect the dexamethasone in sample.
Summary of the invention
It is an object of the invention to provide a kind of dexamethasone hapten and preparation method thereof.
The dexamethasone hapten molecule structural formula that the present invention provides is:
。
The haptenic preparation method of dexamethasone that the present invention provides, comprises the steps:
0.80g dexamethasone is dissolved in 5ml dimethyl sulfoxide (DMSO), it is slowly added dropwise at 60 DEG C into 0.5ml1,3-propane diamine and 0.5ml pyridine are in the mixed liquor of 10mlDMSO, after dropping, continue reaction 15 hours, rotation is evaporated off solvent and unreacted propane diamine, quantitatively obtains the propane diamine list condensation substance of dexamethasone.
Another object of the present invention is the application in immune detection of the above-mentioned dexamethasone hapten, specifically include the dexamethasone antigen obtained by described dexamethasone hapten and carrier protein couplet, and the dexamethasone antibody prepared by gained dexamethasone antigen-immunized animal.
Wherein said carrier protein can be bovine serum albumin, oralbumin, human albumin, thyroprotein, rabbit serum proteins, Mus serum albumin, hemocyanin or Fibrinogen.
Described antibody is dexamethasone monoclonal antibody, and this monoclonal antibody is to be obtained by dexamethasone monoclonal antibody hybridoma cell strain D-4-2CGMCCNo.6068 secretion.
The present invention also provides for the enzyme linked immunological kit prepared by above-mentioned dexamethasone antibody, and the application of dexamethasone residual in detection animal derived food.
The dexamethasone hapten that the present invention provides the most farthest remains the chemical constitution of dexamethasone, introducing further through chemosynthesis transformation can be with the NH of protein molecule, with this hapten as raw material, preparation is adapted for the antigen-immunized animal of animal immune, and the titer of gained antibody, specificity, affinity are relatively good；The antibody of gained is used for preparing enzyme linked immunological kit, and test kit testing cost is low, easy to use, and detection method is accurate, quick, can detect large batch of sample simultaneously, is suitable to on-site supervision and the examination of great amount of samples of dexamethasone residual in animal derived food.The dexamethasone hapten of the present invention plays a significant role in the detection of dexamethasone.
Accompanying drawing explanation
Fig. 1: dexamethasone hapten synthesis route map
Fig. 2: dexamethasone hapten hydrogen nuclear magnetic resonance spectrogram
Fig. 3: dexamethasone ELISA standard curve
Detailed description of the invention
The present invention is expanded on further below in conjunction with specific embodiment.Should be understood that these embodiments are merely to illustrate the present invention, and be not limited to the scope of the present invention.
Embodiment 1: the haptenic synthesis of dexamethasone and qualification (synthetic route such as Fig. 1)
0.80g dexamethasone is dissolved in 5mlDMSO, is slowly added dropwise into 0.5ml1,3-propane diamine and 0.5ml pyridine at 60 DEG C in the mixed liquor of 10mlDMSO, after dropping, continuing reaction 15 hours, rotation is evaporated off solvent and unreacted propane diamine, quantitatively obtains the propane diamine list condensation substance of dexamethasone.
Take above-mentioned product through nuclear magnetic resonance hydrogen spectruming determining, as in figure 2 it is shown, nuclear magnetic spectrum explanation: in collection of illustrative plates, the olefin peaks between 6.5-7.0ppm moves to High-Field, the methylene peak explanation hapten synthesis success increased between 1.0-2.5ppm.
Embodiment 2: dexamethasone antigen
Dexamethasone hapten and carrier protein couplet are obtained dexamethasone antigen.
One, immunogen prepares dexamethasone hapten-bovine serum albumin (BSA) conjugate synthesis
Take EDC50mg 2ml water to be allowed to fully dissolve and obtain solution I；Take dexamethasone hapten 11mg 1.0mlDMF dissolving and obtain solution II；Take BSA60mg and be dissolved in 3ml0.01mol/LPBS(pH=8.0) solution obtains solution III；Solution II is mixed with solution III, is added dropwise over solution I under magnetic stirring；Stirring reaction 24 hours under room temperature.Dialyse 48 hours with tri-distilled water, obtain immunogen.
Two, coating antigen prepares dexamethasone hapten-oralbumin (OVA) conjugate synthesis
Take dexamethasone hapten 15mg 1.0ml dimethylformamide (DMF) dissolving and obtain solution IV；Glutaraldehyde (GA) the 10 μ l taking 50% adds in solution IV, and under room temperature, stirring reaction 18h obtains solution V；Add in solution V after taking the dilution of OVA40mg 3ml water；Reaction adds 24mgNaBH the most afterwards4Reaction 3h；Dialyse 48 hours with tri-distilled water, obtain coating antigen.
Three, the qualification of dexamethasone antigen
Carrier protein, dexamethasone hapten, the PBS of dexamethasone hapten-carrier protein conjugate pH7.4 are made into the solution of 0.5mg/mL, return to zero with 0.01mol/LpH7.4PBS, scan in the range of wavelength 200 ~ 800nm with ultraviolet spectrophotometer, obtain carrier protein, dexamethasone hapten, the absorption curve of dexamethasone hapten-carrier protein conjugate, and calculate its combination ratio.It was found that different absorption curves occurs in three, show dexamethasone hapten and carrier protein couplet success.
Embodiment 3: dexamethasone monoclonal antibody
One, the preparation of dexamethasone monoclonal antibody
Animal immune: immunogen be injected in Balb/c Mice Body, immunizing dose is 150 μ g/ so that it is produce polyclonal antibody.
Cell merges and cloning: after mice serum measurement result is higher, take its splenocyte, in 8:1 ratio and SP2/0 myeloma cell fusion, uses indirect competitive ELISA to measure cell supernatant, the positive hole of screening.Utilize limiting dilution assay that positive hole is carried out cloning, until obtaining the hybridoma cell strain of secrete monoclonal antibody, and find that wherein a strain titer is significantly higher than other hybridoma cell strain, by this dexamethasone named D-4-2 of monoclonal antibody hybridoma cell strain, this cell strain is preserved in (address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, China Committee for Culture Collection of Microorganisms's common micro-organisms center on May 3rd, 2012, Institute of Microorganism, Academia Sinica), preserving number is CGMCCNo.6068.
Cell cryopreservation and recovery: the monoclonal hybridoma strain frozen stock solution of dexamethasone is made 5 × 107The cell suspension of individual/ml, preserves in liquid nitrogen for a long time.Take out cryopreservation tube during recovery, be immediately placed in 37 DEG C of water-bath middling speeds and melt, after centrifugal segregation frozen stock solution, move into and cultivate culture in glassware.
The production of monoclonal antibody and purification: Balb/c mouse peritoneal is injected sterilizing paraffin oil 0.5ml/ only, the monoclonal hybridoma strain 5 × 10 of 7 days pneumoretroperitoneum injection dexamethasone5Individual/only, gather ascites after 7 days.Carry out ascites by octanoic acid-saturated ammonium sulfate method to purify ,-20 DEG C of preservations.This monoclonal antibody specificity is good, and detection sensitivity is up to 0.05 μ g/L.
Two, the mensuration of antibody titer
The titer measuring antibody with indirect competitive ELISA method is 1:80000 ~ 100000.
Indirect competitive ELISA method: with dexamethasone hapten-oralbumin conjugate coated elisa plate, add dexamethasone standard solution and monoclonal antibody working solution, 25 DEG C of reaction 30min, pour out liquid in hole, wash 3 ~ 5 times with cleaning mixture, pat dry with absorbent paper；Add enzyme labelling anti antibody, mixing of vibrating gently, 25 DEG C of reaction 30min, pour out liquid in hole, wash 3 ~ 5 times with cleaning mixture, pat dry with absorbent paper；Add substrate solution, after 25 DEG C of reaction 15min, add stop buffer and terminate reaction；Set microplate reader at wavelength 450nm, measure every hole absorbance.
Embodiment 4: the enzyme linked immunological kit prepared by dexamethasone monoclonal antibody
One, the composition of enzyme linked immunological kit
(1) ELISA Plate of dexamethasone coupled antigen it is coated；
(2) enzyme labelling anti antibody: with the sheep anti mouse anti antibody of horseradish peroxidase-labeled；
(3) dexamethasone monoclonal antibody working solution；
(4) standard solution: concentration is respectively 0 μ g/L, 0.05 μ g/L, 0.15 μ g/L, 0.45 μ g/L, 1.35 μ g/L, 4.05 μ g/L；
(5) substrate nitrite ion is made up of A liquid and B liquid, and A liquid is urea peroxide, and B liquid is tetramethyl biphenyl amine aqueous solution；
(6) stop buffer is the sulfuric acid solution of 2mol/L；
(7) concentrated cleaning solution is the phosphate buffer of 0.1 ~ 0.2mol/LpH7.2 containing 1% tween 20, and described percentage ratio is percent weight in volume；
(8) concentrating redissolution liquid is pH7.2 ~ 7.8, and the phosphate buffer of 0.1 ~ 0.3mol/L, described percentage ratio is percent weight in volume.
The main agents of this test kit provides with the form of working solution, and the method for inspection is convenient and easy, has that specificity is high, highly sensitive, degree of accuracy is high, accuracy high.
Two, the application of enzyme linked immunological kit detection actual sample
1. the pre-treatment of sample
(1) Carnis Gallus domesticus Sample pretreatment method
With homogenizer homogeneous structure sample；Weigh the equal pledge of 2.0g ± 0.05g in 50ml polystyrene centrifuge tube, add 8ml ethyl acetate, vibrate 5min with agitator；Room temperature places 20min, more than 3000g, and room temperature is centrifuged 5min；Taking 4ml supernatant in 50ml polystyrene centrifuge tube, add 2ml2M sodium hydroxide solution, vibration this step of 10min(is more crucial), more than 3000g, room temperature is centrifuged 5min；Take upper strata 1ml organic facies in 10ml teat glass；Flow down nitrogen in 50-60 DEG C of water-bath to dry up.Add 0.5ml redissolution working solution, whirling motion 2～3min, take 100 μ l for analyzing.
(2) milk pre-treating method
Take 200 μ l milk samples add 400 μ l redissolve liquid working solutions fully mix.Take 100 μ l for analyzing.
2. detect with test kit
In the ELISA Plate micropore be coated with coating antigen, add 100 μ l standard substance/sample, add 50 μ l monoclonal antibody working solutions, mixing of vibrating gently, cover cover plate film, lucifuge reaction 30min in 25 DEG C of calorstats.Pour out the liquid in hole, fully wash 4-5 time with wash operating solution (20 × concentrated cleaning solution being diluted by 1:19 volume ratio with deionized water) 250 μ l/ hole, every minor tick 10s, the liquid completely removing in hole with guarantee is patted dry with absorbent paper, add 100 μ l enzyme labelling anti antibodys, vibrate gently mixing, cover cover plate film, lucifuge reaction 30min in 25 DEG C of calorstats.Pouring out the liquid in hole, repeat to wash plate step, add substrate solution A liquid 50 μ l, add substrate solution B liquid 50 μ l, mixing of vibrating gently, with lucifuge colour developing 15min in 25 DEG C of calorstats after cover plate membrane cover plate.Add 50 μ l stop buffers, mixing of vibrating gently, set microplate reader at 450nm, measure the absorbance in every hole.
3. Analysis of test results
By the meansigma methods (diplopore) of the standard substance obtained or the absorbance of sample divided by the absorbance of first standard (0 standard), then it is multiplied by 100%, i.e. obtains the percentage absorptance of standard substance or sample.With dexamethasone standard substance percentage absorptance as vertical coordinate, with the logarithm of dexamethasone standard concentration as abscissa, draw canonical plotting, as shown in Figure 3.The percentage absorptance of sample being substituted in standard curve, read the concentration corresponding to sample from standard curve, the extension rate being multiplied by its correspondence is dexamethasone actual concentrations in sample.
Three, the determination of enzyme linked immunological kit technical parameter
Lowest detectable limit: 20 parts of dummies are detected, the concentration corresponding to each percentage absorptance is found from standard curve, detection limit is represented plus 3 times of standard deviations with the meansigma methods of 20 parts of sample dexamethasone concentrations, result obtains the method and Carnis Gallus domesticus detection is limited to 0.1 μ g/kg, and milk detection is limited to 0.3 μ g/L.
The accuracy that accuracy and precision: ELISA measures represents with the response rate, and precision represents with the coefficient of variation.Take blank Carnis Gallus domesticus sample, with the dexamethasone of 0.2,0.4,0.8 tri-concentration of μ g/kg, it is added recovery test, take blank milk sample, with the dexamethasone of 1.0,2.0,4.0 tri-concentration of μ g/kg, it is added recovery test, it is 80% ± 15% that result obtains the method to the response rate of Carnis Gallus domesticus sample, the response rate to milk sample is 80% ± 15%, variation within batch coefficient ＜ 15%, interassay coefficient of variation ＜ 25%.
Test kit the most of the present invention at least can preserve 12 months at 2 ~ 8 DEG C.
Claims (7)
1. a dexamethasone hapten, it is characterised in that molecular structural formula is:
2. prepare the haptenic method of dexamethasone described in claim 1 for one kind, it is characterised in that comprise the steps:
0.80g dexamethasone is dissolved in 5mLDMSO, is slowly added dropwise into 0.5mL1,3-propane diamine and 0.5mL pyridine at 60 DEG C in the mixed liquor of 10mLDMSO, after dropping, continuing reaction 15 hours, rotation is evaporated off solvent and unreacted propane diamine, quantitatively obtains the propane diamine list condensation substance of dexamethasone.
3. a dexamethasone antigen, it is characterised in that obtained with carrier protein couplet by the dexamethasone hapten described in claim 1, molecular structural formula is:
4. preparing a method for dexamethasone antigen described in claim 3, its preparation method comprises the steps:
Take EDC50mg 2mL water to be allowed to fully dissolve and obtain solution I, take dexamethasone hapten 11mg 1.0mLDMF dissolving and obtain solution II, take carrier protein 60mg to be dissolved in the 0.01mol/LPBS solution that 3mLpH is 8.0 and obtain solution III, solution II is mixed with solution III, it is added dropwise over solution I under magnetic stirring, under room temperature, stirring reaction 24 hours, dialyse 48 hours with tri-distilled water, obtain immunogen；
Take dexamethasone hapten 15mg 1.0mLDMF dissolving and obtain solution IV, the GA10 μ L taking 50% adds in solution IV, under room temperature, stirring reaction 18h obtains solution V, adds in solution V after taking the dilution of carrier protein 40mg 3mL water, and reaction adds 24mgNaBH the most afterwards4Reaction 3h, dialyses 48 hours with tri-distilled water, obtains coating antigen.
5. a dexamethasone antibody, it is characterized in that being prepared by the dexamethasone immunogen immune animal described in claim 3, described dexamethasone antibody is dexamethasone monoclonal antibody, and described dexamethasone monoclonal antibody is to be obtained by dexamethasone monoclonal antibody hybridoma cell strain D-4-2CGMCCNo.6068 secretion.
6. the dexamethasone enzyme linked immunological kit prepared by the antibody described in claim 5.
7. dexamethasone enzyme linked immunological kit as claimed in claim 6 application of dexamethasone residual in detection animal derived food.
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| CN103713122A (en) * | 2014-01-02 | 2014-04-09 | 中山市粤尔生物技术有限公司 | Quick detection kit for dexamethasone |
| CN105044367A (en) * | 2015-08-03 | 2015-11-11 | 武汉上成生物科技有限公司 | Colloidal gold immunochromatographic assay test strip for detecting dexamethasone residues in milk |
| CN105838681B (en) * | 2016-05-30 | 2019-04-30 | 江南大学 | One plant of anti-dexamethasone monoclonal antibody specific hybridoma cell strain C3 and its application |
| CN105907725B (en) * | 2016-07-11 | 2019-07-16 | 江南大学 | One plant of anti-hydrocortisone monoclonal antibody specific hybridoma cell strain YH7 and its application |
| CN106589034A (en) * | 2016-12-12 | 2017-04-26 | 深圳市绿诗源生物技术有限公司 | Dexamethasone artificial hapten and preparing method thereof |
| CN109030838A (en) * | 2018-10-11 | 2018-12-18 | 北京工商大学 | It is a kind of for detecting the colloid gold test paper of dexamethasone |
| CN109293771A (en) * | 2018-10-11 | 2019-02-01 | 北京工商大学 | A kind of method and its special monoclonal antibody detecting dexamethasone |
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