CN106317153A - Preparation method and application of dexamethasone semiantigen - Google Patents
Preparation method and application of dexamethasone semiantigen Download PDFInfo
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- CN106317153A CN106317153A CN201510340100.3A CN201510340100A CN106317153A CN 106317153 A CN106317153 A CN 106317153A CN 201510340100 A CN201510340100 A CN 201510340100A CN 106317153 A CN106317153 A CN 106317153A
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Abstract
The present invention discloses a dexamethasone semiantigen and a corresponding artificial antigen. Meanwhile, the invention also discloses a preparation method and application of the dexamethasone semiantigen and the corresponding artificial antigen. The dexamethasone semiantigen is a product shown as formula 1, and the product shown as the formula 1 is connected with a carrier protein to obtain the dexamethasone antigen. The dexamethasone antigen can be applied to the preparation of a dexamethasone specific antibody. The preparation method is simple and feasible, the cost is low, and the yield of the semiantigen is higher. The dexamethasone artificial antigen can produce specific antibodies against dexamethasone by immune animals, can be used for the preparation of ELISA (enzyme-linked immunosorbent assay) detection kits for detection of dexamethasone residues, and has the advantages of being simple, fast, large in processing amount of samples, high in sensitivity, strong in specificity, and the like.
Description
Technical field
The invention belongs to technical field of food safety detection, be specifically related to a kind of dexamethasone hapten, antigen preparation procedure and application thereof.
Background technology
Dexamethasone (Dexamethasone, DSMS) has another name called dexamethasone, fluorine first meticortelone, Dexamethasone, is the adrenocortical hormone of a kind of synthetic.Being glucocorticosteroid hormones antiinflammatory, Claritin, its pharmacological action is mainly antiinflammatory, antitoxin, antiallergic, rheumatism, and Clinical practice is wide.Additionally, dexamethasone is also as regulant for animal's growth, promotes animal protein synthesis and metabolism, increase meat yield, be once widely used as domestic animal, poultry, use.
But found by long-term laboratory research, dexamethasone can make laboratory animal generation canceration and gene mutation, dexamethasone and residual thereof all have obvious toxic and side effects to people, animal, use the dexamethasone of high dose to may result in the side effect such as amyotrophy, growth inhibited.And thus infer, such medicine of mankind's life-time service or be eaten for a long time the interpolation domestic animal of such growth promoter, poultry, the most also canceration and gene mutation can occur.So this type of drug withdrawal uses in treatment and feedstuff.
China is in 2002 in No. 235 " the animal food herbal medicine MRLs " announced that the Ministry of Agriculture issues, and dexamethasone must not exceed 0.75 μ g/kg in all food animal muscle, must not exceed 2.0 μ g/kg in liver.Therefore, for guaranteeing safety and the development of export abroad trade of animal derived food, setting up accurately and reliably, highly sensitive qualitative-and-quantitative method is the most necessary.
Develop the enzyme linked immunological kit of detection dexamethasone both at home and abroad, but the test kit of domestic production now can't be fully achieved the requirement of detection at aspects such as accuracy, sensitivity, specificitys.Dexamethasone hapten disclosed by the invention, antigen provide raw material for development dexamethasone antibody and dexamethasone enzyme linked immunological kit further.
Summary of the invention
It is an object of the invention to provide a kind of dexamethasone hapten, antigen preparation procedure and application thereof.
The dexamethasone hapten that the present invention provides, is compound shown in formula 1:
Formula 1.
The invention also discloses the preparation method of product shown in formula 1, comprise the steps:
50ml round-bottomed flask adds dexamethasone crude drug 500mg, succinic anhydrides 110mg is added after 5ml pyridinium dissolution, 80 DEG C of reaction 3h, process after TLC detection raw material reaction, remove pyridine under reduced pressure, residue adds 10ml frozen water, separate out white solid, filtering, washing and drying obtains 350mg product, is i.e. hapten.
The dexamethasone antigen that the present invention provides, is conjugate product shown in formula 1 and carrier protein couplet obtained.
The present invention also protects the preparation method of described dexamethasone antigen, comprises the steps:
(1), hapten 22mg is dissolved in 1.5mL DMF, after being completely dissolved, is sequentially added into EDC 25mg, NHS 25mg, room temperature magnetic agitation reaction 3h；
(2), weighing 50mgBSA, be dissolved in 3.5mL 0.1M carbonic acid buffer pH=9.6,400rpm stirs 10min, fully dissolves；
(3), take activated solution in above-mentioned steps 1, be added dropwise in protein solution under ice water bath environment, stirring while adding, room temperature magnetic agitation reaction 24h；
(4), product being loaded 1 the clean bag filter of distilled water flushing (15cm), 1LPBS dialyses 3 days, and dialysis is stirred at room temperature, changing dialysis solution every day 3 times, dialysis product 4500rpm is centrifuged 6min, 0.5ml/ pipe subpackage, being numbered by antigen ,-20 DEG C save backup.
Common carrier albumen all can use, such as bovine serum albumin (BSA), ovalbumin (OVA), human serum albumin (HSA), mouse serum albumin (MSA), thyroprotein (TG) or hemocyanin (KLH) etc..
Described dexamethasone antigen can prepare dexamethasone specific antibody as immunogen immune animal, it is also possible to prepares ELISA Plate as coating antigen.
Described antibody specific can be monoclonal antibody.
Product shown in formula 1, described dexamethasone antigen, described antibody all can be applicable to detect dexamethasone.
The present invention also disclosed the enzyme linked immunological kit applying dexamethasone antigen and dexamethasone monoclonal antibody to prepare.
Described enzyme-linked immunologic detecting kit, is by being coated with the ELISA Plate of dexamethasone antigen, enzyme labelled antibody working solution, dexamethasone serial standards, substrate nitrite ion, stop buffer, concentration redissolution liquid, concentrated cleaning solution.
The present invention relies on immunology, immunochemistry ultimate principle and retention analysis technological means, design, synthesized micromolecule target analytes hapten, and and carrier protein couplet, prepare effective artificial antigen.Preparation method of the present invention is simple and feasible, cost is relatively low, and yield of hapten is higher.The dexamethasone artificial antigen of the present invention, can create the specific antibody for dexamethasone by immune animal, the dexamethasone residual in quickly detection food.
Accompanying drawing explanation
Fig. 1 is dexamethasone haptenic Mass Spectrometer Method result.
Fig. 2 is the Mass Spectrometer Method result of BSA.
Fig. 3 is the Mass Spectrometer Method result of dexamethasone antigen " dexamethasone hapten+BSA ".
Fig. 4 is dexamethasone enzyme-linked immunologic detecting kit standard curve.
Detailed description of the invention
Below example facilitates a better understanding of the present invention, but does not limit the present invention.Experimental technique in following embodiment, if no special instructions, is conventional method.Test material used in following embodiment, if no special instructions, is and is commercially available from routine biochemistry reagent shop.
The haptenic preparation of embodiment 1, dexamethasone
One, the haptenic preparation of dexamethasone
50ml round-bottomed flask adds dexamethasone crude drug 500mg, 5ml pyridine F adds succinic anhydrides 110mg after dissolving, 80 degree of reaction 3h, process after TLC detection raw material reaction, remove pyridine under reduced pressure, residue adds 10ml frozen water, separate out white solid, filtering, washing and drying obtains 350mg product, is hapten.
Reaction equation is as follows:
Two, the haptenic qualification of dexamethasone
Products obtained therefrom is carried out Mass Spectrometric Identification ,-MS:M-1=491.2 ,+MS:M+K=531.7(Fig. 1).
Result shows its chemical structural formula (MW=492.53) shown in formula I, is dexamethasone hapten.
Formula 1.
Embodiment 2, the preparation of dexamethasone artificial antigen and qualification
One, the synthesis of dexamethasone immunizing antigen (DSMS-BSA)
(1), hapten 22mg is dissolved in 1.5mL DMF, after being completely dissolved, is sequentially added into EDC 25mg, NHS 25mg, room temperature magnetic agitation reaction 3h；
(2), weighing 50mgBSA, be dissolved in 3.5mL 0.1M carbonic acid buffer pH=9.6,400rpm stirs 10min, fully dissolves；
(3), take activated solution in above-mentioned steps 1, be added dropwise in protein solution under ice water bath environment, stirring while adding, room temperature magnetic agitation reaction 24h；
(4), product being loaded 1 the clean bag filter of distilled water flushing (15cm), 1LPBS dialyses 3 days, and dialysis is stirred at room temperature, changing dialysis solution every day 3 times, dialysis product 4500rpm is centrifuged 6min, 0.5ml/ pipe subpackage, being numbered by antigen ,-20 DEG C save backup.
Two, the synthesis of dexamethasone envelope antigen (DSMS-OVA)
(1), above-mentioned hapten 22mg is dissolved in 1.5mL DMF, after being completely dissolved, is sequentially added into EDC 25mg, NHS 25mg, room temperature magnetic agitation reaction 3h.
(2), weighing 67.0mgOVA, be dissolved in 3.5mL 0.1M carbonic acid buffer pH=9.6,400rpm stirs 10min, fully dissolves.
(3), take activated solution in above-mentioned steps 1, be added dropwise in protein solution under ice water bath environment, stirring while adding, room temperature magnetic agitation reaction 24h.
(4), product being loaded 1 the clean bag filter of distilled water flushing (15cm), 1LPBS dialyses 3 days, and dialysis is stirred at room temperature, changing dialysis solution every day 3 times, dialysis product 4500rpm is centrifuged 6min, 0.5ml/ pipe subpackage, being numbered by antigen ,-20 DEG C save backup.
Three, the qualification of dexamethasone artificial antigen
Immunogen MALDI-TOF-MS qualification result display coupling ratio is: R=(DMS-HS-BSA)-(BSA)/492.53=(73828.009-67485.901)/492.53=12.88(Fig. 2 and Fig. 3).I.e. in immunogen, described dexamethasone hapten (Formulas I) is 12.88:1 with the mol ratio of bovine serum albumin (BSA) coupling.
Embodiment 3, the preparation of enzyme mark monoclonal antibody and specificity identification
One, the preparation of dexamethasone monoclonal antibody
1, by the above-mentioned immunogen prepared by 100 μ g/ only, mix with Freund's complete adjuvant equal-volume with physiological saline solution immunogen, the female Mus of neck dorsal sc injection immunity 6 ~ 8 week old Balb/c, within after initial immunity the 7th, 14,28 days, mix with incomplete Freund's adjuvant equal-volume with immunogen, each supplementary immunization is once, merge first 3 days with immune complex 100 μ g/ only, be not added with Freund adjuvant supplementary immunization more once.
2, carry out according to a conventional method, the splenocyte taking immune mouse mixes with the murine myeloma cell (SP2/0) being in exponential phase, then the fusion agent (PEG4000) being slowly added to preheating in 45s merges, suspend uniformly by HAT culture medium, add appropriate feeder cells, it is incubated at 96 well culture plates, in 37 DEG C, 5%CO2Incubator is cultivated, partly changes liquid by HT culture medium after 5 days, when 9 days, entirely change liquid.
3, after cell merges, when cell grows to the 1/4 of culture hole area, substep screening method screening hybridoma is used.Primary election uses indirect ELISA method, with envelope antigen (it is most preferably coated concentration and positive serum dilution factor with square formation method conventional titration in advance) coated elisa plate, add measured hole culture supernatant, hatch, add sheep anti-mouse igg-HRP and IgM-HRP, OPD after cleaning and carry out chromogenic reaction.The positive Kong Zaiyong indirect competitive ELISA method screening filtered out, first mixes the dexamethasone equal-volume of cell conditioned medium and 100 μ g/mL, 37 DEG C of water-bath effect 30min, is then added in the ELISA Plate being coated.Replacing dexamethasone with PBS to compare, remaining step is ibid simultaneously.If the OD after dexamethasone blocks450Nm value drops to less than the 50% of control wells, then be judged to the positive, is all positive hole through 2 ~ 3 detections, carries out subcloning with limiting dilution assay immediately.
4,2 ~ 3 sub-clones are built the hybridoma amplification culture after strain, collects supernatant indirect ELISA and measure titer, frozen；And only take 8 ~ 10 week old Balb/c mouse peritoneal injecting fluid paraffin 0.5mL/, lumbar injection hybridoma 1 ~ 2 × 10 after 7 ~ 10 days6/ only, and extract mouse ascites, centrifuging and taking supernatant after 7 ~ 10 days, measure titer, and frozen standby.
Two, the preparation of enzyme labelled antibody
(1) weigh horseradish peroxidase (HRP) 2 mg to be dissolved in 0.5 mL water, add 0.5 mL 0.06 mol/L NaIO4Solution, 4 DEG C of lucifuge effect 30 min；
(2) the ethylene glycol 0.5mL of 160 mmol/L, room temperature effect 30 min are added；
(3) adding dexamethasone monoclonal antibody 2 mg of step one preparation, in the bag filter that after mixing, loading processed, put in the 0.05 mmol/L sodium carbonate buffer of 1000 mL and dialyse, 4 DEG C overnight；
(4), during dialysis solution is drawn to the centrifuge tube of 10 mL, the NaBH of 0.25mL 5g/L is added4Solution, mixes rearmounted 4 DEG C of 2 h；
(5) adding isopyknic saturated ammonium sulfate solution, after 4 DEG C of effect 30 min, at 4 DEG C, 3000 r/min are centrifuged 25 min, abandon supernatant；
(6) precipitation being dissolved in the PBS of 1.5 mL0.02 mol/L pH 7.4, suck in bag filter, at 0.02mol/L pH 7.4 PBS, 4 DEG C overnight (PBS is changed 3 times in midway)；
(7) by during in bag filter, liquid is drawn to microcentrifugal tube, at 4 DEG C, 10000r/min is centrifuged 30min, by supernatant sucking-off, adds equivalent glycerol, and mixing ,-20 DEG C save backup.
Three, the mensuration of enzyme mark dexamethasone antibody titer
Dexamethasone standard substance are purchased from Sigma company.
With the working concentration of monoclonal antibody prepared by square formation titrimetry Sai meter Song envelope antigen definitely and step one, the working concentration of dexamethasone envelope antigen is 1.5 μ g/mL, and the working concentration of monoclonal antibody is 1:50000.
Doing experimental solutions with the dexamethasone standard solution of variable concentrations, its concentration is as follows: 0,0.02,0.06,0.18,0.54,1.62 μ g/L.Use 8 groups of parallel tests (n=8).Indirect Competitive ELISA method:
(1) by the antigen coated ELISA Plate of dexamethasone of above-mentioned working concentration, dexamethasone standard substance experimental solutions and enzyme labelled antibody solution are simultaneously introduced in ELISA Plate micropore, blank well is set simultaneously and (changes the antibody-solutions of interpolation into high purity water, other is consistent) and negative control hole (standard substance experimental solutions PBS solution is replaced, other is consistent), 25 DEG C of light protected environment react 30min；
(2) pour out liquid in hole, wash 3 ~ 5 times with cleaning mixture, ELISA Plate is upside down in absorbent paper and pats dry；
(3) add substrate chromophoric solution in ELISA Plate micropore, 25 DEG C of light protected environment react 15min；
(4) add stop buffer, mixing of vibrating gently, at wavelength 450nm, measure OD value by microplate reader.
With OD value as vertical coordinate, with the log10 value of dexamethasone experimental solutions concentration as abscissa, draw semilog canonical plotting.Standard curve has complete reverse-s shape shape, and has upper mounting plate and lower platform, the parallel assay number of times of standard curve 8 times, and experimental repeatability is good, and relative standard deviation (coefficient of variation) is all within 10%.
Half amount of suppression (IC is drawn according to standard curve50), determine detection sensitivity.
Suppression ratio is with being calculated as follows:
(ODmax-ODmin)-(ODx-ODmin)
Suppression ratio (%)=------------------------------------------------× 100%
(ODmax-ODmin)
In formula: ODmax: for being not added with light absorption value during standard substance, ODx is light absorption value during standard substance x, and ODmin is the light absorption value of blank control wells.
Being calculated dexamethasone antibody half amount of suppression (IC50) in buffer by above-mentioned formula is 0.06 μ g/L.
Embodiment 4, the enzyme linked immunological kit of detection dexamethasone and preparation thereof
One, enzyme linked immunological kit is made up of following substances:
(1) the haptenic ELISA Plate of dexamethasone it is coated；
(2) enzyme mark dexamethasone antibody working solution: enzyme labelled antibody solution described in embodiment 3；
(3) dexamethasone standard substance: dexamethasone standard solution concentration is respectively 0,0.02,0.06,0.18,0.54,1.62 μ g/L；
(4) substrate nitrite ion: be made up of A liquid and B liquid, A liquid is the aqueous solution of 2% urea peroxide, and B liquid is the aqueous solution of 1% tetramethyl benzidine (TMB)；
(5) stop buffer: 0.2M aqueous sulfuric acid；
(6) concentrated cleaning solution: every 1 liter of described cleaning mixture is prepared as follows and obtained: 10mL tween 20,5g Hydrazoic acid,sodium salt and 990mL phosphate buffer are mixed, obtains described cleaning mixture；The concentration of described phosphate buffer be 0.01M pH value be 7.4；
(7) phosphate buffer of redissolution liquid: 0.04mo1/L is concentrated.
Two, ELISA Plate and the preparation thereof of DSMS-OVA it are coated with
It is coated the polystyrene ELISA Plate of DSMS-OVA: with the carbonate solution of 0.05M by antigen diluent to 1.5 μ g/mL, it is coated 96 hole polystyrene ELISA Plate, every hole 100 μ L, 37 DEG C of incubation 2h, incline and be coated liquid, wash 3 times with cleaning mixture, each 10s, pat dry, then in every hole, add 150 μ L confining liquids, 37 DEG C of incubation 2h, liquid in hole of inclining, dried sealing by aluminum film vacuum preserves.
It is coated buffer: the sodium carbonate buffer of pH9.6,0.05mo1/L；
Confining liquid: every 1 liter of confining liquid is prepared as follows: by 5mL horse serum, 1g Hydrazoic acid,sodium salt, the mixing of 30g casein, dissolve with phosphate buffer and be settled to 1000mL, obtaining confining liquid；Wherein, the concentration of phosphate buffer is 0.02M, and pH value is 7.2.
Three, kit test method
(1) sample pre-treatments
(1) Carnis Gallus domesticus, duck meat (coefficient of dilution: 4)
A) 1 ± 0.01 g sample is accurately weighed in 50 mL centrifuge tubes；B) Carnis Gallus domesticus sample: add 3 mL chicken meat sample extracting solution；Duck meat sample: add 3 mL duck meat sample extracting solutions；C) fully whirling motion 1 min；
(note: must whirling motion be completely dispersed to tissue！) d) 4000 g be centrifuged 10 min；E) take 50 mL supernatant to detect；
(2) raw milk, reconstituted milk (coefficient of dilution: 5)
A) reconstituted milk: accurately weigh 1 ± 0.01 fresh milk powder of g in 50 mL centrifuge tubes, adds the abundant whirling motion of 10mL deionized water and dissolves standby；B) 200 mL mixing samples are taken in centrifuge tube；C) 200 mL raw milk extracting solution and 600 mL 15% methanol aqueous solutions, abundant whirling motion 30 s are added；D) take 50 mL to detect.
(3) Carnis Sus domestica (coefficient of dilution: 10)
A) accurately weigh 1 ± 0.01 g sample in 50 mL centrifuge tubes, add 9 mL Carnis Sus domestica sample extracting solutions, abundant whirling motion 1 min；B) 4000 g are centrifuged 10 min；C) take 50 mL to detect.
(4) Hepar Sus domestica, Hepar Gallus domesticus (coefficient of dilution: 5)
A) accurately weighing 1 ± 0.01 g fresh sample in 50mL centrifuge tube, add 1 mL liver specimens extracting solution, abundant whirling motion is to tissue dispersion；B) Pig Liver: add 5 mL Pig Liver extracting solution；Hepar Gallus domesticus sample: add 5 mL Hepar Gallus domesticus sample extracting solutions；C) high speed whirling motion 1 min；D) 4000 g are centrifuged 10 min；
E) 1 mL supernatant is taken in 4 mL centrifuge tubes；F) in 50-60 DEG C of water-bath, nitrogen dries up；G) 2 mL normal hexane, 1 mL liver specimens diluent, high speed whirling motion 1 min it are sequentially added into；H) 4000 g are centrifuged 5 min；I) upper strata normal hexane and intermediate layer impurity are removed；J) take 50 mL to detect.
(2) detect with test kit
1, the making of standard curve
Adding dexamethasone standard solution 50 μ L in the ELISA Plate micropore be coated with DSMS-OVA, be subsequently adding enzyme labelled antibody working solution 100 μ L/ hole, vibrate mix homogeneously gently, with reacting 30min in the rearmounted 25 DEG C of light protected environment of cover plate membrane cover plate.Carefully open cover plate film, liquid in hole is dried, add wash operating solution 250mL/ hole, fully wash 4 ~ 5 times, every minor tick 10s, sprinkle cleaning mixture in board falling hole, pat dry with absorbent paper.Adding substrate A liquid 50 μ L/ hole, substrate B liquid 50 μ L/ hole, mixing of vibrating gently, 25 DEG C of calorstat lucifuge colour developing 15min, every hole adds stop buffer 50 μ L, mixing of vibrating gently, by microplate reader, measures every hole absorbance.
With the absorbance values (B) of the standard solution of each concentration divided by the absorbance (B of first standard solution (0 standard)0) it is multiplied by 100% again, obtain percentage absorbance.With the semilog value of dexamethasone standard concentration (μ g/L) as X-axis, percentage absorbance is Y-axis, draws canonical plotting.The standard curve obtained is as shown in Figure 4.
Percentage absorbance (%)=(B/B0) × 100%
2, the mensuration of dexamethasone concentration in sample
With the absorbance values (B) of each test sample solution divided by the absorbance (B of first standard solution (0 standard)0) it is multiplied by 100% again, obtain percentage absorbance.The percentage absorbance of each test sample solution corresponding, then can read the absorbance of test sample solution from standard curve, concentration value further according to standard solution converses the residual quantity of dexamethasone in sample solution, the last extension rate being multiplied by each sample pretreatment process again, can calculate the concentration of dexamethasone in sample.
Four, test kit Detection results is evaluated
(1) accuracy and precision test
In the raw milk sample without dexamethasone, add dexamethasone standard substance, make dexamethasone standard substance final concentration in the sample be respectively 0.1,0.2,0.4 μ g/L；Sample after adding carries out pre-treatment according to method described in experiment three respectively, obtains test sample solution.
From the test kit of three different batches, 3 test kits of each extraction detect, and detection method is as described in experiment three, and each experiment is repeated 5 times, and calculates the coefficient of variation respectively.Result is shown in Table 1 respectively.
Table 1 accuracy and Precision test result
Variation within batch coefficient: with the coefficient of variation of each parallel samples in once measuring.
Interassay coefficient of variation: same sample, in the coefficient of variation of different batches measurement result, takes its meansigma methods.
Result shows: the average TIANZHU XINGNAO Capsul of raw milk sample is 86.9 ~ 97.3%, and variation within batch coefficient is 6.4 ~ 13.6%, and interassay coefficient of variation is at 9.8 ~ 10.1 %.
(2) test kit storage life
Test kit preservation condition is 2 ~ 8 DEG C, and through the mensuration of 15 months, the maximum absorbance value (0 standard) of test kit, 50% inhibition concentration, dexamethasone add practical measurement value all within normal range.During transport and using, having improper preservation condition and occur, place 9 days, be accelerated senile experiment under conditions of being preserved at 37 DEG C by test kit, result shows that the indices of this test kit complies fully with requirement.Occurring in view of test kit freezing situation, test kit is put into-20 DEG C of refrigerator freezings 9 days, measurement result also indicates that test kit indices is the most normal.Can show that test kit at least can be able to preserve more than 12 months at 2 ~ 8 DEG C from result above.
(3) cross reacting rate test
Select to carry out cross reaction test with DSMS structure or intimate other drug, respectively obtain its 50% inhibition concentration by the standard curve of various medicines.The test kit cross reacting rate to other analog is calculated with following formula.The least with the cross reacting rate of other drug, illustrate that dexamethasone enzyme-linked immunologic detecting kit is the best to the detection specificity of dexamethasone.The results are shown in Table 2.
Cause the dexamethasone concentration of 50% suppression
Cross reacting rate (%)=
------------------------------------------ ×100%
Cause the other drug concentration of 50% suppression
Table 2 dexamethasone test kit cross reacting rate
Result of the test shows, test kit of the present invention to the cross reacting rate of dexamethasone be 100%, the cross reacting rate of triamcinolone be 52%, the cross reacting rate of prednisolone be 14%, the cross reacting rate of betamethasone be 24%, the cross reacting rate of hydrocortisone be 1.5%, so test kit is good to the specificity of dexamethasone, test kit the most of the present invention can detect dexamethasone.
Claims (10)
1. a dexamethasone hapten, for product shown in formula 1:
Formula 1.
2. the preparation method of product shown in formula 1, comprise the steps: 50ml round-bottomed flask adds dexamethasone crude drug 500mg, add succinic anhydrides 110mg after 5ml pyridinium dissolution, process after 80 DEG C of reaction 3h, TLC detection raw material reactions, remove pyridine under reduced pressure, residue adds 10ml frozen water, separates out white solid, filter, washing and drying obtains 350mg product, is i.e. hapten.
3. a dexamethasone antigen, is conjugate product shown in formula 1 and carrier protein couplet obtained.
Dexamethasone antigen the most according to claim 3, it is characterised in that described carrier protein can be bovine serum albumin, ovalbumin, human serum albumin, mouse serum albumin, thyroprotein or hemocyanin.
5. the preparation method of dexamethasone antigen described in claim 3, comprises the steps: (1), is dissolved in 1.5mL DMF by hapten 22mg, after being completely dissolved, is sequentially added into EDC 25mg, NHS 25mg, room temperature magnetic agitation reaction 3h；(2), weighing 50mgBSA, be dissolved in 3.5mL 0.1M carbonic acid buffer pH=9.6,400rpm stirs 10min, fully dissolves；(3), take activated solution in above-mentioned steps 1, be added dropwise in protein solution under ice water bath environment, stirring while adding, room temperature magnetic agitation reaction 24h；(4), product being loaded 1 the clean bag filter of distilled water flushing (15cm), 1LPBS dialyses 3 days, and dialysis is stirred at room temperature, changing dialysis solution every day 3 times, dialysis product 4500rpm is centrifuged 6min, 0.5ml/ pipe subpackage, being numbered by antigen ,-20 DEG C save backup.
6. dexamethasone antigen application in preparing dexamethasone specific antibody described in claim 3.
7. the specific antibody that dexamethasone antigen described in application claim 3 prepares.
8. the application in detection dexamethasone of the antibody described in dexamethasone antigen, claim 7 described in product, claim 3 described in claim 1.
9. the enzyme-linked immunologic detecting kit that specific antibody described in dexamethasone antigen, claim 7 described in product, claim 3 described in application claim 1 prepares.
10. enzyme-linked immunologic detecting kit described in claim 8, it is characterized in that, it includes: be coated with the ELISA Plate of dexamethasone antigen, enzyme labelled antibody working solution, dexamethasone serial standards, substrate nitrite ion, stop buffer, concentration redissolution liquid, concentrated cleaning solution.
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| 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 |
| CN116462729A (en) * | 2023-03-31 | 2023-07-21 | 华南农业大学 | Prednisone acetate hapten, artificial antigen and application thereof |
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