CN106317153B - A kind of dexamethasone haptens preparation method and applications - Google Patents
A kind of dexamethasone haptens preparation method and applications Download PDFInfo
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- CN106317153B CN106317153B CN201510340100.3A CN201510340100A CN106317153B CN 106317153 B CN106317153 B CN 106317153B CN 201510340100 A CN201510340100 A CN 201510340100A CN 106317153 B CN106317153 B CN 106317153B
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Abstract
The invention discloses a kind of dexamethasone haptens and corresponding artificial antigens, while invention also discloses the preparation method and applications of the dexamethasone haptens and corresponding artificial antigen.Dexamethasone haptens provided by the invention is product shown in formula 1, and the product shown in formula 1 connect available dexamethasone antigen with carrier protein.The dexamethasone antigen can be applied to prepare dexamethasone specific antibody.Preparation method of the present invention is simple and feasible, cost is relatively low, and yield of hapten is higher.Dexamethasone artificial antigen of the invention, the specific antibody for dexamethasone is can produce by immune animal, it can be used for preparing the detection remaining enzyme-linked immunologic detecting kit of dexamethasone, there is many advantages, such as simple, quick, processing sample size is big, high sensitivity, high specificity.
Description
Technical field
The invention belongs to technical field of food safety detection, and in particular to a kind of dexamethasone haptens, antigen preparation side
Method and its application.
Background technique
Dexamethasone (Dexamethasone, DSMS) also known as dexamethasone, fluorine first strong pine dragon, Dexamethasone, are a kind of
Artificial synthesized cortex hormone of aadrenaline.It is that glucocorticosteroid steroids is anti-inflammatory, Claritin, pharmacological action is mainly anti-
Scorching, antitoxin, antiallergy, antirheumatic, clinical use are wide.In addition, dexamethasone is also used as regulant for animal's growth, promote dynamic
The synthesis of object protein and metabolism, increase meat yield, were once widely used as domestic animal, poultry, use.
But it being found by long-term laboratory research, dexamethasone can make experimental animal that canceration and gene mutation occur,
Dexamethasone and its residual have apparent toxic side effect to people, animal, using the dexamethasone of high dose can lead to muscular atrophy,
The side effects such as growth inhibition.And thus infer, such drug is used for a long time in the mankind or such growth accelerator is added in long-term consumption
Poultry, canceration and gene mutation can also equally occur for domestic animal.So such drug withdrawal uses in treatment and feed.
China is in " the animal food herbal medicine maximum residue limit " of No. 235 bulletins in Ministry of Agriculture's publication in 2002
In, dexamethasone must not exceed 0.75 μ g/kg in all food animal muscles, must not exceed 2.0 μ g/kg in liver.Therefore,
To ensure the safety of animal derived food and the development of export abroad trade, accurate and reliable, the qualitative, quantitative of high sensitivity is established
Method is very necessary.
The domestic and international enzyme linked immunological kit for having developed detection dexamethasone, but the kit of domestic production now
Accuracy, sensitivity, in terms of the requirement of detection can't be fully achieved.Dexamethasone disclosed by the invention half is anti-
Former, antigen provides raw material for further development dexamethasone antibody and dexamethasone enzyme linked immunological kit.
Summary of the invention
The object of the present invention is to provide a kind of dexamethasone haptens, antigen preparation procedure and its applications.
Dexamethasone haptens provided by the invention, is compound shown in formula 1:
Formula 1.
The invention also discloses the preparation methods of product shown in formula 1, include the following steps:
Succinic anhydride 110mg is added after dexamethasone bulk pharmaceutical chemicals 500mg, 5ml pyridinium dissolution are added in 50ml round-bottomed flask,
80 DEG C of reactions 3h, TLC detect the post-processing of raw material end of reaction, remove pyridine under reduced pressure, 10ml ice water is added in residue, is precipitated white
Color solid, filtering, it is haptens that washing and drying, which obtains 350mg product,.
Dexamethasone antigen provided by the invention is the conjugate for obtaining product shown in formula 1 and carrier protein couplet.
The present invention also protects the preparation method of the dexamethasone antigen, includes the following steps:
(1), haptens 22mg is dissolved in 1.5mL DMF, after being completely dissolved, sequentially adds EDC 25mg, NHS
25mg, room temperature magnetic agitation react 3h；
(2), 50mgBSA is weighed, is dissolved in 3.5mL 0.1M carbonic acid buffer pH=9.6,400rpm stirs 10min, fills
Divide dissolution；
(3), activated solution in above-mentioned steps 1 is taken, is added dropwise in protein solution under ice water bath environment, side edged stirs
It mixes, room temperature magnetic agitation is reacted for 24 hours；
(4), reaction product is packed into 1 clean bag filter of distilled water flushing (15cm), 1LPBS dialyses 3 days, is stirred at room temperature
Dialysis, daily replacement dialyzate 3 times, dialysis product 4500rpm are centrifuged 6min, and the packing of 0.5ml/ pipe numbers antigen, -20 DEG C
It saves backup.
Common carrier albumen can be used, such as bovine serum albumin(BSA) (BSA), ovalbumin (OVA), human serum albumins
(HSA), mouse serum albumin (MSA), thyroprotein (TG) or hemocyanin (KLH) etc..
The dexamethasone antigen can be used as immunogen immune animal and prepare dexamethasone specific antibody, can also make
ELISA Plate is prepared for coating antigen.
The antibody specific can be monoclonal antibody.
Product shown in formula 1, the dexamethasone antigen, the antibody can be applied to detection dexamethasone.
The enzyme linked immunological being prepared using dexamethasone antigen and dexamethasone monoclonal antibody is also disclosed in the present invention
Kit.
The enzyme-linked immunologic detecting kit, be by be coated with the ELISA Plate of dexamethasone antigen, enzyme labelled antibody working solution,
Liquid, concentrated cleaning solution are redissolved in dexamethasone serial standards, substrate developing solution, terminate liquid, concentration.
The present invention relies on immunology, immunochemistry basic principle and retention analysis technological means, design, synthesized micromolecule mesh
Mark analyte haptens, and and carrier protein couplet, prepare effective artificial antigen.Preparation method of the present invention is simple and feasible, cost
Lower, yield of hapten is higher.Dexamethasone artificial antigen of the invention can produce by immune animal for dexamethasone
Specific antibody, for quickly detect the dexamethasone in food residual.
Detailed description of the invention
Fig. 1 is the Mass Spectrometer Method result of dexamethasone haptens.
Fig. 2 is the Mass Spectrometer Method result of BSA.
Fig. 3 is the Mass Spectrometer Method result of dexamethasone antigen " dexamethasone haptens+BSA ".
Fig. 4 is dexamethasone enzyme-linked immunologic detecting kit standard curve.
Specific embodiment
Embodiment below facilitates a better understanding of the present invention, but does not limit the present invention.Experiment in following embodiments
Method is unless otherwise specified conventional method.Test material as used in the following examples is unless otherwise specified certainly
What routine biochemistry reagent shop was commercially available.
The preparation of embodiment 1, dexamethasone haptens
One, the preparation of dexamethasone haptens
Succinic anhydride is added after dexamethasone bulk pharmaceutical chemicals 500mg, 5ml pyridine F dissolution is added in 50ml round-bottomed flask
110mg, 80 degree of reactions 3h, TLC detect the post-processing of raw material end of reaction, remove pyridine under reduced pressure, 10ml ice water is added in residue,
White solid, filtering is precipitated, washing and drying obtains 350mg product, as haptens.
Reaction equation is as follows:
Two, the identification of dexamethasone haptens
Mass Spectrometric Identification ,-MS:M-1=491.2 ,+MS:M+K=531.7(Fig. 1 are carried out to products obtained therefrom).
Its chemical structural formula is (MW=492.53) shown in formula I as the result is shown, as dexamethasone haptens.
Formula 1.
Embodiment 2, the preparation and authentication of dexamethasone artificial antigen
One, the synthesis of dexamethasone immunizing antigen (DSMS-BSA)
(1), haptens 22mg is dissolved in 1.5mL DMF, after being completely dissolved, sequentially adds EDC 25mg, NHS
25mg, room temperature magnetic agitation react 3h；
(2), 50mgBSA is weighed, is dissolved in 3.5mL 0.1M carbonic acid buffer pH=9.6,400rpm stirs 10min, fills
Divide dissolution；
(3), activated solution in above-mentioned steps 1 is taken, is added dropwise in protein solution under ice water bath environment, side edged stirs
It mixes, room temperature magnetic agitation is reacted for 24 hours；
(4), reaction product is packed into 1 clean bag filter of distilled water flushing (15cm), 1LPBS dialyses 3 days, is stirred at room temperature
Dialysis, daily replacement dialyzate 3 times, dialysis product 4500rpm are centrifuged 6min, and the packing of 0.5ml/ pipe numbers antigen, -20 DEG C
It saves backup.
Two, the synthesis of dexamethasone envelope antigen (DSMS-OVA)
(1), above-mentioned haptens 22mg is dissolved in 1.5mL DMF, after being completely dissolved, sequentially adds EDC 25mg, NHS
25mg, room temperature magnetic agitation react 3h.
(2), 67.0mgOVA is weighed, is dissolved in 3.5mL 0.1M carbonic acid buffer pH=9.6,400rpm stirs 10min,
Sufficiently dissolution.
(3), activated solution in above-mentioned steps 1 is taken, is added dropwise in protein solution under ice water bath environment, side edged stirs
It mixes, room temperature magnetic agitation is reacted for 24 hours.
(4), reaction product is packed into 1 clean bag filter of distilled water flushing (15cm), 1LPBS dialyses 3 days, is stirred at room temperature
Dialysis, daily replacement dialyzate 3 times, dialysis product 4500rpm are centrifuged 6min, and the packing of 0.5ml/ pipe numbers antigen, -20 DEG C
It saves backup.
Three, the identification of dexamethasone artificial antigen
Immunogene MALDI-TOF-MS qualification result shows coupling ratio are as follows: R=(DMS-HS-BSA)-(BSA)/492.53=
(73828.009-67485.901)/492.53=12.88(Fig. 2 and Fig. 3).I.e. in immunogene, the dexamethasone haptens (formula
It I is) 12.88:1 with the molar ratio of bovine serum albumin(BSA) (BSA) coupling.
Embodiment 3, the preparation of enzyme mark monoclonal antibody and specificity identification
One, the preparation of dexamethasone monoclonal antibody
1, with the above-mentioned immunogene prepared by 100 μ g/, with physiological saline solution immunogene and Freund's complete adjuvant etc.
Volume mixes, and the nape of the neck is subcutaneously injected immune 6 ~ 8 week old Balb/c female mices, after initial immunity the 7th, 14,28 day with immunogene with
Incomplete Freund's adjuvant mixes in equal volume, and each supplementary immunization is primary, merges first 3 days with 100 μ of immune complex g/, is not added not
Supplementary immunization is primary again for family name's adjuvant.
2, it carries out according to a conventional method, takes the splenocyte of immune mouse and the murine myeloma cell for being in logarithmic growth phase
(SP2/0) it mixes, the fusion agent (PEG4000) that preheating is then slowly added in 45s is merged, and is suspended with HAT culture medium
Uniformly, suitable feeder cells are added, 96 well culture plates are incubated at, in 37 DEG C, 5%CO2It cultivates in incubator, is used after 5 days
HT culture medium partly changes liquid, and 9 days whens carry out changing liquid entirely.
3, thin using substep screening method screening hybridoma when cell grows to the 1/4 of culture hole area after cell fusion
Born of the same parents.Primary election uses indirect ELISA method, (uses its best peridium concentration of square matrix method conventional titration and the positive in advance with envelope antigen
Serum dilution) coated elisa plate, measured hole culture supernatant is added, is incubated for, sheep anti-mouse igg-HRP and IgM- is added after cleaning
HRP, OPD carry out chromogenic reaction.The positive Kong Zaiyong indirect competitive ELISA method screening filtered out, first by cell conditioned medium and 100
The dexamethasone of μ g/mL mixes in equal volume, and 37 DEG C of water-baths act on 30min, is then added in the ELISA Plate being coated with.It uses simultaneously
PBS replaces dexamethasone to compare, remaining step is same as above.If the OD after dexamethasone blocks450Nm value drops to control wells
50% hereinafter, be then judged to the positive, and the hole detected through 2 ~ 3 times all for the positive carries out subcloning with limiting dilution assay immediately.
4, the hybridoma that 2 ~ 3 subclones are built after strain is expanded into culture, collects supernatant indirect ELISA measurement and imitates
Valence freezes；And take 8 ~ 10 week old Balb/c mouse peritoneal injecting fluid paraffin 0.5mL/ only, hybridoma is injected intraperitoneally after 7 ~ 10 days
Cell 1 ~ 2 × 106/ only, mouse ascites are extracted after 7 ~ 10 days, centrifuging and taking supernatant measures potency, and freezes spare.
Two, the preparation of enzyme labelled antibody
(1) it weighs 2 mg of horseradish peroxidase (HRP) to be dissolved in 0.5 mL water, 0.5 mL, 0.06 mol/L is added
NaIO4Solution, 4 DEG C are protected from light 30 min of effect；
(2) the ethylene glycol 0.5mL of 160 mmol/L is added, room temperature acts on 30 min；
(3) 2 mg of dexamethasone monoclonal antibody of step 1 preparation is added, is fitted into after mixing in processed bag filter, sets 1000
It dialyses in the 0.05 mmol/L sodium carbonate buffer of mL, 4 DEG C overnight；
(4) dialyzate is drawn in the centrifuge tube of 10 mL, adds the NaBH of 0.25mL 5g/L4Solution mixes 4 DEG C of 2 h of postposition；
(5) it is added isometric saturated ammonium sulfate solution, 3000 r/min are centrifuged 25 at 4 DEG C after 4 DEG C of 30 min of effect
Min abandons supernatant；
(6) precipitating is dissolved in the PBS of 1.5 mL0.02 mol/L pH 7.4, is sucked in bag filter, in 0.02mol/L
7.4 PBS of pH dialysis, 4 DEG C overnight (midway replacement PBS 3 times)；
(7) liquid in bag filter is drawn in microcentrifugal tube, 10000r/min is centrifuged 30min at 4 DEG C, and supernatant is inhaled
Out, add equivalent glycerol, mix, -20 DEG C save backup.
Three, the measurement of enzyme mark dexamethasone antibody titer
Dexamethasone standard items are purchased from Sigma company.
With the working concentration of the square matrix titration monoclonal antibody that definitely prepared by Sai meter Song envelope antigen and step 1, dexamethasone
The working concentration of envelope antigen is 1.5 μ g/mL, and the working concentration of monoclonal antibody is 1:50000.
Do experimental solutions with the dexamethasone standard solution of various concentration, concentration is as follows: 0,0.02,0.06,0.18,
0.54,1.62μg/L.Using 8 groups of parallel tests (n=8).Indirect Competitive ELISA method:
(1) with the dexamethasone antigen coat ELISA Plate of above-mentioned working concentration, by dexamethasone standard items experimental solutions with
Enzyme labelled antibody solution is added in micropore of enzyme marker plate simultaneously, at the same be arranged blank well (change the antibody-solutions of addition into high purity water,
It is consistent) and negative control hole (replacing standard items experimental solutions with PBS solution, other consistent), in 25 DEG C of light protected environments instead
Answer 30min；
(2) liquid in hole is poured out, is washed 3 ~ 5 times with cleaning solution, ELISA Plate is upside down on blotting paper and is patted dry；
(3) substrate chromophoric solution is added into micropore of enzyme marker plate, reacts 15min in 25 DEG C of light protected environments；
(4) terminate liquid is added, gently oscillation mixes, and OD value is measured at wavelength 450nm with microplate reader.
Using OD value as ordinate, using the log10 value of dexamethasone experimental solutions concentration as abscissa, semilog standard is drawn
Curve graph.Standard curve has complete reverse-s shape shape, and has upper mounting plate and lower platform, the parallel determination number 8 of standard curve
Secondary, experimental repeatability is good, and relative standard deviation (coefficient of variation) is within 10%.
Half amount of suppression (IC is obtained according to standard curve50), determine detection sensitivity.
Inhibiting rate is with being calculated as follows:
(ODmax- ODmin)-(ODx-ODmin)
Inhibiting rate (%)=--- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- × 100%
(ODmax-ODmin)
In formula: ODmax: to be not added light absorption value when standard items, light absorption value when ODx is standard items x, ODmin is blank
The light absorption value of control wells.
By above-mentioned formula calculate half amount of suppression (IC50) of the dexamethasone antibody in buffer be 0.06 μ g/L.
Embodiment 4, the enzyme linked immunological kit for detecting dexamethasone and its preparation
One, enzyme linked immunological kit is made of following substances:
(1) it is coated with the ELISA Plate of dexamethasone haptens；
(2) enzyme mark dexamethasone antibody working solution: enzyme labelled antibody solution described in embodiment 3；
(3) dexamethasone standard items: dexamethasone standard solution concentration is respectively 0,0.02,0.06,0.18,0.54,
1.62μg/L；
(4) substrate developing solution: being made of A liquid and B liquid, and A liquid is the aqueous solution of 2% urea peroxide, and B liquid is 1% tetramethyl connection
The aqueous solution of aniline (TMB)；
(5) terminate liquid: 0.2M aqueous sulfuric acid；
(6) concentrated cleaning solution: every 1 liter of cleaning solution is prepared as follows to be obtained: by 10mL Tween-20,
5g sodium azide and the mixing of 990mL phosphate buffer, obtain the cleaning solution；The concentration of the phosphate buffer is
0.01M pH value is 7.4；
(7) liquid: the phosphate buffer of 0.04mo1/L is redissolved in concentration.
Two, ELISA Plate and its preparation of DSMS-OVA are coated with
It is coated with 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 with 96 hole polystyrene ELISA Plates, 100 μ L of every hole, 37 DEG C of incubation 2h, coating buffer of inclining wash 3 times with cleaning solution, every time
10s is patted dry, and is then added 150 μ L confining liquids in every hole, 37 DEG C of incubation 2h, liquid in hole of inclining, and uses aluminium film vacuum after dry
It is sealed.
It is coated with buffer: the sodium carbonate buffer of pH9.6,0.05mo1/L；
Confining liquid: every 1 liter of confining liquid is prepared as follows: 5mL horse serum, 1g sodium azide, 30g casein are mixed
It closes, 1000mL is dissolved and be settled to phosphate buffer, obtains confining liquid；Wherein, the concentration of phosphate buffer is
0.02M, pH value 7.2.
Three, kit test method
(1) sample pre-treatments
(1) chicken, the duck (coefficient of dilution: 4)
A) 1 ± 0.01 g sample is accurately weighed in 50 mL centrifuge tubes；B) 3 mL chicken samples chicken sample: are added
Product extracting solution；Duck sample: 3 mL duck sample extracting solutions are added；C) sufficiently 1 min of whirling motion；
(note: palpus whirling motion to tissue is completely dispersed！) d) 4000 g be centrifuged 10 min；E) 50 mL supernatants is taken to be examined
It surveys；
(2) former milk, the recombined milk (coefficient of dilution: 5)
A) recombined milk: accurately weighing the fresh milk powder of 1 ± 0.01 g in 50 mL centrifuge tubes, be added 10mL go from
The sub- abundant whirling motion dissolution of water is spare；B) 200 mL is taken to mix sample in centrifuge tube；C) 200 mL original milk extracting solutions are added
With 600 mL, 15% methanol aqueous solution, abundant 30 s of whirling motion；D) 50 mL is taken to be detected.
(3) pork (coefficient of dilution: 10)
A) 1 ± 0.01 g sample is accurately weighed in 50 mL centrifuge tubes, and 9 mL pork sample extracting solutions are added,
1 min of abundant whirling motion；B) 4000 g are centrifuged 10 min；C) 50 mL is taken to be detected.
(4) pork liver, the chicken gizzard (coefficient of dilution: 5)
A) 1 ± 0.01 g fresh sample is accurately weighed in 50mL centrifuge tube, and 1 mL liver specimens extracting solution is added,
Abundant whirling motion is to tissue dispersion；B) 5 mL Pig Liver extracting solutions Pig Liver: are added；Chicken gizzard sample: 5 mL chicken gizzard are added
Sample extracting solution；C) 1 min of high speed whirling motion；D) 4000 g are centrifuged 10 min；
E) take 1 mL supernatant in 4 mL centrifuge tubes；F) it is dried with nitrogen in 50-60 DEG C of water-bath；G) it sequentially adds
2 mL n-hexanes, 1 mL liver specimens dilution, 1 min of high speed whirling motion；H) 4000 g are centrifuged 5 min；I) in removal
Layer n-hexane and middle layer impurity；J) 50 mL is taken to be detected.
(2) it is detected with kit
1, the production of standard curve
50 μ L of dexamethasone standard solution is added into the micropore of enzyme marker plate for be coated with DSMS-OVA, enzyme mark is then added
100 hole μ L/ of antibody working solution, gently oscillation is uniformly mixed, and reacts 30min in cover board membrane cover plate 25 DEG C of light protected environments of postposition.
Cover board film carefully is opened, liquid in hole is dried, the hole wash operating solution 250mL/ is added, is sufficiently washed 4 ~ 5 times, every minor tick
10s sprinkles cleaning solution in board falling hole, is patted dry with blotting paper.50 hole μ L/ of substrate A liquid, 50 hole μ L/ of substrate B liquid is added, gently vibrates
It mixes, 25 DEG C of insulating boxs are protected from light colour developing 15min, and 50 μ L of terminate liquid is added in every hole, and gently oscillation mixes, and with microplate reader, measurement is every
Hole absorbance value.
With the absorbance values (B) of the standard solution of each concentration divided by first standard solution (0 standard)
Absorbance value (B0) multiplied by 100%, obtain percentage absorbance value.With the semilog value of dexamethasone standard concentration (μ g/L)
For X-axis, percentage absorbance value is Y-axis, draws canonical plotting.Obtained standard curve is as shown in Figure 4.
Percentage absorbance value (%)=(B/B0) × 100%
2, in sample dexamethasone concentration measurement
With the absorbance values (B) of each test sample solution divided by the extinction of first standard solution (0 standard)
Angle value (B0) multiplied by 100%, obtain percentage absorbance value.The percentage absorbance value of each corresponding test sample solution, then
The absorbance value that test sample solution can be read from standard curve, further according to the concentration value of standard solution, to converse sample molten
The residual quantity of dexamethasone in liquid, finally multiplied by the extension rate of each sample pretreatment process, with can calculating in sample
The concentration of Sai meter Song.
Four, kit detection effect is evaluated
(1) accuracy and precision test
Dexamethasone standard items are added into the former milk sample without dexamethasone, make dexamethasone standard items in the sample
Final concentration be respectively 0.1,0.2,0.4 μ g/L；Place before sample after addition is carried out according to method described in experiment three respectively
Reason, obtains test sample solution.
It respectively extracts 3 kits from the kit of three different batches to be detected, detection method such as institute in experiment three
It states, each experiment is repeated 5 times, and calculates separately the coefficient of variation.As a result it is shown in Table 1 respectively.
1 accuracy of table and Precision test result
Variation within batch coefficient: with the coefficient of variation of each parallel samples in primary measurement.
Interassay coefficient of variation: same sample takes its average value in the coefficient of variation of different batches measurement result.
The result shows that: the average TIANZHU XINGNAO Capsul of former milk sample 86.9 ~ 97.3%, variation within batch coefficient 6.4 ~
13.6%, interassay coefficient of variation is in 9.8 ~ 10.1 %.
(2) kit storage life
Kit preservation condition is 2 ~ 8 DEG C, by measurement in 15 months, the maximum absorbance value (0 standard) of kit,
50% inhibition concentration, dexamethasone addition actual measured value are within normal range (NR).In view of transport and use process in, meeting
There is improper preservation condition to occur, kit is placed 9 days under conditions of 37 DEG C of preservations, carries out accelerated aging tests, as a result
Show that the indices of the kit comply fully with requirement.In view of kit freezing happens, kit is put into -20 DEG C
Refrigerator freezing 9 days, measurement result also indicated that kit indices are completely normal.It can show that kit can be from result above
2 ~ 8 DEG C can at least save 12 months or more.
(3) cross reacting rate is tested
It selects other drugs similar with DSMS structure or function to carry out cross reaction test, passes through the standard of various drugs
Curve respectively obtains its 50% inhibition concentration.Kit is calculated to the cross reacting rate of other analogs with following formula.With other drugs
Cross reacting rate it is smaller, illustrate that dexamethasone enzyme-linked immunologic detecting kit is better to the detection specificity of dexamethasone.Knot
Fruit is shown in Table 2.
Cause the dexamethasone concentration of 50% inhibition
Cross reacting rate (%)=--- --- --- --- --- --- --- --- --- --- --- --- --- --- × 100%
Cause the other drugs concentration of 50% inhibition
2 dexamethasone kit cross reacting rate of table
Test result shows that kit of the present invention is the intersection of 100%, triamcinolone to the cross reacting rate of dexamethasone
Reactivity is 52%, the cross reacting rate of prednisolone is 14%, the cross reacting rate of betamethasone is 24%, hydrocortisone
Cross reacting rate is 1.5%, so kit is good to the specificity of dexamethasone, i.e., kit of the present invention fills in rice with can detecte
Pine.
Claims (3)
1. a kind of dexamethasone antigen is obtained by dexamethasone haptens and carrier protein couplet, it is characterised in that:
(1) the dexamethasone haptens is product shown in formula 1:
Formula 1；
(2) the dexamethasone haptens is to be made by the following method:
Addition succinic anhydride 110mg after addition dexamethasone bulk pharmaceutical chemicals 500mg, 5ml pyridinium dissolution in 50ml round-bottomed flask, 80 DEG C
3h is reacted, TLC detects the post-processing of raw material end of reaction, removes pyridine under reduced pressure, 10ml ice water is added in residue, it is solid that white is precipitated
Body, filtering, it is haptens that washing and drying, which obtains 350mg product,；
(3) preparation method of the dexamethasone antigen, includes the following steps:
1), haptens 22mg is dissolved in 1.5mL DMF, after being completely dissolved, sequentially adds EDC 25mg, NHS 25mg, room
Warm magnetic agitation reacts 3h；
2) 50mgBSA, is weighed, is dissolved in 3.5mL 0.1M carbonic acid buffer pH=9.6,400rpm stirs 10min, sufficiently molten
Solution；
3) activated solution in above-mentioned steps 1, is taken, is added dropwise in protein solution under ice water bath environment, stirring while adding, room
Warm magnetic agitation reaction is for 24 hours；
4) reaction product, is packed into the clean 15cm bag filter of 1 distilled water flushing, 1LPBS dialyses 3 days, dialysis is stirred at room temperature, often
Its replacement dialyzate 3 times, dialysis product 4500rpm are centrifuged 6min, and the packing of 0.5ml/ pipe numbers antigen, -20 DEG C of preservations are standby
With.
2. dexamethasone antigen described in claim 1 is preparing the application in dexamethasone specific antibody.
3. specific antibody as claimed in claim 2.
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| 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 |
| 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 |
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| 唬铂酸醉-碳二亚胺法制备高结合比利福平人工抗原;李林峰等;《北京医科大学学报》;19941231;第26卷(第2期);136-137 * |
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