CN104788383B - Clonidine haptens, antigen and its preparation method and application - Google Patents
Clonidine haptens, antigen and its preparation method and application Download PDFInfo
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Abstract
The invention discloses a kind of clonidine haptens, corresponding artificial antigen and monoclonal antibody, while invention also discloses the clonidine haptens, the preparation method and applications of corresponding artificial antigen and monoclonal antibody.Clonidine haptens provided by the invention is product shown in formula I, and product is connect with carrier protein shown in formula I can obtain clonidine antigen.The clonidine antigen can be applied to prepare clonidine specific antibody.Preparation method of the present invention is simple and feasible, cost is relatively low, and yield of hapten is higher.The clonidine artificial antigen of the present invention, the specific antibody for clonidine can be produced by immune animal, the remaining enzyme-linked immunologic detecting kit of clonidine is detected available for preparing, there is many advantages, such as simple, quick, processing sample size is big, high sensitivity, high specificity.
Description
Technical field
The present invention relates to a kind of clonidine haptens, antigen, method for preparing monoclonal antibody and its applications, belong to bioid
Work field.
Background technology
Clonidine（Clonidine）Chemical name for 2- [（2,6- dichlorophenyls）Imino group] imidazolidine hydrochloride, alias
Clonidine, clonidine, Clonidine, clonidin, 110 hypertension pills, clonidine, Clonidine, English alias 2,6-dichloro-
N-(2-imidazolin-2-yl)aniline.Molecular formula C9H9Cl2N3；Molecular weight：230.1.Cola is set to white crystalline powder
End；It is odorless.It dissolves in water or in ethyl alcohol, the soluble,very slightly in chloroform is almost insoluble in ether, and fusing point is 305 DEG C.It is laughable
Surely be a kind of drug for hypertension, pharmacological action predominantly by stimulate brain stem α 2- adrenoreceptors cause sympathetic nerve from
The outflow of central nervous system is reduced, so as to reduce peripheral resistance, renal vascular resistance, heart rate and blood pressure.Clonidine takes orally suction
It receives rapidly, about 50% in liver metabolism, and metabolin is to hydroxyl clonidine and glucuronate, and hydroxyl clonidine cannot be led to
Cross blood-brain barrier, no antihypertensive activity；20%~40% is discharged in the form of prototype and metabolin from urine；About 20% arranges from excrement
Go out.It has been found that have the tendency that addition in feed product from 2006 at home, analyzing its mechanism of action should be with the β such as clenbuterol hydrochloride
Receptor stimulating agent is similar, achievees the effect that weightening to improve food conversion ratio.April in 2010 Huairou District, Beijing City on the 23rd Beijing
Acute collective's poisoning by clonidine event that waterfront mountain food and drink Co., Ltd occurs has caused the great attention of the whole society.
At present, clonidine to the effect of promoting animal growth and weightening it has been reported that at home it has been found that adding this drug is illegal
It is added in pannage, No. 1519 bulletins of in December, 2010 Ministry of Agriculture are clearly included in clonidine《Forbid in feed and animal
The substance used in drinking-water》Inventory, the World Health Organization, the U.S., European Union and some developed countries etc. all forbids illegal use can
It is happy fixed.Its common side effect has dry, drowsiness, dizziness, calmness, respiration inhibition etc..It is edible to contain the remaining animal of clonidine
Property product in source has certain harmfulness to health, the effect of children is become apparent.
It is measured mostly using high performance liquid chromatography, gas chromatography both at home and abroad at present, it is these method high specificities, sensitive
Degree is high but sample pre-treatments are complex for operation step, and cost is higher, is not also suitable for the selective mechanisms of batch samples.It is immune
Chemical analysis due to the unique advantage in terms of the qualitative, quantitative of antigen-antibody and quick, at low cost, sensitivity easy to operate compared with
The advantages of height, big analysis sample size, compensates for the deficiency of physico-chemical analysis.The reality for being prepared as this method of clonidine artificial antigen
It applies and has established important foundation.Influence immunochemistry analysis quality basic factor be antibody specificity and compatibility, these property
Matter is decided by the structure of immune hapten molecule again, therefore the MOLECULE DESIGN of immune haptens is exactly that generation specificity is anti-with synthesis
The step of body and most basic and most critical for establishing small molecule residue of veterinary drug Fast Detection Technique.
Invention content
The object of the present invention is to provide a kind of clonidine artificial antigens and preparation method and application.
Clonidine artificial antigen provided by the present invention is resisted what clonidine haptens and carrier protein couplet obtained
It is former.
Clonidine haptens is a kind of compound, and structure is shown in formula I.
Formula I
Clonidine haptens is also to belong to the scope of protection of the present invention.
The synthetic route schematic diagram of the clonidine haptens is shown in Fig. 1.
The invention also discloses the preparation methods of product shown in formula I, include the following steps：
100mg Apraclonldines and 49mg glyoxalic acids are dissolved in ethyl alcohol, 16h is stirred at room temperature, is concentrated under reduced pressure.It is thin using preparing
Layer chromatography isolates and purifies, solvent dichloromethane：Methanol（V/V）=5:1, obtain white solid CLD-ECO.
Clonidine antigen provided by the invention is the conjugate for obtaining product and carrier protein couplet shown in formula I.
The structure diagram of the clonidine antigen is shown in Fig. 3.
The present invention also protects the preparation method of the clonidine antigen, includes the following steps：
（1）Product 13mg shown in modus ponens I, is dissolved in 2mL dimethylformamides, magnetic agitation makes it fully dissolve；
（2）Step 1 solution is taken, adds in 17mg EDC and 15mg NHS, room temperature priming reaction 2h；
（3）Carrier protein 50mg is weighed, adds in 0.1mol/L sodium bicarbonate buffer solution 4ml, stirring makes it fully dissolve；
（4）Activating solution is added in protein solution, room temperature reaction is overnight；
（5）With the phosphate buffer of 0.01mol/L in 4 DEG C of dialysis 3d, 3 dialyzates are changed daily, to remove unreacted
Small-molecule substance；
（6）Packing, saves backup in -20 DEG C.
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）Deng.
The clonidine antigen can prepare clonidine specific antibody as immunogene, can also be used as coating antigen preparation
ELISA Plate.
The antibody specific can be monoclonal antibody.
Product shown in formula I, the clonidine antigen, the antibody can be applied to detection clonidine.
The ELISA reagent being prepared using clonidine antigen and clonidine monoclonal antibody is also disclosed in the present invention
Box.
The enzyme-linked immunologic detecting kit, be by be coated with the ELISA Plate of clonidine antigen, enzyme labelled antibody working solution, can
It is happy to determine serial standards, substrate developing solution, terminate liquid, concentration redissolution liquid, concentrated cleaning solution.
The present invention is by immunology, immunochemistry basic principle and retention analysis technological means, design, synthesized micromolecule mesh
Mark analyte haptens, and and carrier protein couplet, effective artificial antigen is prepared, animal is immunized and prepares for small molecule analyte
Specific antibody.It is reacted using the specificity immunology of antigen-antibody, micro small molecule target point in quantitative detection sample
Analyse object, have the characteristics that it is special, sensitive, accurate, quick, conveniently, it is cheap.Preparation method of the present invention is simple and feasible, cost is relatively low,
Yield of hapten is higher.The present invention overcomes complicated, time-consuming to clonidine sample pretreatment in existing detection technique and needs are big
It the extraction of amount organic solvent and the detecting instrument of accurate costliness is used in detection process and is unsuitable for promoting the use of etc. lacks
Point.The clonidine artificial antigen of the present invention can generate the specific antibody for clonidine, for quickly examining by immune animal
The clonidine residual in food is surveyed, there is many advantages, such as simple, quick, processing sample size is big, high sensitivity, high specificity.
Description of the drawings
Fig. 1 is clonidine hapten synthesis route map；
Fig. 2 is clonidine haptens mass spectrogram；
Fig. 3 is clonidine kit standard curve graph.
Specific embodiment
Following embodiment facilitates a better understanding of the present invention, but does not limit the present invention.Experiment in following embodiments
Method is conventional method unless otherwise specified.Test material used in following embodiments is certainly unless otherwise specified
What routine biochemistry reagent shop was commercially available.Quantitative test in following embodiment is respectively provided with three repeated experiments, as a result makes even
Mean value.
The preparation and identification of embodiment 1, clonidine haptens
First, the preparation of clonidine haptens
100mg Apraclonldines and 49mg glyoxalic acids are dissolved in ethyl alcohol, 16h is stirred at room temperature, is concentrated under reduced pressure.It is thin using preparing
Layer chromatography isolates and purifies, solvent dichloromethane：Methanol（V/V）=5:1, obtain product shown in formula 1, i.e. haptens CLD-ECO.
2nd, the identification of clonidine haptens
The clonidine haptens that step 1 is prepared carries out Mass Spectrometric Identification.
Mass spectrogram is shown in Fig. 2.MS characterization results show that-MS results are 298.9, and+MS results are 301, illustrate hapten synthesis
Success.
Embodiment 2, the preparation and identification of clonidine artificial antigen
First, clonidine immunizing antigen（CLD-BSA）Synthesis
（1）Product 13mg shown in modus ponens 1, is dissolved in 2mL dimethylformamides, magnetic agitation makes it fully dissolve；
（2）Step 1 solution is taken, adds in 17mg EDC and 15mg NHS, room temperature priming reaction 2h；
（3）Weigh bovine serum albumin(BSA)（BSA）50mg, adds in 0.1mol/L sodium bicarbonate buffer solution 4ml, and stirring makes it
Fully dissolving；
（4）Activating solution is added in protein solution, room temperature reaction is overnight；
（5）With the phosphate buffer of 0.01mol/L in 4 DEG C of dialysis 3d, 3 dialyzates are changed daily, to remove unreacted
Small-molecule substance；
（6）Packing, saves backup in -20 DEG C.
2nd, clonidine envelope antigen（CLD-OVA）Synthesis
（1）Product 13mg shown in modus ponens 1, is dissolved in 2mL dimethylformamides, magnetic agitation makes it fully dissolve；
（2）Step 1 solution is taken, adds in 17mg EDC and 15mg NHS, room temperature priming reaction 2h；
（3）Weigh ovalbumin（OVA）50mg, adds in 0.1mol/L sodium bicarbonate buffer solution 4ml, and stirring makes it fully
Dissolving；
（4）Activating solution is added in protein solution, room temperature reaction is overnight；
（5）With the phosphate buffer of 0.01mol/L in 4 DEG C of dialysis 3d, 3 dialyzates are changed daily, to remove unreacted
Small-molecule substance；
（6）Packing, saves backup in -20 DEG C.
3rd, the identification of clonidine artificial antigen
By the ratio of synthesis clonidine immunizing antigen and envelope antigen reaction haptens used, carrier protein and coupled product
Example, carries out ultraviolet respectively（200nm~400nm）Scanning identification, and calculate it by comparing light absorption value of the three under Same Wavelength
With reference to than.The ultraviolet spectrogram of product is changed compared with carrier protein, illustrates that haptens is successful with carrier protein
Clonidine artificial antigen is made in coupling.It is computed, the combination ratio of clonidine hapten molecule and BSA molecules is 16.5:1, it is laughable
The combination ratio for determining hapten molecule and OVA molecules is 12.2:1.
Embodiment 3, the preparation of enzyme mark monoclonal antibody and specificity identification
First, the preparation of clonidine monoclonal antibody
1st, with the above-mentioned immunogene prepared（CLD-BSA）By 100 μ g/ only, with physiological saline solution immunogene and Freund
The isometric mixing of Freund's complete adjuvant, nape part, which is subcutaneously injected, is immunized 6 ~ 8 week old BALB/c female mices, the 7th, 14,28 day after initial immunity
With immunogene and the isometric mixing of incomplete Freund's adjuvant, each supplementary immunization is primary, before fusion 3 days with 100 μ g/ of immune complex
Only, being not added with Freund's adjuvant, supplementary immunization is primary again.
2nd, it carries out according to a conventional method, the splenocyte and the murine myeloma cell in exponential phase for taking immune mouse
（SP2/0）Then mixing is slowly added to the fusion agent of preheating in 45s（PEG4000）It is merged, is suspended with HAT culture mediums
Uniformly, suitable feeder cells are added, are incubated at 96 well culture plates, in 37 DEG C, 5%CO2It is cultivated in incubator, HT is used after 5 days
Culture medium partly changes liquid, and 9 days whens carry out changing liquid entirely.
3rd, it is 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, with envelope antigen（Its best coating concentration and the positive with square formation method conventional titration in advance
Serum dilution）Coated elisa plate adds in measured hole culture supernatant, is incubated, and sheep anti-mouse igg-HRP and IgM- is added in after cleaning
HRP, OPD carry out chromogenic reaction.The positive Kong Zaiyong indirect competitive ELISAs method screening filtered out, first by cell conditioned medium and 100
The clonidine of μ g/mL mixes in equal volume, and 37 DEG C of water-baths act on 30min, are then added in the ELISA Plate being coated with.Use PBS simultaneously
Substitution clonidine compares, remaining step is same as above.If the OD after clonidine blocks450nmValue drop to the 50% of control wells hereinafter,
The positive is then judged to, is all positive hole through 2 ~ 3 detections, carries out subcloning with limiting dilution assay immediately.
4th, the hybridoma that 2 ~ 3 subclones are built after strain is expanded into culture, collects supernatant indirect ELISA measure and imitate
Valency freezes；And take 8 ~ 10 week old BALB/c mouses intraperitoneal injection atoleine 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.
2nd, the preparation of enzyme labelled antibody
（1）Weigh horseradish peroxidase（HRP）2 mg are dissolved in 0.5 mL water, add in 0.5 mL, 0.06 mol/L
NaIO4 solution, 4 DEG C are protected from light 30 min of effect；
（2）The ethylene glycol 0.5mL of 160 mmol/L is added in, room temperature acts on 30 min；
（3）2 mg of clonidine monoclonal antibody prepared by step 1 is added in, is fitted into processed bag filter after mixing, puts 1000 mL
0.05 mmol/L sodium carbonate buffers in dialyse, 4 DEG C overnight；
（4）Dialyzate is drawn in the centrifuge tube of 10 mL, adds the NaBH4 solution of 0.25mL 5g/L, 4 DEG C of 2 h of mixing postposition；
（5）Add in isometric saturated ammonium sulfate solution, 3000 r/min centrifugations 25 at 4 DEG C after 4 DEG C of 30 min of effect
Min abandons supernatant；
（6）Precipitation 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 dialyse, and 4 DEG C overnight（PBS is replaced 3 times in midway）；
（7）Liquid in bag filter is drawn in microcentrifugal tube, 10000r/min centrifuges 30min at 4 DEG C, and supernatant is inhaled
Go out, add equivalent glycerine, mixing, -20 DEG C save backup.
3rd, the measure of enzyme mark clonidine antibody titer
Clonidine standard items are purchased from Sigma companies.
Clonidine envelope antigen and the working concentration of monoclonal antibody prepared by step 1, clonidine coating are determined with square formation titration
The working concentration of antigen is 0.5 μ g/mL, and the working concentration of monoclonal antibody is 1:10000.
Experimental solutions are done with the clonidine standard solution of various concentration, concentration is as follows：0、0.2、0.6、1.8、5.4、
16.2μg/L.Using 8 groups of parallel tests（n=8）.Indirect Competitive ELISA method：
（1）With the clonidine antigen of above-mentioned working concentration（CLD-OVA）Coated elisa plate tests clonidine standard items molten
Liquid adds in ELISA Plate micropore, while set blank well simultaneously with enzyme labelled antibody solution（The antibody-solutions of addition are changed into high-purity
Water, it is other consistent）And negative control hole（Standard items experimental solutions are replaced with PBS solution, it is other consistent）, 25 DEG C of light protected environments
Middle reaction 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）It adds in substrate chromophoric solution to ELISA Plate micropore, reacts 15min in 25 DEG C of light protected environments；
（4）Terminate liquid is added in, mixing is gently vibrated, OD values is measured at wavelength 450nm with microplate reader.
Using OD values as ordinate, using the log10 values of clonidine experimental solutions concentration as abscissa, it is bent to draw semilog standard
Line chart.Standard curve has complete reverse-s shape shape, and with 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 (IC50) is obtained according to standard curve, determines detection sensitivity.
Inhibiting rate is calculated with the following formula：
In formula：ODmax：For the light absorption value being not added with during standard items, ODxFor the light absorption value of standard items x, ODminFor blank control
The light absorption value in hole.
Declot is calculated by above-mentioned formula and determines half amount of suppression (IC of the antibody in buffer solution50) it is 0.78 μ g/L.
Embodiment 4, the enzyme linked immunological kit for detecting clonidine and its preparation
First, enzyme linked immunological kit is made of following substances：
（1）It is coated with clonidine haptens（CLD-OVA）ELISA Plate；
（2）Enzyme mark clonidine antibody working solution：Enzyme labelled antibody solution described in embodiment 3；
（3）Clonidine standard items：Clonidine standard solution concentration is respectively 0,0.2,0.6,1.8,5.4,16.2 μ g/L；
（4）Substrate developing solution：It is made of A liquid and B liquid, A liquid is the aqueous solution of 2% urea peroxide, and B liquid joins for 1% tetramethyl
Aniline（TMB）Aqueous solution；
（5）Terminate liquid：0.2M aqueous sulfuric acids；
（6）Concentrated cleaning solution：Every 1 liter of cleaning solution is prepared as follows to be obtained：By 10mL Tween-20s,
5g sodium azide and the mixing of 990mL phosphate buffers, obtain the cleaning solution；The phosphate buffer it is a concentration of
0.01M, pH value 7.4；
（7）Liquid is redissolved in concentration：The phosphate buffer of 0.04mo1/L.
2nd, ELISA Plate and its preparation of CLD-OVA are coated with
It is coated with the polystyrene ELISA Plate of CLD-OVA：With the carbonate solution of 0.05M by antigen diluent to 0.5 μ g/mL,
96 hole polystyrene ELISA Plates are coated with, per 100 μ L of hole, 37 DEG C of incubation 2h, coating buffer of inclining washs 3 times, every time with cleaning solution
10s is patted dry, and then adds in 150 μ L confining liquids in every hole, 37 DEG C of incubation 2h, and liquid in hole of inclining uses aluminium film vacuum after dry
It is sealed.
It is coated with buffer solution：The sodium carbonate buffer of pH9.6,0.05mo1/L；
Confining liquid：Every 1 liter of confining liquid is prepared as follows：5mL horse serums, 1g sodium azide, 30g caseins are mixed
It closes, is dissolved with phosphate buffer and be settled to 1000mL, obtain confining liquid；Wherein, phosphate buffer is a concentration of
0.02M, pH value 7.2.
3rd, kit test method
(1) urine sample pre-treatment
（1）It measures in urine specimen 400mL to 2mL polystyrene centrifuge tubes, 3000g centrifugations 10min；
（2）It draws supernatant and presses 1 with sample diluting liquid：1 volume mixture；
（3）50mL mixed liquors are taken for detecting.
(2) it is detected with kit
1st, the making of standard curve
50 μ L of clonidine standard solution are added in into the ELISA Plate micropore for be coated with CLD-OVA, then in every Kong Zhongjia
Enter 50 μ L ELIAS secondary antibodies, add 50 μ L antibody working solutions, gently oscillation is uniformly mixed, and is kept away for 25 DEG C with cover board membrane cover plate postposition
30min is reacted in luminous environment.Cover board film carefully is opened, outwells liquid in plate hole, 260 μ L wash operating solutions, leaching are added in every hole
15-30s is steeped, is repeated 3 times, is outwelled liquid in plate hole, ELISA Plate is inverted on blotting paper.100 μ L are added in every hole immediately
Substrate A, B gently vibrate mixing, and 25 DEG C of insulating boxs are protected from light colour developing 15min, 50 μ L of terminate liquid are added in per hole, gently vibrate mixing,
With reading ELISA Plate absorbance value under 450 nm of microplate reader.
With the absorbance values of the standard solution of each concentration（B）Divided by first standard solution（0 standard）'s
Absorbance value（B0）Multiplied by with 100%, percentage absorbance value is obtained.With clonidine standard concentration（μg/L）Semilog value be X
Axis, percentage absorbance value are Y-axis, draw canonical plotting.Obtained standard curve is as shown in Figure 3.
2nd, sample medium coke determines the measure of concentration
With the absorbance values of each test sample solution（B）Divided by first standard solution（0 standard）Extinction
Angle value（B0）Multiplied by with 100%, percentage absorbance value is obtained.The percentage absorbance value of each corresponding test sample solution, then
The absorbance value of test sample solution can be read from standard curve, to converse sample molten further according to the concentration value of standard solution
The fixed residual quantity of liquid medium coke, finally multiplied by with the extension rate of each sample pretreatment process, you can calculate sample medium coke
Fixed concentration.
4th, kit detection result is evaluated
(1) accuracy and precision test
Clonidine standard items are added into the pig urine samples without clonidine, make the end of clonidine standard items in the sample dense
Degree is respectively 0.5,1,2 μ g/L；Sample after addition is subjected to pre-treatment according to method described in experiment three respectively, is detected
Sample solution.
It respectively extracts 3 kits from the kit of three different batches to be detected, institute in detection method such as experiment three
It states, each experiment is repeated 5 times, and calculates the coefficient of variation respectively.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 measure.
Interassay coefficient of variation：Same sample is in the coefficient of variation of different batches measurement result.
The result shows that：The average TIANZHU XINGNAO Capsul of pig urine samples 83.9 ~ 109.4%, variation within batch coefficient 6.4 ~
8.6%, interassay coefficient of variation is in 8.9 ~ 10.1 %.
(2) kit storage life
Kit preservation condition is 2 ~ 8 DEG C, by the measure of 15 months, the maximum absorbance value of kit（0 standard）,
50% inhibition concentration, clonidine add actual measured value within normal range (NR).During transport and use, have
Improper preservation condition occurs, and is placed 9 days under conditions of kit is preserved at 37 DEG C, carries out accelerated aging tests, as a result table
The indices of the bright kit comply fully with requirement.It is happened in view of kit freezing, kit is put into -20 DEG C of ice
Case freezes 9 days, and measurement result also indicates that kit indices are completely normal.From result above can obtain kit can 2 ~
8 DEG C can at least preserve 12 months or more.
(3) cross reacting rate is tested
The selection other drugs similar to CLD structure or functions carry out cross reaction experiment, pass through the standard of various drugs
Curve respectively obtains its 50% inhibition concentration.Cross reacting rate of the kit to other analogs is calculated with following formula.With other drugs
Cross reacting rate it is smaller, illustrate that clonidine enzyme-linked immunologic detecting kit is better to the detection specificity of clonidine.As a result see
Table 2.
2 clonidine kit cross reacting rate of table
Result of the test shows that kit of the present invention is non-to moxonidine, Rilmenidine, Dexmedetomidine, Tizanidine, Lip river
Western fixed cross reacting rate is respectively less than 1%, so kit is good to the specificity of clonidine, i.e., kit of the present invention can detect
Clonidine.
Claims (8)
1. a kind of clonidine haptens is compound shown in formula I：
Formula I；
The preparation method of compound, includes the following steps shown in formula I：
100mg Apraclonldines and 49mg glyoxalic acids are dissolved in ethyl alcohol, 16h is stirred at room temperature, is concentrated under reduced pressure, using preparing thin layer color
Spectrum isolates and purifies, solvent dichloromethane：Methanol（V/V）=5:1, obtain white solid, the clonidine haptens as shown in formula I.
2. a kind of clonidine antigen is the coupling for obtaining compound and carrier protein couplet shown in formula I described in claim 1
Object.
3. the preparation method of clonidine antigen, includes the following steps described in claim 2：
（1）Compound 13mg shown in formula I described in claim 1 is taken, is dissolved in 2mL dimethylformamides, magnetic agitation makes
It is fully dissolved；
（2）Step 1 solution is taken, adds in 17mg 1- ethyls -3- (3- dimethyl aminopropyls)-carbodiimides（EDC）And 15mg
N-hydroxysuccinimide（NHS）, room temperature priming reaction 2h；
（3）Carrier protein 50mg is weighed, adds in 0.1mol/L sodium bicarbonate buffer solution 4ml, stirring makes it fully dissolve；
（4）Activating solution is added in protein solution, room temperature reaction is overnight；
（5）It is dialysed with the phosphate buffer of 0.01mol/L in 4 DEG C 3d, changes 3 dialyzates daily, it is unreacted small to remove
Molecular substance；
（6）Packing, saves backup in -20 DEG C.
4. application of the clonidine antigen in clonidine specific antibody is prepared described in claim 2.
5. the specific antibody that clonidine antigen described in application claim 2 is prepared.
6. antibody described in clonidine haptens described in claim 1, clonidine antigen described in claim 2, claim 5 is being examined
Survey the application in clonidine.
It is 7. special described in clonidine haptens described in application claim 1, clonidine antigen described in claim 2, claim 5
The enzyme-linked immunologic detecting kit that property Antibody preparation obtains.
8. enzyme-linked immunologic detecting kit according to claim 7, it is characterised in that including：It is coated with clonidine antigen
Liquid, thickening and washing are redissolved in ELISA Plate, enzyme labelled antibody working solution, clonidine serial standards, substrate developing solution, terminate liquid, concentration
Liquid.
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| US4094964A (en) * | 1977-05-10 | 1978-06-13 | Hoffmann-La Roche Inc. | Clonidine assay |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4094964A (en) * | 1977-05-10 | 1978-06-13 | Hoffmann-La Roche Inc. | Clonidine assay |
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