CN102768284A - Immunodetection reagent of carbamazepine homogeneous enzyme and detection method thereof - Google Patents
Immunodetection reagent of carbamazepine homogeneous enzyme and detection method thereof Download PDFInfo
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Abstract
The invention discloses a carbamazepine immunogen, a carbamazepine specific antibody directly obtained through the immunogen, a detection reagent containing the specific antibody and a method for detecting the content of the carbamazepine in samples to be tested. The immunodetection reagent and the detection method have the advantages that the carbamazepine immunogen is strong in specificity and high in immunogenicity; the prepared carbamazepine immunogen is strong in specificity and high in titer and has no cross reaction with 45 common drugs; the homogeneous enzyme immunodetection reagent containing the carbamazepine specific antibody can conveniently, fast and accurately determine the content of the carbamazepine in the samples and can measure a plurality of samples simultaneously on a full-automatic biochemical analyzer, so that high-throughput and fast measuring of the carbamazepine are achieved, the accuracy is high, the specificity is strong, the accuracy and the detection efficiency are greatly improved compared with the prior art, full automation in the detection process is achieved simultaneously, the requirements on detection staff is not high, and the immunodetection reagent and the detection method are easy to achieve, popularize and use.
Description
Technical field
The present invention relates to a kind of detectable and detection method, be specifically related to a kind of carbamazepine homogeneous enzyme immunoassay detectable and detection method thereof.
Background technology
Carbamazepine (Carbamazepine) structural formula is shown in (III):
Formula (III)
Carbamazepine is a kind of common spirit type medicine, has anti-epileptic, anti-trigeminal neuralgia, anti-glossopharyngeal neuralgia and effects such as prophylactic treatment manic-depressive psychosis and anti-arrhythmia.But since its effectively treatment concentration range is narrower, concentration does not reach result of treatment when low, and can cause spinoffs such as dizzy drowsiness, weak, vomiting, alpastic anemia, toxic hepatitis and shock after the overdose.Therefore, in therapeutic process, carrying out monitor drug concentration is very important.
At present, the method that carbamazepine carries out monitor drug concentration mainly contains high performance liquid chromatography, puts the method for exempting from and fluorescence polarization method.The high performance liquid chromatography consumption time is long, and sample pre-treatments and operating process and complicacy thereof require high to testing staff's technical merit; The radioactive ray of putting the method for exempting from has produced great harm to operating personnel's health, seldom uses in the world at present; Main dependence on import of reagent that fluorescence polarization method needs and expense are extremely expensive, need the expensive analytical instrument of acquisition price simultaneously.
Summary of the invention
For solving the deficiency of prior art; The object of the present invention is to provide a kind of not only safety but also can be fast, efficient, sensitivity, accurately detect the detectable of carbamazepine content in the sample to be tested, and can be with various types of automatic biochemistry analyzer couplings, to the less demanding detection method of testing staff.
In order to realize above-mentioned target, the technical scheme that the present invention adopts is:
A kind of carbamazepine homogeneous enzyme immunoassay detectable is characterized in that, comprising: anti-carbamazepine specific antibody is used to detect the indicator of anti-carbamazepine specific antibody-carbamazepine compound; Above-mentioned anti-carbamazepine specific antibody is obtained by carbamazepine immunogen immune animal, and the immunogenic structural formula of above-mentioned carbamazepine is suc as formula shown in (I):
Formula (I)
In the formula, R is a linking group, and carrier has immunogenicity; Above-mentioned indicator is selected from enzyme reagent, radioactive isotope reagent, fluorescent reagent and chemical illuminating reagent.
Aforesaid carbamazepine homogeneous enzyme immunoassay detectable is characterized in that above-mentioned R is-(CH
2)
n-COO-, n are the integer between 1 to 20; Preferably, above-mentioned R is-(CH
2)
4-COO-.
Aforesaid carbamazepine homogeneous enzyme immunoassay detectable is characterized in that, above-mentioned carrier is for having immunogenic protein, and preferred, carrier is a bovine serum albumin(BSA).
Aforesaid carbamazepine homogeneous enzyme immunoassay detectable is characterized in that above-mentioned indicator is selected from enzyme reagent, comprising: the substrate of enzyme mark conjugate and enzyme; Above-mentioned enzyme mark conjugate comprises glucose-6-phosphate dehydrogenase (G6PD)-haptens enzyme mark conjugate; The substrate of above-mentioned enzyme is a G-6-P.
Aforesaid carbamazepine homogeneous enzyme immunoassay detectable; It is characterized in that; Above-mentioned glucose-6-phosphate dehydrogenase (G6PD)-haptens enzyme mark conjugate is formed by glucose-6-phosphate dehydrogenase (G6PD) and carbamazepine derivative coupling, and the structural formula of above-mentioned carbamazepine derivative is suc as formula shown in (II):
Formula (II)
Above-mentioned R is-(CH
2)
n-COO-, n are the integer between 1 to 20.
Aforesaid carbamazepine homogeneous enzyme immunoassay detectable is characterized in that above-mentioned R is-(CH
2)
4-COO-.
Utilize aforesaid detectable to detect the method for carbamazepine, it is characterized in that, may further comprise the steps:
1) sample to be tested is contacted with above-mentioned anti-carbamazepine specific antibody;
2), utilize the content of carbamazepine in the indicator judgement sample according to the situation that combines of carbamazepine in the sample to be tested with above-mentioned anti-carbamazepine specific antibody.
The method of aforesaid detection carbamazepine is characterized in that, above-mentioned sample to be tested is the physiology sample.
The method of aforesaid detection carbamazepine is characterized in that, above-mentioned physiology sample is serum or blood plasma.
Usefulness of the present invention is: carbamazepine immunogene high specificity of the present invention, immunogenicity are high, the anti-carbamazepine specific antibody high specificity of preparing, the height of tiring, and do not have any cross reaction with common 45 kinds of medicines; The homogeneous enzyme immunoassay detectable that contains above-mentioned anti-carbamazepine specific antibody can be confirmed the carbamazepine content in the sample easily and fast, exactly; And can on automatic clinical chemistry analyzer, measure a plurality of samples simultaneously; Realize the rapid mensuration of high flux of carbamazepine, accuracy is high, high specificity; Degree of accuracy all is enhanced before comparing with detection efficiency; Realized the full-automation of testing process simultaneously, less demanding to the testing staff is easy to realize and promote the use of.
Description of drawings
Fig. 1 is a carbamazepine homogeneous enzyme immunoassay reaction normal curve map;
Fig. 2 is carbamazepine homogeneous enzyme immunoassay correlation analysis figure.
Embodiment
The technical scheme that the present invention taked is:
The carbamazepine immunogene, its structural formula is suc as formula shown in (I):
Formula (I)
In the formula, R is a linking group, can be-(CH
2)
n-COO-, n is the integer between 1 to 20, and is special, R is-(CH
2)
4-COO-; Carrier has immunogenicity, and preferred, carrier is for having immunogenic protein.Though the immunogenic material that possesses that other are enough big also can be used as carrier, generally selects for use protein as carrier.The most frequently used immunogenic carrier comprises haemocyanin, hemocyanin (KLH) and thyroglobulin.The selection of carrier is those skilled in the art's a basic general knowledge.
A kind of anti-carbamazepine specific antibody is obtained by the carbamazepine immunogen immune animal shown in the formula (I).
" antibody " of indication not only refers to complete antibody molecule among the present invention, also comprises the antibody fragment or the derivant that keep the complete antibody specific binding capacity.Antibody of the present invention can be that polyclonal antibody can be a monoclonal antibody also, is preferably polyclonal antibody.
The method that obtains polyclonal antibody is to use the carbamazepine immunogene shown in the formula (I), after adding or not adding adjuvant, carries out immunity at one or more position of animal, and host animal comprises: rabbit, goat, mouse, sheep, cavy or horse.Lasting immunity is carried out always, reaches the highest until antibody titer.Animal is regularly taken a blood sample and obtains an amount of specific corrosioning anteserum, and antiserum can purifying.
Monoclonal antibody can be made through somatocyte hybriding technology.
A kind of carbamazepine homogeneous enzyme immunoassay detectable comprises: above-mentioned anti-carbamazepine specific antibody, be used to detect the indicator of anti-carbamazepine specific antibody-carbamazepine compound.Indicator is selected from enzyme reagent, radioactive isotope reagent, fluorescent reagent and chemical illuminating reagent.Preferably, indicator is an enzyme reagent, comprising: the substrate of enzyme mark conjugate and enzyme.Wherein, enzyme mark conjugate comprises glucose-6-phosphate dehydrogenase (G6PD)-haptens enzyme mark conjugate, and it can obtain through chemical synthesis process.
Detect the method for carbamazepine, may further comprise the steps:
1) sample to be tested is contacted with above-mentioned anti-carbamazepine specific antibody;
2), utilize the content of carbamazepine in the indicator judgement sample according to the situation that combines of carbamazepine in the sample to be tested with above-mentioned anti-carbamazepine specific antibody.
Sample to be tested is various physiology samples, for example serum, blood plasma, urine, saliva etc.Preferably, sample to be tested is serum or blood plasma.
Embodiment below in conjunction with concrete further specifies the present invention.
Embodiment one: the synthetic and structural confirmation of carbamazepine derivative
The carbamazepine derivative chemical constitution of using in following examples is suc as formula shown in (IV):
Formula (IV)
The synthetic route of this carbamazepine derivative is following:
Concrete synthesis step is following:
Synthesizing of compound 2
1) accurately take by weighing 10.0g (51.8mmol) compound 1 5H-dibenzo [b, f] azatropylidene, compound 1 is purchased the company in Sigma;
2) compound 1 is dissolved in the 1500mL methylene chloride, adds 5.4g (18.1mmol) triphosgene and handle, add 100ml (126.6mmol) pyrimidine again, stirred overnight at room temperature obtains potpourri;
3) make water and bittern flushing said mixture, add Na
2SO
4, vacuum drying concentrates and carries out purifying through the chromatographic column that silica gel is filled, and it is the petrol ether/ethyl acetate mixed flow phase of 10:1 that moving phase adopts volume ratio; Final drying obtains 12.9g white solid compound 2; Be 5H-dibenzo [b, f] azatropylidene-5-phosgene, productive rate 95%.
Synthesizing of carbamazepine derivative
1) takes by weighing 4.65g (39.6mmol) epsilon-amino valeric acid, be dissolved in the mixed solution of 2800mL toluene and 120mL triethylamine;
2) adding 2,90 ℃ of stirring reactions of 9.22g (36mmol) compound under the room temperature spends the night;
3) temperature is reduced to room temperature, and vacuum concentrates, and adds the 500mL methylene chloride, and water and bittern flushing add Na
2SO
4Drying concentrates, and obtains yellow compound;
4) above-mentioned yellow compound is used the ethyl acetate/petroleum ether crystallization of volume ratio as 1:10, obtain 1.636g white solid purified product after the drying, be i.e. carbamazepine derivative shown in the formula (IV), productive rate 12%.
Above-mentioned white solid purified product is carried out structure to be identified
1, utilizes Bruker Avance III plus400MHz that above-mentioned white solid compound is carried out NMR spectrum scanning, adopt TMS as interior mark.The result is following:
1H NMR (400MHz, DMSO-d
6): δ 1.39-1.30 (m, 4H), 2.14 (2H, t, J=7.4Hz), 2.93 (2H, q, J=6.0Hz), 5.53 (2H, t, J=5.6Hz), 7.00 (s, 1H), 7.36-7.48 (m, 8H).Be characterized by the carbamazepine derivative shown in the formula (IV).
2, utilize chromatogram/mass-spectrometric technique (LCMS) that the derivant that obtains is carried out Analysis and Identification, adopt the series connection level Four bar mass spectrometer LC/MSD1200 series of Agilent company, ion gun adopts positive ion or negative ionization pattern.The chromatographic column specification is: Welchrom XB-C18 (50 * 4.6mm, 5 μ m), and column temperature is 30 ℃, and flow velocity is 1.5mL/min, and moving phase is 95% water and 5% acetonitrile, 6min, the last 0.5min that continues under this condition.
LCMS result shows: purity is 98.62%; Retention time 2.682min; Molecular weight 336.4; Molecule is from 337.2 ([M+1]
+).
Comprehensive The above results can confirm that this white solid compound is the carbamazepine derivative shown in the formula (IV).
Similarly, when the analog that adopts the epsilon-amino valeric acid is participated in reaction, can obtain the analog suc as formula the carbamazepine derivative shown in (IV), its difference only is-CH
2-the difference of number n.At this, epsilon-amino valeric acid and analog thereof all remember and do compd A, when n gets different value, adds the amount of compd A and the relation of the amount of the carbamazepine derivative that obtains is seen table 1.
The compound quality and the yield that need during the different n value of table 1
Embodiment two: the BSA-carbamazepine is immunogenic synthetic
BSA-carbamazepine immunogene by the carbamazepine derivative shown in bovine serum albumin(BSA) BSA and the formula (II)-(CH
2)
n-COO-group is formed by connecting, and in the present embodiment, is that example specifies this immunogenic synthetic method with n=4, and concrete steps are following:
1) 20mg BSA is dissolved in 5ml 0.2M, the phosphate buffer of pH8.5 (Phosphate buffer solution, PBS) in, above-mentioned solution places beaker A;
2) following chemicals is joined stirring and dissolving among the beaker B: the 20mg carbamazepine derivative, the 0.35ml dimethylformamide (dimethylformamide, DMF), 0.35ml ethanol, the kaliumphosphate buffer of 0.7ml 10mM pH5.0.40mg1-ethyl-3-(3-dimethylaminopropyl) carbodiimide, and 5mg N-hydroxy thiosuccinimide (N-hydroxysuccinimide, Sulfo-NHS), stirring and dissolving under room temperature was reacted 30 minutes;
3) drips of solution among the beaker B is added among the beaker A, obtain mixed solution, 2～8 ℃ of following stirred overnight; (4 * 4L) purifying obtain BSA-carbamazepine immunogene, are stored in-20 ℃ with the dialysis of the process of the mixed solution after above-mentioned stirring neutral phosphor phthalate buffer.
Similarly, when n gets other integers in 1～20 scope, use the same method and to prepare carbamazepine immunogene suc as formula shown in (I).Certainly, carrier for having immunogenic protein, can be a haemocyanin still, hemocyanin (KLH) and thyroglobulin.Preferably, carrier is a bovine serum albumin(BSA).
Embodiment three: the preparation of anti-carbamazepine specific antibody
The above-mentioned BSA-carbamazepine immunogene that makes is adopted conventional method inoculation experiments animal rabbit, get antiserum behind the booster immunization, concrete steps are following:
With PBS above-mentioned synthetic BSA-carbamazepine immunogene is diluted to 1.0mg/ml, obtains antigenic solution, mix with Freund's complete adjuvant with the 1.0ml antigenic solution then, rabbit is injected;
After 2～3 weeks, with 1.0ml identical antigenic solution and incomplete Freund rabbit is injected once again, every afterwards at a distance from all around once, inject altogether 4 times.
Above-mentioned animal used as test rabbit is got blood, and separation and purification obtains anti-carbamazepine specific antibody, and through measuring, tiring of this antibody is 1:30000.
Embodiment four: the preparation of glucose-6-phosphate dehydrogenase (G6PD)-hapten conjugation thing
1. the preparation of glucose-6-phosphate dehydrogenase (G6PD) (G6PDH) solution:
1) take by weighing the G6PDH that the 15mg specification is 100KU, accurately take by weighing the G6PDH that the 15mg specification is 100KU, room temperature is dissolved in 12mL and contains 72.6mg (0.05M) Tris, 8mg MgCl
2(3.3mM) with the solution of 100mg NaCl in, this pH value of solution=9.0.
2) in above-mentioned beaker C, add 225mg also the NADH NADH of ortho states, 135mg G-6-P G-6-P and 0.75mL carbitol (Carbitol).
3) in above-mentioned beaker C, dropwise add again the 2mL dimethyl sulfoxide (DMSO) (dimethy sulfoxide, DMSO).
2. the activation of carbamazepine derivative
1) under anhydrous state, takes by weighing the above-mentioned carbamazepine derivative of 10mg, be dissolved among the 600 μ L DMF.
2) make above-mentioned solution temperature drop to-2 ~-8 ℃.
3) add 3 μ L tri-n-butylamines (tributylamine).
4) add 1.5 μ L isobutyl chlorocarbonates (isobutylchloroformate).
5)-2 ~-8 ℃ stirring is 30 minutes.
3.G6PDH with being connected of carbamazepine derivative
1) the carbamazepine derivative solution with above-mentioned activation dropwise joins in the G6PDH solution of above-mentioned dissolving.
2) 2-8 ℃ of stirred overnight.
4. purified product
Through the solution in the G-25 gel chromatography column purification step 3, the final product of acquisition is glucose-6-phosphate dehydrogenase (G6PD)-hapten conjugation thing, stores down in 2-8 ℃.
Embodiment five: the preparation of carbamazepine homogeneous enzyme immunoassay detectable
Carbamazepine homogeneous enzyme immunoassay detectable comprises: above-mentioned anti-carbamazepine specific antibody is used to detect the indicator of anti-carbamazepine specific antibody-carbamazepine compound.Indicator is selected from enzyme reagent, radioactive isotope reagent, fluorescent reagent and chemical illuminating reagent.Preferably, indicator is an enzyme reagent, comprising: the substrate of enzyme mark conjugate and enzyme.Wherein, enzyme mark conjugate comprises glucose-6-phosphate dehydrogenase (G6PD)-haptens enzyme mark conjugate, and it obtains through above-mentioned chemical synthesis process.
Carbamazepine homogeneous enzyme immunoassay detectable is before using; The enzyme mark conjugate in the indicator and the substrate of enzyme react; The substrate of enzyme mark conjugate and enzyme is placed apart, does not mix, so the substrate and the above-mentioned anti-carbamazepine specific antibody of enzyme mixed.That is to say that carbamazepine homogeneous enzyme immunoassay detectable comprises the reagent that two kinds of branches are arranged, and is specific as follows:
1. the preparation of reagent A: NADH NAD, 1.711g (11.25mM) the G-6-P G6P of 4.036g (11.25mM) oxidation state are placed beaker D, process the homogeneous phase zymolyte with the Tris damping fluid dissolving of 1L 55mM, pH=8.0; The anti-carbamazepine specific antibody of above-mentioned preparation is added in the above-mentioned homogeneous phase zymolyte, and the volume ratio of antibody and homogeneous phase zymolyte can be 1:100～1:10000, and concrete in the present embodiment ratio is 1:2000.
2. the preparation of reagent B: the glucose-6-phosphate dehydrogenase (G6PD)-hapten conjugation thing of above-mentioned preparation is added in the Tris damping fluid of 120mM, pH=8.2; The volume ratio of above-mentioned conjugate and Tris damping fluid can be 1:100～1:10000, and concrete in the present embodiment ratio is 1:1500.
Utilize above-mentioned homogeneous enzyme immunoassay detectable to detect the method for carbamazepine, may further comprise the steps:
1) sample to be tested is contacted with above-mentioned anti-carbamazepine specific antibody;
2), utilize indicator to judge the content of carbamazepine in the sample to be tested according to the situation that combines of carbamazepine in the sample to be tested with above-mentioned anti-carbamazepine specific antibody.
Concrete, during detection, sample to be tested is added in the reagent A, the carbamazepine in the sample to be tested combines with anti-carbamazepine specific antibody generation specificity in the reagent A, generates anti-carbamazepine specific antibody-carbamazepine compound; Add reagent B again; The substrate mixing of the enzyme in the glucose-6-phosphate dehydrogenase (G6PD)-hapten conjugation thing among this moment reagent B and the reagent A, contact; Enzymatic reaction takes place; Constitute the indicator that detects anti-carbamazepine specific antibody-carbamazepine compound, indicator is judged the content of carbamazepine in the sample to be tested according to carbamazepine in the sample to be tested with the situation that combines of above-mentioned anti-carbamazepine specific antibody.
Combine anti-carbamazepine specific antibody owing to glucose-6-phosphate dehydrogenase (G6PD)-hapten conjugation thing and the carbamazepine in the sample to be tested are competitive; So; The amount of carbamazepine is many more in the sample to be tested; The amount of free glucose-6-phosphate dehydrogenase (G6PD)-hapten conjugation thing is many more in the homogeneous phase enzyme solutions, and enzymatic reaction is fast, causes OD
340Rise.
Above-mentioned sample to be tested is the physiology sample, for example serum, blood plasma, urine, saliva etc.
As a kind of preferred scheme, above-mentioned sample to be tested is serum or blood plasma.
Embodiment six: the check of carbamazepine homogeneous enzyme immunoassay
1, obtain typical curve: be provided with and step auspicious BS200 automatic clinical chemistry analyzer response parameter (seeing table 2), operating process is: add reagent A earlier, add standard items again, add reagent B at last.After adding reagent B, measure the OD of different time points
340Light absorption value, the reaction rate when calculating various criterion article concentration needs the continuous volume ratio of adjusting reagent A and reagent B in the actual mechanical process, adjust the photometry point simultaneously, draws comparatively ideal reaction normal curve map at last, and is as shown in Figure 1.
Table 2 steps auspicious BS200 automatic clinical chemistry analyzer response parameter
The typical curve that obtains through homogeneous enzyme immunoassay detectable of the present invention; The basic, normal, high concentration Quality Control of replication sample 10 times, above-mentioned Quality Control sample is: the carbamazepine standard items are dissolved in human serum or the blood plasma, are respectively 1.75 to concentration; 3.75,15.0 μ g/ml.Detect data and data analysis and see table 3.
Table 3 sample determination and precision and recovery assessment
| Blood sample | Low | In | High |
| Sample concentration (μ l/ml) | 1.75 | 3.75 | 15.0 |
| 1 | 1.68 | 3.46 | 14.90 |
| 2 | 1.80 | 3.43 | 15.46 |
| 3 | 1.77 | 3.57 | 15.12 |
| 4 | 1.76 | 3.67 | 14.39 |
| 5 | 1.76 | 3.71 | 15.05 |
| 6 | 1.76 | 3.47 | 15.57 |
| 7 | 1.76 | 3.55 | 15.25 |
| 8 | 1.74 | 3.58 | 15.10 |
| 9 | 1.76 | 3.76 | 14.81 |
| 10 | 1.75 | 3.64 | 15.11 |
| Mean value (μ g/ml) | 1.75 | 3.58 | 15.08 |
| Standard deviation (SD) | 0.0303 | 0.1110 | 0.3160 |
| Precision (CV%) | 1.73 | 3.10 | 2.10 |
| Recovery % | 100.0 | 95.5 | 100.5 |
Testing result: the accuracy that homogeneous enzyme immunoassay detectable of the present invention is measured is high, and the recovery reaches 95%-105%, and precision is high, and CV all is lower than 4%.
Embodiment seven: the medicine interference test
Choose 45 kinds of common drugs, adjusting its concentration is 10.0 μ g/ml, carries out interference test and measures, and common 45 kinds of medicines and determination data are referring to table 4.
Table 4 common interference medicine and mensuration result
Testing result: all less than 0.1 μ g/ml.It is thus clear that antibody of the present invention is the specific antibody of anti-carbamazepine.
Embodiment eight: correlation analysis
Use the fluorescence polarization method of Abbott Laboratories and homogeneous enzyme immunoassay method of the present invention to carry out correlation analysis respectively to the 83 routine clinical samples that comprise 66 routine positive samples and 17 routine negative samples, the data of mensuration are referring to table 5.
Table 5 authentic specimen measured value
To above-mentioned data mapping, referring to Fig. 2, the linear equation that obtains is: y=0.9947x-0.0154, coefficient R
2=0.9938, show the carbamazepine clinical samples accuracy height that detectable of the present invention is measured.
Because testing process of the present invention is to accomplish by instrument is full-automatic, so less demanding to the testing staff is easy to realize and promote the use of.
Need to prove; The above is merely embodiments of the invention; Be not so limit claim of the present invention; Every equivalent structure or equivalent flow process conversion that utilizes instructions of the present invention and accompanying drawing content to be done, or directly or indirectly be used in other correlative technology fields, all in like manner be included in the scope of patent protection of the present invention.
Claims (10)
1. a carbamazepine homogeneous enzyme immunoassay detectable is characterized in that, comprising: anti-carbamazepine specific antibody is used to detect the indicator of anti-carbamazepine specific antibody-carbamazepine compound; Above-mentioned anti-carbamazepine specific antibody is obtained by carbamazepine immunogen immune animal, and the immunogenic structural formula of above-mentioned carbamazepine is suc as formula shown in (I):
Formula (I)
In the formula, R is a linking group, and carrier has immunogenicity; Above-mentioned indicator is selected from enzyme reagent, radioactive isotope reagent, fluorescent reagent and chemical illuminating reagent.
2. carbamazepine homogeneous enzyme immunoassay detectable according to claim 1 is characterized in that above-mentioned R is-(CH
2)
n-COO-, n are the integer between 1 to 20.
3. carbamazepine homogeneous enzyme immunoassay detectable according to claim 2 is characterized in that above-mentioned R is-(CH
2)
4-COO-.
4. carbamazepine homogeneous enzyme immunoassay detectable according to claim 1 is characterized in that, above-mentioned carrier is for having immunogenic protein, and preferred, carrier is a bovine serum albumin(BSA).
5. carbamazepine homogeneous enzyme immunoassay detectable according to claim 1 is characterized in that above-mentioned indicator is selected from enzyme reagent, comprising: the substrate of enzyme mark conjugate and enzyme; Above-mentioned enzyme mark conjugate comprises glucose-6-phosphate dehydrogenase (G6PD)-haptens enzyme mark conjugate; The substrate of above-mentioned enzyme is a G-6-P.
6. carbamazepine homogeneous enzyme immunoassay detectable according to claim 5; It is characterized in that; Above-mentioned glucose-6-phosphate dehydrogenase (G6PD)-haptens enzyme mark conjugate is formed by glucose-6-phosphate dehydrogenase (G6PD) and carbamazepine derivative coupling, and the structural formula of above-mentioned carbamazepine derivative is suc as formula shown in (II):
Formula (II)
Above-mentioned R is-(CH
2)
n-COO-, n are the integer between 1 to 20.
7. carbamazepine homogeneous enzyme immunoassay detectable according to claim 6 is characterized in that above-mentioned R is-(CH
2)
4-COO-.
8. utilize the described detectable of any claim of claim 1 to 7 to detect the method for carbamazepine, it is characterized in that, may further comprise the steps:
1) sample to be tested is contacted with above-mentioned anti-carbamazepine specific antibody;
2), utilize the content of carbamazepine in the indicator judgement sample according to the situation that combines of carbamazepine in the sample to be tested with above-mentioned anti-carbamazepine specific antibody.
9. the method for detection carbamazepine according to claim 8 is characterized in that, above-mentioned sample to be tested is the physiology sample.
10. the method for detection carbamazepine according to claim 9 is characterized in that, above-mentioned physiology sample is serum or blood plasma.
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Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4058511A (en) * | 1976-01-12 | 1977-11-15 | Syva Company | Tegretol antigens and antibodies |
| JPS5716866A (en) * | 1980-07-03 | 1982-01-28 | Chugai Pharmaceut Co Ltd | Novel 5h-dibenz b,f azepine and its production |
| EP0583820A1 (en) * | 1992-08-07 | 1994-02-23 | Johnson & Johnson Clinical Diagnostics, Inc. | Novel carbamazepine hapten analogues |
| CN1971270A (en) * | 2005-11-24 | 2007-05-30 | 复旦大学附属华山医院 | Method for detecting blood concentration of multiple antiepileptic drugs simultaneously |
| CN102183659A (en) * | 2011-03-02 | 2011-09-14 | 广州金域医学检验中心有限公司 | Method for detecting carbamazepine |
| CN102180965A (en) * | 2011-03-02 | 2011-09-14 | 广州金域医学检验中心有限公司 | Carbamazepine immunogen, anti-carbamazepine specific antibody, detection reagent and detection kit |
| CN102331499A (en) * | 2011-01-27 | 2012-01-25 | 无锡安迪生物工程有限公司 | Bi-path detecting card capable of simultaneously detecting carbamazepine and sodium valproate and detecting method of same |
-
2012
- 2012-08-01 CN CN201210272607.6A patent/CN102768284B/en active Active
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4058511A (en) * | 1976-01-12 | 1977-11-15 | Syva Company | Tegretol antigens and antibodies |
| JPS5716866A (en) * | 1980-07-03 | 1982-01-28 | Chugai Pharmaceut Co Ltd | Novel 5h-dibenz b,f azepine and its production |
| EP0583820A1 (en) * | 1992-08-07 | 1994-02-23 | Johnson & Johnson Clinical Diagnostics, Inc. | Novel carbamazepine hapten analogues |
| CN1971270A (en) * | 2005-11-24 | 2007-05-30 | 复旦大学附属华山医院 | Method for detecting blood concentration of multiple antiepileptic drugs simultaneously |
| CN102331499A (en) * | 2011-01-27 | 2012-01-25 | 无锡安迪生物工程有限公司 | Bi-path detecting card capable of simultaneously detecting carbamazepine and sodium valproate and detecting method of same |
| CN102183659A (en) * | 2011-03-02 | 2011-09-14 | 广州金域医学检验中心有限公司 | Method for detecting carbamazepine |
| CN102180965A (en) * | 2011-03-02 | 2011-09-14 | 广州金域医学检验中心有限公司 | Carbamazepine immunogen, anti-carbamazepine specific antibody, detection reagent and detection kit |
Non-Patent Citations (3)
| Title |
|---|
| A. M. SIDKI的民航: "Influence of the Hapten-Fluorophore Bridge on Binding Parameters in a Fluoroimmunoassay for Carbamazepine", 《CLINICAL CHEMISTRY》 * |
| TAKASHI NISHIKAWA: "Nephelometric Immunoassay for Therapeutic Drug Level Monitoring", 《PHARMACEUTICAL RESEARCH》 * |
| 蒋中华、张津辉主编: "《生物分子固定化技术及应用》", 31 July 1998, 化学工业出版社 * |
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