CN104447745B - A kind of theophylline homogeneous enzyme immunoassay detects tests test kit and preparation method thereof - Google Patents
A kind of theophylline homogeneous enzyme immunoassay detects tests test kit and preparation method thereof Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D473/00—Heterocyclic compounds containing purine ring systems
- C07D473/02—Heterocyclic compounds containing purine ring systems with oxygen, sulphur, or nitrogen atoms directly attached in positions 2 and 6
- C07D473/04—Heterocyclic compounds containing purine ring systems with oxygen, sulphur, or nitrogen atoms directly attached in positions 2 and 6 two oxygen atoms
- C07D473/06—Heterocyclic compounds containing purine ring systems with oxygen, sulphur, or nitrogen atoms directly attached in positions 2 and 6 two oxygen atoms with radicals containing only hydrogen and carbon atoms, attached in position 1 or 3
- C07D473/08—Heterocyclic compounds containing purine ring systems with oxygen, sulphur, or nitrogen atoms directly attached in positions 2 and 6 two oxygen atoms with radicals containing only hydrogen and carbon atoms, attached in position 1 or 3 with methyl radicals in positions 1 and 3, e.g. theophylline
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/76—Albumins
- C07K14/765—Serum albumin, e.g. HSA
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/795—Porphyrin- or corrin-ring-containing peptides
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
Abstract
The object of this invention is to provide a kind of theophylline derivative, artificial antigen of theophylline, homogeneous phase enzyme reagent kit and preparation method thereof of easy, quick, highly sensitive and full-automatic theophylline detection of drug concentration, in order to solve the problems of the technologies described above, the invention provides a kind of theophylline homogeneous enzyme immunoassay kit and preparation method thereof, comprise the synthesis of Theophylline immunogen, the preparation of anti-theophylline specific antibody, the preparation of enzyme mark conjugate and the mensuration of sample.The present invention is highly sensitive, stability and reproducible.The present invention applies on automatic clinical chemistry analyzer, easy and simple to handle, can carry out the rapid sample detection of high-throughput, is applicable to the clinical detection of conventional treatment drug concentration.The high specificity of the theophylline antibody provided in this invention, in medicine cross reaction test, to 31 kinds of Common drugs of test and compound almost without any cross reaction, is applicable to the Plasma Concentration of Clinical Laboratory theophylline.
Description
Technical field
The invention belongs to field of medical examination, be specifically related to the detection of a kind of theophylline homogeneous enzyme immunoassay and test test kit and preparation method thereof.
Background technology
Theophylline is the derivative of methyl xanthine class, is usually used in treatment bronchial asthma, asthma type chronic bronchitis and cardiac asthma etc. clinically.But theophylline also has a lot of toxic side effect, comprising: feel sick, headache, diarrhoea, vomiting, gastrointestinal haemorrhagia, epilepsy and irregular pulse etc.Due to effective concentration treatment window narrow (therapeutic blood serum concentrations term of reference is 10-20 μ g/mL) of theophylline, and toxic side effects incidence and its Plasma Concentration closely related, therefore carry out detection in the serum theophylline concentrations level of treatments period to patient extremely important, the concrete structure formula of theophylline is as follows:
The method of current mensuration serum theophylline concentration mainly contains high performance liquid chromatography (HPLC) and fluorescence polarization immunoassay (FPIA).Supermatic FPIA method becomes the most frequently used method of clinical SERUM THEOPHYLLINE CONCENTRATION MONITORING fast because it is easy, but its test kit dependence on import, expensive and validity period is shorter is its unavoidable shortcoming.And HPLC Fa Changyin complex operation, efficiency is low, and the shortcoming that determination period is long and analysis cost is high limits its widespread use in clinical medicine concentration monitor.
EP0077896 discloses a kind of anti-theophylline specific antibody and theophylline homogeneous enzyme immunoassay detection reagent, and its immunogen structural formula obtaining antibody is as follows:
CN201210258850 discloses a kind of anti-theophylline specific antibody and theophylline homogeneous enzyme immunoassay detection reagent, and its immunogen structural formula obtaining antibody is as follows:
And the difference of EP0077896 and CN201210258850 is that R substituent is slightly different.
CN201310285736 also discloses a kind of artificial antigen of theophylline, and it also can be applicable in theophylline homogeneous enzyme immunoassay detection reagent, and disclosed in it, artificial antigen structural formula is as follows:
US4230805 also discloses a kind of artificial antigen of theophylline, and it also can be applicable in theophylline homogeneous enzyme immunoassay detection reagent, and disclosed in it, artificial antigen structural formula is as follows:
As everyone knows, in theophylline homogeneous phase enzyme detects, the Theophylline immunogen structure of employing directly can determine its immunogenicity, thus affects sensitivity and the specificity of test kit.And Theophylline immunogen disclosed in above-mentioned prior art all can not embody the overall immunity of theophylline, thus cause the sensitivity of test kit and specificity not good.
Summary of the invention
An object of the present invention is to provide a kind of theophylline derivative, it can be used for preparing artificial antigen of theophylline, and its structural formula is as follows:
R is selected from following group :-CO-(CH
2)
n-COOH ,-O-(CH
2)
n-COOH ,-CS-(CH
2)
n-COOH or-(CH
2)
n-NH-COOH etc., n are the integers between 1 to 20; R is preferably-(CH
2)
6-NH-COOH.
In embodiment further, the present invention also provides a kind of artificial antigen of theophylline detected for homogeneous phase enzyme, and it is obtained by described theophylline derivative and carrier conjugation, and its structural formula is as follows:
In formula, carrier is for having immunogenic protein.
In the present invention further embodiment, carrier is preferably serum protein, hemocyanin or thyroglobulin; Be more preferably bovine serum albumin.
Another object of the present invention is to provide a kind of easy, quick, highly sensitive and full-automatic theophylline detection of drug concentration test kit and preparation method.
Described theophylline homogeneous enzyme immunoassay kit, comprises following component:
Reagent R1: the polyclonal antibody that described theophylline derivative is special;
Reagent R2: the theophylline derivative of G6PD mark;
Reagent R3: calibration solution;
It is obtained that described calibration solution is that theophylline calibration object is dissolved in blank serum;
The theophylline derivative of described G6PD mark is described theophylline derivative and G6PD coupled product, the theophylline derivative that described G6PD marks can with theophylline derivative specific antibody specific binding;
The special polyclonal antibody of described theophylline derivative is for artificial antigen obtains with the product of described theophylline derivative and bovine serum albumin coupling.
Preferably, described calibration solution is the solution of 6 kinds of different theophylline mass concentrations, and described calibration solution theophylline mass concentration is respectively 0 μ g/mL, 2.5 μ g/mL, 5.0 μ g/mL, 10.0 μ g/mL, 20.0 μ g/mL, 40.0 μ g/mL.
Preferably, the theophylline derivative extent of dilution of described G6PD mark is 1:1000-1:6000, and the special polyclonal antibody extent of dilution of theophylline derivative is 1:400-1:3000.
In order to solve the problems of the technologies described above, another technical scheme of the present invention is to provide a kind of making method of theophylline homogeneous enzyme immunoassay kit, said method comprising the steps of: the preparation of the mono-clonal that theophylline derivative is special or polyclonal antibody, the preparation of the theophylline derivative of G6PD mark, the preparation of calibration solution.
Preferably, the preparation of the polyclonal antibody that described theophylline derivative is special comprises the following steps:
Step a:
PH bovine serum albumin being dissolved in 0.2mol/L is in the phosphoric acid buffer of 8.5;
The activation of theophylline derivative: described theophylline derivative 100mg is placed in container, and add 2.5mL dimethylformamide, 4.5mL ethanol, 7mL potassium phosphate buffer, 400mg1-ethyl-3-(-3-dimethylaminopropyl) carbodiimide and 50mgN-hydroxy thiosuccinimide successively; Described potassium phosphate buffer concentration is 10mmol/L, and pH value is 5.5;
Step b: the coupling of theophylline derivative immunizing antigen and purifying: the theophylline derivative that step a activates is added drop-wise in the bovine serum albumin solution of step a gained, under 2 ~ 8 DEG C of conditions, stir 12-16 hour, the antigen of coupling is carried out the dialysis tubing dialysis purifying that aperture is 8KD; Thus obtained artificial antigen of theophylline.
Step c: the theophylline derivative immunizing antigen adopting step b to obtain prepares theophylline derivative antibody.
Preferably, described step c is:
By the theophylline immunizing antigen 10mmol/L that step b synthesizes, the phosphoric acid buffer of pH7.4 is diluted to 1.0mg/mL, then mix with Freund's complete adjuvant with theophylline immunizing antigen solution, rabbit is injected, after 14-21 days, then with identical antigenic solution and Freund's incomplete adjuvant, rabbit is injected once, afterwards every injection in 28 days once, from rabbit carries out initial injection, the antibody obtained after 4 months.
Preferably, the preparation of the theophylline derivative of described G6PD mark comprises the following steps:
A, 15mg G6PD is dissolved in 12mLTris damping fluid, then adds 225mg reduced coenzyme Ⅰ, 135mg G-6-P, 0.75mL Trivalin SF and 2.25mL dimethyl sulfoxide (DMSO) successively; Described Tris pH of buffer is 9.0, and each concentration of component is: 0.05mol/LTris, 3.3mmol/L magnesium chloride, 145.4mmol/L sodium-chlor;
The activation of b, theophylline derivative: special theophylline derivative 8.64mg is dissolved in the mixing solutions of 420 μ L dimethyl sulfoxide (DMSO) and 180 μ L dimethylformamides composition, described mixing solutions adds 6 μ L Tributylamines and 3 μ L isobutyl chlorocarbonates, stirs 30 minutes under 2 ~ 8 DEG C of conditions;
C, described step b gained dropwise is joined in step a gained solution, by magnetic stirrer 60 minutes under 2 ~ 8 DEG C of conditions, gained enzyme-labelled antigen is carried out dialysis purifying, obtains the theophylline derivative that G6PD marks.
Beneficial effect of the present invention is that theophylline drug level homogeneous enzyme immunoassay test kit of the present invention is highly sensitive, stability and reproducible.
1, high specificity of the present invention, as caffeine, closely similar with the result of theophylline, the present invention almost without any cross reaction, is applicable to the Plasma Concentration of Clinical Laboratory theophylline to 31 kinds of Common drugs of test and compound.
2, sensitivity of the present invention can reach 0.01 μ g/mL, far below the clinical application scope 10-20 μ g/mL of theophylline.
3, the present invention applies on automatic clinical chemistry analyzer, easy and simple to handle, only need sample be joined in detection reagent, the content of sample can be calculated by the change of OD340nm light absorption value, the high-throughput of clinical sample, rapid detection can be realized on automatic clinical chemistry analyzer; Meanwhile, detection reagent production domesticization can solve the problem of domestic clinical medicine Concentration Testing reagent dependence on import, cost intensive, is applicable to the clinical detection of conventional treatment drug concentration.
Embodiment
By describing technology contents of the present invention, structural attitude in detail, being realized object and effect, be explained in detail below in conjunction with embodiment.
Specific antibody containing anti-theophylline in homogeneous enzyme immunoassay detection reagent R1 of the present invention, after adding the liquid sample containing theophylline, add the R2 reagent marking theophylline derivative containing glucose-6-phosphate dehydrogenase (G6PD) (G6PDH) again, composition homogeneous enzyme immunoassay reaction system.In reaction system, antibody is combined the inactivation that can cause enzyme with the theophylline derivative of enzyme labelling, and theophylline in sample can the enzyme mark theophylline derivative of emulative replacement and antibodies, and make it discharge from the binding site of antibody, thus make enzyme activity recovery.NAD (NAD+) in reagent can be converted into nicotinamide adenine dinucleotide reduced (NADH) by active G6PDH enzyme, NADH has absorption peak at 340nm, measures tea paper mill wastewater by the change measuring light absorption value.Therefore, the marker enzyme in reaction solution is active relevant to theophylline concentration in sample, and in liquid sample, the content of theophylline is more, and free G6PDH enzyme mark theophylline is biological more, thus can obtain stronger signal.
Content of the present invention comprises the synthesis of Theophylline immunogen, the preparation of anti-theophylline specific antibody, the preparation of enzyme mark conjugate and the mensuration of sample.Method respectively by chemosynthesis prepares G6PDH-theophylline conjugate and BSA-Theophylline immunogen, and immunogen immune animal thus, prepare specific anti-theophylline polyclonal antibody.The antibody prepared and enzyme mark conjugate are mixed with homogeneous enzyme immunoassay reagent R1 and R2, automatic clinical chemistry analyzer carries out the mensuration of theophylline sample.
Core technology of the present invention is homogeneous enzyme immunoassay detection method, and wherein reagent and testing sample are liquid phase, there is not separating step in mensuration process, and the antigen adding mark after sample and antibody response again reacts.
embodiment 1the synthesis of theophylline derivative
Step 1: first use 150mL anhydrous alcohol solution 100mmol compound 1 and 120mmol compound 2, add 150mmol triethyl aluminum again and obtain the first reaction soln, by the first reaction soln heating reflux reaction 16 hours, after cooling, product is vacuumized concentrated, recrystallization and obtain the compound 3 that productive rate is 33%.
Compound 3:
ESI-MS：344.08[M-H]
-
Ultimate analysis: theoretical value/measured value, C (48.85/48.97), H (7.41/7.34), N (20.29/20.40), O (23.45/23.30).
Step 2: the NaOH solution adding the 2N of 15ml in previous step product, back flow reaction 1.5 hours in 75 DEG C of oil baths, after reaction terminates, cooling, adjusts PH=3 with 6N hydrochloric acid, occurs precipitating in a large number, filtered and recycled solid.Then 17mg compound 4(theophylline derivative is obtained through hot water recrystallization).
Utilize BrukerAvanceIIIplus300MHz to carry out NMR (Nuclear Magnetic Resonance) spectrum scanning to compound 4, adopt TMS as interior mark.Result is as follows: 1HNMR (DMSO-d6,300MHz): 12.09 (s, 1H), 6.76 (s, 1H), 6.48 (s, 1H), 5.14 (s, 1H), 3.18 (t, 2H), 3.02-3.06 (m, 6H), 2.98 (s, 1H), 2.25 (t, 2H, J=7.2Hz), 1.47-1.52 (m, 4H), 1.28-1.32 (m, 4H).
ESI-MS：324.17[M-H]
-
Ultimate analysis: theoretical value/measured value, C (51.61/51.68), H (7.21/7.13), N (21.47/21.52), O (19.71/19.67).
embodiment 2
1. the synthesis of artificial antigen of theophylline
A, bovine serum albumin (BSA) 250mg is dissolved in 100mL0.1mol/L, in the phosphoric acid buffer of pH8.5;
B, following component is joined stirring and dissolving in beaker: theophylline derivative, 2.5mL dimethylformamide (DMF), 4.5mL ethanol, 7.0mL(10mmol/L prepared by 100mg embodiment 1, pH5.0) potassium phosphate buffer, 200mg1-ethyl-3-(-3-dimethylaminopropyl) carbodiimide, 50mgN-hydroxy thiosuccinimide (Sulfo-NHS), stirring and dissolving reaction 30min; PH5.5;
The solution dissolved in above-mentioned b is dropped in the BSA solution in above-mentioned a, and stir at 2-8 DEG C and spend the night, obtain antigen;
Synthetic antigen is carried out purifying through dialysis, obtains artificial antigen of theophylline.
2. the preparation of anti-theophylline specific antibody
With PBS phosphoric acid buffer, the artificial antigen of theophylline of synthesis is diluted to 1.0mg/mL, then mixes with Freund's complete adjuvant with the antigenic solution of 1.0mL, rabbit is injected;
After 14-21 days, then inject once rabbit with the identical antigenic solution of 1.0mL and Freund's incomplete adjuvant, afterwards every surrounding once, after 4 months, the antibody titer of acquisition is about 1:320000.
3. glucose-6-phosphate dehydrogenase (G6PD) (G6PDH) marks the preparation of embodiment 1 theophylline derivative
1) G6PDH enzyme solution preparation: by 15mgG6PDH(100ku) be dissolved in 12mLTris damping fluid (0.05MTris, 3.3mmol/LMgCl2,145.4mmol/LNaCl, pH9.0).Then 225mgNADH, 135mg G-6-P (G6P) and 0.75mL Trivalin SF is added successively.Then 2.25mL methyl-sulphoxide (DMSO) is dropwise added.
2) activation of theophylline derivative: 8.64mg theophylline derivative is dissolved in the mixing solutions of 420 μ LDMSO and 180 μ LDMF.Then, after solution being cooled to 2 ~ 8 DEG C, adding 6 μ L Tributylamines and 3 μ L isobutyl chlorocarbonates, at 2 ~ 8 DEG C, stir 30min.
3) coupling of G6PDH and theophylline derivative: by step 2) in dropwise be added in solution prepared by step 1), stir 60 minutes at 2 ~ 8 DEG C, obtain conjugate.
4) conjugate is carried out purifying through dialysis, obtain the theophylline derivative of glucose-6-phosphate dehydrogenase (G6PD) enzyme labelling.
4. the preparation of homogeneous enzyme immunoassay reagent
1) preparation of R1 reagent
In 1LTris damping fluid (55mmol/LTris, 0.1%BSA, 145.4mmol/LNaCl, 3mmol/LMgCl2, pH8.0), add 2.02gNAD and 0.86gG6P successively, at room temperature stir 10min.Then the antibody obtained in being prepared by antibody adds in solution, and Dilution ratio is 1:1000-1:6000.
2) preparation of R2 reagent
In 1LTris damping fluid (120mmol/LTris, 0.1%BSA, 145.4mmol/LNaCl, 3mmol/LMgCl2, pH8.2), add G6PDH enzyme labelling theophylline derivative, Dilution ratio is 1:400-1:3000.
Adjustable antibody and the concentration of enzyme mark conjugate in damping fluid, measure working curve scope to optimize.
embodiment 3 utilizes automatic clinical chemistry analyzer to carry out test sample
(1). the collection of serum specimen, conventionally collect serum specimen;
(2). according to the operation instructions of Hitachi 7180 type full automatic biochemical apparatus, open instrument, carry out instrument optical density(OD) and detect and the cleaning of probe, detecting instrument whether normal operation;
(3). after instrument detects normal operation; reagent R1, R2 are put into R1, R2 agent bin successively; serum specimen puts into sample disc 1 (S1), and the theophylline calibration solution of 0 μ g/mL, 2.5 μ g/mL, 5.0 μ g/mL, 10.0 μ g/mL, 20.0 μ g/mL, 40.0 μ g/mL puts into the specified location of sample disc 2 (S2);
(4). instrument is when Standby state, and set schedule of operation and the inspection parameter of theophylline, concrete inspection parameter is as table one:
Table one Theophylline In Serum detection of drug concentration parameter
(5) according to the inspection parameter of setting, first carry out scaling experiment, set up the calibration curve of theophylline, then according to the calibration curve set up, detect tea paper mill wastewater in serum specimen.
(6) detect tea paper mill wastewater in serum specimen, the absorbancy velocity of variation recorded is converted into drug level according to typical curve by instrument, and prints examining report by terminal demonstration.
embodiment 4
1. theophylline homogeneous enzyme immunoassay calibration curve makes
Automatic clinical chemistry analyzer is adopted to carry out the homogeneous enzyme immunoassay inspection of theophylline, by adjusting the ratio of reagent R1 and reagent R2, can optimizing reaction system further, calibration solution theophylline mass concentration is respectively 0 μ g/mL, 2.5 μ g/mL, 5.0 μ g/mL, 10.0 μ g/mL, 20.0 μ g/mL, 40.0 μ g/mL.The working volume of calibration solution is 10-35 μ L, then 100-200 μ L reagent R1 and 100-200 μ L reagent R2 is added, adopt two point rate assay, the absorbancy velocity of variation that detection predominant wavelength is 340nm, commplementary wave length is 405nm, set up and optimize theophylline homogeneous enzyme immunoassay calibration curve, foundation and the optimization of calibration curve complete on Hitachi 7180 type automatic clinical chemistry analyzer.Obtain Working calibration curve, this curve within the scope of 2.5-40.0 μ g/mL, linearly well, R=0.99.
2. sensitivity test
The theophylline standard substance of inequality are added in blank serum, its concentration is made to be respectively 0.0 μ g/mL, 0.01 μ g/mL, 0.02 μ g/mL and 0.04 μ g/mL, each sample concentration is measured, calculating mean value and standard deviation continuous 5 times by theophylline homogeneous enzyme immunoassay detection reagent.Result shows, and the sample of the test result of 0.01 μ g/mL sample (degree of confidence 99.73%) and 0.0 μ g/mL and 0.02 μ g/mL within the scope of 3 times of standard deviations is all without intersecting.
Table two sensitivity test result
The sensitivity of the theophylline homogeneous enzyme immunoassay detection reagent that table two result shows in the present invention can reach 0.01 μ g/mL.
3. interfering effects of drug test
Choose 31 kinds of common compounds and medicine, adjusting its concentration is 10 μ g/mL, carries out interference test mensuration, test-results as shown in Table 3:
Table three interfering effects of drug test-results
Result shows the high specificity of the theophylline antibody provided in this invention, in medicine cross reaction test, to 31 kinds of Common drugs of test and compound almost without any cross reaction, is applicable to the Plasma Concentration of Clinical Laboratory theophylline.
4. precision test
In blank serum, add theophylline standard substance, it is low that preparation concentration is respectively 2.5(), in 10.0(), 40.0(is high) serum sample of μ g/mL, often kind of sample theophylline homogeneous enzyme immunoassay detection reagent replication 5 times, METHOD FOR CONTINUOUS DETERMINATION 5 days, calculates 3 kinds of concentration respectively and criticizes interior, betweenrun precision.
Table four Precision test result
5. recovery test
In blank serum, add theophylline standard substance, make its concentration be respectively 2.5,5.0,10.0,20.0 and 40.0 μ g/mL, measure each sample concentration continuous 5 times by theophylline homogeneous enzyme immunoassay detection reagent, calculate the rate of recovery.
Table five recovery test result
simultaneous test 1
In order to the superiority of theophylline homogeneous phase enzyme detection kit of the present invention is described, adopt difference but the theophylline derivative of structure and similar of the present invention, corresponding artificial antigen, polyclonal antibody and homogeneous phase enzyme reagent kit is prepared according to the method described in embodiment 1 and 2, and the experiment listed by embodiment 4 is carried out to prepared homogeneous phase enzyme reagent kit, the theophylline derivant structure that each comparative example adopts is as follows:
Comparative example 1 comparative example 2
Comparative example 3: from theophylline derivative synthesized in the step 1 of the specification sheets embodiment 1 of patent documentation CN201310285736,
Comparative example 4: from the structural formula of compound II of the 025th section, the specification sheets of patent documentation CN201210258850.
Concrete outcome is as follows:
(1) sensitivity test
According to the testing method of embodiment 4, test theophylline homogeneous phase enzyme reagent kit prepared by each comparative example, the sensitivity results of each comparative example is as follows:
(2) interfering effects of drug test
(3) precision test
simultaneous test 2
The theophylline derivative prepared by comparative example 1-4 in simultaneous test 1 is when with BSA coupling, measure the coupling ratio of each comparative example, in addition in this simultaneous test, the present invention also added comparative example 5 and 6, the theophylline derivative that comparative example 5 and 6 adopts is theophylline derivative prepared by embodiment 1, but slightly different, specific as follows from the step 1 of embodiment 2 in some parameters of preparation method:
Except upper table parameter, other preparation methods are with embodiment 2 for comparative example 5 and 6
The measuring method of coupling ratio: see the specification sheets 07-09 section of patent documentation 201310285736, concrete outcome is as follows:
The foregoing is only embodiments of the invention; not thereby the scope of the claims of the present invention is limited; every utilize specification sheets of the present invention to do equivalent structure or the conversion of equivalent flow process, or be directly or indirectly used in other relevant technical fields, be all in like manner included in scope of patent protection of the present invention.
Claims (7)
1. a theophylline derivative, it can be used for preparing artificial antigen of theophylline, and its structural formula is as follows:
R is-(CH
2)
6-NH-COOH.
2., for the artificial antigen of theophylline that homogeneous phase enzyme detects, it is obtained by theophylline derivative according to claim 1 and carrier conjugation, and its structural formula is as follows:
In formula, carrier is serum protein, hemocyanin or thyroglobulin.
3. artificial antigen of theophylline according to claim 2, is characterized in that, carrier is bovine serum albumin
4. a theophylline homogeneous enzyme immunoassay kit, is characterized in that, it is composed of the following components:
Reagent R1: the polyclonal antibody that theophylline derivative according to claim 1 is special;
Reagent R2: theophylline derivative described in the claim 1 of G6PD mark;
Reagent R3: calibration solution.
5. prepare a method for theophylline homogeneous enzyme immunoassay kit according to claim 4, it is characterized in that, concrete steps are as follows:
The preparation of the mono-clonal that theophylline derivative is special or polyclonal antibody, the preparation of the theophylline derivative of G6PD mark, the preparation of calibration solution.
6. preparation method according to claim 5, is characterized in that, the preparation of the polyclonal antibody that described theophylline derivative is special comprises the following steps:
Step a:
PH bovine serum albumin being dissolved in 0.2mol/L is in the phosphoric acid buffer of 8.5;
The activation of theophylline derivative: described theophylline derivative 100mg is placed in container, and add 2.5mL dimethylformamide, 4.5mL ethanol, 7mL potassium phosphate buffer, 400mg1-ethyl-3-(-3-dimethylaminopropyl) carbodiimide and 50mgN-hydroxy thiosuccinimide successively; Described potassium phosphate buffer concentration is 10mmol/L, and pH value is 5.5;
Step b: the coupling of theophylline derivative immunizing antigen and purifying: the theophylline derivative that step a activates is added drop-wise in the bovine serum albumin solution of step a gained, under 2 ~ 8 DEG C of conditions, stir 12-16 hour, the antigen of coupling is carried out the dialysis tubing dialysis purifying that aperture is 8KD; Thus obtained artificial antigen of theophylline.
Step c: the theophylline immunizing antigen 10mmol/L that step b is synthesized, the phosphoric acid buffer of pH7.4 is diluted to 1.0mg/mL, then mix with Freund's complete adjuvant with theophylline immunizing antigen solution, rabbit is injected, after 14-21 days, then with identical antigenic solution and Freund's incomplete adjuvant, rabbit is injected once, afterwards every injection in 28 days once, from rabbit carries out initial injection, the antibody obtained after 4 months.
7. preparation method according to claim 6, is characterized in that, the preparation of the theophylline derivative of described G6PD mark comprises the following steps:
A, 15mg G6PD is dissolved in 12mLTris damping fluid, then adds 225mg reduced coenzyme Ⅰ, 135mg G-6-P, 0.75mL Trivalin SF and 2.25mL dimethyl sulfoxide (DMSO) successively; Described Tris pH of buffer is 9.0, and each concentration of component is: 0.05mol/LTris, 3.3mmol/L magnesium chloride, 145.4mmol/L sodium-chlor;
The activation of b, theophylline derivative: special theophylline derivative 8.64mg is dissolved in the mixing solutions of 420 μ L dimethyl sulfoxide (DMSO) and 180 μ L dimethylformamides composition, described mixing solutions adds 6 μ L Tributylamines and 3 μ L isobutyl chlorocarbonates, stirs 30 minutes under 2 ~ 8 DEG C of conditions;
C, described step b gained dropwise is joined in step a gained solution, by magnetic stirrer 60 minutes under 2 ~ 8 DEG C of conditions, gained enzyme-labelled antigen is carried out dialysis purifying, obtains the theophylline derivative that G6PD marks.
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| CN106405069A (en) * | 2016-04-06 | 2017-02-15 | 李松羊 | Preparation method for homogeneous enzyme immunodiagnosis reagent used for glycocholic acid |
| CN106596917B (en) * | 2016-12-19 | 2018-04-20 | 苏州博源医疗科技有限公司 | Homovanillic acid homogeneous enzyme immunoassay detection reagent, preparation method and detection method |
| CN108794620A (en) * | 2017-05-04 | 2018-11-13 | 南开大学 | Conjugate of theophylline and the preparation method and application thereof |
| CN109517056B (en) * | 2018-11-05 | 2022-02-11 | 杭州旭科生物技术有限公司 | Synthetic method and application of theophylline artificial antigen |
| CN110174363A (en) * | 2019-01-09 | 2019-08-27 | 北京九强生物技术股份有限公司 | Glucose-6-phosphate dehydrogenase mutant and its purposes in preparation detection reagent |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4302438A (en) * | 1979-01-13 | 1981-11-24 | Byk Gulden Lomberg Chemische Fabrik Gmbh | Antigen, antiserum and immunoassay for theophylline |
| CN102253215A (en) * | 2011-06-07 | 2011-11-23 | 济南金域医学检验中心有限公司 | Theophylline homogeneous enzyme immunoassay kit and preparation method thereof |
| CN103242446A (en) * | 2012-07-25 | 2013-08-14 | 苏州博源医疗科技有限公司 | Theophylline immunogen and preparation method and application thereof |
| CN103360488A (en) * | 2013-07-05 | 2013-10-23 | 杭州博林生物技术有限公司 | Preparation method for artificial antigen of theophylline |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4302438A (en) * | 1979-01-13 | 1981-11-24 | Byk Gulden Lomberg Chemische Fabrik Gmbh | Antigen, antiserum and immunoassay for theophylline |
| CN102253215A (en) * | 2011-06-07 | 2011-11-23 | 济南金域医学检验中心有限公司 | Theophylline homogeneous enzyme immunoassay kit and preparation method thereof |
| CN103242446A (en) * | 2012-07-25 | 2013-08-14 | 苏州博源医疗科技有限公司 | Theophylline immunogen and preparation method and application thereof |
| CN103360488A (en) * | 2013-07-05 | 2013-10-23 | 杭州博林生物技术有限公司 | Preparation method for artificial antigen of theophylline |
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