CN104597238B - A kind of mycophenolic acid homogeneous enzyme immunoassay detectable and preparation thereof and detection method - Google Patents
A kind of mycophenolic acid homogeneous enzyme immunoassay detectable and preparation thereof and detection method Download PDFInfo
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Abstract
The present invention relates to a kind of mycophenolic acid detectable and preparation thereof and detection method, it is specifically related to a kind of mycophenolic acid homogeneous enzyme immunoassay detectable and preparation thereof and detection method, including: anti-mildew phenolic acid specific antibody, for detecting the indicator of anti-mildew phenolic acid specific antibody mycophenolic acid complex；Above-mentioned anti-mildew phenolic acid specific antibody is obtained by mycophenolic acid immunogens immune animal.The invention have benefit that: the mycophenolic acid immunogens high specificity of the present invention, immunogenicity are high, anti-mildew phenolic acid specific antibody high specificity, the titer prepared are high, and with 62 kinds of common medicines without any cross reaction；Homogeneous enzyme immunoassay detectable containing above-mentioned anti-mildew phenolic acid specific antibody can easily and fast, the mycophenolic acid content that accurately determines in sample, and multiple sample can be measured on automatic clinical chemistry analyzer simultaneously, realize the rapid mensuration of high flux of mycophenolic acid, accuracy is high, high specificity, degree of accuracy and detection efficiency are enhanced.
Description
Technical field
The present invention relates to a kind of mycophenolic acid detectable and preparation thereof and detection method, be specifically related to a kind of mycophenolic acid homogeneous
Enzyme immunologic function test reagent and preparation thereof and detection method.
Background technology
Mycophenolic acid (Mycophenolic acid) structural formula is as shown in formula III:
Mycophenolic acid, as a kind of immunosuppressant, is widely used in suppressing the organs such as kidney, liver and heart to move clinically
Rejection during planting.Mycophenolic acid can combine hypoxanthine mononucleotide dehydrogenase (IMPDH) noncompetitive, then
Person is the key enzyme of guanylic acid de novo synthesis during T, B lymphocyte proliferation.Its clinical effective blood drug concentration is narrower
(clinical effective blood drug concentration is 1.0-3.5 μ g/mL), cannot play immunosuppressive action less than its effective blood drug concentration, reach not
To corresponding curative effect；Then can produce serious nephrotoxicity higher than its effective blood drug concentration, other side reactions also include Nausea and vomiting,
Have a stomachache, lose weight, heating etc., also result in patient's immunosuppressant time serious and excessively infect death.Therefore, to using mould phenol
The patient of acid treatment carries out therapeutic drug monitoring for instructing clinical safety rational use of drug and personalized medicine significant.
Monitoring mycophenolic acid blood drug level mainly uses high performance liquid chromatography, enzyme to amplify immunoassay, chemistry both at home and abroad
The method such as luminescence method, immunoturbidimetry, but these methods all have certain defective in clinical practice.Siemens, Roche
Although the mycophenolic acid that the external producer such as diagnosis can be applicable to biochemistry analyzer measures test kit listing, but far from meeting
Growing Clinical detection demand.The most still deficient in stability mycophenolic acid good, highly sensitive, high specificity detects
Reagent, especially matter measured Automated inspection reagent.Therefore, development & production quality reaches clinical requirement, practical, sexual valence
Ratio is high, and the mycophenolic acid mensuration reagent that can be applicable to automatic clinical chemistry analyzer has become the heat of domestic and international external diagnosis reagent industry
Point.The mycophenolic acid homogeneous enzyme immunoassay detectable of the present invention can be implemented in the high pass on automatic clinical chemistry analyzer to mycophenolic acid
Amount, rapid detection, and there is the advantages such as easy and simple to handle, highly sensitive, high specificity, result are accurate, moreover it is possible to effectively reduce mould
Phenolic acid testing cost, beneficially clinical expansion use.
Summary of the invention
For solving the deficiencies in the prior art, it is an object of the invention to provide one not only safety but also can be quick, efficient, clever
Quick, mycophenolic acid homogeneous enzyme immunoassay detectable accurately detecting mycophenolic acid content in sample to be tested and preparation method thereof, and
Can be combined with various types of automatic biochemistry analyzers, mycophenolic acid homogeneous enzyme immunoassay detection side less demanding to testing staff
Method.
Another object of the present invention is for providing a kind of mycophenolic acid contributing to preparing rapid and accurate determination mycophenolic acid content
The derivatives of mycophenolic acid of homogeneous enzyme immunoassay detectable.
In order to realize above-mentioned target, the technical solution used in the present invention is:
A kind of mycophenolic acid homogeneous enzyme immunoassay detectable, including: anti-mildew phenolic acid specific antibody, is used for detecting anti-mildew phenolic acid
The indicator of specific antibody-mycophenolic acid complex；Above-mentioned anti-mildew phenolic acid specific antibody is moved by mycophenolic acid immunogens immunity
Thing obtains, and the structural formula of mycophenolic acid immunogens is as shown in formula I:
In formula, carrier is for having immunogenic protein；Above-mentioned indicator is selected from enzyme reagent, radiosiotope examination
Agent, fluorometric reagent or chemical illuminating reagent.
Above-mentioned indicator is selected from enzyme reagent, including: enzyme mark conjugate and the substrate of enzyme；Above-mentioned enzyme mark conjugate includes Portugal
Glucose-6-phosphate dehydrogenase-hapten enzyme mark conjugate；The substrate of above-mentioned enzyme is G-6-P；
Described glucose-6-phosphate dehydrogenase (G6PD)-hapten enzyme mark conjugate is by glucose-6-phosphate dehydrogenase (G6PD) and mycophenolic acid
Derivatives reaction is formed.
The structural formula of above-mentioned derivatives of mycophenolic acid is as shown in formula II:
The synthetic method of derivatives of mycophenolic acid, it is characterised in that synthetic route is:
The preparation method of a kind of mycophenolic acid homogeneous enzyme immunoassay detectable, it is characterised in that comprise the steps:
(1) synthesis of derivatives of mycophenolic acid and purification, and carry out Structural Identification；
(2) synthesis of mycophenolic acid immunogens: make the terminal carboxyl group of mycophenolic acid connect with having immunogenic protein carrier
Connect；
(3) mycophenolic acid immunogens immune animal, preparation purification anti-mildew phenolic acid specific antibody are used；
(4) preparation of glucose-6-phosphate dehydrogenase (G6PD)-hapten conjugation thing: prepare glucose-6-phosphate dehydrogenase (G6PD) molten
Liquid, activates derivatives of mycophenolic acid, makes glucose-6-phosphate dehydrogenase (G6PD) be connected with the terminal carboxyl group of mycophenolic acid, and purification connects product；
(5) preparation of mycophenolic acid homogeneous enzyme immunoassay detectable:
The preparation of reagent A: mixed by anti-mildew phenolic acid specific antibody and homogeneous zymolyte；
The preparation of reagent B: mixed with Tris buffer by glucose-6-phosphate dehydrogenase (G6PD)-hapten conjugation thing.
The preparation method of aforesaid a kind of mycophenolic acid homogeneous enzyme immunoassay detectable, in described step (2), protein carries
Body is BSA, and concrete synthesis step is as follows:
A. weigh 2.72g potassium dihydrogen phosphate, 4.26g disodium hydrogen phosphate, 8.5g sodium chloride, 0.95g magnesium chloride, jointly dissolve
In 1L deionized water, regulate pH to 7.4, make buffer solution A；
B. weigh and be dissolved under 3mg BSA, room temperature in 3mL above-mentioned buffer solution A, make BSA solution；
C. weigh 3mg derivatives of mycophenolic acid, be dissolved in 300ul above-mentioned buffer solution A, make derivatives of mycophenolic acid molten
Liquid；
D., when above-mentioned derivatives of mycophenolic acid solution just becomes clarification, it is added dropwise in above-mentioned BSA solution, then by this
Mixed solution stirs 1 hour at 2-8 DEG C；
E. being dialysed with above-mentioned buffer solution A by reacted above-mentioned mixed solution, after dialysis, gained solution is mould
Phenolic acid immunogen solution, adds the NaN of mass fraction 0.1% in mycophenolic acid immunogens solution3, store at-20 DEG C.
The preparation method of aforesaid a kind of mycophenolic acid homogeneous enzyme immunoassay detectable, described step (4) detailed process is:
A. weigh 1.09g potassium dihydrogen phosphate, 1.70g disodium hydrogen phosphate, 8.5g sodium chloride, 0.95g magnesium chloride, jointly dissolve
In 1L deionized water, regulate pH to 7.4, make buffer solution B；
B. weigh and be dissolved under 3mg glucose-6-phosphate dehydrogenase (G6PD), room temperature in 3mL above-mentioned buffer solution B, make Fructus Vitis viniferae
Sugar-6-phosphate dehydrogenase enzymatic solution；
C. weigh 3mg derivatives of mycophenolic acid, be dissolved in 300ul above-mentioned buffer solution B, make derivatives of mycophenolic acid molten
Liquid；
D., when above-mentioned derivatives of mycophenolic acid solution just becomes clarification, it is added dropwise over above-mentioned G-6-P dehydrogenation
In enzymatic solution, then this mixed solution is stirred 1 hour at 2-8 DEG C；
E. being dialysed with above-mentioned buffer solution B by reacted above-mentioned mixed solution, after dialysis, gained solution is Portugal
Glucose-6-phosphate dehydrogenase-hapten conjugation thing solution, adds in glucose-6-phosphate dehydrogenase (G6PD)-hapten conjugation thing solution
Enter the BSA and the NaN of mass fraction 0.1% of mass fraction 0.5%3, store at 2-8 DEG C.
The preparation method of aforesaid a kind of mycophenolic acid homogeneous enzyme immunoassay detectable, the detailed process of step (5) is as follows:
The preparation of reagent A: by the nicotinamide adenine dinucleotide (NAD) of 4.036g (11.25mM) oxidation state, 1.711g
(11.25mM) G-6-P (G-6-P) the Tris buffer solution of 1L55mM, pH=8.0 makes homogeneous zymolyte；
The anti-mildew phenolic acid specific antibody of preparation being added in above-mentioned homogeneous zymolyte, antibody is 1 with the volume ratio of homogeneous zymolyte:
100～1:10000；
The preparation of reagent B: the glucose-6-phosphate dehydrogenase (G6PD) of preparation-hapten conjugation thing is added to 120mM, pH=8.2
Tris buffer in, the volume ratio of above-mentioned conjugate and Tris buffer is 1:100～1:10000.
Utilize the detection method of mycophenolic acid homogeneous enzyme immunoassay detectable, it is characterised in that comprise the following steps:
1) sample to be tested is contacted with anti-mildew phenolic acid specific antibody；
2) according to the combination situation of mycophenolic acid in sample to be tested Yu anti-mildew phenolic acid specific antibody,
Utilize the content of mycophenolic acid in indicator judgment sample；
Described sample to be tested is serum, blood plasma, saliva or urine；
Described indicator is selected from enzyme reagent, including: enzyme mark conjugate and the substrate of enzyme；Above-mentioned enzyme mark conjugate includes
Glucose-6-phosphate dehydrogenase (G6PD)-hapten enzyme mark conjugate；The substrate of above-mentioned enzyme is G-6-P；Described glucose-
6-phosphate dehydrogenase-hapten enzyme mark conjugate is reacted with derivatives of mycophenolic acid by glucose-6-phosphate dehydrogenase (G6PD) and is formed.
The invention have benefit that: the mycophenolic acid immunogens high specificity of the present invention, immunogenicity are high, prepare
Anti-mildew phenolic acid specific antibody high specificity, titer are high, and with 62 kinds of common medicines without any cross reaction；Containing above-mentioned
The homogeneous enzyme immunoassay detectable of anti-mildew phenolic acid specific antibody can easily and fast, the mycophenolic acid accurately determined in sample
Content, and multiple sample can be measured on automatic clinical chemistry analyzer simultaneously, it is achieved the rapid survey of high flux of mycophenolic acid
Fixed, accuracy is high, and high specificity, degree of accuracy is all enhanced before comparing with detection efficiency, is simultaneously achieved detection
The full-automation of process, less demanding to testing staff, it is easy to accomplish and promote the use of.
Accompanying drawing explanation
Fig. 1 is mycophenolic acid homogeneous enzyme immunoassay reaction normal curve chart；
Fig. 2 is mycophenolic acid homogeneous enzyme immunoassay correlation analysis figure.
Detailed description of the invention
The technical solution used in the present invention is:
Mycophenolic acid immunogens, its structural formula is as shown in formula I:
In formula, carrier has immunogenicity, it is preferred that carrier is for having immunogenic protein.Although other are enough
Big possess immunogenic material and as carrier, but protein can also be selected under normal circumstances as carrier.The most frequently used
Immunogenic carrier includes serum albumin, hemocyanin (KLH) and Elityran.Carrier in the present invention is preferably serum
Albumen.
A kind of anti-mildew phenolic acid specific antibody, is obtained by the mycophenolic acid immunogens immune animal shown in formula I.
In the present invention, " antibody " of indication refers not only to complete antibody molecule, also includes retaining complete antibody specificity knot
The antibody fragment of conjunction ability or derivant.The antibody of the present invention can be polyclonal antibody can also be monoclonal antibody, excellent
Elect polyclonal antibody as.
The method obtaining polyclonal antibody is to use the mycophenolic acid immunogens shown in formula I, after adding or being not added with adjuvant,
Carrying out immunity at one or more position of animal, host animal includes: rabbit, goat, mice, sheep, Cavia porcellus or horse.Continue
Immunity is carried out always, until antibody titer reaches the highest.Animal timing blood sampling obtains appropriate specific corrosioning anteserum, and antiserum is permissible
Purification.
Monoclonal antibody can be made by somatocyte hybriding technology.
A kind of mycophenolic acid homogeneous enzyme immunoassay detectable, including: above-mentioned anti-mildew phenolic acid specific antibody, is used for detecting anti-mildew
The indicator of phenolic acid specific antibody-mycophenolic acid complex.Indicator is selected from enzyme reagent, radiosiotope reagent, glimmering
Light reagent and chemical illuminating reagent.Preferably, indicator is enzyme reagent, including: enzyme mark conjugate and the substrate of enzyme.Wherein,
Enzyme mark conjugate includes glucose-6-phosphate dehydrogenase (G6PD)-hapten enzyme mark conjugate, and it can be obtained by chemical synthesis process.
The using method of above-mentioned mycophenolic acid homogeneous enzyme immunoassay detectable, comprises the following steps:
1) sample to be tested is contacted with above-mentioned anti-mildew phenolic acid specific antibody；
2) according to the combination situation of mycophenolic acid in sample to be tested Yu above-mentioned anti-mildew phenolic acid specific antibody, indicator is utilized
The content of mycophenolic acid in judgment sample.
Sample to be tested is various physiology samples, such as serum, blood plasma, urine, saliva etc..Preferably, sample to be tested is blood
Clear or blood plasma.
Below in conjunction with specific embodiment, further illustrate the present invention.
Embodiment one: the synthesis of derivatives of mycophenolic acid and structural confirmation thereof
The derivatives of mycophenolic acid chemical constitution used in following example is as shown in formula II:
The synthetic route of this derivatives of mycophenolic acid is as follows:
Concrete synthesis step is as follows:
The synthesis of compound 2
Weigh 5.0g compound 1, be dissolved in the MeOH of 50mL, at 0 DEG C, be added dropwise over 4.0g (34.1mmol) SOCl2,
Then this reaction mixture is stirred overnight at 70 DEG C, makes solvent evaporate by decompression method, by residue by 200mL's
The NaHCO of DCM and 200mL3Saturated aqueous solution separates, and is rinsed by organic facies salt, then passes through Na2SO4Do
Dry, and carry out being concentrated to give 4.9g compound as white solid 2, productivity 91%.
The synthesis of compound 3
Weigh 4.9g compound 2, be dissolved in the MeOH of 100mL, at 0 DEG C, add 1.3g (33.3mmol) several times
NaBH4, then this reaction mixture is stirred at room temperature overnight, reacted mixture is concentrated, obtains after concentration
Residue be purified by silica dehydrator post (DCM:MeOH=10:1), obtain the compound 3 of 4.4g colorless oil, productivity
95%.
The synthesis of derivatives of mycophenolic acid
Weigh 2.2g compound 3, be dissolved in the THF of 40mL, under 0 DEG C and nitrogen protective condition, add 1.2g
(12.6mmol) maleimide, 3.8g (14.56mmol) PPh3And 2.9g (14.56mmol) DIAD, then by mixed for this reaction
Closing liquid to be stirred overnight at 70 DEG C, concentrated by reacted mixture, the residue obtained after concentration passes through silica dehydrator post
(DCM:MeOH=50:1) it is purified, finally gives 400mg tan solid Compound 4, productivity 12%.This compound 4 is i.e.
For the derivatives of mycophenolic acid shown in formula II.
The synthesis of embodiment two: BSA-mycophenolic acid immunogens
BSA-mycophenolic acid immunogens is connected with the terminal carboxyl group of the mycophenolic acid shown in formula III by bovine serum albumin (BSA)
Forming, this immunogenic concrete synthesis step is as follows:
A. weigh 2.72g potassium dihydrogen phosphate, 4.26g disodium hydrogen phosphate, 8.5g sodium chloride, 0.95g magnesium chloride, jointly dissolve
In 1L deionized water, regulate pH to 7.4, make buffer solution A；
B. weigh and be dissolved under 3mg BSA, room temperature in 3mL above-mentioned buffer solution A, make BSA solution；
C. weigh 3mg derivatives of mycophenolic acid, be dissolved in 300ul above-mentioned buffer solution A, make derivatives of mycophenolic acid molten
Liquid；
D., when above-mentioned derivatives of mycophenolic acid solution just becomes clarification, it is added dropwise in above-mentioned BSA solution, then by this
Mixed solution stirs 1 hour at 2-8 DEG C；
E. being dialysed with above-mentioned buffer solution A by reacted above-mentioned mixed solution, after dialysis, gained solution is mould
Phenolic acid immunogen solution, adds the NaN of 0.1% in mycophenolic acid immunogens solution3, store at-20 DEG C.The NaN of 0.1%3It is
Referring to that addition accounts for the mass percent of the immunogen solution of final gained, concrete addition is according to the immunogen of gained after dialysis
Depending on the concrete quality of solution.
Embodiment three: the preparation of anti-mildew phenolic acid specific antibody
Above-mentioned prepared BSA-mycophenolic acid immunogens is used conventional method inoculation experiments animal rabbit, takes anti-after booster immunization
Serum, specifically comprises the following steps that
With PBS, the BSA-mycophenolic acid immunogens of above-mentioned synthesis is diluted to 1.0mg/ml, obtains antigenic solution, then use
1.0ml antigenic solution mixes with Freund's complete adjuvant, injects experimental animal rabbit.
After 2～3 weeks, more above-mentioned experimental animal rabbit is injected with incomplete Freund's adjuvant with antigenic solution identical for 1.0ml
Once, inject once every surrounding afterwards, amount to injection 4 times.
Above-mentioned experimental animal rabbit takes blood, and isolated and purified to obtain the anti-mildew phenolic acid that titer is 1: 30000-1: 50000 special
Property antibody.
Embodiment four: the preparation of glucose-6-phosphate dehydrogenase (G6PD)-hapten conjugation thing
A. weigh 1.09g potassium dihydrogen phosphate, 1.70g disodium hydrogen phosphate, 8.5g sodium chloride, 0.95g magnesium chloride, jointly dissolve
In 1L deionized water, regulate pH to 7.4, make buffer solution B；
B. weigh and be dissolved under 3mg glucose-6-phosphate dehydrogenase (G6PD), room temperature in 3mL above-mentioned buffer solution B, make Fructus Vitis viniferae
Sugar-6-phosphate dehydrogenase enzymatic solution；
C. weigh 3mg derivatives of mycophenolic acid, be dissolved in 300ul above-mentioned buffer solution B, make derivatives of mycophenolic acid molten
Liquid；
D., when above-mentioned derivatives of mycophenolic acid solution just becomes clarification, it is added dropwise over above-mentioned G-6-P dehydrogenation
In enzymatic solution, then this mixed solution is stirred 1 hour at 2-8 DEG C；
E. being dialysed with above-mentioned buffer solution B by reacted above-mentioned mixed solution, after dialysis, gained solution is Portugal
Glucose-6-phosphate dehydrogenase-hapten conjugation thing solution, adds in glucose-6-phosphate dehydrogenase (G6PD)-hapten conjugation thing solution
Enter the BSA of the 0.5% and NaN of 0.1%3, store at 2-8 DEG C.The BSA of the 0.5% and NaN of 0.1%3Refer to that addition accounts for
The mass percent of the conjugate solution of whole gained, concrete addition is according to the concrete matter of the conjugate solution of gained after dialysis
Depending on amount.
Embodiment five: the preparation of mycophenolic acid homogeneous enzyme immunoassay detectable
Mycophenolic acid homogeneous enzyme immunoassay detectable, including: above-mentioned anti-mildew phenolic acid specific antibody, it is used for detecting anti-mildew phenolic acid
The indicator of specific antibody-mycophenolic acid complex.Indicator is selected from enzyme reagent, radiosiotope reagent, fluorescence examination
Agent and chemical illuminating reagent.Preferably, indicator is enzyme reagent, including: enzyme mark conjugate and the substrate of enzyme.Wherein, enzyme mark
Conjugate includes glucose-6-phosphate dehydrogenase (G6PD)-hapten enzyme mark conjugate, and it is obtained by above-mentioned chemical synthesis process.
Mycophenolic acid homogeneous enzyme immunoassay detectable before the use, in order to avoid the enzyme mark conjugate in indicator and enzyme
Substrate react, the substrate of enzyme mark conjugate and enzyme is separated, does not mixes, so the substrate of enzyme is anti-with above-mentioned
Mycophenolic acid specific antibody mixes.It is to say, mycophenolic acid homogeneous enzyme immunoassay detectable includes that two kinds are provided separately
Reagent, specific as follows:
1. the preparation of reagent A: by the nicotinamide adenine dinucleotide (NAD) of 4.036g (11.25mM) oxidation state,
1.711g (11.25mM) G-6-P (G-6-P) is placed in beaker D, molten with the Tris buffer of 1L55mM, pH=8.0
Solution makes homogeneous zymolyte；The anti-mildew phenolic acid specific antibody of above-mentioned preparation being added in above-mentioned homogeneous zymolyte, antibody is with equal
The volume ratio of phase zymolyte can be 1:100～1:10000, and the most concrete ratio is 1:400.
2. the preparation of reagent B: the glucose-6-phosphate dehydrogenase (G6PD) of above-mentioned preparation-hapten conjugation thing is added to 120mM,
In the Tris buffer of pH=8.2, above-mentioned conjugate can be 1:100～1:10000 with the volume ratio of Tris buffer, at this
Ratio concrete in embodiment is 1:1500.
The using method of above-mentioned mycophenolic acid homogeneous enzyme immunoassay detectable, comprises the following steps:
1) sample to be tested is contacted with above-mentioned anti-mildew phenolic acid specific antibody；
2) according to the combination situation of mycophenolic acid in sample to be tested Yu above-mentioned anti-mildew phenolic acid specific antibody, indicator is utilized
Judge the content of mycophenolic acid in sample to be tested.
Concrete, during detection, sample to be tested is added in reagent A, the mycophenolic acid in sample to be tested and the anti-mildew in reagent A
Phenolic acid specific antibody occurs specific binding, generates anti-mildew phenolic acid specific antibody-mycophenolic acid complex；Add reagent B,
Now the glucose-6-phosphate dehydrogenase (G6PD) in reagent B-hapten conjugation thing mixes with the substrate of the enzyme in reagent A, contacts, and sends out
Raw enzymatic reaction, constitutes the indicator of detection anti-mildew phenolic acid specific antibody-mycophenolic acid complex, and indicator is according to be measured
In sample, mycophenolic acid judges the content of mycophenolic acid in sample to be tested with the combination situation of above-mentioned anti-mildew phenolic acid specific antibody.
Owing to glucose-6-phosphate dehydrogenase (G6PD)-hapten conjugation thing resists with the mycophenolic acid competitive binding in sample to be tested
Mycophenolic acid specific antibody, so, in sample to be tested, the amount of mycophenolic acid is the most, glucose-6-phosphorus free in homogeneous enzymatic solution
The amount of acidohydrogenase-hapten conjugation thing is the most, and enzymatic reaction is the fastest, causes OD340Rise.
Above-mentioned sample to be tested is physiology sample, such as serum, blood plasma, urine, saliva etc..
As the preferred scheme of one, above-mentioned sample to be tested is serum or blood plasma.
Embodiment six: mycophenolic acid homogeneous enzyme immunoassay is checked
1, obtain standard curve: auspicious BS200 automatic clinical chemistry analyzer response parameter (being shown in Table 1) advanced in years, operating process are set
For: first reagent adding A, add standard substance, be eventually adding reagent B.After adding reagent B, measure the OD of different time points340Extinction
Value, calculates reaction rate during various criterion product concentration, needs constantly to adjust reagent A and the volume of reagent B in actual mechanical process
Ratio, adjusts light-metering point simultaneously, finally draws comparatively ideal reaction normal curve chart, as shown in Figure 1.
Table 1 steps auspicious BS200 automatic clinical chemistry analyzer response parameter
The standard curve obtained by the homogeneous enzyme immunoassay detectable of the present invention, replication basic, normal, high concentration Quality Control
Sample 10 times, above-mentioned Quality Control sample is: be dissolved in human serum by mycophenolic acid standard substance, to concentration be respectively 1.00,5.00,
20.00μg/ml.Detection data and data analysis are shown in Table 2.
Table 2 sample determination and precision and the response rate are assessed
| Blood sample | Low | In | High |
| Sample concentration (μ g/ml) | 1.00 | 5.00 | 20.00 |
| 1 | 0.96 | 5.16 | 19.90 |
| 2 | 0.95 | 5.21 | 20.45 |
| 3 | 1.04 | 4.96 | 20.16 |
| 4 | 0.97 | 5.27 | 19.79 |
| 5 | 1.03 | 5.15 | 20.73 |
| 6 | 1.05 | 4.89 | 20.52 |
| 7 | 0.98 | 5.12 | 19.25 |
| 8 | 1.03 | 4.88 | 19.90 |
| 9 | 1.00 | 5.14 | 21.33 |
| 10 | 1.02 | 4.83 | 19.41 |
| Meansigma methods (μ g/ml) | 1.00 | 5.06 | 20.14 |
| Standard deviation (SD) | 0.0359 | 0.1560 | 0.6296 |
| Precision (CV%) | 3.59 | 3.08 | 3.13 |
| Response rate % | 100.0 | 101.2 | 100.7 |
Testing result: the accuracy that the homogeneous enzyme immunoassay detectable of the present invention measures is high, and the response rate reaches 95%-
105%, precision is high, and CV is below 5%.
Embodiment seven: interfering effects of drug is tested
Choosing 62 kinds of Common drugs, adjustment concentration, to 10.0 μ g/ml, carries out interference test mensuration.62 kinds of common medicines
And measurement result is referring specifically to table 3.
Table 3 common interference medicine and measurement result
Measurement result: the concentration that above-mentioned 62 kinds of Common drugs are equivalent to mycophenolic acid is respectively less than 0.1 μ g/ml.Visible, the present invention
Antibody be the specific antibody of anti-mildew phenolic acid.
Embodiment eight: correlation analysis
100 example clinical samples are used respectively the mycophenolic acid detection examination of medical diagnostic prods (Shanghai) Co., Ltd. of Siemens
Agent (using enzyme to amplify immunoassay) and the homogeneous enzyme immunoassay reagent of the present invention carry out correlation analysis, and the data of mensuration see
Table 4.
Table 4 clinical sample measured value
Mapping above-mentioned data, see Fig. 2, the linear equation obtained is: y=0.9882x+0.0399, coefficient R2
=0.9994, show that the detectable of the present invention measures the accuracy height of mycophenolic acid clinical samples.
Owing to the detection process of the present invention is to be completed by instrument full-automation, so less demanding, easily to testing staff
In realizing and promoting the use of.
It should be noted that the foregoing is only embodiments of the invention, not thereby limit the scope of the claims of the present invention,
Every equivalent structure utilizing description of the invention and accompanying drawing content to be done or equivalence flow process conversion, or be directly or indirectly used in
Other correlative technology fields, are the most in like manner included in the scope of patent protection of the present invention.
Claims (6)
1. the synthetic method of derivatives of mycophenolic acid, it is characterised in that derivatives of mycophenolic acid, structural formula is as shown in formula II:
Synthetic route is:
2. the preparation method of a mycophenolic acid homogeneous enzyme immunoassay detectable, it is characterised in that comprise the steps:
(1) synthesis of the derivatives of mycophenolic acid described in claim 1 and purification, and carry out Structural Identification；Derivatives of mycophenolic acid
Synthetic route is:
(2) synthesis of mycophenolic acid immunogens: make the terminal carboxyl group of mycophenolic acid be connected with having immunogenic protein carrier；
(3) mycophenolic acid immunogens immune animal, preparation purification anti-mildew phenolic acid specific antibody are used；
(4) preparation of glucose-6-phosphate dehydrogenase (G6PD)-hapten conjugation thing: prepare glucose-6-phosphate dehydrogenase (G6PD) solution, swashs
Derivatives of mycophenolic acid alive, makes glucose-6-phosphate dehydrogenase (G6PD) be connected with derivatives of mycophenolic acid, and purification connects product；
(5) preparation of mycophenolic acid homogeneous enzyme immunoassay detectable:
The preparation of reagent A: mixed by anti-mildew phenolic acid specific antibody and homogeneous zymolyte；
The preparation of reagent B: mixed with Tris buffer by glucose-6-phosphate dehydrogenase (G6PD)-hapten conjugation thing.
The preparation method of a kind of mycophenolic acid homogeneous enzyme immunoassay detectable the most according to claim 2, it is characterised in that institute
In the step (2) stated, protein carrier is BSA, and concrete synthesis step is as follows:
A. weigh 2.72g potassium dihydrogen phosphate, 4.26g disodium hydrogen phosphate, 8.5g sodium chloride, 0.95g magnesium chloride, be jointly dissolved in 1L
In deionized water, regulate pH to 7.4, make buffer solution A；
B. weigh and be dissolved under 3mg BSA, room temperature in 3mL above-mentioned buffer solution A, make BSA solution；
C. weigh 3mg mycophenolic acid, be dissolved in 300ul above-mentioned buffer solution A, make mycophenolic acid solution；
D., when above-mentioned mycophenolic acid solution just becomes clarification, it is added dropwise in above-mentioned BSA solution, then this mixed solution is existed
Stir 1 hour at 2-8 DEG C；
E. being dialysed with above-mentioned buffer solution A by reacted above-mentioned mixed solution, after dialysis, gained solution is mycophenolic acid
Immunogen solution, adds the NaN of mass fraction 0.1% in mycophenolic acid immunogens solution3, store at-20 DEG C.
The preparation method of a kind of mycophenolic acid homogeneous enzyme immunoassay detectable the most according to claim 2, it is characterised in that institute
Step (4) detailed process stated is:
A. weigh 1.09g potassium dihydrogen phosphate, 1.70g disodium hydrogen phosphate, 8.5g sodium chloride, 0.95g magnesium chloride, be jointly dissolved in 1L
In deionized water, regulate pH to 7.4, make buffer solution B；
B. weigh and be dissolved under 3mg glucose-6-phosphate dehydrogenase (G6PD), room temperature in 3mL above-mentioned buffer solution B, make glucose-6-
Phosphate dehydrogenase enzymatic solution；
C. weigh 3mg derivatives of mycophenolic acid, be dissolved in 300ul above-mentioned buffer solution B, make derivatives of mycophenolic acid solution；
D., when above-mentioned derivatives of mycophenolic acid solution just becomes clarification, it is added dropwise over above-mentioned glucose-6-phosphate dehydrogenase (G6PD) molten
In liquid, then this mixed solution is stirred 1 hour at 2-8 DEG C；
E. reacted above-mentioned mixed solution is dialysed with above-mentioned buffer solution B, after dialysis gained solution be glucose-
6-phosphate dehydrogenase-hapten conjugation thing solution, adds matter in glucose-6-phosphate dehydrogenase (G6PD)-hapten conjugation thing solution
The BSA and the NaN of mass fraction 0.1% of amount mark 0.5%3, store at 2-8 DEG C.
The preparation method of a kind of mycophenolic acid homogeneous enzyme immunoassay detectable the most according to claim 2, it is characterised in that step
Suddenly the detailed process of (5) is as follows:
The preparation of reagent A: by the nicotinamide adenine dinucleotide of the oxidation state of 4.036g, the G-6-P of 1.711g
Homogeneous zymolyte is made with the Tris buffer solution of 1L 55mM, pH=8.0；The anti-mildew phenolic acid specific antibody of preparation is added
In above-mentioned homogeneous zymolyte, antibody is 1:100～1:10000 with the volume ratio of homogeneous zymolyte；
The preparation of reagent B: the glucose-6-phosphate dehydrogenase (G6PD) of preparation-hapten conjugation thing is added to 120mM, pH=8.2
In Tris buffer, above-mentioned conjugate is 1:100～1:10000 with the volume ratio of Tris buffer.
6. the detection method of mycophenolic acid homogeneous enzyme immunoassay detectable, it is characterised in that comprise the following steps:
(1) sample to be tested is contacted with anti-mildew phenolic acid specific antibody；
(2) according to the combination situation of mycophenolic acid in sample to be tested Yu anti-mildew phenolic acid specific antibody, indicator is utilized to judge sample
The content of mycophenolic acid in Ben；
Described sample to be tested is serum, blood plasma, saliva or urine；
Described indicator is selected from enzyme reagent, including: enzyme mark conjugate and the substrate of enzyme；Above-mentioned enzyme mark conjugate includes Fructus Vitis viniferae
Sugar-6-phosphate dehydrogenase-hapten enzyme mark conjugate；The substrate of above-mentioned enzyme is G-6-P；Described glucose-6-phosphorus
Acidohydrogenase-hapten enzyme mark conjugate is reacted with derivatives of mycophenolic acid by glucose-6-phosphate dehydrogenase (G6PD) and is formed；Described mould phenol
The synthetic route of acid derivative is:
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