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CN104597238A - Mycophenolic acid homogeneous phase enzyme immunological detection reagent as well as preparation and detection methods thereof - Google Patents

Mycophenolic acid homogeneous phase enzyme immunological detection reagent as well as preparation and detection methods thereof Download PDF

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CN104597238A
CN104597238A CN201510039618.3A CN201510039618A CN104597238A CN 104597238 A CN104597238 A CN 104597238A CN 201510039618 A CN201510039618 A CN 201510039618A CN 104597238 A CN104597238 A CN 104597238A
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mycophenolic acid
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g6pd
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CN104597238B (en
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虞留明
伍勇
邓拥军
封飒飒
成志鹏
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SUZHOU EVERMED CO Ltd
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    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/544Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being organic
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    • C07KPEPTIDES
    • C07K16/00Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/44Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material not provided for elsewhere, e.g. haptens, metals, DNA, RNA, amino acids

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Abstract

The invention relates to a mycophenolic acid detection reagent as well as preparation and detection methods thereof and particularly relates to a mycophenolic acid homogeneous phase enzyme immunological detection reagent as well as preparation and detection methods thereof. The mycophenolic acid homogeneous phase enzyme immunological detection reagent comprises an anti-mycophenolic acid specific antibody, and an indication reagent for detecting an anti-mycophenolic acid specific antibody-mycophenolic acid compound; and the anti-mycophenolic acid specific antibody is prepared from mycophenolic acid immunogen immunological animals. The mycophenolic acid detection reagent has the beneficial effects that mycophenolic acid immunogen has strong specificity and high immunogenicity; the prepared anti-mycophenolic acid specific antibody has strong specificity and high valence and has no crossed reaction with common 62 types of medicines; the homogeneous phase enzyme immunological detection reagent containing the anti-mycophenolic acid specific antibody can be used for conveniently, rapidly and accurately determining the content of mycophenolic acid in a sample, and determining a plurality of samples on a full-automatic biochemical analyzer at the same time, so that the high-flux rapid determination for the mycophenolic acid is realized; and the accuracy is high, the specificity is strong, and the accuracy and the detection efficiency can be relatively greatly improved.

Description

A kind of mycophenolic acid homogeneous enzyme immunoassay detects reagent and preparation and determination methods method thereof
Technical field
The present invention relates to a kind of mycophenolic acid and detect reagent and preparation and determination methods method thereof, be specifically related to a kind of mycophenolic acid homogeneous enzyme immunoassay and detect reagent and preparation and determination methods method thereof.
Background technology
Mycophenolic acid (Mycophenolic acid) structural formula is such as formula shown in (III):
Mycophenolic acid, as a kind of immunodepressant, is widely used in suppressing the rejection in the organ transplant processes such as kidney, liver and heart clinically.Mycophenolic acid can noncompetitive in conjunction with hypoxanthine mononucleotide dehydrogenasa (IMPDH), and the latter is the key enzyme of guanylic acid de novo formation in T, B lymphocyte proliferation process.Its clinical effective blood drug concentration narrower (clinical effective blood drug concentration is 1.0-3.5 μ g/mL), cannot play immunosuppressive action lower than its effective blood drug concentration, not reach corresponding curative effect; Then can produce serious renal toxicity higher than its effective blood drug concentration, other subsidiary reactions also comprise Nausea and vomiting, have a stomachache, lose weight, heating etc., patient's immunosupress also can be caused time serious excessively to infect dead.Therefore, to use mycophenolic acid treatment patient carry out therapeutic drug monitoring for instruct the clinical safety rational use of medicines and personalized medicine significant.
Domestic and international monitoring mycophenolic acid blood concentration mainly uses high performance liquid chromatography, enzyme to amplify the methods such as immunoassay, chemoluminescence method, immunoturbidimetry, but these methods all have certain defective in clinical practice.Although the existing mycophenolic acid that can be applicable to Biochemical Analyzer of the external producer such as Siemens, Roche Diagnistics measures kit listing, growing clinical detection demand far can not be met.Still deficient in stability mycophenolic acid that is good, highly sensitive, high specificity detects reagent, the especially measured Automated inspection reagent of matter in the market.Therefore, development & production quality reaches clinical requirement, practical, cost performance is high, and the mycophenolic acid that can be applicable to automatic clinical chemistry analyzer measures the focus that reagent has become domestic and international external diagnosis reagent industry.Mycophenolic acid homogeneous enzyme immunoassay of the present invention detects reagent and can be implemented on automatic clinical chemistry analyzer the high flux of mycophenolic acid, rapid detection, and there is the advantages such as easy and simple to handle, highly sensitive, high specificity, result are accurate, can also effectively reduce mycophenolic acid testing cost, be conducive to clinical expansion and use.
Summary of the invention
For solving the deficiencies in the prior art, the object of the present invention is to provide a kind of not only safe but also quick, efficient, sensitive, accurately can detect that the mycophenolic acid homogeneous enzyme immunoassay of mycophenolic acid content in sample to be tested detects reagent and preparation method thereof, and can with various types of automatic biochemistry analyzer coupling, to testing staff's less demanding mycophenolic acid homogeneous enzyme immunoassay detection method.
Another object of the present invention detects the derivatives of mycophenolic acid of reagent for providing a kind of mycophenolic acid homogeneous enzyme immunoassay contributing to preparing rapid and accurate determination mycophenolic acid content.
In order to realize above-mentioned target, the technical solution used in the present invention is:
A kind of mycophenolic acid homogeneous enzyme immunoassay detects reagent, comprising: anti-mildew phenolic acid specific antibody, for detecting the indicator of anti-mildew phenolic acid specific antibody-mycophenolic acid compound; Above-mentioned anti-mildew phenolic acid specific antibody is obtained by mycophenolic acid immunogens immune animal, and the structural formula of mycophenolic acid immunogens is such as formula shown in (I):
In formula, carrier is for having immunogenic protein; Above-mentioned indicator is selected from enzyme reagent, radioactive isotope reagent, fluorescent reagent or chemical illuminating reagent.
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 G-6-P;
Described glucose-6-phosphate dehydrogenase (G6PD)-haptens enzyme mark conjugate is reacted by glucose-6-phosphate dehydrogenase (G6PD) and derivatives of mycophenolic acid and is formed.
The structural formula of above-mentioned derivatives of mycophenolic acid is such as formula shown in (II):
The synthetic method of derivatives of mycophenolic acid, is characterized in that, synthetic route is:
Mycophenolic acid homogeneous enzyme immunoassay detects a preparation method for reagent, it is characterized in that, comprises the steps:
(1) synthesis of derivatives of mycophenolic acid and purifying, and carry out Structural Identification;
(2) synthesis of mycophenolic acid immunogens: make the terminal carboxyl group of mycophenolic acid and there is immunogenic protein carrier be connected;
(3) use mycophenolic acid immunogens immune animal, preparation is purifying anti-mildew phenolic acid specific antibody also;
(4) preparation of glucose-6-phosphate dehydrogenase (G6PD)-hapten conjugation thing: prepare glucose-6-phosphate dehydrogenase (G6PD) solution, activates derivatives of mycophenolic acid, glucose-6-phosphate dehydrogenase (G6PD) is connected with the terminal carboxyl group of mycophenolic acid, and purifying connects product;
(5) mycophenolic acid homogeneous enzyme immunoassay detects the preparation of reagent:
The preparation of reagent A: mixed by anti-mildew phenolic acid specific antibody and homogeneous phase zymolyte;
The preparation of reagent B: mixed by glucose-6-phosphate dehydrogenase (G6PD)-hapten conjugation thing and Tris damping fluid.
Aforesaid a kind of mycophenolic acid homogeneous enzyme immunoassay detects the preparation method of reagent, and in described step (2), protein carrier is BSA, and concrete synthesis step is as follows:
A. take 2.72g potassium dihydrogen phosphate, 4.26g sodium hydrogen phosphate, 8.5g sodium chloride, 0.95g magnesium chloride, be jointly dissolved in 1L deionized water, regulate pH to 7.4, make buffer solution A;
B. take 3mg BSA, be dissolved under room temperature in the above-mentioned buffer solution A of 3mL, make BSA solution;
C. take 3mg derivatives of mycophenolic acid, be dissolved in the above-mentioned buffer solution A of 300ul, make derivatives of mycophenolic acid solution;
D., when above-mentioned derivatives of mycophenolic acid solution has just become clarification, it is dropwise added in above-mentioned BSA solution, then this mixed solution is stirred 1 hour at 2-8 DEG C;
E. dialysed by the above-mentioned buffer solution A of reacted above-mentioned mixed solution, after dialysis, gained solution is mycophenolic acid immunogens solution, adds the NaN of massfraction 0.1% in mycophenolic acid immunogens solution 3, store at-20 DEG C.
Aforesaid a kind of mycophenolic acid homogeneous enzyme immunoassay detects the preparation method of reagent, and described step (4) detailed process is:
A. take 1.09g potassium dihydrogen phosphate, 1.70g sodium hydrogen phosphate, 8.5g sodium chloride, 0.95g magnesium chloride, be jointly dissolved in 1L deionized water, regulate pH to 7.4, make buffer solution B;
B. take 3mg glucose-6-phosphate dehydrogenase (G6PD), be dissolved under room temperature in the above-mentioned buffer solution B of 3mL, make glucose-6-phosphate dehydrogenase (G6PD) solution;
C. take 3mg derivatives of mycophenolic acid, be dissolved in the above-mentioned buffer solution B of 300ul, make derivatives of mycophenolic acid solution;
D., when above-mentioned derivatives of mycophenolic acid solution has just become clarification, it is dropwise added in above-mentioned glucose-6-phosphate dehydrogenase (G6PD) solution, then this mixed solution is stirred 1 hour at 2-8 DEG C;
E. the above-mentioned buffer solution B of reacted above-mentioned mixed solution is dialysed, after dialysis, gained solution is glucose-6-phosphate dehydrogenase (G6PD)-hapten conjugation thing solution, adds the BSA of the massfraction 0.5% and NaN of massfraction 0.1% in glucose-6-phosphate dehydrogenase (G6PD)-hapten conjugation thing solution 3, store at 2-8 DEG C.
Aforesaid a kind of mycophenolic acid homogeneous enzyme immunoassay detects the preparation method of reagent, and the detailed process of step (5) is as follows:
The preparation of reagent A: 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 are made homogeneous phase zymolyte; Be added in above-mentioned homogeneous phase zymolyte by the anti-mildew phenolic acid specific antibody of preparation, the volume ratio of antibody and homogeneous phase zymolyte is 1:100 ~ 1:10000;
The preparation of reagent B: be added in the Tris damping fluid of 120mM, pH=8.2 by the glucose-6-phosphate dehydrogenase (G6PD) of preparation-hapten conjugation thing, the volume ratio of above-mentioned conjugate and Tris damping fluid is 1:100 ~ 1:10000.
Utilize mycophenolic acid homogeneous enzyme immunoassay to detect the detection method of reagent, it is characterized in that, comprise the following steps:
1) sample to be tested is contacted with anti-mildew phenolic acid specific antibody;
2) according to mycophenolic acid in sample to be tested and anti-mildew phenolic acid specific antibody in conjunction with situation,
Utilize the content of mycophenolic acid in indicator judgement sample;
Described sample to be tested is serum, blood plasma, saliva or urine;
Described 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 G-6-P; Described glucose-6-phosphate dehydrogenase (G6PD)-haptens enzyme mark conjugate is reacted by glucose-6-phosphate dehydrogenase (G6PD) and derivatives of mycophenolic acid and is formed.
Usefulness of the present invention is: mycophenolic acid immunogens high specificity of the present invention, immunogenicity are high, the anti-mildew phenolic acid specific antibody high specificity prepared, height of tiring, and with common 62 kinds of medicines without any cross reaction; Homogeneous enzyme immunoassay detection reagent containing above-mentioned anti-mildew phenolic acid specific antibody can determine the mycophenolic acid content in sample easily and fast, exactly, and can on automatic clinical chemistry analyzer the multiple sample of Simultaneously test, realize the rapid mensuration of high flux of mycophenolic acid, accuracy is high, high specificity, degree of accuracy is all enhanced before comparing with detection efficiency, achieves the full-automation of testing process simultaneously, less demanding to testing staff, is easy to realize and promote the use of.
Accompanying drawing explanation
Fig. 1 is mycophenolic acid homogeneous enzyme immunoassay reaction normal curve map;
Fig. 2 is mycophenolic acid homogeneous enzyme immunoassay correlation analysis figure.
Embodiment
The technical solution used in the present invention is:
Mycophenolic acid immunogens, its structural formula is such as formula shown in (I):
In formula, carrier has immunogenicity, and preferably, carrier is for having immunogenic protein.Also can, as carrier, select protein as carrier under normal circumstances although what other were enough large possesses immunogenic material.The most frequently used immunogenic carrier comprises haemocyanin, hemocyanin (KLH) and thyroglobulin.Carrier in the present invention is preferably haemocyanin.
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 not only refers to complete antibody molecule, also comprises the antibody fragment or derivant that retain complete antibody specific binding capacity.Antibody of the present invention can be polyclonal antibody also can be monoclonal antibody, is preferably polyclonal antibody.
The method obtaining polyclonal antibody is the mycophenolic acid immunogens shown in use formula (I), and after adding or not adding adjuvant, carry out immunity at one or more position of animal, host animal comprises: rabbit, goat, mouse, sheep, cavy or horse.Persistent immunological carries out always, until antibody titer reaches the highest.Animal timing blood sampling obtains appropriate specific corrosioning anteserum, and antiserum can purifying.
Monoclonal antibody makes by somatocyte hybriding technology.
A kind of mycophenolic acid homogeneous enzyme immunoassay detects reagent, comprising: above-mentioned anti-mildew phenolic acid specific antibody, for detecting the indicator of anti-mildew phenolic acid specific antibody-mycophenolic acid compound.Indicator is selected from enzyme reagent, radioactive isotope reagent, fluorescent reagent and chemical illuminating reagent.Preferably, indicator is 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 by chemical synthesis process.
Above-mentioned mycophenolic acid homogeneous enzyme immunoassay detects the using method of reagent, comprises the following steps:
1) sample to be tested is contacted with above-mentioned anti-mildew phenolic acid specific antibody;
2) according to mycophenolic acid in sample to be tested and above-mentioned anti-mildew phenolic acid specific antibody in conjunction with situation, utilize the content of mycophenolic acid in indicator judgement sample.
Sample to be tested is various physiology samples, such as serum, blood plasma, urine, saliva etc.Preferably, sample to be tested is serum 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 examples is such as formula shown in (II):
The synthetic route of this derivatives of mycophenolic acid is as follows:
Concrete synthesis step is as follows:
The synthesis of compound 2
Take 5.0g compound 1, be dissolved in the MeOH of 50mL, at 0 DEG C, dropwise add 4.0g (34.1mmol) SOCl 2, then this reaction mixture is stirred at 70 DEG C and spends the night, by decompression method, solvent is evaporated, by the NaHCO of residue by DCM and 200mL of 200mL 3saturated aqueous solution is separated, and is rinsed by organic phase bittern, then passes through Na 2sO 4carry out drying, and carry out concentrated obtaining 4.9g compound as white solid 2, productive rate 91%.
The synthesis of compound 3
Take 4.9g compound 2, be dissolved in the MeOH of 100mL, at 0 DEG C, add 1.3g (33.3mmol) NaBH several times 4then at room temperature stirred by this reaction mixture and spend the night, concentrated by reacted potpourri, the residue obtained after concentrated carries out purifying by silica dehydrator post (DCM:MeOH=10:1), obtain the compound 3 of 4.4g colorless oil, productive rate 95%.
The synthesis of derivatives of mycophenolic acid
Take 2.2g compound 3, be dissolved in the THF of 40mL, under 0 DEG C and nitrogen protection condition, add 1.2g (12.6mmol) maleimide, 3.8g (14.56mmol) PPh 3and 2.9g (14.56mmol) DIAD, then this reaction mixture is stirred at 70 DEG C and spend the night, reacted potpourri is concentrated, the residue obtained after concentrated carries out purifying by silica dehydrator post (DCM:MeOH=50:1), finally obtain 400mg tan solid Compound 4, productive rate 12%.This compound 4 is the derivatives of mycophenolic acid shown in formula (II).
The synthesis of embodiment two: BSA-mycophenolic acid immunogens
BSA-mycophenolic acid immunogens is formed by connecting by the terminal carboxyl group of the mycophenolic acid shown in bovine serum albumin(BSA) (BSA) Yu formula (III), and this immunogenic concrete synthesis step is as follows:
A. take 2.72g potassium dihydrogen phosphate, 4.26g sodium hydrogen phosphate, 8.5g sodium chloride, 0.95g magnesium chloride, be jointly dissolved in 1L deionized water, regulate pH to 7.4, make buffer solution A;
B. take 3mg BSA, be dissolved under room temperature in the above-mentioned buffer solution A of 3mL, make BSA solution;
C. take 3mg derivatives of mycophenolic acid, be dissolved in the above-mentioned buffer solution A of 300ul, make derivatives of mycophenolic acid solution;
D., when above-mentioned derivatives of mycophenolic acid solution has just become clarification, it is dropwise added in above-mentioned BSA solution, then this mixed solution is stirred 1 hour at 2-8 DEG C;
E. dialysed by the above-mentioned buffer solution A of reacted above-mentioned mixed solution, after dialysis, gained solution is mycophenolic acid immunogens solution, adds the NaN of 0.1% in mycophenolic acid immunogens solution 3, store at-20 DEG C.The NaN of 0.1% 3refer to that addition accounts for the mass percent of the immunogen solution of final gained, concrete addition is determined according to the concrete quality of the immunogen solution of gained after dialysis.
Embodiment three: the preparation of anti-mildew phenolic acid specific antibody
Above-mentioned obtained BSA-mycophenolic acid immunogens is adopted conventional method inoculation experiments animal rabbit, get antiserum after booster immunization, concrete steps are as follows:
With PBS, the BSA-mycophenolic acid immunogens of above-mentioned synthesis is diluted to 1.0mg/ml, obtains antigenic solution, then mix with Freund's complete adjuvant with 1.0ml antigenic solution, experimental animal rabbit is injected.
After 2 ~ 3 weeks, then with the identical antigenic solution of 1.0ml and incomplete Freund's adjuvant, above-mentioned experimental animal rabbit is injected once, afterwards every surrounding injection once, amount to injection 4 times.
Blood is got to above-mentioned experimental animal rabbit, separation and purification obtain tiring be 1: 30000-1: 50000 anti-mildew phenolic acid specific antibody.
Embodiment four: the preparation of glucose-6-phosphate dehydrogenase (G6PD)-hapten conjugation thing
A. take 1.09g potassium dihydrogen phosphate, 1.70g sodium hydrogen phosphate, 8.5g sodium chloride, 0.95g magnesium chloride, be jointly dissolved in 1L deionized water, regulate pH to 7.4, make buffer solution B;
B. take 3mg glucose-6-phosphate dehydrogenase (G6PD), be dissolved under room temperature in the above-mentioned buffer solution B of 3mL, make glucose-6-phosphate dehydrogenase (G6PD) solution;
C. take 3mg derivatives of mycophenolic acid, be dissolved in the above-mentioned buffer solution B of 300ul, make derivatives of mycophenolic acid solution;
D., when above-mentioned derivatives of mycophenolic acid solution has just become clarification, it is dropwise added in above-mentioned glucose-6-phosphate dehydrogenase (G6PD) solution, then this mixed solution is stirred 1 hour at 2-8 DEG C;
E. the above-mentioned buffer solution B of reacted above-mentioned mixed solution is dialysed, after dialysis, gained solution is glucose-6-phosphate dehydrogenase (G6PD)-hapten conjugation thing solution, in glucose-6-phosphate dehydrogenase (G6PD)-hapten conjugation thing solution, add 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 final gained, concrete addition is determined according to the concrete quality of the conjugate solution of gained after dialysis.
Embodiment five: mycophenolic acid homogeneous enzyme immunoassay detects the preparation of reagent
Mycophenolic acid homogeneous enzyme immunoassay detects reagent, comprising: above-mentioned anti-mildew phenolic acid specific antibody, for detecting the indicator of anti-mildew phenolic acid specific antibody-mycophenolic acid compound.Indicator is selected from enzyme reagent, radioactive isotope reagent, fluorescent reagent and chemical illuminating reagent.Preferably, indicator is 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 is obtained by above-mentioned chemical synthesis process.
Mycophenolic acid homogeneous enzyme immunoassay detects reagent before the use, in order to avoid the substrate of the enzyme mark conjugate in indicator and enzyme reacts, the substrate of enzyme mark conjugate and enzyme is separated, does not mix, so the substrate of enzyme and above-mentioned anti-mildew phenolic acid specific antibody are mixed.That is, mycophenolic acid homogeneous enzyme immunoassay detects reagent and comprises two kinds of reagent be provided separately, specific as follows:
1. the preparation of reagent A: the nicotinamide adenine dinucleotide (NAD) of 4.036g (11.25mM) oxidation state, 1.711g (11.25mM) G-6-P (G-6-P) are placed in beaker D, make homogeneous phase zymolyte with the Tris buffer solution of 1L55mM, pH=8.0; Be added in above-mentioned homogeneous phase zymolyte by the anti-mildew phenolic acid specific antibody of above-mentioned preparation, the volume ratio of antibody and homogeneous phase zymolyte can be 1:100 ~ 1:10000, and ratio concrete is in the present embodiment 1:400.
2. the preparation of reagent B: the glucose-6-phosphate dehydrogenase (G6PD) of above-mentioned preparation-hapten conjugation thing 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 ratio concrete is in the present embodiment 1:1500.
Above-mentioned mycophenolic acid homogeneous enzyme immunoassay detects the using method of reagent, comprises the following steps:
1) sample to be tested is contacted with above-mentioned anti-mildew phenolic acid specific antibody;
2) according to mycophenolic acid in sample to be tested and above-mentioned anti-mildew phenolic acid specific antibody in conjunction with situation, utilize indicator to 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 phenolic acid specific antibody generation specific binding in reagent A, generates anti-mildew phenolic acid specific antibody-mycophenolic acid compound; Add reagent B again, glucose-6-phosphate dehydrogenase (G6PD) now in reagent B-hapten conjugation thing mixes with the substrate of the enzyme in reagent A, contacts, there is enzymatic reaction, form the indicator detecting anti-mildew phenolic acid specific antibody-mycophenolic acid compound, indicator is according to the content judging mycophenolic acid in sample to be tested in conjunction with situation of mycophenolic acid in sample to be tested and above-mentioned anti-mildew phenolic acid specific antibody.
Due to the mycophenolic acid competitive binding anti-mildew phenolic acid specific antibody in glucose-6-phosphate dehydrogenase (G6PD)-hapten conjugation thing and sample to be tested, so, in sample to be tested, the amount of mycophenolic acid is more, the amount of glucose-6-phosphate dehydrogenase (G6PD)-hapten conjugation thing free in homogeneous phase enzyme solutions is more, enzymatic reaction is faster, causes OD 340rise.
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, typical curve is obtained: arrange auspicious BS200 automatic clinical chemistry analyzer response parameter (see table 1) advanced in years, operating process is: first reagent adding A, then adds standard items, finally adds reagent B.After adding reagent B, measure the OD of different time points 340light absorption value, calculates reaction rate during various criterion product concentration, needs the volume ratio constantly adjusting reagent A and reagent B in actual mechanical process, adjusts light-metering point simultaneously, finally draws comparatively ideal reaction normal curve map, as shown in Figure 1.
Table 1 steps auspicious BS200 automatic clinical chemistry analyzer response parameter
By the typical curve that homogeneous enzyme immunoassay detection reagent of the present invention obtains, 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 items, is respectively 1.00,5.00,20.00 μ g/ml to concentration.Detection data and data analysis are in table 2.
Table 2 sample determination and precision and recovery assessment
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
Mean value (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
Recovery % 100.0 101.2 100.7
Testing result: the accuracy that homogeneous enzyme immunoassay of the present invention detects reagent mensuration is high, and the recovery reaches 95%-105%, and precision is high, and CV is all lower than 5%.
Embodiment seven: interfering effects of drug is tested
Choose 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 are specifically see 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 all less than 0.1 μ g/ml.Visible, antibody of the present invention is the specific antibody of anti-mildew phenolic acid.
Embodiment eight: correlation analysis
Use the mycophenolic acid of medical diagnostic prods (Shanghai) Co., Ltd. of Siemens detection reagent (adopting enzyme to amplify immunoassay) and homogeneous enzyme immunoassay reagent of the present invention to carry out correlation analysis respectively to 100 routine clinical samples, the data of mensuration are see table 4.
Table 4 clinical sample measured value
Map to above-mentioned data, see Fig. 2, the linear equation obtained is: y=0.9882x+0.0399, coefficient R 2=0.9994, show that the accuracy of detection reagent of the present invention mensuration mycophenolic acid clinical samples is high.
Because testing process of the present invention is completed by instrument full-automation, so less demanding to testing staff, be easy to realize and promote the use of.
It should be noted that; the foregoing is only embodiments of the invention; not thereby the scope of the claims of the present invention is limited; every utilize instructions of the present invention and accompanying drawing content to do equivalent structure or equivalent flow process conversion; or be directly or indirectly used in other correlative technology fields, be all in like manner included in scope of patent protection of the present invention.

Claims (10)

1. mycophenolic acid homogeneous enzyme immunoassay detects a reagent, comprising: anti-mildew phenolic acid specific antibody, for detecting the indicator of anti-mildew phenolic acid specific antibody-mycophenolic acid compound; Above-mentioned anti-mildew phenolic acid specific antibody is obtained by mycophenolic acid immunogens immune animal, and the structural formula of mycophenolic acid immunogens is such as formula shown in (I):
In formula, carrier is for having immunogenic protein; Above-mentioned indicator is selected from enzyme reagent, radioactive isotope reagent, fluorescent reagent or chemical illuminating reagent.
2. a kind of mycophenolic acid homogeneous enzyme immunoassay according to claim 1 detects reagent, and it 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 G-6-P; Described glucose-6-phosphate dehydrogenase (G6PD)-haptens enzyme mark conjugate is reacted by glucose-6-phosphate dehydrogenase (G6PD) and derivatives of mycophenolic acid and is formed.
3. mycophenolic acid homogeneous enzyme immunoassay according to claim 2 detects reagent, and it is characterized in that, the structural formula of above-mentioned derivatives of mycophenolic acid is such as formula shown in (II):
4. derivatives of mycophenolic acid, structural formula is such as formula shown in (II):
5. derivatives of mycophenolic acid according to claim 4, is characterized in that, synthetic route is:
6. mycophenolic acid homogeneous enzyme immunoassay detects a preparation method for reagent, it is characterized in that, comprises the steps:
(1) synthesis of the derivatives of mycophenolic acid described in claim 3,4 or 5 and purifying, and carry out Structural Identification;
(2) synthesis of mycophenolic acid immunogens: make the terminal carboxyl group of mycophenolic acid and there is immunogenic protein carrier be connected;
(3) use mycophenolic acid immunogens immune animal, preparation is purifying anti-mildew phenolic acid specific antibody also;
(4) preparation of glucose-6-phosphate dehydrogenase (G6PD)-hapten conjugation thing: prepare glucose-6-phosphate dehydrogenase (G6PD) solution, activates derivatives of mycophenolic acid, glucose-6-phosphate dehydrogenase (G6PD) is connected with the terminal carboxyl group of mycophenolic acid, and purifying connects product;
(5) mycophenolic acid homogeneous enzyme immunoassay detects the preparation of reagent:
The preparation of reagent A: mixed by anti-mildew phenolic acid specific antibody and homogeneous phase zymolyte;
The preparation of reagent B: mixed by glucose-6-phosphate dehydrogenase (G6PD)-hapten conjugation thing and Tris damping fluid.
7. a kind of mycophenolic acid homogeneous enzyme immunoassay according to claim 6 detects the preparation method of reagent, and it is characterized in that, in described step (2), protein carrier is BSA, and concrete synthesis step is as follows:
A. take 2.72g potassium dihydrogen phosphate, 4.26g sodium hydrogen phosphate, 8.5g sodium chloride, 0.95g magnesium chloride, be jointly dissolved in 1L deionized water, regulate pH to 7.4, make buffer solution A;
B. take 3mg BSA, be dissolved under room temperature in the above-mentioned buffer solution A of 3mL, make BSA solution;
C. take 3mg derivatives of mycophenolic acid, be dissolved in the above-mentioned buffer solution A of 300ul, make derivatives of mycophenolic acid solution;
D., when above-mentioned derivatives of mycophenolic acid solution has just become clarification, it is dropwise added in above-mentioned BSA solution, then this mixed solution is stirred 1 hour at 2-8 DEG C;
E. dialysed by the above-mentioned buffer solution A of reacted above-mentioned mixed solution, after dialysis, gained solution is mycophenolic acid immunogens solution, adds the NaN of massfraction 0.1% in mycophenolic acid immunogens solution 3, store at-20 DEG C.
8. a kind of mycophenolic acid homogeneous enzyme immunoassay according to claim 6 detects the preparation method of reagent, and it is characterized in that, described step (4) detailed process is:
A. take 1.09g potassium dihydrogen phosphate, 1.70g sodium hydrogen phosphate, 8.5g sodium chloride, 0.95g magnesium chloride, be jointly dissolved in 1L deionized water, regulate pH to 7.4, make buffer solution B;
B. take 3mg glucose-6-phosphate dehydrogenase (G6PD), be dissolved under room temperature in the above-mentioned buffer solution B of 3mL, make glucose-6-phosphate dehydrogenase (G6PD) solution;
C. take 3mg derivatives of mycophenolic acid, be dissolved in the above-mentioned buffer solution B of 300ul, make derivatives of mycophenolic acid solution;
D., when above-mentioned derivatives of mycophenolic acid solution has just become clarification, it is dropwise added in above-mentioned glucose-6-phosphate dehydrogenase (G6PD) solution, then this mixed solution is stirred 1 hour at 2-8 DEG C;
E. the above-mentioned buffer solution B of reacted above-mentioned mixed solution is dialysed, after dialysis, gained solution is glucose-6-phosphate dehydrogenase (G6PD)-hapten conjugation thing solution, adds the BSA of the massfraction 0.5% and NaN of massfraction 0.1% in glucose-6-phosphate dehydrogenase (G6PD)-hapten conjugation thing solution 3, store at 2-8 DEG C.
9. a kind of mycophenolic acid homogeneous enzyme immunoassay according to claim 6 detects the preparation method of reagent, and it is characterized in that, the detailed process of step (5) is as follows:
The preparation of reagent A: the Tris buffer solution of G-6-P 1L 55mM, pH=8.0 of the nicotinamide adenine dinucleotide of the oxidation state of 4.036g 11.25mM, 1.711g 11.25mM is made homogeneous phase zymolyte; Be added in above-mentioned homogeneous phase zymolyte by the anti-mildew phenolic acid specific antibody of preparation, the volume ratio of antibody and homogeneous phase zymolyte is 1:100 ~ 1:10000;
The preparation of reagent B: be added in the Tris damping fluid of 120mM, pH=8.2 by the glucose-6-phosphate dehydrogenase (G6PD) of preparation-hapten conjugation thing, the volume ratio of above-mentioned conjugate and Tris damping fluid is 1:100 ~ 1:10000.
10. utilize the mycophenolic acid homogeneous enzyme immunoassay described in claim 1 to 3 any one to detect the detection method of reagent, it is characterized in that, comprise the following steps:
(1) sample to be tested is contacted with anti-mildew phenolic acid specific antibody;
(2) according to mycophenolic acid in sample to be tested and anti-mildew phenolic acid specific antibody in conjunction with situation, utilize the content of mycophenolic acid in indicator judgement sample;
Described sample to be tested is serum, blood plasma, saliva or urine;
Described 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 G-6-P; Described glucose-6-phosphate dehydrogenase (G6PD)-haptens enzyme mark conjugate is reacted by glucose-6-phosphate dehydrogenase (G6PD) and derivatives of mycophenolic acid and is formed.
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