CN108956971A - A kind of valproic acid immunologic function test reagent and its preparation and detection method - Google Patents
A kind of valproic acid immunologic function test reagent and its preparation and detection method Download PDFInfo
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- CN108956971A CN108956971A CN201810484514.7A CN201810484514A CN108956971A CN 108956971 A CN108956971 A CN 108956971A CN 201810484514 A CN201810484514 A CN 201810484514A CN 108956971 A CN108956971 A CN 108956971A
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Abstract
The invention discloses a kind of valproic acid immunologic function test reagent and its preparation and detection methods.It include: enzyme mark valproic acid, for detecting valproic acid antibody-enzyme mark valproic acid compound indicator；Above-mentioned enzyme mark valproic acid is coupled by valproic acid and glucose dehydrogenase.Valproic acid immunologic function test reagent of the invention can accurately and quickly determine content of valproic acid in the samples such as blood of human body.Compared with existing detection reagent in the market, detection reagent of the present invention has many advantages, such as fast and easy, high sensitivity, high specificity, quantitative accurate, is conducive to clinical popularization and use.
Description
Technical field
The present invention relates to field of biological detection, specifically a kind of valproic acid immunologic function test reagent and its preparation and detection side
Method.
Background technique
Valproic acid (valproate, VPA) is a kind of common spectrum antiepileptic, and can be used for treating and double feelings
The relevant maniac access of obstacle is felt, clinically usually using its sodium salt.It, can due to the advantages that its is significant in efficacy, safety is good
For treating the epileptic attack of systemic or partial type, including various epilepsies, anxiety disorder and rare major depressive disorder.
But over the course for the treatment of, the big drug interaction of pharmacokinetics individual difference is more in vivo for valproic acid, and dosage and
Blood concentration correlation is unstable, and treats curative effect and toxic reaction and blood concentration related intimate.Therefore, to valproic acid
Internal blood concentration detection is very important.
The common method of detection valproic acid has at present: efficient liquid phase chromatographic analysis (HPLC), enzyme linked immunosorbent assay (ELISA)
(ELISA) and fluorescence polarization method (FPIA) etc..Wherein, HPLC is time-consuming and is not easy to automate；ELISA method dosing accuracy is poor, grasps
Make that the time is long, the degree of automation is low, is chiefly used in qualitative detection；FPIA method reagent relies on import, and expensive and validity period is shorter,
It clinically can not large-scale use.The method that the present invention uses is homogeneous enzyme immunoassay detection method, advantage are as follows: easy to operate, fast
Speed, high sensitivity, accuracy are good, are suitable for automating, and are widely used, and with automatic clinical chemistry analyzer to small-molecule substance
It can high-throughput quickly measurement with macromolecular substances.
Summary of the invention
It is an object of the invention to solve, valproic acid detection process is complicated for operation in the prior art and accuracy of measurement is low
The problem of, the present invention provides one kind quickly, high sensitivity, accurately detects that the valproic acid of content of valproic acid in sample to be tested is equal
Phase enzyme immunologic function test reagent and preparation method thereof.
To achieve the above object, the invention provides the following technical scheme:
A kind of valproic acid immunologic function test reagent and its preparation and detection method, it is characterised in that: enzyme mark valproic acid, for detecting third
Valeric acid antibody-enzyme mark valproic acid compound indicator；Above-mentioned enzyme mark valproic acid is coupled by valproic acid and glucose dehydrogenase
It forms.
As a further solution of the present invention, the indicator is selected from enzymatic reagent, comprising: the bottom of enzyme mark conjugate and enzyme
Object；Above-mentioned enzyme mark conjugate includes glucose dehydrogenase-valproic acid conjugate；The substrate of above-mentioned enzyme is glucose.
As a further solution of the present invention, a kind of valproic acid immunologic function test reagent and preparation method thereof, it is special
Sign is, includes the following steps:
(1) glucose dehydrogenase-valproic acid conjugate preparation: preparation glucose dehydrogenase (GDH) solution, the work of valproic acid
Change, GDH and valproic acid are coupled, the enzyme-labelled antigen of coupling is purified；
(2) preparation of valproic acid homogeneous enzyme immunoassay detection reagent:
The preparation of reagent 1: it is mixed by valproic acid antibody and homogeneous zymolyte；
The preparation of reagent 2: it is mixed by glucose dehydrogenase-antigen conjugates with phosphate buffer.
As a further solution of the present invention, the preparation method of a kind of valproic acid immunologic function test reagent, feature
It is, the step (1) detailed process are as follows:
1) preparation of glucose dehydrogenase (GDH) solution:
A. the GDH that 5-20 mg specification is 100-300 KU is weighed, room-temperature dissolution is in 6-10 mL PBS buffer solution；
2) activation of valproic acid
Following chemicals is added to stirring and dissolving in beaker: 10-30 mg valproic acid, 10-40 mg 1- ethyl -3- (- 3- two
First aminopropyl) carbodiimide, 2-10 mg N- hydroxy thiosuccinimide, it is dissolved in morpholino b acid (MES) solution, in
Stirring and dissolving at room temperature is reacted 15-60 minutes；
3) coupling of GDH and valproic acid
A. the valproic acid solution of above-mentioned activation is added dropwise in the GDH solution of above-mentioned dissolution；
B 2-8 DEG C is stirred overnight；
4) enzyme-labelled antigen of purifying coupling
The enzyme-labelled antigen being coupled by G-25 gel chromatography column purification, obtains glucose dehydrogenase-valproic acid conjugate, in 2-8
It is stored at DEG C.
As a further solution of the present invention, the preparation method of a kind of valproic acid immunologic function test reagent, feature
It is, detailed process is as follows for step (2):
The preparation of reagent 1: by nicotinamide adenine dinucleotide NAD, the 0.5-3 g glucose 0.5-2L of 2-5 g oxidation state
Homogeneous zymolyte is made in phosphate buffer dissolution；Valproic acid antibody is added in above-mentioned homogeneous zymolyte, antibody and homogeneous enzyme
The volume ratio of substrate is 1:100~1:10000；
The preparation of reagent 2: the glucose dehydrogenase of preparation-valproic acid conjugate is added in phosphate buffer, above-mentioned coupling
The volume ratio of object and phosphate buffer is 1:100~1:10000.
As a further solution of the present invention, the detection method of the valproic acid immunologic function test reagent, which is characterized in that
The following steps are included:
1) sample to be tested is contacted with valproic acid antibody；
2) according to the combination situation of enzyme mark valproic acid in sample to be tested and valproic acid antibody, in indicator judgement sample third is utilized
The content of valeric acid；The sample to be tested is serum, blood plasma, saliva or urine.
The principle of the present invention is antigen and enzyme is combined into enzyme-labelled antigen, retains the bioactivity of antigen and enzyme, when enzyme mark is anti-
After original is in conjunction with antibody, zymoprotein and antibody close contact on antigen molecule make the activated centre of enzyme be affected, the work of enzyme
Property is suppressed.For antigen, enzyme-labelled antigen when measurement in sample in conjunction with antibody competition, the antigenic content in sample is higher,
Add its OD value after substrate higher.
The present invention has the advantages that valproic acid immunologic function test reagent of the invention can accurately and quickly determine blood of human body
Content of valproic acid in equal samples.Compared with existing detection reagent in the market, detection reagent of the present invention has fast and easy, sensitive
It spends height, high specificity, quantify the advantages that accurate, be conducive to clinical popularization and use.
Detailed description of the invention
Fig. 1 is valproic acid homogeneous enzyme immunoassay reaction calibration graph.
Fig. 2 is valproic acid homogeneous enzyme immunoassay range of linearity figure.
Specific embodiment
The present invention provides a kind of valproic acid immunologic function test reagent and its preparation and detection method, for make the object of the invention,
Technical solution and effect are clearer, clear, and the present invention is described in detail below.
The present invention provides a kind of valproic acid immunologic function test reagent and its preparation and detection methods.Include: enzyme mark valproic acid,
For detecting valproic acid antibody-enzyme mark valproic acid compound indicator；Above-mentioned enzyme mark valproic acid is by valproic acid and glucose
Dehydrogenase is coupled.
Signified " valproic acid " refers not only to complete valproic acid molecule in the present invention, also includes that reservation intact antigen is special
The valproic acid segment or derivative of property binding ability.
A kind of valproic acid homogeneous enzyme immunoassay detection reagent, comprising: enzyme mark valproic acid, for detecting valproic acid antibody-enzyme mark
The indicator of valproic acid compound.Indicator is selected from enzymatic reagent, radioactive isotope reagent, fluorescent reagent and chemiluminescence
Reagent.Preferably, indicator is enzymatic reagent, comprising: the substrate of enzyme mark conjugate and enzyme.Wherein, enzyme mark conjugate includes Portugal
Grape glucocorticoid dehydrogenase-antigen conjugates, can be obtained by chemical synthesis process.
The application method of above-mentioned valproic acid immunologic function test reagent, comprising the following steps:
1) sample to be tested is contacted with valproic acid antibody；
2) according to the combination situation of enzyme mark valproic acid in sample to be tested and valproic acid antibody, in indicator judgement sample third is utilized
The content of valeric acid；The sample to be tested is serum, blood plasma, saliva or urine etc..Preferably, sample to be tested is serum or blood plasma.
Below by specific embodiment, the present invention is described in detail.
Embodiment one: glucose dehydrogenase-antigen conjugates preparation
1) preparation of glucose dehydrogenase (GDH) solution:
A. weigh 12 mg specifications be 100 KU GDH, room-temperature dissolution in 8 mL concentration be 10 mM PBS solution in, pH=
7.4；
2) activation of valproic acid
Following chemicals is added to stirring and dissolving in beaker: 30 mg valproic acids, 25 mg 1- ethyl -3- (- 3- diformazan ammonia third
Base) carbodiimide, 10 mg N- hydroxy thiosuccinimides, it is dissolved in 800 μ L morpholino b acid (MES) solution, in room
The lower stirring and dissolving of temperature, reacts 20 minutes；
3) coupling of GDH and valproic acid
A. the valproic acid solution of above-mentioned activation is added dropwise in the GDH solution of above-mentioned dissolution；
B 2-8 DEG C is stirred overnight；
4) enzyme-labelled antigen of purifying coupling
The enzyme-labelled antigen being coupled by G-25 gel chromatography column purification, obtains glucose dehydrogenase-valproic acid conjugate, in 2-8
It is stored at DEG C.
Embodiment two: the preparation of valproic acid homogeneous enzyme immunoassay detection reagent
Valproic acid homogeneous enzyme immunoassay detection reagent, comprising: enzyme mark valproic acid is answered for detecting valproic acid antibody-enzyme mark valproic acid
Close the indicator of object.Indicator is selected from enzymatic reagent, radioactive isotope reagent, fluorescent reagent and chemical illuminating reagent.It is excellent
Choosing, indicator is enzymatic reagent, comprising: the substrate of enzyme mark conjugate and enzyme.Wherein, enzyme mark conjugate includes glucose dehydrogenation
Enzyme-antigen conjugates can be obtained by chemical synthesis process.
Valproic acid homogeneous enzyme immunoassay detection reagent before the use, in order to avoid the enzyme mark conjugate and enzyme in indicator
Substrate react, the substrate of enzyme mark conjugate and enzyme is separated, therefore valproic acid homogeneous enzyme immunoassay detection reagent
The reagent being provided separately including two kinds, specific as follows:
1. the preparation of reagent 1: by nicotinamide adenine dinucleotide NAD, 1.802 g(10 of 3.588 g(10 mM) oxidation state
MM) homogeneous zymolyte is made with the phosphate buffer dissolution of 50 mM, pH 8.0 of 1L in glucose；Valproic acid antibody is added to
It states in homogeneous zymolyte, the volume ratio of antibody and homogeneous zymolyte is 1:100~1:10000, in the present embodiment specific ratio
Example is 1:600.
2. the preparation of reagent 2: the glucose dehydrogenase of preparation-valproic acid conjugate is added to the phosphorus of 50 mM, pH 8.0
In phthalate buffer, the volume ratio of above-mentioned conjugate and phosphate buffer is 1:100~1:10000, is had in the present embodiment
The ratio of body is 1:1800.
The application method of above-mentioned valproic acid homogeneous enzyme immunoassay detection reagent, comprising the following steps:
1) sample to be tested is contacted with valproic acid antibody；
2) according to the combination situation of enzyme mark valproic acid in sample to be tested and valproic acid antibody, in indicator judgement sample third is utilized
The content of valeric acid；
Specifically, sample to be tested is added in reagent 1 when detection, the valproic acid in sample to be tested and the valproic acid in reagent 1 are anti-
Body is specifically bound, and generates anti-valproic acid antibody-valproic acid compound；Reagent 2 is added, at this time the grape in reagent 2
Glucocorticoid dehydrogenase-valproic acid conjugate is mixed with the substrate of the enzyme in reagent 1, is contacted, and enzymatic reaction occurs, and constitutes detection valproic acid
Antibody-enzyme mark valproic acid compound indicator, indicator is according to valproic acid in sample to be tested and above-mentioned valproic acid antibody
Combination situation judge the content of valproic acid in sample to be tested.
Due to the valproic acid competitive binding valproic acid antibody in glucose dehydrogenase-antigen conjugates and sample to be tested,
So the amount of valproic acid is more in sample to be tested, the glucose dehydrogenase-antigen conjugates amount dissociated in homogeneous enzyme solutions is got over
More, enzymatic reaction is faster, leads to OD340 Rise.
Above-mentioned sample to be tested is physiology sample, such as serum, blood plasma, urine, saliva etc., as a preferred solution,
Above-mentioned sample to be tested is serum or blood plasma.
Embodiment three: valproic acid homogeneous enzyme immunoassay detection reagent reacts calibration curve.
1) calibration object is prepared: commercially available people's valproic acid recombinant protein is dissolved in the solution (NaCl of similar human serum matrix
0.9%, BSA 0.2%, NaN3 0.1%, Tris-HCl pH 7.4) in, the calibration object of various concentration is made.With Suzhou Bo Yuan medical treatment
The valproic acid calibration object of Science and Technology Ltd. is primary standard, is distinguished using calibration object of its valproic acid kit to various concentration
Detection 10 times, finds out mean value, obtains the concentration of valproic acid calibration object: 5,10,20,40,80,160 μ g/mL.
2) Biochemical Analyzer detects: by taking Hitachi 7170 operates as an example: measurement wavelength is 340 nm, takes various concentration respectively
Valproic acid R is added in calibration object solution (15 μ L)1Reagent (160 μ L) mixes, adds valproic acid R2Reagent (40 μ L), after mixing,
Measure the OD of different time points340Light absorption value calculates reaction rate when different calibration object concentration, needs not in actual mechanical process
The volume ratio of disconnected adjustment reagent 1 and reagent 2, while survey luminous point is adjusted, finally obtain comparatively ideal reaction normal curve graph,
Every pipe replication 3 times, using the average value of the pipe 3 times absorbance difference Δ A measured of each calibration as ordinate, corresponding calibration object
Concentration is abscissa, draws " concentration-absorbance difference " calibration curve (see figure 1).
Test serum or plasma sample are taken, the absorbance difference of sample is measured in the same method, calibration curve is substituted into, can calculate
The content of valproic acid in sample to be tested.If the concentration of valproic acid exceeds calibration curve range in serum or blood plasma, need to be to sample
The accuracy to guarantee testing result is detected after being diluted again.
This detection reagent is applicable not only to Hitachi 7170, applies also for semi-automatic, the full-automatic life of other brands and model
Change analyzer, design parameter can be adjusted according to instrument.
Example IV: the range of linearity determines
With the valproic acid high concentration sample (153.6 μ g/mL) close to the range of linearity upper limit, with above-mentioned similar human serum matrix
It by 1/2,1/4,1/8,1/16,1/32,1/64 dilution, is configured to the solution of 6 diluted concentrations (xi) altogether, uses institute by solution
It states Biochemical Analyzer detection method and measures each diluted sample concentration.Each diluted concentration is tested 3 times, and it is dense to find out each dilution respectively
Spend the mean value (yi) of testing result.It is that dependent variable finds out linear return to measure mean value (yi) with diluted concentration (xi) for independent variable
Return equation, the correlation coefficient r of linear regression is calculated according to formula (1), regression equation is y=0.9773x+0.7718 as the result is shown,
Correlation coefficient r=0.9998, show reagent of the present invention in the 2.4 μ g/mL-153.6 μ g/mL ranges of linearity good relationship (see
Fig. 2).
... ... ... ... ... ... (1)
Since detection process of the invention is completed by instrument full-automation, so being easy to real to the of less demanding of testing staff
Now and promote the use of.
It should be noted that obviously invention is not limited to the details of the above exemplary embodiments, the scope of the present invention is by institute
Attached claim rather than above description limit, it is intended that will fall within the meaning and scope of the equivalent elements of the claims
All changes are included in scope of patent protection of the invention.
In addition, above-described is only the preferred embodiments of the invention, for the technical staff in this technology neck city,
Without departing from the principle of the present invention, several modifications and adaptations can also be done, these improved adjustment also should be regarded as this
The protection scope of invention.
Claims (8)
1. a kind of valproic acid immunologic function test reagent and its preparation and detection method, it is characterised in that: enzyme mark valproic acid, for detecting
Valproic acid antibody-enzyme mark valproic acid compound indicator.
2. the immunologic function test reagent of valproic acid according to claim 1, it is characterised in that: the enzyme mark valproic acid is by the third penta
Acid and glucose dehydrogenase are coupled.
3. valproic acid immunologic function test reagent according to claim 1, it is characterised in that: the indicator is tried selected from enzyme
Agent, comprising: the substrate of enzyme mark conjugate and enzyme；Above-mentioned enzyme mark conjugate includes glucose dehydrogenase-valproic acid conjugate；It is above-mentioned
The substrate of enzyme is glucose.
4. a kind of valproic acid immunologic function test reagent and preparation method thereof, which comprises the steps of:
(1) glucose dehydrogenase-valproic acid conjugate preparation: preparation glucose dehydrogenase (GDH) solution, the work of valproic acid
Change, GDH and valproic acid are coupled, the enzyme-labelled antigen of coupling is purified.
5.(2) the preparation of valproic acid homogeneous enzyme immunoassay detection reagent:
The preparation of reagent 1: it is mixed by valproic acid antibody and homogeneous zymolyte；
The preparation of reagent 2: it is mixed by glucose dehydrogenase-valproic acid conjugate with phosphate buffer.
6. a kind of preparation method of valproic acid immunologic function test reagent according to claim 5, which is characterized in that the step
Suddenly (1) detailed process are as follows:
1) preparation of glucose dehydrogenase (GDH) solution
The GDH that 5-20 mg specification is 100-300 KU is weighed, room-temperature dissolution is in 6-10 mL PBS buffer solution；
2) activation of valproic acid
Following chemicals is added to stirring and dissolving in beaker: 10-30 mg valproic acid, 10-40 mg1- ethyl -3- (- 3- two
First aminopropyl) carbodiimide, 2-10 mg N- hydroxy thiosuccinimide, it is dissolved in morpholino b acid (MES) solution, in
Stirring and dissolving at room temperature is reacted 15-60 minutes；
3) coupling of GDH and valproic acid
A. the valproic acid solution of above-mentioned activation is added dropwise in the GDH solution of above-mentioned dissolution；
B. it is stirred overnight for 2-8 DEG C；
4) enzyme-labelled antigen of purifying coupling
The enzyme-labelled antigen being coupled by G-25 gel chromatography column purification, obtains glucose dehydrogenase-valproic acid conjugate, in 2-8
It is stored at DEG C.
7. a kind of preparation method of valproic acid immunologic function test reagent according to claim 5, which is characterized in that step (2)
Detailed process is as follows:
The preparation of reagent 1: by nicotinamide adenine dinucleotide NAD, the 0.5-3 g glucose 0.5-2 L of 2-5 g oxidation state
Homogeneous zymolyte is made in phosphate buffer dissolution；Valproic acid antibody is added in above-mentioned homogeneous zymolyte, antibody and homogeneous enzyme
The volume ratio of substrate is 1:100~1:10000；
The preparation of reagent 2: the glucose dehydrogenase of preparation-valproic acid conjugate is added in phosphate buffer, above-mentioned coupling
The volume ratio of object and phosphate buffer is 1:100~1:10000.
8. utilizing the detection method of valproic acid immunologic function test reagent described in 4 any one of Claims 1-4, which is characterized in that
The following steps are included:
1) sample to be tested is contacted with valproic acid antibody；
2) according to the combination situation of valproic acid in sample to be tested and valproic acid antibody, valproic acid in indicator judgement sample is utilized
Content；The sample to be tested is serum, blood plasma, saliva or urine.
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| CN110954707A (en) * | 2019-07-04 | 2020-04-03 | 北京九强生物技术股份有限公司 | Valproic acid derivatives and their use in immunoassays |
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| CN110954707A (en) * | 2019-07-04 | 2020-04-03 | 北京九强生物技术股份有限公司 | Valproic acid derivatives and their use in immunoassays |
| CN110954707B (en) * | 2019-07-04 | 2022-12-13 | 北京九强生物技术股份有限公司 | Valproic acid derivatives and their use in immunoassays |
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