CN102507917A - Valproic acid homogeneous-phase enzyme immunity rapid detection kit - Google Patents
Valproic acid homogeneous-phase enzyme immunity rapid detection kit Download PDFInfo
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Abstract
The invention relates to the technical field of valproic acid blood drug level monitoring, and specifically discloses a valproic acid homogeneous-phase enzyme immunity rapid detection kit. With derivative synthesis, valproic acid is connected onto bovine serum albumin (BSA), such that an immunizing antigen is obtained. An obtained polyclonal antibody has valproic acid specificity. Experiment data shows that when the reagent is used for detecting valproic acid, no cross reaction occurs between the reagent and other commonly used medicines; an analysis sensitivity is better than 25ng/ml; within-run precision and within-run precision coefficient of variability (CV) are both smaller than 4.7%; and compared with a fluorescence polarization immunoassay (EPIA) method, an R2 value reaches 0.98. The kit can be used in various routine analysis instruments. Because of characteristics of simple operation, high speed, low cost, and high sensitivity, with the kit, automatic detection can easily be realized. The kit assists in guiding clinical customized medication. With the kit, toxic or side effect of medicines is reduced, and curative effect optimization is realized.
Description
Technical field
The invention belongs to valproic acid clinical sample detection technique field, be specifically related to a kind of kit of fast detecting valproic acid concentration.
Technical background
In at present numerous antiepileptics, valproic acid is a first-selection that clinical practice is wide, curative effect is high and security is good, broad-spectrum medicinal.Valproic acid not only can be used to treat each epileptoid generalized seizures, especially absence seizure and tonic-clonic seizures, also can be used for partial seizures.In addition, valproic acid seldom influences patient's cognitive function, and causes that seldom induced seizures increases the weight of.In therapeutic process, because individual patients pharmacokinetics difference, cause between valproic acid dosage and the blood concentration correlativity relatively poor.The blood concentration that records by the routine dose administration; Usually have 50% be lower than or exceed valproic acid effective therapeutic domain (50～100 μ g/ml); Therefore patient's valproic acid blood concentration is monitored; To timely adjustment dosage, the safety and effectiveness that improves valproic acid treatment epilepsy has the important clinical directive significance.
At present, apply to the method that blood concentration detects both at home and abroad and mainly contain red, orange, green, blue, yellow (ROGBY) and immunoassay.Chromatography comprises high performance liquid chromatography (HPLC), LC-MS (LC-MSMS) etc.; This method has high degree of specificity to determinand; Handle but need carry out early stage to sample, complicated operating process, cycle are longer, and instrument and equipment is expensive and operating personnel are had relatively high expectations.And advantage such as that immunoassay has is simple to operate, sensitivity height, the domestic immunization that is applied to the valproic acid analysis at present mainly contains electrochemiluminescent immunoassay, enzyme-linked immunosorbent assay (ELISAA) etc.The ELISA detection method is highly sensitive, but accuracy is not enough, can only sxemiquantitative.The simultaneously domestic electrochemiluminescent immunoassay reagent that does not still have independent development at present mainly detects on the enclosed type light-emitting appearance through buying external assay kit.Limited by instrument and reagent cost, this method is unfavorable for promoting in China.
Summary of the invention
The present invention is based on the valproic acid drug concentration detection method that the homogeneous enzyme immunoassay method is set up, a kind of easy, quick, high sensitivity, full-automatic detection kit are provided, have advantages such as high specificity, degree of accuracy height and high flux check.Can remedy the deficiency of existing detection technique, satisfy domestic valproic acid drug concentration and detect needs, have great social significance and using value.
In order to solve the problems of the technologies described above, the technical scheme that the present invention adopted is: a kind of valproic acid homogeneous enzyme immunoassay quick detection kit is characterized in that being made up of following several kinds of components:
Reactant liquor R1 is the anti-valproic acid specific antibody of enzyme reaction substrate dilution;
Reactant liquor R2 is valproic acid enzyme mark conjugate;
Calibration solution is dissolved in the blank buffer solution by the valproic acid calibration object and makes.
Said anti-valproic acid specific antibody is monoclonal or polyclone body.
The total amount of said reactant liquor R1 and reactant liquor R2 is 75～150 μ l, and calibration solution is 5～20 μ l.
The preparation process of said reactant liquor R1 is: make the activation of valproic acid derivant through adding 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDAc) and N-hydroxy thiosuccinimide (Sulfo-NHS) in the valproic acid derivative solution earlier, at room temperature carry out coupling with carrier protein subsequently and prepare immunizing antigen; With producing the valproic acid specific antibody behind the immunizing antigen immune animal of preparation; Make by 1: 300～1: 5000 dilution specific antibody with enzyme reaction substrate at last.
Said carrier protein comprises seralbumin (BSA), the pure albumen of ovum gallinaceum (OVA) or keyhole limpet hemocyanin (KLH).
Said enzyme reaction substrate contains NADH (NAD) and 6 glucose 1-phosphate1-s (G6P), and both concentration total amounts are 2.81～11.25mmol/L.
Said reactant liquor R2 is made up of the valproic acid of glucose 6 phosphate dehydrogenases (G6PDH) mark.
The preparation process of said reactant liquor R2 is: earlier through in the valproic acid derivative solution, adding tri-n-butylamine and isobutyl chlorocarbonate activation valproic acid derivant; Under-2～-8 ℃ of conditions, carry out coupling with glucose 6 phosphate dehydrogenases (G6PDH) subsequently, utilize gel chromatography column to carry out purifying, obtain valproic acid enzyme mark conjugate; Make by 1: 300～1: 5000 dilution valproic acid enzyme mark conjugate with buffer solution at last.
In the process that valproic acid is measured, reaction system is carried out in the liquid phase of homogeneous, need not separating step.
Valproic acid detection kit provided by the invention can be applicable to the accurate quantification analysis of blood valproic acid concentration, aspect clinical practice, has significant advantage.
1, accuracy is good, precision is high, specificity and sensitivity are strong.Reagent checking result shows that utilization homogeneous enzyme immunoassay detection kit provided by the invention carries out the valproic acid analysis; The average recovery rate of sample (Recovery) is 95.7 ± 3.2%; In batch, betweenrun precision related coefficient deviation (CV) is less than 4.7%, detection sensitivity reaches below the 25ng/ml.In the high specificity of the valproic acid antibody that provides in this kit, medicine cross reaction test, 32 kinds of common medicines of test there is not any cross reaction with compound, the blood concentration of suitable clinical examination valproic acid.
2, easy and simple to handle, robotization fast detecting, lower to the detecting instrument requirement.Detection kit designs to the relatively poor present situation of domestic majority hospital inspection equipment, on general Biochemical Analyzer, can carry out.On the analyser that sets operational factor, calibration solution, sample and reagent are put into corresponding sample disc and reagent dish, click working procedure and can accomplish detection automatically.Get rid of the experimental error that manual operation causes, testing result accurately and reliably.
3, practice thrift reagent cost.The valproic acid detection kit can apply to open Biochemical Analyzer, realizes the production of detectable autonomy-oriented.Realize that according to a preliminary estimate biochemical investigation changes to style of opening from enclosed type, reduce by 50% reagent cost expense at least.
Description of drawings
Fig. 1 is a valproic acid immunity quick detection kit calibration curve.
Fig. 2 detects and the comparison of immunofluorescence polarizing method for the valproic acid homogeneous enzyme immunoassay.
Embodiment
The present invention is based on the homogeneous enzyme immunoassay method and set up the valproic acid detection method, its principle has mainly been used the competitive immunization reaction principle, and course of reaction is a valproic acid and the competitive binding specificity antibody of valproic acid that is coupled on the G6PDH free in the liquid sample.G6PDH-valproic acid conjugate has immune response and enzymatic activity, can react by catalytic oxidation-reduction, transforms NADH (NAD) reduction and generates NADH.This process causes the variation of absorbance, can under the 340nm wavelength light source, be detected.In case conjugate and antibodies, the enzymatic activity of conjugate promptly is suppressed, so valproic acid content is many more in the liquid sample, makes more G6PDH-valproic acid conjugate dissociate out, causes absorption value to rise.
By specifying technology contents of the present invention, structural attitude, being realized purpose and effect, explain below in conjunction with embodiment and conjunction with figs..
One, the preparation of reactant liquor R1 reagent
1, the valproic acid immunizing antigen is synthetic
At first prepare BSA solution: 20mg BSA is dissolved in 5ml phosphate buffer (0.2mol/LNa
2HPO
4, pH8.5) in; Prepare the valproic acid derivative solution simultaneously; With 20mg valproic acid derivant, 350 μ l dimethyl formamides (DMF), 350 μ l ethanol, 7.0ml (10mmol/L; PH 5.0) EDAc of kaliumphosphate buffer, 40mg, the Sulfo-NHS of 5mg add stirring and dissolving in the beaker, stirring and dissolving reaction 30min; The valproic acid drips of solution for preparing is added in the BSA solution under 2～8 ℃ of temperature, continues to stir 12 hours, carries out purifying through dialysis subsequently, obtain the valproic acid immunizing antigen.
2, the preparation of anti-valproic acid specific antibody
With the PBS phosphate buffer synthetic valproic acid immunogene is diluted to 1.0mg/ml, the antigenic solution with 1.0ml mixes with Freund's complete adjuvant then, and rabbit is injected; After 14～21 days, again to the rabbit injection once with the identical antigenic solution of 1.0ml and incomplete Freund, every afterwards at a distance from around once, the antibody that obtains after 4 months is about 1: 300～1: 5000.
3, the preparation of reactant liquor R1 reagent
Measure 1L trishydroxymethylaminomethane (Tris) damping fluid (55mmol/L Tris, 0.1%BSA, 145.4mmol/L NaCl, 3mmol/L MgCl2 pH8.0), adds 2.02～8.07gNAD and 0.86～3.42gG6P successively, at room temperature stirs 10min.Then the valproic acid specific antibody is added in the above-mentioned solution by dilution ratio, dilution ratio is 1: 300～1: 5000.
Two, the preparation of reactant liquor R2 reagent
1, valproic acid enzyme mark conjugate is synthetic
G6PDH formulations prepared from solutions: the G6PDH of 10mg is dissolved in the 12ml Tris damping fluid (pH7.5), adds NADH, 90mg G6P and the 0.5ml carbitol of 150mg then successively, dropwise add 1.5ml dimethyl sulfoxide (DMSO) subsequently.
Valproic acid derivative solution preparation: 8mg valproic acid derivant is dissolved in 600 μ l dimethyl formamides (DMF), then solution is cooled to-2～-8 ℃ after, add 4 μ l tri-n-butylamines and 2 μ l isobutyl chlorocarbonates, continue to stir 30min down at-2～-8 ℃; At last the valproic acid derivative solution for preparing is dropwise added in the G6PDH solution and continue down to stir 1 hour at-2～-8 ℃.
Conjugate is crossed gel chromatography column carry out purifying, obtained valproic acid enzyme mark conjugate.
2, the preparation of reactant liquor R2 reagent
Valproic acid enzyme mark conjugate is added in the 1L Tris damping fluid (pH8.0) by dilution ratio, and dilution ratio is 1: 300～1: 5000.
Three, the preparation of calibration solution
Calibration solution is the solution of 6 kinds of different valproic acid mass concentrations, is dissolved in the blank buffer solution by the valproic acid calibration object making.
Valproic acid mass concentration in the calibration solution is respectively 0 μ g/ml, 12.5 μ g/ml, 25.0 μ g/ml, 50.0 μ g/ml, 100.0 μ g/ml, 150.0 μ g/ml.
Four, the use of valproic acid detection kit on automatic clinical chemistry analyzer
1, the operational factor of instrument is set: the cumulative volume of reactant liquor R1 and reactant liquor R2 reagent is 75～150 μ l; The cumulative volume of sample and calibration solution is 5～20 μ l; Analytical approach is two point velocity methods, and detection predominant wavelength is that 340nm, commplementary wave length are 405nm, and photometric range is 10 to 20 points.Specify the position and the corresponding mass concentration of reactant liquor R1 and reactant liquor R2 reagent position, valproic acid calibration solution.
2, kit is put into biochemical instruments by classification, the operation instrument is tested and the match of typical curve automatically, and calibration is the result be stored in the instrument.Subsequently clinical blood sample to be measured is added sample cup and puts into sample disc, the operation instrument is tested automatically after the sample test application, and instrument is converted into drug concentration with the absorbance rate of change that records according to typical curve, and is shown and printing by the terminal.
Five, valproic acid detection kit method validation
1, precision analysis
The valproic acid standard items are dissolved in the buffer solution, prepare concentration respectively and be 37.5 μ g/ml (low), 75 μ g/ml (in), the sample of 125 μ g/ml (height).With 5 parts of every kind of sample packing, to measure with valproic acid homogeneous enzyme immunoassay kit, calculating mean value, standard deviation obtain withinrun precision (CV%); Got the basic, normal, high sample METHOD FOR CONTINUOUS DETERMINATION of valproic acid simultaneously 5 days, calculating mean value, standard deviation obtain withinrun precision.The result is as shown in table 1, and valproic acid homogeneous enzyme immunoassay detection kit has good precision, wherein criticizes interior, betweenrun precision CV all less than 4.7%.
Table 1 valproic acid homogeneous enzyme immunoassay detects the precision analysis
2, reclaim experiment
Utilize the valproic acid calibration curve of setting up, measure the valproic acid sample of 6 concentration from low to high, each sample METHOD FOR CONTINUOUS DETERMINATION 10 times.The result is as shown in table 2, and the recovery scope of sample test is between 90.9%～98.7%, and average recovery rate is 95.7 ± 3.2%.Show that the accuracy of valproic acid homogeneous enzyme immunoassay detection kit is higher.
Table 2 valproic acid homogeneous enzyme immunoassay detects and reclaims experiment
3, lowest detection lower bound analysis
The valproic acid standard items are dissolved in the buffer solution, and the preparation sample concentration is 0ng/ml, 25ng/ml, 50ng/ml, 100ng/ml, 200ng/ml.With valproic acid homogeneous enzyme immunoassay detection kit METHOD FOR CONTINUOUS DETERMINATION 5 times, calculating mean value and standard deviation.(mean value ± 2SD) and 0ng/ml sample do not have and intersect (table 3) test result that the result shows the 25ng/ml sample in 95% fiducial interval.
Table 3 valproic acid homogeneous enzyme immunoassay detection sensitivity is analyzed
4, medicine interference experiment
Choose 32 kinds of common compounds and medicine (table 4), adjusting its working concentration is 10 μ g/ml, utilizes the valproic acid homogeneous enzyme immunoassay reagent of having set up to carry out interference test and measures.All detection of drugs of experimental result demonstration all do not have any intersection with valproic acid, and the valproic acid antibody that the present invention obtains has very strong specificity.
Table 4 valproic acid homogeneous enzyme immunoassay detection of drugs interference experiment
| Acetylsalicylic acid | (L)-ephedrine | Chlordiazepoxide | Procaine |
| Phenobarbital | Lidocaine | The d-crystal methamphetamine | Quinindium |
| Amytal | Naproxen | Fenoprofen | The U.S. acid of assistant |
| Ampicillin | Niacinamide | Gemfibrozil | The ecgonine methyl ester |
| Phenyl ethylamine | Penicillin | Gentianic acid | Ecgonine |
| Caffeine | Phyenlephrinium | Dihydrocodeinone | Stable |
| Bent | Phenylpropanolamine | Brufen | Cotinine |
| Chlorpromazine | Procainamide | Imipramine | Cis-4-cotinine carboxylic acid |
5, with FPIA method correlation analysis
Obtain 40 routine patients serum's samples, use homogeneous enzyme immunoassay detection method and Abbott's immunofluorescence polarization (FPIA) detection method to measure respectively.Valproic acid homogeneous enzyme immunoassay detection method as shown in Figure 2 and FPIA have good correlativity, and equation of linear regression is y=0.889x-2.9915, R
2Value reaches greater than 0.98.Further verified the accuracy of valproic acid homogeneous enzyme immunoassay detection kit provided by the invention.
Claims (9)
1. valproic acid homogeneous enzyme immunoassay quick detection kit is characterized in that being made up of following several kinds of components:
Reactant liquor R1 is the anti-valproic acid specific antibody of enzyme reaction substrate dilution;
Reactant liquor R2 is valproic acid enzyme mark conjugate;
Calibration solution is dissolved in the blank buffer solution by the valproic acid calibration object and makes.
2. detection kit according to claim 1 is characterized in that, said anti-valproic acid specific antibody is monoclonal or polyclone body.
3. detection kit according to claim 1 is characterized in that, the total amount of said reactant liquor R1 and reactant liquor R2 is 75～150 μ l, and calibration solution is 5～20 μ l.
4. according to each described detection kit of claim 1 to 3; It is characterized in that; The preparation process of said reactant liquor R1 is: make the activation of valproic acid derivant through adding 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDAc) and N-hydroxy thiosuccinimide (Sulfo-NHS) in the valproic acid derivative solution earlier, at room temperature carry out coupling with carrier protein subsequently and prepare immunizing antigen; With producing the valproic acid specific antibody behind the immunizing antigen immune animal of preparation; Make by 1: 300～1: 5000 dilution specific antibody with enzyme reaction substrate at last.
5. detection kit according to claim 4 is characterized in that, said carrier protein comprises seralbumin (BSA), the pure albumen of ovum gallinaceum (OVA) or keyhole limpet hemocyanin (KLH).
6. detection kit according to claim 4 is characterised in that, said enzyme reaction substrate contains NADH (NAD) and 6 glucose 1-phosphate1-s (G6P), and both concentration total amounts are 2.81～11.25mmol/L.
7. according to each described valproic acid homogeneous enzyme immunoassay detection kit of claim 1 to 3, it is characterized in that said reactant liquor R2 is made up of the valproic acid of glucose 6 phosphate dehydrogenases (G6PDH) mark.
8. valproic acid homogeneous enzyme immunoassay detection kit according to claim 7 is characterized in that, the preparation process of said reactant liquor R2 is: earlier through in the valproic acid derivative solution, adding tri-n-butylamine and isobutyl chlorocarbonate activation valproic acid derivant; Under-2～-8 ℃ of conditions, carry out coupling with glucose 6 phosphate dehydrogenases (G6PDH) subsequently, utilize gel chromatography column to carry out purifying, obtain valproic acid enzyme mark conjugate; Make by 1: 300～1: 5000 dilution valproic acid enzyme mark conjugate with buffer solution at last.
9. according to the application of the said detection kit of claim 1, it is characterized in that in the process that valproic acid is measured, reaction system is carried out, and need not separating step in the liquid phase of homogeneous.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103242445A (en) * | 2012-07-25 | 2013-08-14 | 苏州博源医疗科技有限公司 | Valproic acid immunogen and preparation method and application thereof |
| TWI473998B (en) * | 2013-01-28 | 2015-02-21 | Univ Nat Taiwan | Valproic acid concentration sensor and method for measuring the concentration of valproic acid |
| CN106190996A (en) * | 2016-08-30 | 2016-12-07 | 宁波美康生物科技股份有限公司 | A kind of G 6 PD mutant |
| CN106405069A (en) * | 2016-04-06 | 2017-02-15 | 李松羊 | Preparation method for homogeneous enzyme immunodiagnosis reagent used for glycocholic acid |
| 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 |
| CN115728353A (en) * | 2022-08-05 | 2023-03-03 | 四川大学华西医院 | Application of MscCG in preparation of kit for detecting valproic acid drugs |
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| TWI473998B (en) * | 2013-01-28 | 2015-02-21 | Univ Nat Taiwan | Valproic acid concentration sensor and method for measuring the concentration of valproic acid |
| CN106405069A (en) * | 2016-04-06 | 2017-02-15 | 李松羊 | Preparation method for homogeneous enzyme immunodiagnosis reagent used for glycocholic acid |
| CN106190996A (en) * | 2016-08-30 | 2016-12-07 | 宁波美康生物科技股份有限公司 | A kind of G 6 PD mutant |
| 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 |
| CN115825455A (en) * | 2019-07-04 | 2023-03-21 | 北京九强生物技术股份有限公司 | Application of valproic acid derivative in immunoassay |
| CN115825455B (en) * | 2019-07-04 | 2024-07-16 | 北京九强生物技术股份有限公司 | Use of valproic acid derivatives in immunoassays |
| CN115728353A (en) * | 2022-08-05 | 2023-03-03 | 四川大学华西医院 | Application of MscCG in preparation of kit for detecting valproic acid drugs |
| CN115728353B (en) * | 2022-08-05 | 2023-08-11 | 四川大学华西医院 | Application of MscCG in preparation of kit for detecting valproic acid medicines |
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Application publication date: 20120620 |