CN108061796A - A kind of amphetamine immunologic function test reagent and its preparation and detection method - Google Patents
A kind of amphetamine immunologic function test reagent and its preparation and detection method Download PDFInfo
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Abstract
The invention discloses a kind of amphetamine immunologic function test reagent and its preparation and detection methods.Including：Enzyme mark amphetamine, the indicator for detecting amphetamine enzyme-labeled antibody amphetamine compound；Above-mentioned enzyme mark amphetamine is coupled by amphetamine and glucose dehydrogenase.The amphetamine immunologic function test reagent of the present invention can accurately and quickly determine amphetamine content 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 promote the use of.
Description
Technical field
The present invention relates to field of medical examination, are specifically a kind of amphetamine immunologic function test reagent and its preparation and detection side
Method.
Background technology
Amphetamine (Amphetamine, AMPH), is called amphetamine, is a kind of clinically common central excitation and anti-
Antidepressant agents.AMPH can cause profound mental to act on, and be improved including alertness, initiative and confidence, floaty euphoria, feeling of fatigue subtract
Ability enhancing low, that language increases and focuses on.AMPH can also cause adverse reaction, can generate long-term brain tissue
Damage, is most commonly overexcited, have it is uneasy, have a sleepless night, tremble, the symptoms such as nervous and agitation.It takes after heavy dose of AMPH most
Serious consequence is exactly a kind of toxicity neuropathy, therefore the blood substance level of patient is monitored extremely important.
The common method of detection amphetamine has at present：Efficient liquid phase chromatographic analysis, radiommunoassay（RIA）And fluorescence
Polarization Method etc..Wherein, efficient liquid phase chromatographic analysis is time-consuming and is not easy to automate；Longer the time required to RIA methods, testing result is unstable
It is fixed and dangerous there are radioactive pollution.Fluorescence polarization method needs expensive instrument and equipment and veteran operating personnel.
The method that the present invention uses is for homogeneous enzyme immunoassay detection method, advantage：Easy to operate, quick, high sensitivity, standard
Really property is good, is suitable for automating, and is widely used, and with automatic clinical chemistry analyzer to small-molecule substance and macromolecular substances all
It can high-throughput quick measure.
The content of the invention
It is an object of the invention to solve, amphetamine detection process in the prior art is complicated for operation and accuracy of measurement
The problem of low, the present invention provides a kind of quick, high sensitivity, accurately detects that the peace of amphetamine content in sample to be tested is non-
His bright homogeneous enzyme immunoassay detection reagent and preparation method thereof.
To achieve the above object, the present invention provides following technical solution：
A kind of amphetamine immunologic function test reagent and its preparation and detection method, it is characterised in that：Enzyme mark amphetamine, for examining
Survey the indicator of amphetamine antibody-enzyme mark amphetamine compound；Above-mentioned enzyme mark amphetamine is by amphetamine and grape
Glucocorticoid dehydrogenase is coupled.
As further embodiment of the present invention, the indicator is selected from enzymatic reagent, including：The bottom of enzyme mark conjugate and enzyme
Object；Above-mentioned enzyme mark conjugate includes glucose dehydrogenase-amphetamine conjugate；The substrate of above-mentioned enzyme is glucose.
As further embodiment of the present invention, described a kind of amphetamine immunologic function test reagent and preparation method thereof,
It is characterized in that, includes the following steps：
（1）The preparation of glucose dehydrogenase-amphetamine conjugate：Glucose dehydrogenase（GDH）It is coupled with amphetamine, purifying
The enzyme-labelled antigen of coupling；
（2）The preparation of amphetamine homogeneous enzyme immunoassay detection reagent：
The preparation of reagent 1：It is mixed by amphetamine antibody and homogeneous zymolyte；
The preparation of reagent 2：It is mixed by glucose dehydrogenase-antigen conjugates with phosphate buffer.
As further embodiment of the present invention, a kind of preparation method of amphetamine immunologic function test reagent is special
Sign is, the step（1）Detailed process is：
1）Glucose dehydrogenase（GDH）With the coupling of amphetamine
A. 5-30 mg amphetamines are dissolved in 100-500 μ L dimethylformamides（DMF）In, slight oscillatory dissolving；Meanwhile
The GDH that 10-40 mg specifications is taken to be 100-300 KU is dissolved in concussion, uniform dissolution in PBS buffer solution；
B. amphetamine solution is added slowly with stirring in GDH solution, while it is molten to be slowly dropped into 10-40 μ L glutaraldehydes
Solution is placed in 4 DEG C after being added dropwise and is stirred to react overnight by liquid；
2）Purify the enzyme-labelled antigen of coupling
The enzyme-labelled antigen being coupled by G-25 gel chromatographies column purification, obtains glucose dehydrogenase-amphetamine conjugate, in 2-
It is stored at 8 DEG C.
As further embodiment of the present invention, a kind of preparation method of amphetamine immunologic function test reagent is special
Sign is, step（2）Detailed process it is as follows：
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 dissolving；Amphetamine antibody is added in above-mentioned homogeneous zymolyte, antibody with it is homogeneous
The volume ratio of zymolyte is 1:100~1:10000；
The preparation of reagent 2：The glucose dehydrogenase of preparation-amphetamine conjugate is added in phosphate buffer, above-mentioned idol
The volume ratio for joining object and phosphate buffer is 1:100~1:10000.
As further embodiment of the present invention, the detection method of the amphetamine immunologic function test reagent, feature exists
In comprising the following steps：
1）Sample to be tested is contacted with amphetamine antibody；
2）According to the combination situation of enzyme mark amphetamine in sample to be tested and amphetamine antibody, indicator judgement sample is utilized
The content of middle amphetamine；The sample to be tested is serum, blood plasma, saliva or urine.
The principle of the present invention is that antigen is combined into enzyme-labelled antigen with enzyme, retains the bioactivity of antigen and enzyme, when enzyme mark resists
After original is combined 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.Antigen, enzyme-labelled antigen during measure in sample are combined with antibody competition, and the antigenic content in sample is higher,
Its OD value is higher after adding substrate.
The advantage of the invention is that：The amphetamine immunologic function test reagent of the present invention can accurately and quickly determine human body blood
Amphetamine content in the samples such as liquid.Compared with existing detection reagent in the market, detection reagent of the present invention have fast and easy,
High sensitivity, high specificity quantify the advantages that accurate, are conducive to clinical promote the use of.
Description of the drawings
Fig. 1 is amphetamine homogeneous enzyme immunoassay reaction calibration graph.
Fig. 2 is amphetamine homogeneous enzyme immunoassay range of linearity figure.
Specific embodiment
The present invention provides a kind of amphetamine immunologic function test reagent and its preparation and detection method, to make mesh of the present invention
, technical solution and effect it is clearer, clear and definite, the present invention is described in detail below.
The present invention provides a kind of amphetamine immunologic function test reagent and its preparation and detection methods.Including：Enzyme mark peace is non-
His indicator bright, for detecting amphetamine antibody-enzyme mark amphetamine compound；Above-mentioned enzyme mark amphetamine is non-by pacifying
He is bright and glucose dehydrogenase is coupled.
Signified " amphetamine " refers not only to complete amphetamine molecule in the present invention, also includes retaining intact antigen
The amphetamine segment or derivative of specific binding capacity.
A kind of amphetamine homogeneous enzyme immunoassay detection reagent, including：Enzyme mark amphetamine resists for detecting amphetamine
The indicator of body-enzyme mark amphetamine compound.Indicator is selected from enzymatic reagent, radio isotope reagent, fluorometric reagent
And chemical illuminating reagent.Preferably, indicator is enzymatic reagent, including：The substrate of enzyme mark conjugate and enzyme.Wherein, enzyme mark is even
Joining object includes glucose dehydrogenase-antigen conjugates, can be obtained by chemical synthesis process.
The application method of above-mentioned amphetamine immunologic function test reagent, comprises the following steps：
1）Sample to be tested is contacted with amphetamine antibody；
2）According to the combination situation of enzyme mark amphetamine in sample to be tested and amphetamine antibody, indicator judgement sample is utilized
The content of middle amphetamine；The sample to be tested is serum, blood plasma, saliva or urine etc..Preferably, sample to be tested for serum or
Blood plasma.
Below by specific embodiment, the present invention is described in detail.
Embodiment one：The preparation of glucose dehydrogenase-antigen conjugates
1）Glucose dehydrogenase（GDH）With the coupling of amphetamine
A. 15mg amphetamines are dissolved in 250 μ L dimethylformamides（DMF）In, slight oscillatory dissolving；Meanwhile take 30.0 mg
Specification is that the GDH of 100 KU is dissolved in the PBS (pH7.4 of 3mL 0.01mol/L）In buffer solution, concussion, uniform dissolution；
B. amphetamine solution is added slowly with stirring in GDH solution, while is slowly dropped into 20 μ L25% glutaraldehyde solutions,
Solution is placed in 4 DEG C after being added dropwise to be stirred to react overnight.
2）Purify the enzyme-labelled antigen of coupling
The enzyme-labelled antigen being coupled by G-25 gel chromatographies column purification, obtains glucose dehydrogenase-amphetamine conjugate, in 2-
It is stored at 8 DEG C.
Embodiment two：The preparation of amphetamine homogeneous enzyme immunoassay detection reagent
Amphetamine homogeneous enzyme immunoassay detection reagent, including：Enzyme mark amphetamine is pacified for detecting amphetamine antibody-enzyme mark
The indicator of Fei Taming compounds.Indicator is selected from enzymatic reagent, radio isotope reagent, fluorometric reagent and chemiluminescence
Reagent.Preferably, indicator is enzymatic reagent, including：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.
Amphetamine homogeneous enzyme immunoassay detection reagent before the use, in order to avoid the enzyme mark conjugate in indicator and
The substrate of enzyme reacts, and the substrate of enzyme mark conjugate and enzyme is separated, therefore amphetamine homogeneous enzyme immunoassay detects
Reagent includes two kinds of reagents being provided separately, specific as follows：
1. the preparation of reagent 1：By 3.588g（10mM）Nicotinamide adenine dinucleotide NAD, 1.802g of oxidation state（10mM）
Homogeneous zymolyte is made with the phosphate buffer dissolving of 50 mM, pH 8.0 of 1L in glucose；Amphetamine antibody is added to
It states in homogeneous zymolyte, the volume ratio of antibody and homogeneous zymolyte is 1:100~1:10000, it is specific in the present embodiment to compare
Example is 1:400.
2. the preparation of reagent 2：The glucose dehydrogenase of preparation-amphetamine conjugate is added to the phosphorus of 50mM, pH 8.0
In phthalate buffer, the volume ratio of above-mentioned conjugate and phosphate buffer is 1:100~1:10000, have in the present embodiment
The ratio of body is 1:1500.
The application method of above-mentioned amphetamine homogeneous enzyme immunoassay detection reagent, comprises the following steps：
1）Sample to be tested is contacted with amphetamine antibody；
2）According to the combination situation of enzyme mark amphetamine in sample to be tested and amphetamine antibody, indicator judgement sample is utilized
The content of middle amphetamine；
Specifically, sample to be tested is added in reagent 1 during detection, the amphetamine in sample to be tested and the An Feita in reagent 1
Bright antibody is specifically bound, and generates anti-amphetamine antibody-amphetamine compound；Reagent 2 is added, at this time reagent 2
In glucose dehydrogenase-amphetamine conjugate and the enzyme in reagent 1 substrate mixing, contact, enzymatic reaction occurs, forms
The indicator of amphetamine antibody-enzyme mark amphetamine compound is detected, indicator is according to amphetamine in sample to be tested
And the combination situation of above-mentioned amphetamine antibody judges the content of amphetamine in sample to be tested.
Since glucose dehydrogenase-antigen conjugates and the amphetamine competitive binding amphetamine in sample to be tested resist
Body, so, the amount of amphetamine is more in sample to be tested, the glucose dehydrogenase-antigen conjugates to dissociate in homogeneous enzyme solutions
Amount it is more, enzymatic reaction is faster, causes OD340Rise.
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：Amphetamine homogeneous enzyme immunoassay detection reagent reacts calibration curve.
1）AMPH calibration objects are prepared：Commercially available people's amphetamine recombinant protein is dissolved in the solution of similar human serum matrix
（NaCl 0.9%, BSA 0.2%, NaN3 0.1%, Tris-HCl pH 7.4）In, the calibration object of various concentration is made.With Thymopetidum Injection
Object science（China）Co., Ltd's amphetamine calibration object is primary standard, using its amphetamine kit to various concentration
Calibration object detects 10 times respectively, and average is obtained, and obtains the concentration of amphetamine calibration object：10,50,100,200,400,800
ng/mL。
2）Biochemical Analyzer detects：By taking Hitachi 7170 operates as an example：Measure wavelength is 340nm, takes various concentration respectively
Calibration object solution（15μl）, add in amphetamine R1Reagent（200μl）, mixing adds amphetamine R2Reagent（50μl）, mix
After even, the OD of different time points is measured340Light absorption value calculates reaction rate during different calibration object concentration, actual mechanical process
It is middle constantly to adjust the volume ratio of reagent 1 and reagent 2, while survey luminous point is adjusted, finally show that comparatively ideal reaction normal is bent
Line chart, often pipe replication 3 times are corresponding using the average value of 3 absorbance difference Δ A measured of each calibration pipe as ordinate
Calibration object concentration is abscissa, draws " concentration-absorbance difference " calibration curve（See Fig. 1）.
Test serum or plasma sample are taken, is measured in the same method the absorbance difference of sample, substitutes into calibration curve, you can calculate
The content of amphetamine in sample to be tested.If the concentration of amphetamine exceeds calibration curve scope, need pair in serum or blood plasma
Sample detects to ensure the accuracy of testing result 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 amphetamine high concentration sample close to the range of linearity upper limit（768 ng/mL）, 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 6 diluted concentrations by solution altogether（xi）Solution, use institute
It states Biochemical Analyzer detection method and measures each diluted sample concentration.Each diluted concentration is tested 3 times, and it is dense that each dilution is obtained respectively
Spend the average of testing result（yi）.With diluted concentration（xi）For independent variable, to measure average（yi）Linear return is obtained for dependent variable
Return equation, according to formula（1）The correlation coefficient r of linear regression is calculated, the results show regression equation is y=0.9706x+0.3721,
Correlation coefficient r=0.9999 shows reagent of the present invention good relationship in the 12 ng/mL-768 ng/mL ranges of linearity（See figure
2）.
Since the detection process of the present invention is completed by instrument is full-automatic, so to the of less demanding of testing staff, easily
In realizing and promote the use of.
It should be noted that obviously the invention is not restricted to the details of above-mentioned exemplary embodiment, 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 requirements of the claims
All changes are included in the scope of patent protection of the present 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 domain of invention.
Claims (7)
1. a kind of amphetamine immunologic function test reagent and its preparation and detection method, it is characterised in that：Enzyme mark amphetamine is used for
Detect the indicator of amphetamine antibody-enzyme mark amphetamine compound.
2. the immunologic function test reagent of amphetamine according to claim 1, it is characterised in that：The enzyme mark amphetamine by
Amphetamine and glucose dehydrogenase are coupled.
3. amphetamine immunologic function test reagent according to claim 1, it is characterised in that：The indicator is tried selected from enzyme
Agent, including：The substrate of enzyme mark conjugate and enzyme；Above-mentioned enzyme mark conjugate includes glucose dehydrogenase-amphetamine conjugate；On
The substrate for stating enzyme is glucose.
4. a kind of amphetamine immunologic function test reagent and preparation method thereof, which is characterized in that include the following steps：
（1）The preparation of glucose dehydrogenase-amphetamine conjugate：Glucose dehydrogenase（GDH）It is coupled with amphetamine, purifying
The enzyme-labelled antigen of coupling；
（2）The preparation of amphetamine homogeneous enzyme immunoassay detection reagent：
The preparation of reagent 1：It is mixed by amphetamine antibody and homogeneous zymolyte；
The preparation of reagent 2：It is mixed by glucose dehydrogenase-amphetamine conjugate with phosphate buffer.
5. the preparation method of a kind of amphetamine immunologic function test reagent according to claim 5, which is characterized in that described
Step（1）Detailed process is：
1）Glucose dehydrogenase（GDH）With the coupling of amphetamine
A. 5-30 mg amphetamines are dissolved in 100-500 μ L dimethylformamides（DMF）In, slight oscillatory dissolving；Meanwhile
The GDH that 10-40 mg specifications is taken to be 100-300 KU is dissolved in concussion, uniform dissolution in PBS buffer solution；
B. amphetamine solution is added slowly with stirring in GDH solution, while it is molten to be slowly dropped into 10-40 μ L glutaraldehydes
Solution is placed in 4 DEG C after being added dropwise and is stirred to react overnight by liquid；
2）Purify the enzyme-labelled antigen of coupling
The enzyme-labelled antigen being coupled by G-25 gel chromatographies column purification, obtains glucose dehydrogenase-amphetamine conjugate, in 2-
It is stored at 8 DEG C.
A kind of 6. preparation method of amphetamine immunologic function test reagent according to claim 5, which is characterized in that step
（2）Detailed process it is as follows：
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 dissolving；Amphetamine antibody is added in above-mentioned homogeneous zymolyte, antibody with it is homogeneous
The volume ratio of zymolyte is 1:100~1:10000；
The preparation of reagent 2：The glucose dehydrogenase of preparation-amphetamine conjugate is added in phosphate buffer, above-mentioned idol
The volume ratio for joining object and phosphate buffer is 1:100~1:10000.
7. using the detection method of the amphetamine immunologic function test reagent described in 4 any one of Claims 1-4, feature exists
In comprising the following steps：
1）Sample to be tested is contacted with amphetamine antibody；
2）According to the combination situation of amphetamine in sample to be tested and amphetamine antibody, using pacifying in indicator judgement sample
The content of Fei Taming；The sample to be tested is serum, blood plasma, saliva or urine.
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101048660A (en) * | 2004-08-27 | 2007-10-03 | 灵芝国际股份有限公司 | Homogeneous enzyme immunoassay for oral fluid |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101048660A (en) * | 2004-08-27 | 2007-10-03 | 灵芝国际股份有限公司 | Homogeneous enzyme immunoassay for oral fluid |
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