CN108089001A - A kind of morphine immunologic function test reagent and its preparation and detection method - Google Patents
A kind of morphine immunologic function test reagent and its preparation and detection method Download PDFInfo
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- CN108089001A CN108089001A CN201711392424.7A CN201711392424A CN108089001A CN 108089001 A CN108089001 A CN 108089001A CN 201711392424 A CN201711392424 A CN 201711392424A CN 108089001 A CN108089001 A CN 108089001A
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/94—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving narcotics or drugs or pharmaceuticals, neurotransmitters or associated receptors
- G01N33/9486—Analgesics, e.g. opiates, aspirine
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- G—PHYSICS
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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- G01N33/581—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with enzyme label (including co-enzymes, co-factors, enzyme inhibitors or substrates)
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Abstract
The invention discloses a kind of morphine immunologic function test reagent and its preparation and detection methods.Including：Enzyme mark morphine, the indicator for detecting morphine Abs enzyme mark morphine compound；Above-mentioned enzyme mark morphine is coupled by morphine and glucose dehydrogenase.The morphine immunologic function test reagent of the present invention can accurately and quickly determine morphine 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 morphine immunologic function test reagent and its preparation and detection method.
Background technology
Opium is narcotic analgeiscs medically, is extract from a kind of herbaceous plant opium poppy.Morphine is crow
Main alkaloid in piece has analgesia and syngignoscism, can medically be used as analgesics utilization, is considered as releasing severe pain most
Effective tradition anodyne.Morphine also has sedation, can protect body because of traumatic shock, internal haemorrhage, congestive heart failure
And the failure caused by some deelines.If but long-term consumption morphine can habituation and occur it is weak, feel sleepy, feel cold, face it is yellow
Flesh is thin or even damages nervous system, digestive system, causes endocrine disorder.Human body just generates floaty euphoria, illusion after taking morphine
Sense, may also lead to rely on or even habituation, be caused great harm to personal and society, the detection of morphine caused social
Extensive concern.
The common method of detection morphine has at present：Enzyme linked immunosorbent assay (ELISA)（ELISA）, radiommunoassay（RIA）, gas
Phase chromatograph-mass spectrometer coupling method, liquid chromatogram and Liquid Chromatography-Mass Spectrometry etc..Wherein, ELISA method dosing accuracy it is poor,
Operating time is long, the degree of automation is low, is chiefly used in qualitative detection；Longer the time required to RIA methods, testing result is unstable, repeats
Property it is poorer than ELISA, and there are radioactive pollution is dangerous.Gas chromatographic technique is more mature, and application is also commonplace, but is subject to
Mobile phase must be the limitation of gas, and high temperature often makes morphine acid decarboxylation higher so as to cause its quantitative analysis results.It is high
Effect liquid phase chromatogram method analysis cost is high, and liquid chromatograph price and regular maintenance expense are expensive, and analysis time is generally longer than gas phase.
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, morphine detection process is complicated for operation in the prior art and accuracy of measurement is low
Problem the present invention provides a kind of quick, high sensitivity, accurately detects that the homogeneous enzyme of morphine of morphine content in sample to be tested is exempted from
Epidemic disease detection reagent and preparation method thereof.
To achieve the above object, the present invention provides following technical solution：
A kind of morphine immunologic function test reagent and its preparation and detection method, it is characterised in that：Enzyme mark morphine resists for detecting morphine
The indicator of body-enzyme mark morphine compound；Above-mentioned enzyme mark morphine is coupled by morphine and glucose dehydrogenase.
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-morphine conjugate；The substrate of above-mentioned enzyme is glucose.
As further embodiment of the present invention, the glucose dehydrogenase-morphine conjugate by glucose dehydrogenase with
Morphine is coupled to be formed.
As further embodiment of the present invention, described a kind of morphine immunologic function test reagent and preparation method thereof, feature
It is, includes the following steps：
（1）The preparation of glucose dehydrogenase-morphine conjugate：Glucose dehydrogenase（GDH）With the coupling of morphine, coupling is purified
Enzyme mark morphine；
（2）The preparation of morphine homogeneous enzyme immunoassay detection reagent：
The preparation of reagent 1：It is mixed by morphine Abs 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 morphine immunologic function test reagent, feature exists
In the step（1）Detailed process is：
1）Glucose dehydrogenase（GDH）With the coupling of morphine
A. 10-50 mg GDH are accurately weighed, and are dissolved in 5-15 mL sodium-acetate buffers in round-bottomed flask；
B. 100-500 mg morphines are dissolved in dimethyl sulfoxide (DMSO), and be added dropwise in above-mentioned round-bottomed flask；
C. the formalin of 10-100 μ L is rapidly joined in above-mentioned b solution, when 20-50 DEG C of water-bath jog 6-12 is small.
2）Purify the enzyme mark morphine of coupling
The enzyme mark morphine being coupled by G-25 gel chromatographies column purification, obtains glucose dehydrogenase-morphine conjugate, and in 2-8
It is stored at DEG C.
As further embodiment of the present invention, a kind of preparation method of morphine immunologic function test reagent, feature exists
In step（2）Detailed process it is as follows：
The preparation of reagent 1：By nicotinamide adenine dinucleotide NAD, the 0.5-3 g glucose 0.5-2 of 2-5 g oxidation state
Homogeneous zymolyte is made in the dissolving of L phosphate buffers；Morphine Abs are added in above-mentioned homogeneous zymolyte, antibody and homogeneous enzyme bottom
The volume ratio of object is 1:100~1:10000；
The preparation of reagent 2：The glucose dehydrogenase of preparation-morphine conjugate is added in phosphate buffer, above-mentioned conjugate
Volume ratio with phosphate buffer is 1:100~1:10000.
As further embodiment of the present invention, the detection method of the morphine immunologic function test reagent, which is characterized in that bag
Include following steps：
1）Sample to be tested is contacted with morphine Abs；
2）According to the combination situation of enzyme mark morphine in sample to be tested and morphine Abs, morphine in indicator judgement sample is utilized
Content；The sample to be tested is serum, blood plasma, saliva or urine.
The principle of the present invention is that haptens is combined into enzyme mark haptens with enzyme, retains the bioactivity of haptens and enzyme, when
After enzyme mark haptens is combined with antibody, zymoprotein and antibody close contact on hapten molecule are subject to the activated centre of enzyme
It influences, the activity of enzyme is suppressed.Antigen, enzyme mark haptens during measure in sample are combined with antibody competition, in sample
Antigenic content is higher, its OD value is higher after adding substrate.
The advantage of the invention is that：The morphine immunologic function test reagent of the present invention can accurately and quickly determine blood of human body etc.
Morphine content in sample.Compared with existing detection reagent in the market, detection reagent of the present invention has fast and easy, sensitivity
Height, high specificity quantify the advantages that accurate, are conducive to clinical promote the use of.
Description of the drawings
Fig. 1 is morphine homogeneous enzyme immunoassay reaction calibration graph；
Fig. 2 is morphine homogeneous enzyme immunoassay range of linearity figure.
Specific embodiment
The present invention provides a kind of morphine immunologic function test reagent and its preparation and detection method, to make the object of the invention, skill
Art scheme and effect are clearer, clear and definite, and the present invention is described in detail below.
The present invention provides a kind of morphine immunologic function test reagent and its preparation and detection methods.Including：Enzyme mark morphine is used for
Detect the indicator of morphine Abs-enzyme mark morphine compound；Above-mentioned enzyme mark morphine by morphine and glucose dehydrogenase coupling and
Into.
Signified " morphine " refers not only to complete morphine molecule in the present invention, also includes retaining intact antigen specificity knot
The morphine segment or derivative of conjunction ability.
A kind of morphine homogeneous enzyme immunoassay detection reagent, including：Enzyme mark morphine is answered for detecting morphine Abs-enzyme mark morphine
Close the indicator of object.Indicator is selected from enzymatic reagent, radio isotope reagent, fluorometric reagent and chemical illuminating reagent.It is excellent
Choosing, indicator is enzymatic reagent, including：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.
The application method of above-mentioned morphine immunologic function test reagent, comprises the following steps：
1）Sample to be tested is contacted with morphine Abs；
2）According to the combination situation of enzyme mark morphine in sample to be tested and morphine Abs, morphine in indicator judgement sample is utilized
Content；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：The preparation of glucose dehydrogenase-morphine conjugate
1）Glucose dehydrogenase（GDH）With the coupling of morphine
A. 25 mg GDH are accurately weighed, and are dissolved in the sodium-acetate buffer of 7 mL 0.1M in round-bottomed flask；
B. 250 mg morphines are dissolved in the dimethyl sulfoxide (DMSO) of 3 mL, and be added dropwise in above-mentioned round-bottomed flask；
C. the formalin of 75 μ L is rapidly joined in above-mentioned b solution, when 30 DEG C of water-bath jogs 10 are small.
2）Purify the enzyme mark morphine of coupling
The enzyme mark morphine being coupled by G-25 gel chromatographies column purification, obtains glucose dehydrogenase-morphine conjugate, and in 2-8
It is stored at DEG C.
Embodiment two：The preparation of morphine homogeneous enzyme immunoassay detection reagent
Morphine homogeneous enzyme immunoassay detection reagent, including：Enzyme mark morphine, the finger for detecting morphine Abs-enzyme mark morphine compound
Show reagent.Indicator is selected from enzymatic reagent, radio isotope reagent, fluorometric reagent and chemical illuminating reagent.Preferably, indicate
Reagent is enzymatic reagent, including：The substrate of enzyme mark conjugate and enzyme.Wherein, it is even to include glucose dehydrogenase-antigen for enzyme mark conjugate
Join object, can be obtained by chemical synthesis process.
Morphine homogeneous enzyme immunoassay detection reagent before the use, in order to avoid the enzyme mark conjugate in indicator and enzyme
Substrate reacts, and the substrate of enzyme mark conjugate and enzyme is separated, therefore morphine homogeneous enzyme immunoassay detection reagent includes
Two kinds of reagents being provided separately, it is specific as follows：
1. the preparation of reagent 1：By 3.588g（10 mM）Nicotinamide adenine dinucleotide NAD, 1.802g of oxidation state（10
mM）Homogeneous zymolyte is made with the phosphate buffer dissolving of 50 mM, pH 8.0 of 1L in glucose；Morphine Abs are added to above-mentioned
In homogeneous zymolyte, the volume ratio of antibody and homogeneous zymolyte is 1:100~1:10000, specific ratio in the present embodiment
For 1:500.
2. the preparation of reagent 2：The glucose dehydrogenase of preparation-morphine conjugate is added to the phosphoric acid of 50 mM, pH 8.0
In salt buffer, the volume ratio of above-mentioned conjugate and phosphate buffer is 1:100~1:10000, in the present embodiment specifically
Ratio be 1:1600.
The application method of above-mentioned morphine homogeneous enzyme immunoassay detection reagent, comprises the following steps：
1）Sample to be tested is contacted with morphine Abs；
2）According to the combination situation of enzyme mark morphine in sample to be tested and morphine Abs, morphine in indicator judgement sample is utilized
Content.
Specifically, sample to be tested is added in reagent 1 during detection, the morphine in sample to be tested resists with the morphine in reagent 1
Body is specifically bound, generation anti-morphine ab antibody-morphine compound；Reagent 2 is added, the glucose in reagent 2 takes off at this time
Hydrogen enzyme-morphine conjugate is mixed with the substrate of the enzyme in reagent 1, contacted, and enzymatic reaction occurs, and forms detection morphine Abs-enzyme
The indicator of morphine compound is marked, indicator judges according to the combination situation of morphine in sample to be tested and above-mentioned morphine Abs
The content of morphine in sample to be tested.
Due to the morphine competitive binding morphine Abs in glucose dehydrogenase-morphine conjugate and sample to be tested, so,
The amount of morphine is more in sample to be tested, and the amount of the glucose dehydrogenase-morphine conjugate to dissociate in homogeneous enzyme solutions is more, enzymatic
Reaction is faster, causes 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：Morphine homogeneous enzyme immunoassay detection reagent reacts calibration curve.
1）Morphine calibration object is prepared：Commercially available people's morphine is dissolved in normal saline solution, the calibration object of various concentration is made.
Using Beijing Core Technology Co., Ltd.'s morphine calibration object as primary standard, using its morphine kit to the calibration object of various concentration
It detects 10 times respectively, average is obtained, obtains the concentration of morphine 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 340 nm, takes various concentration respectively
Calibration object solution（15 μL）, add in morphine R1Reagent（160 μL）, mixing adds morphine R2Reagent（40 μL）, after mixing,
Measure the OD of different time points340Light absorption value calculates reaction rate during different calibration object concentration, is needed not in actual mechanical process
The volume ratio of disconnected adjustment reagent 1 and reagent 2, while survey luminous point is adjusted, finally draw comparatively ideal reaction normal graph,
Often pipe replication 3 times, using the average value of 3 absorbance difference Δ A measured of each calibration pipe as ordinate, corresponding 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 morphine in sample to be tested.If the concentration of morphine exceeds calibration curve scope in serum or blood plasma, sample need to be carried out
Detect to ensure the accuracy of testing result after dilution 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 morphine high concentration sample close to the range of linearity upper limit（832 ng/mL）, it is pressed 1/2,1/4,1/ with physiological saline
8,1/16,1/32,1/64 dilutions, are configured to 6 diluted concentrations altogether（xi）Solution, with the Biochemical Analyzer detection method
Measure each diluted sample concentration.Each diluted concentration is tested 3 times, and the average of each diluted concentration testing result is obtained respectively
（yi）.With diluted concentration（xi）For independent variable, to measure average（yi）Equation of linear regression is obtained for dependent variable, according to formula
（1）Calculate linear regression correlation coefficient r, the results show regression equation be y=1.0087x-0.4106, correlation coefficient r=
0.9998, show reagent of the present invention good relationship in the 13 ng/mL -832 ng/mL ranges of linearity（See Fig. 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 morphine immunologic function test reagent and its preparation and detection method, it is characterised in that：Enzyme mark morphine, for detecting morphine
The indicator of antibody-enzyme mark morphine compound.
2. the immunologic function test reagent of morphine according to claim 1, it is characterised in that：The enzyme mark morphine is by morphine and Portugal
Grape glucocorticoid dehydrogenase is coupled.
3. morphine immunologic function test reagent according to claim 1, it is characterised in that：The indicator is selected from enzymatic reagent,
Including：The substrate of enzyme mark conjugate and enzyme；Above-mentioned enzyme mark conjugate includes glucose dehydrogenase-morphine conjugate；Above-mentioned enzyme
Substrate is glucose.
4. a kind of morphine immunologic function test reagent and preparation method thereof, which is characterized in that include the following steps：
（1）The preparation of glucose dehydrogenase-morphine conjugate：Glucose dehydrogenase（GDH）With the coupling of morphine, coupling is purified
Enzyme mark morphine；
（2）The preparation of morphine homogeneous enzyme immunoassay detection reagent：
The preparation of reagent 1：It is mixed by morphine Abs and homogeneous zymolyte；
The preparation of reagent 2：It is mixed by glucose dehydrogenase-morphine conjugate with phosphate buffer.
A kind of 5. preparation method of morphine immunologic function test reagent according to claim 5, which is characterized in that the step
（1）Detailed process is：
1）Glucose dehydrogenase（GDH）With the coupling of morphine
A. 10-50 mg GDH are accurately weighed, and are dissolved in 5-15 mL sodium-acetate buffers in round-bottomed flask；
B. 100-500 mg morphines are dissolved in dimethyl sulfoxide (DMSO), and be added dropwise in above-mentioned round-bottomed flask；
C. the formalin of 10-100 μ L is rapidly joined in above-mentioned b solution, when 20-50 DEG C of water-bath jog 6-12 is small；
2）Purify the enzyme mark morphine of coupling
The enzyme mark morphine being coupled by G-25 gel chromatographies column purification, obtains glucose dehydrogenase-morphine conjugate, and in 2-8
It is stored at DEG C.
A kind of 6. preparation method of morphine immunologic function test reagent according to claim 5, which is characterized in that step（2）'s
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 dissolving；Morphine Abs are added in above-mentioned homogeneous zymolyte, antibody and homogeneous enzyme bottom
The volume ratio of object is 1:100~1:10000；
The preparation of reagent 2：The glucose dehydrogenase of preparation-morphine conjugate is added in phosphate buffer, above-mentioned conjugate
Volume ratio with phosphate buffer is 1:100~1:10000.
7. utilize the detection method of the morphine immunologic function test reagent described in 4 any one of Claims 1-4, which is characterized in that bag
Include following steps：
1）Sample to be tested is contacted with morphine Abs；
2）According to the combination situation of morphine in sample to be tested and morphine Abs, contained using morphine in indicator judgement sample
Amount；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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