CN108107203A - A kind of gentamicin immunologic function test reagent and its preparation and detection method - Google Patents
A kind of gentamicin immunologic function test reagent and its preparation and detection method Download PDFInfo
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- CN108107203A CN108107203A CN201711383185.9A CN201711383185A CN108107203A CN 108107203 A CN108107203 A CN 108107203A CN 201711383185 A CN201711383185 A CN 201711383185A CN 108107203 A CN108107203 A CN 108107203A
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- G—PHYSICS
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- 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
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
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- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/5308—Immunoassay; Biospecific binding assay; Materials therefor for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2430/00—Assays, e.g. immunoassays or enzyme assays, involving synthetic organic compounds as analytes
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Abstract
The invention discloses a kind of gentamicin immunologic function test reagent and its preparation and detection methods.Including：Enzyme mark gentamicin, the indicator for detecting gentamicin antibody enzyme mark gentamicin compound；Above-mentioned enzyme mark gentamicin is coupled by gentamicin and glucose dehydrogenase.The gentamicin immunologic function test reagent of the present invention can accurately and quickly determine Study on Determination of Gentamycin 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 biological detection, are specifically a kind of gentamicin immunologic function test reagent and its preparation and detection side
Method.
Background technology
Gentamicin（Gentamicin, GM）It is a kind of aminoglycoside antibiotics, antimicrobial spectrum is wider.Its mechanism of action is
The in vivo ribosomes of bacterium is acted on, protein synthesis is inhibited bacteria, and destroys the integrality of bacterial cell membrane.GM is extensive
Applied to the prevention and control of plant diseases, pest control of agriculture, poultry, fowl, woods and aquatic products, usually occur that GM residual quantities are big to ask due to the use of the abuse of person
Topic.Also, gentamicin has apparent toxic action to the mankind, and the damage to cranial nerve, the sense of hearing and kidney is serious.Therefore
It is very important to the limitation and detection of the residual quantity of gentamicin in animal derived food.
The common method of detection gentamicin has at present：Efficient liquid phase chromatographic analysis, enzyme linked immunosorbent assay (ELISA)
（ELISA）And radiommunoassay（RIA）Deng.Wherein, efficient liquid phase chromatographic analysis is time-consuming and is not easy to automate；ELISA methods
Dosing accuracy is poor, the operating time is long, the degree of automation is low, is chiefly used in qualitative detection；It is longer the time required to RIA methods, detection knot
Fruit is unstable, and repeatability is poorer than ELISA, and dangerous there are radioactive pollution.
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, gentamicin 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 celebrating of Study on Determination of Gentamycin in sample to be tested is big
Mycin homogeneous enzyme immunoassay detection reagent and preparation method thereof.
To achieve the above object, the present invention provides following technical solution：
A kind of gentamicin immunologic function test reagent and its preparation and detection method, it is characterised in that：Enzyme mark gentamicin, for examining
Survey the indicator of gentamicin antibody-enzyme mark gentamicin compound；Above-mentioned enzyme mark gentamicin is by gentamicin 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-gentamicin conjugate；The substrate of above-mentioned enzyme is glucose.
As further embodiment of the present invention, described a kind of gentamicin immunologic function test reagent and preparation method thereof,
It is characterized in that, includes the following steps：
（1）The preparation of glucose dehydrogenase-gentamicin conjugate：Prepare glucose dehydrogenase（GDH）Solution, gentamicin
GDH and gentamicin are coupled by activation, purify the enzyme-labelled antigen of coupling；
（2）The preparation of gentamicin homogeneous enzyme immunoassay detection reagent：
The preparation of reagent 1：It is mixed by gentamicin 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 gentamicin immunologic function test reagent is special
Sign is, the step（1）Detailed process is：
1）Glucose dehydrogenase（GDH）The preparation of solution：
A. the GDH that 5-20 mg specifications are 100-300 KU is weighed, room-temperature dissolution is in 6-10 ml PBS buffer solution；
2）The activation of gentamicin
Following chemicals is added to stirring and dissolving in beaker：10-30 mg gentamicins, 10-40 mg1- ethyl -3- (- 3-
Dimethylaminopropyl) carbodiimide, 2-10 mg N- hydroxy thiosuccinimides, be dissolved in morpholino b acid（MES）In solution,
Stirring and dissolving at room temperature is reacted 15-60 minutes；
3）The coupling of GDH and gentamicin
A. the gentamycin solution of above-mentioned activation is added dropwise in the GDH solution of above-mentioned dissolving；
B 2-8 DEG C are stirred overnight；
4）Purify the enzyme-labelled antigen of coupling
The enzyme-labelled antigen being coupled by G-25 gel chromatographies column purification, obtains glucose dehydrogenase-gentamicin conjugate, in 2-
It is stored at 8 DEG C.
As further embodiment of the present invention, a kind of preparation method of gentamicin 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；Gentamicin 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-gentamicin 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 gentamicin immunologic function test reagent, feature exists
In comprising the following steps：
1）Sample to be tested is contacted with gentamicin antibody；
2）According to the combination situation of enzyme mark gentamicin in sample to be tested and gentamicin antibody, indicator judgement sample is utilized
The content of middle gentamicin；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 gentamicin immunologic function test reagent of the present invention can accurately and quickly determine human body blood
Study on Determination of Gentamycin 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 gentamicin homogeneous enzyme immunoassay reaction calibration graph.
Fig. 2 is gentamicin homogeneous enzyme immunoassay range of linearity figure.
Fig. 3 is that gentamicin homogeneous enzyme immunoassay is related to the enzyme-linked immunosorbent assay testing result of Suzhou Jiang Lai
Property analysis chart.
Specific embodiment
The present invention provides a kind of gentamicin 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 gentamicin immunologic function test reagent and its preparation and detection methods.Including：The celebrating of enzyme mark is big
Mycin, the indicator for detecting gentamicin antibody-enzyme mark gentamicin compound；Above-mentioned enzyme mark gentamicin is big by celebrating
Mycin and glucose dehydrogenase are coupled.
Signified " gentamicin " refers not only to complete gentamicin molecule in the present invention, also includes retaining intact antigen
The gentamicin segment or derivative of specific binding capacity.
A kind of gentamicin homogeneous enzyme immunoassay detection reagent, including：Enzyme mark gentamicin resists for detecting gentamicin
The indicator of body-enzyme mark gentamicin 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 gentamicin immunologic function test reagent, comprises the following steps：
1）Sample to be tested is contacted with gentamicin antibody；
2）According to the combination situation of enzyme mark gentamicin in sample to be tested and gentamicin antibody, indicator judgement sample is utilized
The content of middle gentamicin；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）The preparation of solution：
A. weigh 10 mg specifications be 100KU GDH, room-temperature dissolution in 8 ml concentration be 10mM PBS solution in, pH=
7.4；
2）The activation of gentamicin
Following chemicals is added to stirring and dissolving in beaker：25 mg gentamicins, 20 mg1- ethyls -3- (- 3- diformazan ammonia
Propyl) carbodiimide, 5 mg N- hydroxy thiosuccinimides, be dissolved in 600 μ L morpholino b acids（MES）In solution, in room
The lower stirring and dissolving of temperature, reacts 30 minutes；
3）The coupling of GDH and gentamicin
A. the gentamycin solution of above-mentioned activation is added dropwise in the GDH solution of above-mentioned dissolving；
B 2-8 DEG C are stirred overnight；
4）Purify the enzyme-labelled antigen of coupling
The enzyme-labelled antigen being coupled by G-25 gel chromatographies column purification, obtains glucose dehydrogenase-gentamicin conjugate, in 2-
It is stored at 8 DEG C.
Embodiment two：The preparation of gentamicin homogeneous enzyme immunoassay detection reagent
Gentamicin homogeneous enzyme immunoassay detection reagent, including：Enzyme mark gentamicin is celebrated for detecting gentamicin antibody-enzyme mark
The indicator of big mycin compound.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.
Gentamicin 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 gentamicin 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；Gentamicin 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-gentamicin conjugate is added to the phosphorus of 50mM, pH8.0
In phthalate buffer, the volume ratio of above-mentioned conjugate and phosphate buffer is 1:100~1:10000, in the present embodiment
Specific ratio is 1:1500.
The application method of above-mentioned gentamicin homogeneous enzyme immunoassay detection reagent, comprises the following steps：
1）Sample to be tested is contacted with gentamicin antibody；
2）According to the combination situation of enzyme mark gentamicin in sample to be tested and gentamicin antibody, indicator judgement sample is utilized
The content of middle gentamicin；
Specifically, sample to be tested is added in reagent 1 during detection, the gentamicin in sample to be tested and the celebrating in reagent 1 are mould greatly
Plain antibody is specifically bound, and generates anti-gentamicin antibody-gentamicin compound；Reagent 2 is added, at this time reagent 2
In glucose dehydrogenase-gentamicin conjugate and the enzyme in reagent 1 substrate mixing, contact, enzymatic reaction occurs, forms
The indicator of gentamicin antibody-enzyme mark gentamicin compound is detected, indicator is according to gentamicin in sample to be tested
And the combination situation of above-mentioned gentamicin antibody judges the content of gentamicin in sample to be tested.
Since glucose dehydrogenase-antigen conjugates and the gentamicin competitive binding gentamicin in sample to be tested resist
Body, so, the amount of gentamicin 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 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：Gentamicin homogeneous enzyme immunoassay detection reagent reacts calibration curve.
1）Calibration object is prepared：Commercially available people's gentamicin recombinant protein is dissolved in the solution of similar human serum matrix（NaCl
0.9%, BSA0.2%, NaN30.1%, Tris-HCl pH 7.4）In, the calibration object of various concentration is made.With Suzhou river Lay biology
The gentamicin calibration object of research institute is primary standard, and the calibration object of various concentration is examined respectively using its gentamicin kit
It surveys 10 times, average is obtained, obtains the concentration of gentamicin calibration object：1,5,10,20,40,80 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 gentamicin R1Reagent（160μl）, mixing adds gentamicin R2Reagent（40μl）, 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 gentamicin in sample to be tested.If the concentration of gentamicin 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 gentamicin high concentration sample close to the range of linearity upper limit（76.8 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=1.0233x+0.0027,
Correlation coefficient r=0.9996 shows reagent of the present invention good relationship in the 1.2 ng/mL -76.8 ng/mL ranges of linearity（See
Fig. 2）.
Embodiment five：Correlation analysis
To 80 clinical samples respectively using the enzyme-linked immunosorbent assay of Suzhou Jiang Lai and the homogeneous enzyme immunoassay method of the present invention
Respectively to sample replication 2 times, average value is calculated respectively, and linear regression analyses are carried out to 80 pattern detection results, are calculated
The related coefficient of two kinds of reagent testing results.
The results show that correlation coefficient r=0.9987 of two kinds of kit testing results, equation of linear regression are
y=1.0079x+0.0016（See Fig. 3）, show gentamicin homogeneous enzyme immunoassay method detection reagent of the present invention and commercially available Suzhou
The enzyme-linked immunosorbent assay detection reagent of Jiang Lai has good uniformity.
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 gentamicin immunologic function test reagent and its preparation and detection method, it is characterised in that：Enzyme mark gentamicin is used for
Detect the indicator of gentamicin antibody-enzyme mark gentamicin compound.
2. the immunologic function test reagent of gentamicin according to claim 1, it is characterised in that：The enzyme mark gentamicin by
Gentamicin and glucose dehydrogenase are coupled.
3. gentamicin 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-gentamicin conjugate；On
The substrate for stating enzyme is glucose.
4. a kind of gentamicin immunologic function test reagent and preparation method thereof, which is characterized in that include the following steps：
（1）The preparation of glucose dehydrogenase-gentamicin conjugate：Prepare glucose dehydrogenase（GDH）Solution, gentamicin
GDH and gentamicin are coupled by activation, purify the enzyme-labelled antigen of coupling；
（2）The preparation of gentamicin homogeneous enzyme immunoassay detection reagent：
The preparation of reagent 1：It is mixed by gentamicin antibody and homogeneous zymolyte；
The preparation of reagent 2：It is mixed by glucose dehydrogenase-gentamicin conjugate with phosphate buffer.
5. the preparation method of a kind of gentamicin immunologic function test reagent according to claim 5, which is characterized in that described
Step（1）Detailed process is：
1）Glucose dehydrogenase（GDH）The preparation of solution
The GDH that 5-20 mg specifications are 100-300 KU is weighed, room-temperature dissolution is in 6-10 mL PBS buffer solution；
2）The activation of gentamicin
Following chemicals is added to stirring and dissolving in beaker：10-30 mg gentamicins, 10-40 mg1- ethyl -3- (- 3-
Dimethylaminopropyl) carbodiimide, 2-10 mg N- hydroxy thiosuccinimides, be dissolved in morpholino b acid（MES）In solution,
Stirring and dissolving at room temperature is reacted 15-60 minutes；
3）The coupling of GDH and gentamicin
A. the gentamycin solution of above-mentioned activation is added dropwise in the GDH solution of above-mentioned dissolving；
B. it is stirred overnight for 2-8 DEG C；
4）Purify the enzyme-labelled antigen of coupling
The enzyme-labelled antigen being coupled by G-25 gel chromatographies column purification, obtains glucose dehydrogenase-gentamicin conjugate, in 2-
It is stored at 8 DEG C.
A kind of 6. preparation method of gentamicin 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 0.5-2 L of 2-5 g oxidation state
Homogeneous zymolyte is made in phosphate buffer dissolving；Gentamicin 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-gentamicin 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 gentamicin 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 gentamicin antibody；
2）According to the combination situation of gentamicin in sample to be tested and gentamicin antibody, using being celebrated in indicator judgement sample
The content of big mycin；The sample to be tested is serum, blood plasma, saliva or urine.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109884317A (en) * | 2019-03-12 | 2019-06-14 | 杭州博谱医药科技有限公司 | Application of the oxidized form Thio-NAD+ I in homogeneous enzyme immunoassay diagnostic reagent |
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 |
-
2017
- 2017-12-22 CN CN201711383185.9A patent/CN108107203A/en active Pending
Patent 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 |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109884317A (en) * | 2019-03-12 | 2019-06-14 | 杭州博谱医药科技有限公司 | Application of the oxidized form Thio-NAD+ I in homogeneous enzyme immunoassay diagnostic reagent |
| CN109884317B (en) * | 2019-03-12 | 2022-03-08 | 杭州博谱医药科技有限公司 | Application of oxidized thio-coenzyme I in homogeneous enzyme immunodiagnosis reagent |
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