CN108508194A - A kind of tobramycin immunologic function test reagent and its preparation and detection method - Google Patents
A kind of tobramycin immunologic function test reagent and its preparation and detection method Download PDFInfo
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- CN108508194A CN108508194A CN201810495292.9A CN201810495292A CN108508194A CN 108508194 A CN108508194 A CN 108508194A CN 201810495292 A CN201810495292 A CN 201810495292A CN 108508194 A CN108508194 A CN 108508194A
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- Prior art keywords
- tobramycin
- enzyme
- preparation
- reagent
- function test
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- G—PHYSICS
- G01—MEASURING; TESTING
- 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
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
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- G—PHYSICS
- G01—MEASURING; TESTING
- 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
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
Abstract
The invention discloses a kind of tobramycin immunologic function test reagent and its preparation and detection methods.Including：Enzyme mark tobramycin, the indicator for detecting tobramycin enzyme-labeled antibody tobramycin compound；Above-mentioned enzyme mark tobramycin is coupled by tobramycin and glucose dehydrogenase.The tobramycin immunologic function test reagent of the present invention can accurately and quickly determine tobramycin 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, specifically a kind of tobramycin immunologic function test reagent and its preparation and detection side
Method.
Background technology
Tobramycin（Tobramycin, TOB）It is second generation aminoglycoside antibiotics, opposite first generation aminoglycoside
For class antibiotic, better efficacy, toxicity are lower, are commonly used in drug administration by injection, treat serious gram positive bacterial infection,
As one of current most commonly used aminoglycosides antibiotics of world's clinical application.Tobramycin is also obtained in field of veterinary
Wide use, therefore it also has presence in the common food of the people such as meat, milk.Due to the treatment concentration of its safety
For 4 mg/L-8 mg/L, long-time blood concentration, which is more than 8 mg/L, may cause serious renal toxicity and ototoxicity.So
Tobramycin quickly, sensitively measure in the samples such as related food necessary.
The common method of detection tobramycin has at present：Efficient liquid phase chromatographic analysis, microbial method and capillary electrophoresis
Deng.Wherein, efficient liquid phase chromatographic analysis is time-consuming and is not easy to automate, and using ultraviolet detection when needs to derive；Microbial method
Required time is longer, and testing result is less reproducible；Capillary electrophoresis sensitivity is relatively low and reproducibility is poor.
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 high-throughput can quickly measure.
Invention content
It is an object of the invention to solve, tobramycin detection process in the prior art is complicated for operation and accuracy of measurement
Low problem, the present invention provides a kind of quick, high sensitivity, the accurate appropriate cloth for detecting tobramycin content in sample to be tested
Mycin homogeneous enzyme immunoassay detection reagent and preparation method thereof.
To achieve the above object, the present invention provides the following technical solutions：
A kind of tobramycin immunologic function test reagent and its preparation and detection method, it is characterised in that：Enzyme mark tobramycin, for examining
Survey the indicator of tobramycin antibody-enzyme mark tobramycin compound；Above-mentioned enzyme mark tobramycin is by tobramycin and grape
Glucocorticoid dehydrogenase is coupled.
As a further solution 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-tobramycin conjugate；The substrate of above-mentioned enzyme is glucose.
As a further solution of the present invention, a kind of tobramycin immunologic function test reagent and preparation method thereof,
It is characterized in that, includes the following steps：
（1）The preparation of glucose dehydrogenase-tobramycin conjugate：Glucose dehydrogenase（GDH）It is coupled with tobramycin, purifying
The enzyme-labelled antigen of coupling.
（2）The preparation of tobramycin homogeneous enzyme immunoassay detection reagent：
The preparation of reagent 1：It is mixed by tobramycin antibody and homogeneous zymolyte；
The preparation of reagent 2：It is mixed with phosphate buffer by glucose dehydrogenase-antigen conjugates.
As a further solution of the present invention, the preparation method of a kind of tobramycin immunologic function test reagent, it is special
Sign is, the step（1）Detailed process is：
1）Glucose dehydrogenase（GDH）With the coupling of tobramycin
A. 5-30 mg tobramycin is dissolved in 100-500 μ L dimethylformamides（DMF）In, slight oscillatory dissolving；Meanwhile
It is that the GDH of 100-300 KU is dissolved in concussion, uniform dissolution in PBS buffer solution to take 10-40 mg specifications.
B. tobramycin solution is added slowly with stirring in GDH solution, while is 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 solution.
2）Purify the enzyme-labelled antigen of coupling
The enzyme-labelled antigen being coupled by G-25 gel chromatography column purifications, obtains glucose dehydrogenase-tobramycin conjugate, in 2-
It is stored at 8 DEG C.
As a further solution of the present invention, the preparation method of a kind of tobramycin immunologic function test reagent, it 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 0.5-2 L of 2-5 g oxidation state
Homogeneous zymolyte is made in phosphate buffer dissolving；Tobramycin 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-tobramycin conjugate is added in phosphate buffer, above-mentioned idol
The volume ratio for joining object and phosphate buffer is 1:100~1:10000.
As a further solution of the present invention, the detection method of the tobramycin immunologic function test reagent, feature exist
In including the following steps：
1）Sample to be tested is contacted with tobramycin antibody；
2）According to the combination situation of enzyme mark tobramycin in sample to be tested and tobramycin antibody, indicator judgement sample is utilized
The content of middle tobramycin；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 is anti-
After original is combined with antibody, the zymoprotein on antigen molecule and antibody close contact make the activated centre of enzyme be affected, the work of enzyme
Property is suppressed.Antigen, enzyme-labelled antigen when measurement 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 tobramycin immunologic function test reagent of the present invention can accurately and quickly determine human body blood
Tobramycin 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, quantifies the advantages that accurate at high specificity, is conducive to clinical promote the use of.
Description of the drawings
Fig. 1 is tobramycin homogeneous enzyme immunoassay reaction calibration graph.
Fig. 2 is tobramycin homogeneous enzyme immunoassay range of linearity figure.
Specific implementation mode
The present invention provides a kind of tobramycin immunologic function test reagent and its preparation and detection methods, to make mesh of the present invention
, technical solution and effect it is clearer, clear, the present invention is described in detail below.
The present invention provides a kind of tobramycin immunologic function test reagent and its preparation and detection methods.Including：The appropriate cloth of enzyme mark
Mycin, the indicator for detecting tobramycin antibody-enzyme mark tobramycin compound；Above-mentioned enzyme mark tobramycin is by appropriate cloth
Mycin and glucose dehydrogenase are coupled.
Signified " tobramycin " refers not only to complete tobramycin molecule in the present invention, also includes retaining intact antigen
The tobramycin segment or derivative of specific binding capacity.
A kind of tobramycin homogeneous enzyme immunoassay detection reagent, including：Enzyme mark tobramycin resists for detecting tobramycin
The indicator of body-enzyme mark tobramycin compound.Indicator is selected from enzymatic reagent, radioactive isotope reagent, fluorescent reagent
And chemical illuminating reagent.Preferably, indicator is enzymatic reagent, including：The substrate of enzyme mark conjugate and enzyme.Wherein, enzyme mark is even
It includes glucose dehydrogenase-antigen conjugates to join object, can be obtained by chemical synthesis process.
The application method of above-mentioned tobramycin immunologic function test reagent, includes the following steps：
1）Sample to be tested is contacted with tobramycin antibody；
2）According to the combination situation of enzyme mark tobramycin in sample to be tested and tobramycin antibody, indicator judgement sample is utilized
The content of middle tobramycin；The sample to be tested is serum, blood plasma, saliva or urine etc..Preferably, sample to be tested be 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 tobramycin
A. 20 mg tobramycin are dissolved in 250 μ L dimethylformamides（DMF）In, slight oscillatory dissolving；Meanwhile taking 25.0
Mg specifications are that the GDH of 100 KU is dissolved in the PBS (pH7.4 of 3 mL 0.01mol/L）In buffer solution, concussion, uniform dissolution；
B. tobramycin solution is added slowly with stirring in GDH solution, while it is molten to be slowly dropped into 20 μ L, 25% 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 chromatography column purifications, obtains glucose dehydrogenase-tobramycin conjugate, in 2-
It is stored at 8 DEG C.
Embodiment two：The preparation of tobramycin homogeneous enzyme immunoassay detection reagent
Tobramycin homogeneous enzyme immunoassay detection reagent, including：It is enzyme mark tobramycin, appropriate for detecting tobramycin antibody-enzyme mark
The indicator of Obramycin compound.Indicator is selected from enzymatic reagent, radioactive isotope reagent, fluorescent 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.
Tobramycin homogeneous enzyme immunoassay detection reagent before the use, in order to avoid in indicator enzyme mark conjugate and
The substrate of enzyme reacts, and the substrate of enzyme mark conjugate and enzyme is separated, therefore tobramycin homogeneous enzyme immunoassay detects
Reagent includes two kinds of reagents being provided separately, specific as follows：
1. the preparation of reagent 1：By 3.588 g（10 mM）Nicotinamide adenine dinucleotide NAD, 1.802 g of oxidation state（10
mM）Homogeneous zymolyte is made with the phosphate buffer dissolving of 50 mM, pH 8.0 of 1L in glucose；Tobramycin antibody is added to
In above-mentioned homogeneous zymolyte, the volume ratio of antibody and homogeneous zymolyte is 1:100~1:10000, in the present embodiment specifically
Ratio is 1:600.
2. the preparation of reagent 2：The glucose dehydrogenase of preparation-tobramycin conjugate is added to the phosphorus of 50 mM, pH 8.0
In phthalate buffer, the volume ratio of above-mentioned conjugate and phosphate buffer is 1:100~1:10000, have in the present embodiment
The ratio of body is 1:1700.
The application method of above-mentioned tobramycin homogeneous enzyme immunoassay detection reagent, includes the following steps：
1）Sample to be tested is contacted with tobramycin antibody；
2）According to the combination situation of enzyme mark tobramycin in sample to be tested and tobramycin antibody, indicator judgement sample is utilized
The content of middle tobramycin；
Specifically, sample to be tested is added in reagent 1 when detection, the tobramycin in sample to be tested and the appropriate cloth in reagent 1 are mould
Plain antibody is specifically bound, and generates tobramycin antibody-tobramycin compound；Reagent 2 is added, at this time in reagent 2
Glucose dehydrogenase-tobramycin conjugate and the enzyme in reagent 1 substrate mixing, contact, enzymatic reaction occurs, constitutes inspection
Survey the indicator of tobramycin antibody-enzyme mark tobramycin compound, indicator according to tobramycin in sample to be tested with
The combination situation of above-mentioned tobramycin antibody judges the content of tobramycin in sample to be tested.
Since the tobramycin competitive binding tobramycin in glucose dehydrogenase-antigen conjugates and sample to be tested is anti-
Body, so, the amount of tobramycin 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, leads to 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：Tobramycin homogeneous enzyme immunoassay detection reagent reacts calibration curve.
1）Calibration object is prepared：Commercially available people's tobramycin 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 Xiamen Hui Jia biologies
Science and Technology Ltd.'s tobramycin calibration object is primary standard, using its tobramycin kit to the calibration object point of various concentration
Jian Ce not be 10 times, mean value is found out, the concentration of tobramycin calibration object is obtained：0.5,1,5,10,20,40 ng/mL.
2）Biochemical Analyzer detects：By taking Hitachi 7170 operates as an example：Measurement wavelength is 340 nm, takes various concentration respectively
Calibration object solution（15μL）, tobramycin R is added1Reagent（200μL）, mixing adds tobramycin R2Reagent（50μL）, mix
After even, the OD of different time points is measured340Light absorption value calculates reaction rate when 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
A concentration of abscissa of calibration object draws " concentration-absorbance difference " calibration curve（See Fig. 1）.
Test serum or plasma sample are taken, the absorbance difference of sample is measured in the same method, substitutes into calibration curve, you can calculate
The content of tobramycin in sample to be tested.If the concentration of tobramycin exceeds calibration curve range, need pair in serum or blood plasma
Sample detects after being diluted again to ensure the accuracy of testing result.
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 tobramycin high concentration sample close to the range of linearity upper limit（38.42 ng/mL）, with above-mentioned similar human serum matrix
Solution by its by 1/2,1/4,1/8,1/16,1/32,1/64 dilution, be configured to 6 diluted concentrations altogether（xi）Solution, use
The Biochemical Analyzer detection method measures each diluted sample concentration.Each diluted concentration is tested 3 times, finds out each dilution respectively
The mean value of Concentration Testing result（yi）.With diluted concentration（xi）For independent variable, to measure mean value（yi）It is found out linearly for dependent variable
Regression equation, according to formula（1）As a result the correlation coefficient r for calculating linear regression shows that regression equation is y=0.9750x+
0.0550, correlation coefficient r=0.9998 shows that reagent of the present invention is related in the 0.63 ng/mL-38.42 ng/mL ranges of linearity
Property is preferable（See Fig. 2）.
……………………………（1）
Since the detection process of the present invention is completed by instrument full-automation, so to the of less demanding of testing staff, it is easy to real
Now and promote the use of.
It should be noted that obviously invention is not limited to the details of the above exemplary embodiments, the scope of the present invention is by institute
Attached claim rather than above description limit, it is intended that will fall within the meaning and scope of the equivalent 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 that this technology leads 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 tobramycin immunologic function test reagent and its preparation and detection method, it is characterised in that：Enzyme mark tobramycin is used for
Detect the indicator of tobramycin antibody-enzyme mark tobramycin compound.
2. the immunologic function test reagent of tobramycin according to claim 1, it is characterised in that：The enzyme mark tobramycin by
Tobramycin and glucose dehydrogenase are coupled.
3. tobramycin 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-tobramycin conjugate；On
The substrate for stating enzyme is glucose.
4. a kind of tobramycin immunologic function test reagent and preparation method thereof, which is characterized in that include the following steps：
(1) preparation of glucose dehydrogenase-tobramycin conjugate：Glucose dehydrogenase (GDH) is coupled with tobramycin, purifying
The enzyme-labelled antigen of coupling.
(2) preparation of tobramycin homogeneous enzyme immunoassay detection reagent：
The preparation of reagent 1：It is mixed by tobramycin antibody and homogeneous zymolyte；
The preparation of reagent 2：It is mixed with phosphate buffer by glucose dehydrogenase-tobramycin conjugate.
5. a kind of preparation method of tobramycin immunologic function test reagent according to claim 5, which is characterized in that described
Step (1) detailed process is：
1) coupling of glucose dehydrogenase (GDH) and tobramycin
A. 5-30mg tobramycin is dissolved in 100-500 μ L dimethylformamides (DMF), slight oscillatory dissolving；Meanwhile it taking
10-40mg specifications are that the GDH of 100-300KU is dissolved in concussion, uniform dissolution in PBS buffer solution.
B. tobramycin solution is added slowly with stirring in GDH solution, while is slowly dropped into 10-40 μ L glutaraldehyde solutions,
Solution 4 DEG C are placed in after being added dropwise to be stirred to react overnight.
2) enzyme-labelled antigen of purifying coupling
The enzyme-labelled antigen being coupled by G-25 gel chromatography column purifications, obtains glucose dehydrogenase-tobramycin conjugate, in 2-
It is stored at 8 DEG C.
6. a kind of preparation method of tobramycin immunologic function test reagent according to claim 5, which is characterized in that step
(2) detailed process is as follows：
The preparation of reagent 1：By nicotinamide adenine dinucleotide NAD, the 0.5-3g glucose 0.5-2L phosphorus of 2-5g oxidation state
Homogeneous zymolyte is made in phthalate buffer dissolving；Tobramycin antibody is added in above-mentioned homogeneous zymolyte, antibody and homogeneous enzyme
The volume ratio of substrate is 1:100~1:10000；
The preparation of reagent 2：The glucose dehydrogenase of preparation-tobramycin 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 tobramycin immunologic function test reagent described in 4 any one of Claims 1-4, feature exists
In including the following steps：
1) sample to be tested is contacted with tobramycin antibody；
2) according to the combination situation of tobramycin in sample to be tested and tobramycin antibody, using appropriate in indicator judgement sample
The content of Obramycin；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 |
|---|---|---|---|---|
| CN109884318A (en) * | 2019-03-20 | 2019-06-14 | 杭州博谱医药科技有限公司 | A kind of homogeneous enzyme immunoassay conjugate and its preparation method and application |
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- 2018-05-23 CN CN201810495292.9A patent/CN108508194A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109884318A (en) * | 2019-03-20 | 2019-06-14 | 杭州博谱医药科技有限公司 | A kind of homogeneous enzyme immunoassay conjugate and its preparation method and application |
| CN109884318B (en) * | 2019-03-20 | 2022-02-18 | 杭州博谱医药科技有限公司 | Homogeneous enzyme immunoconjugate and preparation method and application thereof |
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Application publication date: 20180907 |