CN108717117A - A kind of vancomycin immunologic function test reagent and its preparation and detection method - Google Patents
A kind of vancomycin immunologic function test reagent and its preparation and detection method Download PDFInfo
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- CN108717117A CN108717117A CN201810495293.3A CN201810495293A CN108717117A CN 108717117 A CN108717117 A CN 108717117A CN 201810495293 A CN201810495293 A CN 201810495293A CN 108717117 A CN108717117 A CN 108717117A
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- vancomycin
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- 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
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/535—Production of labelled immunochemicals with enzyme label or co-enzymes, co-factors, enzyme inhibitors or enzyme substrates
Abstract
The invention discloses a kind of vancomycin immunologic function test reagent and its preparation and detection methods.Including：Enzyme mark vancomycin, the indicator for detecting vancomycin antibody-enzyme mark vancomycin compound；Above-mentioned enzyme mark vancomycin is coupled by vancomycin and glucose dehydrogenase.The vancomycin immunologic function test reagent of the present invention can accurately and quickly determine vancomycin 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 vancomycin immunologic function test reagent and its preparation and detection side
Method.
Background technology
Vancomycin（Vancomycin, VCM）Also known as Vancomycin, Vancomycin, it is a kind of polypeptide antibiotics, leads to
Often exist with hydrochloride form, it in the solution can be by various heavy precipitation of salts.Vancomycin divides as one kind from microorganism
From obtained polypeptide antibiotics, it has good inhibiting effect to gram-positive bacteria, but because it cannot penetrate external ooze
Permeable barriers, it is of no curative effect to Gram-negative bacteria.But vancomycin is excessively used and Norvancomycin drug will produce
Drug resistance, and there is serious ototoxicity and renal toxicity.The Ministry of Agriculture of China provide vancomycin be disabling veterinary drug, forbid production,
It manages.So to vancomycin quickly, sensitively measure in the samples such as related food necessary.
The common method of detection vancomycin 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, vancomycin detection process in the prior art is complicated for operation and accuracy of measurement
Low problem, the present invention provides it is a kind of quickly, high sensitivity, accurately detect that vancomycin content is through the ages 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 vancomycin immunologic function test reagent and its preparation and detection method, it is characterised in that：Enzyme mark vancomycin, for examining
Survey the indicator of vancomycin antibody-enzyme mark vancomycin compound；Above-mentioned enzyme mark vancomycin is by vancomycin 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-vancomycin conjugate；The substrate of above-mentioned enzyme is glucose.
As a further solution of the present invention, a kind of vancomycin immunologic function test reagent and preparation method thereof,
It is characterized in that, includes the following steps：
（1）The preparation of glucose dehydrogenase-vancomycin conjugate：Glucose dehydrogenase（GDH）It is coupled with vancomycin, purifying
The enzyme-labelled antigen of coupling.
（2）The preparation of vancomycin homogeneous enzyme immunoassay detection reagent：
The preparation of reagent 1：It is mixed by vancomycin 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 vancomycin immunologic function test reagent, it is special
Sign is, the step（1）Detailed process is：
1）Glucose dehydrogenase（GDH）With the coupling of vancomycin
A. 5-30 mg vancomycins are 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. vancomycin solution is added slowly with stirring in GDH solution, while is slowly dropped into 10-50 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-vancomycin 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 vancomycin 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；Vancomycin 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-vancomycin 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 vancomycin immunologic function test reagent, feature exist
In including the following steps：
1）Sample to be tested is contacted with vancomycin antibody；
2）According to the combination situation of enzyme mark vancomycin in sample to be tested and vancomycin antibody, indicator judgement sample is utilized
The content of middle vancomycin；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 vancomycin immunologic function test reagent of the present invention can accurately and quickly determine human body blood
Vancomycin 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 vancomycin homogeneous enzyme immunoassay reaction calibration graph.
Fig. 2 is vancomycin homogeneous enzyme immunoassay range of linearity figure.
Specific implementation mode
The present invention provides a kind of vancomycin 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 vancomycin immunologic function test reagent and its preparation and detection methods.Including：Enzyme mark is through the ages
Mycin, the indicator for detecting vancomycin antibody-enzyme mark vancomycin compound；Above-mentioned enzyme mark vancomycin is by through the ages
Mycin and glucose dehydrogenase are coupled.
Signified " vancomycin " refers not only to complete vancomycin molecule in the present invention, also includes retaining intact antigen
The vancomycin segment or derivative of specific binding capacity.
A kind of vancomycin homogeneous enzyme immunoassay detection reagent, including：Enzyme mark vancomycin resists for detecting vancomycin
The indicator of body-enzyme mark vancomycin 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 vancomycin immunologic function test reagent, includes the following steps：
1）Sample to be tested is contacted with vancomycin antibody；
2）According to the combination situation of enzyme mark vancomycin in sample to be tested and vancomycin antibody, indicator judgement sample is utilized
The content of middle vancomycin；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 vancomycin
A. 25 mg vancomycins are dissolved in 200 μ L dimethylformamides（DMF）In, slight oscillatory dissolving；Meanwhile taking 30 mg
Specification is 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. vancomycin 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-vancomycin conjugate, in 2-
It is stored at 8 DEG C.
Embodiment two：The preparation of vancomycin homogeneous enzyme immunoassay detection reagent
Vancomycin homogeneous enzyme immunoassay detection reagent, including：Enzyme mark vancomycin, for detecting vancomycin antibody-enzyme mark ten thousand
The indicator of ancient mycin 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.
Vancomycin 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 vancomycin 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；Vancomycin 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 1:700.
2. the preparation of reagent 2：The glucose dehydrogenase of preparation-vancomycin 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 1:1900.
The application method of above-mentioned vancomycin homogeneous enzyme immunoassay detection reagent, includes the following steps：
1）Sample to be tested is contacted with vancomycin antibody；
2）According to the combination situation of enzyme mark vancomycin in sample to be tested and vancomycin antibody, indicator judgement sample is utilized
The content of middle vancomycin；
Specifically, detection when sample to be tested is added in reagent 1, the vancomycin in sample to be tested with it is mould through the ages in reagent 1
Plain antibody is specifically bound, and generates vancomycin antibody-vancomycin compound；Reagent 2 is added, at this time in reagent 2
Glucose dehydrogenase-vancomycin conjugate and the enzyme in reagent 1 substrate mixing, contact, enzymatic reaction occurs, constitutes inspection
Survey the indicator of vancomycin antibody-enzyme mark vancomycin compound, indicator according to vancomycin in sample to be tested with
The combination situation of above-mentioned vancomycin antibody judges the content of vancomycin in sample to be tested.
Since the vancomycin competitive binding vancomycin in glucose dehydrogenase-antigen conjugates and sample to be tested is anti-
Body, so, the amount of vancomycin 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：Vancomycin homogeneous enzyme immunoassay detection reagent reacts calibration curve.
1）Calibration object is prepared：Commercially available people's vancomycin 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 Shenyang Li Dong medicine
Science and Technology Ltd.'s vancomycin calibration object is primary standard, using its vancomycin kit to the calibration object point of various concentration
Jian Ce not be 10 times, mean value is found out, the concentration of vancomycin calibration object is obtained：5,10,20,50,100 μ g/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）, vancomycin R is added1Reagent（240μL）, mixing adds vancomycin R2Reagent（60μL）, 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 vancomycin in sample to be tested.If the concentration of vancomycin 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 vancomycin high concentration sample close to the range of linearity upper limit（96.4 μg/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 to find out each dilution respectively
Spend the mean value of testing result（yi）.With diluted concentration（xi）For independent variable, to measure mean value（yi）Linear return is found out for dependent variable
Return equation, according to formula（1）As a result the correlation coefficient r for calculating linear regression shows that regression equation is y=0.9862x+0.1673,
Correlation coefficient r=0.9997 shows reagent of the present invention good relationship in the 1.6 μ g/mL-96.4 μ g/mL ranges of linearity（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 vancomycin immunologic function test reagent and its preparation and detection method, it is characterised in that：Enzyme mark vancomycin is used for
Detect the indicator of vancomycin antibody-enzyme mark vancomycin compound.
2. the immunologic function test reagent of vancomycin according to claim 1, it is characterised in that：The enzyme mark vancomycin by
Vancomycin and glucose dehydrogenase are coupled.
3. vancomycin 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-vancomycin conjugate；On
The substrate for stating enzyme is glucose.
4. a kind of vancomycin immunologic function test reagent and preparation method thereof, which is characterized in that include the following steps：
(1) preparation of glucose dehydrogenase-vancomycin conjugate：Glucose dehydrogenase (GDH) is coupled with vancomycin, purifying
The enzyme-labelled antigen of coupling.
(2) preparation of vancomycin homogeneous enzyme immunoassay detection reagent：
The preparation of reagent 1：It is mixed by vancomycin antibody and homogeneous zymolyte；
The preparation of reagent 2：It is mixed with phosphate buffer by glucose dehydrogenase-vancomycin conjugate.
5. a kind of preparation method of vancomycin 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 vancomycin
A. 5-30mg vancomycins are 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. vancomycin solution is added slowly with stirring in GDH solution, while is slowly dropped into 10-50 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-vancomycin conjugate, in 2-
It is stored at 8 DEG C.
6. a kind of preparation method of vancomycin 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；Vancomycin 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-vancomycin 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 vancomycin 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 vancomycin antibody；
2) according to the combination situation of vancomycin in sample to be tested and vancomycin antibody, in indicator judgement sample ten thousand are utilized
The content of ancient mycin；The sample to be tested is serum, blood plasma, saliva or urine.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111487206A (en) * | 2019-01-09 | 2020-08-04 | 北京九强生物技术股份有限公司 | 6-phosphoglucose dehydrogenase mutant and application thereof in preparation of vancomycin detection reagent |
| CN111620931A (en) * | 2020-06-12 | 2020-09-04 | 苏州博源医疗科技有限公司 | Vancomycin derivative and preparation method and application thereof |
| CN112500496A (en) * | 2020-12-03 | 2021-03-16 | 浙江准策生物技术有限公司 | Vancomycin complete antigen and preparation method and application thereof |
-
2018
- 2018-05-23 CN CN201810495293.3A patent/CN108717117A/en active Pending
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111487206A (en) * | 2019-01-09 | 2020-08-04 | 北京九强生物技术股份有限公司 | 6-phosphoglucose dehydrogenase mutant and application thereof in preparation of vancomycin detection reagent |
| CN111487206B (en) * | 2019-01-09 | 2023-11-10 | 北京九强生物技术股份有限公司 | Glucose 6-phosphate dehydrogenase mutant and application thereof in preparation of vancomycin detection reagent |
| CN111620931A (en) * | 2020-06-12 | 2020-09-04 | 苏州博源医疗科技有限公司 | Vancomycin derivative and preparation method and application thereof |
| CN111620931B (en) * | 2020-06-12 | 2021-12-17 | 苏州博源医疗科技有限公司 | Vancomycin derivative and preparation method and application thereof |
| CN112500496A (en) * | 2020-12-03 | 2021-03-16 | 浙江准策生物技术有限公司 | Vancomycin complete antigen and preparation method and application thereof |
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