CN108761062A - A kind of procainamide immunologic function test reagent and its preparation and detection method - Google Patents
A kind of procainamide immunologic function test reagent and its preparation and detection method Download PDFInfo
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- CN108761062A CN108761062A CN201810484515.1A CN201810484515A CN108761062A CN 108761062 A CN108761062 A CN 108761062A CN 201810484515 A CN201810484515 A CN 201810484515A CN 108761062 A CN108761062 A CN 108761062A
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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
- G01N33/53—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
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- 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
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
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Abstract
The invention discloses a kind of procainamide immunologic function test reagent and its preparation and detection methods.Including：Enzyme mark procainamide, the indicator for detecting procainamide antibody-enzyme mark procaine amine compound；Above-mentioned enzyme mark procainamide is coupled by procainamide and glucose dehydrogenase.The procainamide immunologic function test reagent of the present invention can accurately and quickly determine procaine amine 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 procainamide immunologic function test reagent and its preparation and detection
Method.
Background technology
Procainamide（Procainamide）It is a kind of Ia class antiarrhythmic drugs applied for many years, the anti-rhythm of the heart loses
Normal effect essentially consists in the electrical conduction for slowing down cardiac muscle, extends the Repolarization time of cardiac muscular tissue, the electro physiology mechanism of effect is suppression
Na processed+、K+、Ca2+Channel.Influence of the procainamide to ventricular bipolar is usually expressed as Action Potential Duration and effective refractory period
Extension, stablize myocardial cell membrane, reduce irritability and conductibility, inhibit autorhythmia of cardiac.Originally with quinindium refractory patient
Product may be effectively.Studies have shown that procainamide, which also has, inhibits platelet aggregation.But large dosage is oral to dislike
The gastrointestinal reactions such as the heart, vomiting, diarrhea, long-term administration can cause systemic loupus erythematosus sample to integrate sample syndrome, and patient occurs
Anti-DNA antibody is positive, and dosage is excessive can also to lead to blood leukocytes reduction etc..So to procaine in the samples such as related food
Amine quickly, sensitively measure necessary.
The common method of detection procainamide has at present：Efficient liquid phase chromatographic analysis, spectrophotometry and capillary electricity
Swimming method etc..Wherein, efficient liquid phase chromatographic analysis is time-consuming and is not easy to automate, and using ultraviolet detection when needs to derive；Light splitting
The accuracy of photometry testing result is not relatively high；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, procainamide detection process in the prior art is complicated for operation and measurement is accurate
Spend low problem, the present invention provides it is a kind of quickly, high sensitivity, accurately detect procaine amine content in sample to be tested
Procainamide 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 procainamide immunologic function test reagent and its preparation and detection method, it is characterised in that：Enzyme mark procainamide is used
In the indicator of detection procainamide antibody-enzyme mark procaine amine compound；Above-mentioned enzyme mark procainamide is by general Shandong
Cacaine amine and glucose dehydrogenase are 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-procainamide conjugate；The substrate of above-mentioned enzyme is glucose.
As a further solution of the present invention, a kind of procainamide immunologic function test reagent and preparation method thereof,
It is characterised in that it includes following steps：
（1）The preparation of glucose dehydrogenase-procainamide conjugate：Glucose dehydrogenase（GDH）With procaine amine coupling,
Purify the enzyme-labelled antigen of coupling.
（2）The preparation of procainamide homogeneous enzyme immunoassay detection reagent：
The preparation of reagent 1：It is mixed by procainamide 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 procainamide immunologic function test reagent,
It is characterized in that, the step（1）Detailed process is：
1）Glucose dehydrogenase（GDH）With the coupling of procainamide
A. 5-30 mg procainamides are dissolved in 100-500 μ L dimethylformamides（DMF）In, slight oscillatory dissolving；Together
When, 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. procaine amine aqueous solution is added slowly with stirring in GDH solution, while is slowly dropped into 10-40 μ L penta 2
Solution is placed in 4 DEG C after being added dropwise and is stirred to react overnight by aldehyde 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-procainamide conjugate, in
It is stored at 2-8 DEG C.
As a further solution of the present invention, the preparation method of a kind of procainamide immunologic function test reagent,
It 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；Procainamide antibody is added in above-mentioned homogeneous zymolyte, antibody with
The volume ratio of phase zymolyte is 1:100~1:10000；
The preparation of reagent 2：The glucose dehydrogenase of preparation-procainamide conjugate is added in phosphate buffer, it is above-mentioned
The volume ratio of conjugate and phosphate buffer is 1:100~1:10000.
As a further solution of the present invention, the detection method of the procainamide immunologic function test reagent, feature
It is, includes the following steps：
1）Sample to be tested is contacted with procainamide antibody；
2）According to the combination situation of enzyme mark procainamide in sample to be tested and procainamide antibody, judged using indicator
The content of procainamide in sample；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 procainamide immunologic function test reagent of the present invention can accurately and quickly determine human body
Procaine amine content in the samples such as blood.Compared with existing detection reagent in the market, detection reagent of the present invention has convenient
Quickly, high sensitivity, high specificity, it is quantitative accurate the advantages that, be conducive to clinical promote the use of.
Description of the drawings
Fig. 1 is procainamide homogeneous enzyme immunoassay reaction calibration graph.
Fig. 2 is procainamide homogeneous enzyme immunoassay range of linearity figure.
Specific implementation mode
The present invention provides a kind of procainamide 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 procainamide immunologic function test reagent and its preparation and detection methods.Including：Enzyme mark is general
Shandong cacaine amine, the indicator for detecting procainamide antibody-enzyme mark procaine amine compound；Above-mentioned enzyme mark Proca
Because amine is coupled by procainamide and glucose dehydrogenase.
Signified " procainamide " refers not only to complete procaine amine molecule in the present invention, also includes retaining completely
The procainamide segment or derivative of antigentic specificity binding ability.
A kind of procainamide homogeneous enzyme immunoassay detection reagent, including：Enzyme mark procainamide, for detecting procaine
The indicator of amine antibody-enzyme mark procaine amine compound.Indicator is selected from enzymatic reagent, radioactive isotope reagent, glimmering
Light reagent and chemical illuminating reagent.Preferably, indicator is enzymatic reagent, including：The substrate of enzyme mark conjugate and enzyme.Wherein,
Enzyme mark conjugate includes glucose dehydrogenase-antigen conjugates, can be obtained by chemical synthesis process.
The application method of above-mentioned procainamide immunologic function test reagent, includes the following steps：
1）Sample to be tested is contacted with procainamide antibody；
2）According to the combination situation of enzyme mark procainamide in sample to be tested and procainamide antibody, judged using indicator
The content of procainamide in sample；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-antigen conjugates
1）Glucose dehydrogenase（GDH）With the coupling of procainamide
A. 30 mg procainamides are dissolved in 350 μ L dimethylformamides（DMF）In, slight oscillatory dissolving；Meanwhile it taking
30.0 mg specifications are that the GDH of 100 KU is dissolved in the PBS (pH7.4 of 5 mL 0.01mol/L）It in buffer solution, shakes, is uniformly molten
Solution；
B. procaine amine aqueous solution is added slowly with stirring in GDH solution, while is slowly dropped into 30 μ L, 25% 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-procainamide conjugate, in
It is stored at 2-8 DEG C.
Embodiment two：The preparation of procainamide homogeneous enzyme immunoassay detection reagent
Procainamide homogeneous enzyme immunoassay detection reagent, including：Enzyme mark procainamide, for detecting procainamide antibody-
The indicator of enzyme mark procaine amine compound.Indicator be 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 coupled
Object includes glucose dehydrogenase-antigen conjugates, can be obtained by chemical synthesis process.
Procainamide homogeneous enzyme immunoassay detection reagent before the use, in order to avoid the enzyme mark conjugate in indicator
It reacts with the substrate of enzyme, the substrate of enzyme mark conjugate and enzyme is separated, therefore procainamide homogeneous enzyme immunoassay
Detection 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；Procainamide antibody is added
Into above-mentioned homogeneous zymolyte, the volume ratio of antibody and homogeneous zymolyte is 1:100~1:10000, in the present embodiment specifically
Ratio be 1:650.
2. the preparation of reagent 2：The glucose dehydrogenase of preparation-procainamide conjugate is added to 50 mM, pH's 8.0
In phosphate buffer, the volume ratio of above-mentioned conjugate and phosphate buffer is 1:100~1:10000, in the present embodiment
Specific ratio is 1:1900.
The application method of above-mentioned procainamide homogeneous enzyme immunoassay detection reagent, includes the following steps：
1）Sample to be tested is contacted with procainamide antibody；
2）According to the combination situation of enzyme mark procainamide in sample to be tested and procainamide antibody, judged using indicator
The content of procainamide in sample；
Specifically, sample to be tested is added in reagent 1 when detection, the procainamide in sample to be tested and the Pu Lu in reagent 1
Cacaine amine antibody is specifically bound, and generates procainamide antibody-procaine amine compound；Reagent 2 is added, at this time
Glucose dehydrogenase-procainamide conjugate in reagent 2 is mixed with the substrate of the enzyme in reagent 1, is contacted, and it is anti-that enzymatic occurs
It answers, constitutes the indicator of detection procainamide antibody-enzyme mark procaine amine compound, indicator is according to sample to be tested
The combination situation of middle procainamide and above-mentioned procainamide antibody judges the content of procainamide in sample to be tested.
Due to the procainamide competitive binding procaine in glucose dehydrogenase-antigen conjugates and sample to be tested
Amine antibody, so, the amount of procainamide is more in sample to be tested, the glucose dehydrogenase-antigen to dissociate in homogeneous enzyme solutions
The amount of conjugate is more, and 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：Procainamide homogeneous enzyme immunoassay detection reagent reacts calibration curve.
1）Calibration object is prepared：Commercially available people's procainamide 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 Luo Zhe（Shanghai）It is raw
Object Science and Technology Ltd. procainamide calibration object is primary standard, using its procainamide kit to the school of various concentration
Quasi- product detect 10 times respectively, find out mean value, obtain the concentration of procainamide calibration object：0.5,1,5,10,20,40 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）, procainamide R is added1Reagent（160μL）, mixing adds procainamide R2Reagent（40μ
L）, after mixing, measure the OD of different time points340Light absorption value calculates reaction rate when different calibration object concentration, practical operation
The volume ratio of constantly adjustment reagent 1 and reagent 2 is needed in the process, while adjusting survey luminous point, finally obtains comparatively ideal reaction mark
Directrix curve figure, often pipe replication 3 times are right using the average value of 3 absorbance difference Δ A measured of each calibration pipe as ordinate
A concentration of abscissa of calibration object answered 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 procainamide in sample to be tested.If the concentration of procainamide exceeds calibration curve range in serum or blood plasma,
It is detected again after need to being diluted to sample 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 procainamide high concentration sample close to the range of linearity upper limit（38.68μg/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=0.9793x+
0.0142, correlation coefficient r=0.9997 shows reagent of the present invention correlation in the 0.63 μ g/mL-38.68 μ g/mL ranges of linearity
Preferably（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 (10)
1. a kind of procainamide immunologic function test reagent and its preparation and detection method, it is characterised in that：Enzyme mark procainamide,
Indicator for detecting procainamide antibody-enzyme mark procaine amine compound.
2. the immunologic function test reagent of procainamide according to claim 1, it is characterised in that：The enzyme mark procaine
Amine is coupled by procainamide and glucose dehydrogenase.
3. procainamide immunologic function test reagent according to claim 1, it is characterised in that：The indicator is selected from enzyme
Reagent, including：The substrate of enzyme mark conjugate and enzyme；Above-mentioned enzyme mark conjugate includes glucose dehydrogenase-procaine amine coupling
Object；The substrate of above-mentioned enzyme is glucose.
4. a kind of procainamide immunologic function test reagent and preparation method thereof, which is characterized in that include the following steps：
（1）The preparation of glucose dehydrogenase-procainamide conjugate：Glucose dehydrogenase（GDH）With procaine amine coupling,
Purify the enzyme-labelled antigen of coupling.
5.（2）The preparation of procainamide homogeneous enzyme immunoassay detection reagent：
The preparation of reagent 1：It is mixed by procainamide antibody and homogeneous zymolyte；
The preparation of reagent 2：It is mixed with phosphate buffer by glucose dehydrogenase-procainamide conjugate.
6. a kind of preparation method of procainamide immunologic function test reagent according to claim 5, which is characterized in that described
The step of（1）Detailed process is：
1）Glucose dehydrogenase（GDH）With the coupling of procainamide
A. 5-30 mg procainamides are dissolved in 100-500 μ L dimethylformamides（DMF）In, slight oscillatory dissolving；Together
When, 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.
Procaine amine aqueous solution is added slowly with stirring in GDH solution by 7.b., while being 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.
8.2）Purify the enzyme-labelled antigen of coupling
The enzyme-labelled antigen being coupled by G-25 gel chromatography column purifications, obtains glucose dehydrogenase-procainamide conjugate, in
It is stored at 2-8 DEG C.
9. a kind of preparation method of procainamide 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；Procainamide antibody is added in above-mentioned homogeneous zymolyte, antibody with
The volume ratio of phase zymolyte is 1:100~1:10000；
The preparation of reagent 2：The glucose dehydrogenase of preparation-procainamide conjugate is added in phosphate buffer, it is above-mentioned
The volume ratio of conjugate and phosphate buffer is 1:100~1:10000.
10. using the detection method of the procainamide immunologic function test reagent described in 4 any one of Claims 1-4, feature
It is, includes the following steps：
1）Sample to be tested is contacted with procainamide antibody；
2）According to the combination situation of procainamide in sample to be tested and procainamide antibody, indicator judgement sample is utilized
The content of middle procainamide；The sample to be tested is serum, blood plasma, saliva or urine.
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