CN108037297A - A kind of Procalcitonin immunologic function test reagent and its preparation and detection method - Google Patents
A kind of Procalcitonin immunologic function test reagent and its preparation and detection method Download PDFInfo
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- CN108037297A CN108037297A CN201711389669.4A CN201711389669A CN108037297A CN 108037297 A CN108037297 A CN 108037297A CN 201711389669 A CN201711389669 A CN 201711389669A CN 108037297 A CN108037297 A CN 108037297A
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Abstract
The invention discloses a kind of Procalcitonin immunologic function test reagent and its preparation and detection method.Including：Enzyme mark Procalcitonin, the indicator for detecting Procalcitonin enzyme-labeled antibody Procalcitonin compound；Above-mentioned enzyme mark Procalcitonin is formed by Procalcitonin and glucose dehydrogenase coupling.The Procalcitonin immunologic function test reagent of the present invention can accurately and quickly determine Procalcitonin content in the samples such as blood of human body.Compared with the existing detection reagent of in the market, detection reagent of the present invention has the advantages that fast and easy, high sensitivity, high specificity, quantitative accurate, is conducive to promoting the use of for clinic.
Description
Technical field
The present invention relates to field of biological detection, is specifically a kind of Procalcitonin immunologic function test reagent and its preparation and detection side
Method.
Background technology
Procalcitonin (procaLcitonin, PCT) is the propetide of calcitonin, a kind of glycoprotein of no hormonal activity, by
116 amino acid compositions, molecular weight 13KD, when half-life period is 25-30 small, stability is fine in vivo, from hormone in vivo
Horizontal influence.In normal healthy people body PCT levels it is very low (<0.05 ng/mL).Bacterium infection (particularly septicopyemia,
Gram negative bacilli) caused by systemic inflammatory response when blood-serum P CT horizontal Chang Xianzhu increase, it increases degree with infecting serious journey
Degree and the death rate are proportionate.Compare c reactive protein in the rise of Systemic bacterial infection patients serum PCT concentration（CRP）And other inflammation
Early, raising infecting 2 h occurs in sex factor, and 6 h steeply rise, and 8~24 h maintain high level, and thin with part to whole body
The discriminating of the sexy dye of bacterium has special value.It is now recognized that PCT is probably a kind of endogenous Nonsteroidal anti-inflammatory material, exist more
Induce and produce during bacterium infection, played an important role in regulating cell cytokine network, be to be used to detect severe bacterial infections
An important diagnostic mark, a kind of and index of sensitive judgement inflammation classification and active situation.
The common method of detection Procalcitonin has at present：Gel chromatography and efficient liquid phase chromatographic analysis, Enzyme-linked Immunosorbent Assay
Measure（ELISA）, radiommunoassay（RIA）, immunoluminescence method and colloidal gold chromatography.Wherein, gel chromatography and efficient liquid
Analysis of hplc is time-consuming and is not easy to automate；ELISA method dosing accuracy is poor, the operating time is long, the degree of automation is low, is chiefly used in
Qualitative detection；Longer the time required to RIA methods, testing result is unstable, and repeatability is poorer than ELISA, and there are radioactive pollution danger
Danger.Immunoluminescence method high specificity, sensitiveness is high, but needs expensive instrument and equipment and veteran operating personnel, generally
Used more in specific medical mechanism.Though colloidal gold chromatography stability is preferable, sensitivity is relatively low, generally can only be qualitative, it is impossible to
It is quantitative.
For homogeneous enzyme immunoassay detection method, its advantage is the method that the present invention uses：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
Energy high throughput quickly measures.
The content of the invention
It is an object of the invention to solve, Procalcitonin detection process in the prior art is complicated and accuracy of measurement
The problem of low, the present invention provides a kind of quick, high sensitivity, the accurate drop calcium for detecting Procalcitonin content in sample to be tested
Plain original homogeneous enzyme immunoassay detection reagent and preparation method thereof.
To achieve the above object, the present invention provides following technical solution：
A kind of Procalcitonin immunologic function test reagent and its preparation and detection method, it is characterised in that：Enzyme mark Procalcitonin, for examining
Survey the indicator of Procalcitonin antibody-enzyme mark Procalcitonin compound；Above-mentioned enzyme mark Procalcitonin is by Procalcitonin and grape
Glucocorticoid dehydrogenase coupling forms.
As the further scheme of the present invention, the indicator is selected from enzymatic reagent, including：The bottom of enzyme mark conjugate and enzyme
Thing；Above-mentioned enzyme mark conjugate includes glucose dehydrogenase-Procalcitonin conjugate；The substrate of above-mentioned enzyme is glucose.
As the further scheme of the present invention, a kind of described Procalcitonin immunologic function test reagent and preparation method thereof, its
It is characterized in that, includes the following steps：
（1）The preparation of glucose dehydrogenase-Procalcitonin conjugate：Prepare glucose dehydrogenase（GDH）Solution, Procalcitonin
Activation, GDH and Procalcitonin are coupled, and purify the enzyme-labelled antigen of coupling；
（2）The preparation of Procalcitonin homogeneous enzyme immunoassay detection reagent：
The preparation of reagent 1：Mixed by Procalcitonin antibody and homogeneous zymolyte；
The preparation of reagent 2：Mixed by glucose dehydrogenase-antigen conjugates with phosphate buffer.
As the further scheme of the present invention, a kind of preparation method of Procalcitonin immunologic function test reagent, it 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 solutions, pH 7.0-
10.0；
2）The activation of Procalcitonin
Following chemicals is added to stirring and dissolving in beaker：10-30 mg Procalcitonins, 10-40 mg1- ethyl -3- (- 3-
Dimethylaminopropyl) carbodiimide, 2-10 mg N- hydroxy thiosuccinimides, be dissolved in 2- (N- morpholines) ethyl sulfonic acid
（MES）In solution, stirring and dissolving, reacts 15-60 minutes at room temperature；
3）The coupling of GDH and Procalcitonin
A. the Procalcitonin solution of above-mentioned activation is added dropwise in the GDH solution in above-mentioned stirring；
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-Procalcitonin conjugate, in 2-
Stored at 8 DEG C.
As the further scheme of the present invention, a kind of preparation method of Procalcitonin 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；Procalcitonin 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-Procalcitonin conjugate is added in phosphate buffer, above-mentioned idol
The volume ratio for joining thing and phosphate buffer is 1:100~1:10000.
Exist as the further scheme of the present invention, the detection method of the Procalcitonin immunologic function test reagent, its feature
In comprising the following steps：
1）Sample to be tested is contacted with Procalcitonin antibody；
2）According to the combination situation of enzyme mark Procalcitonin in sample to be tested and Procalcitonin antibody, indicator judgement sample is utilized
The content of middle Procalcitonin；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 former and antibody binding, 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 Procalcitonin immunologic function test reagent of the present invention can accurately and quickly determine human body blood
Procalcitonin content in the samples such as liquid.Compared with the existing detection reagent of in the market, detection reagent of the present invention have fast and easy,
High sensitivity, high specificity, quantify the advantages that accurate, is conducive to promoting the use of for clinic.
Brief description of the drawings
Fig. 1 is Procalcitonin homogeneous enzyme immunoassay reaction calibration graph.
Fig. 2 is Procalcitonin homogeneous enzyme immunoassay range of linearity figure.
Fig. 3 is the correlation analysis figure of the immuno-fluorescence assay result of Procalcitonin homogeneous enzyme immunoassay and norman.
Embodiment
The present invention provides a kind of Procalcitonin 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 Procalcitonin immunologic function test reagent and its preparation and detection method.Including：Calcium drops in enzyme mark
The plain former, indicator for detecting Procalcitonin antibody-enzyme mark Procalcitonin compound；Above-mentioned enzyme mark Procalcitonin is by drop calcium
Plain former and glucose dehydrogenase coupling forms.
Signified " Procalcitonin " refers not only to complete Procalcitonin molecule in the present invention, also includes retaining intact antigen
The Procalcitonin segment or derivative of specific binding capacity.
A kind of Procalcitonin homogeneous enzyme immunoassay detection reagent, including：Enzyme mark Procalcitonin, resist for detecting Procalcitonin
The indicator of body-enzyme mark Procalcitonin 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
Connection thing includes glucose dehydrogenase-antigen conjugates, it can be obtained by chemical synthesis process.
The application method of above-mentioned Procalcitonin immunologic function test reagent, comprises the following steps：
1）Sample to be tested is contacted with Procalcitonin antibody；
2）According to the combination situation of enzyme mark Procalcitonin in sample to be tested and Procalcitonin antibody, indicator judgement sample is utilized
The content of middle Procalcitonin；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. the GDH that 10 mg specifications are 100 KU is weighed, room-temperature dissolution is in the solution of 8 mL 50mM PBS, pH=7.4；
2）The activation of Procalcitonin
Following chemicals is added to stirring and dissolving in beaker：25 mg Procalcitonins, 20mg 1- ethyls -3- (- 3- diformazan ammonia
Propyl group) carbodiimide, 5mg N- hydroxy thiosuccinimides, be dissolved in 2 mL 2- (N- morpholines) ethyl sulfonic acid, in room temperature
Lower stirring and dissolving, reacts 30 minutes；
3）The coupling of GDH and Procalcitonin
A. the Procalcitonin 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-Procalcitonin conjugate, in 2-
Stored at 8 DEG C.
Embodiment two：The preparation of Procalcitonin homogeneous enzyme immunoassay detection reagent
Procalcitonin homogeneous enzyme immunoassay detection reagent, including：Enzyme mark Procalcitonin, for detecting Procalcitonin antibody-enzyme mark drop
The indicator of the former compound of calcium element.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, it can be obtained by chemical synthesis process.
Procalcitonin 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 Procalcitonin 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；Procalcitonin antibody is added to
State 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 1:600.
2. the preparation of reagent 2：The glucose dehydrogenase of preparation-Procalcitonin 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, have in the present embodiment
The ratio of body is 1:1500.
The application method of above-mentioned Procalcitonin homogeneous enzyme immunoassay detection reagent, comprises the following steps：
1）Sample to be tested is contacted with Procalcitonin antibody；
2）According to the combination situation of enzyme mark Procalcitonin in sample to be tested and Procalcitonin antibody, indicator judgement sample is utilized
The content of middle Procalcitonin；
Specifically, sample to be tested is added in reagent 1 during detection, the Procalcitonin in sample to be tested and the calcitonin in reagent 1
Original antibody is specifically bound, and generates anti-Procalcitonin antibody-Procalcitonin compound；Reagent 2 is added, at this time reagent 2
In glucose dehydrogenase-Procalcitonin conjugate and the enzyme in reagent 1 substrate mixing, contact, enzymatic reaction occurs, forms
The indicator of Procalcitonin antibody-enzyme mark Procalcitonin compound is detected, indicator is according to Procalcitonin in sample to be tested
And the combination situation of above-mentioned Procalcitonin antibody judges the content of Procalcitonin in sample to be tested.
Since glucose dehydrogenase-antigen conjugates and the Procalcitonin competitive binding Procalcitonin in sample to be tested resist
Body, so, the amount of Procalcitonin 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：Procalcitonin homogeneous enzyme immunoassay detection reagent reacts calibration curve.
1）PCT calibration objects are prepared：Commercially available human calcitonin original weight histone 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 objects of various concentrations is made.With Shanghai Yuan Sheng companies
Procalcitonin calibration object is primary standard, and the calibration object of various concentrations is detected 10 times respectively using its Procalcitonin kit,
Average is obtained, obtains the concentration of Procalcitonin calibration object：0.2,1,5,25,50,100 ng/mL.
2）Biochemical Analyzer detects：By taking Hitachi 7170 operates as an example：Measure wavelength is 340 nm, takes various concentrations respectively
Calibration object solution（15μL）, add Procalcitonin R1Reagent（200μL）, mix, add Procalcitonin R2Reagent（50μL）, 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 Procalcitonin PCT in sample to be tested.If the concentration of Procalcitonin exceeds calibration curve scope in serum or blood plasma, need
Detected again after 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 Procalcitonin high concentration sample close to the range of linearity upper limit（90.60 ng/mL）, it is pressed 1/2 with physiological saline,
1/4,1/8,1/16,1/32,1/64 dilution, is configured to 6 diluted concentrations altogether（xi）Solution, with the Biochemical Analyzer examine
Survey method measures each diluted sample concentration.Each diluted concentration is tested 3 times, obtains the equal of each diluted concentration testing result respectively
Value（yi）.With diluted concentration（xi）For independent variable, to measure average（yi）Equation of linear regression is obtained for dependent variable, according to formula
（1）The correlation coefficient r of linear regression is calculated, the results show regression equation is y=0.9904x-0.0368, correlation coefficient r=
0.9998, show reagent of the present invention good relationship in the 0.2 ng/mL-90.6 ng/mL ranges of linearity（See Fig. 2）.
Embodiment five：Correlation analysis
To 86 clinical samples including 65 positive samples and 21 ' negative ' specimens respectively using the immune glimmering of norman
The homogeneous enzyme immunoassay method of light method and the present invention to sample replication 2 times, calculate average value, to 86 pattern detections respectively respectively
As a result linear regression analysis is carried out, calculates the related coefficient of two kinds of reagent testing results.
The results show that correlation coefficient r=0.9989 of two kinds of kit testing results, equation of linear regression are
y=0.9905x+0.0156（See Fig. 3）, show Procalcitonin homogeneous enzyme immunoassay method detection reagent of the present invention and commercially available Nore
Graceful immuno-fluorescence assay reagent 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 one exemplary embodiment, the scope of the present invention is by institute
Attached claim rather than described above limit, it is intended that will fall in the implication and scope of the equivalency of claim
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, some 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 Procalcitonin immunologic function test reagent and its preparation and detection method, it is characterised in that：Enzyme mark Procalcitonin, be used for
Detect the indicator of Procalcitonin antibody-enzyme mark Procalcitonin compound.
2. the immunologic function test reagent of Procalcitonin according to claim 1, it is characterised in that：The enzyme mark Procalcitonin by
Procalcitonin and glucose dehydrogenase coupling form.
3. Procalcitonin 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-Procalcitonin conjugate；On
The substrate for stating enzyme is glucose.
4. a kind of Procalcitonin immunologic function test reagent and preparation method thereof, it is characterised in that include the following steps：
（1）The preparation of glucose dehydrogenase-Procalcitonin conjugate：Prepare glucose dehydrogenase（GDH）Solution, Procalcitonin
Activation, GDH and Procalcitonin are coupled, and purify the enzyme-labelled antigen of coupling；
（2）The preparation of Procalcitonin homogeneous enzyme immunoassay detection reagent：
The preparation of reagent 1：Mixed by Procalcitonin antibody and homogeneous zymolyte；
The preparation of reagent 2：Mixed by glucose dehydrogenase-Procalcitonin conjugate with phosphate buffer.
5. the preparation method of a kind of Procalcitonin immunologic function test reagent according to claim 5, it is characterised in that described
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 solutions, pH 7.0-
10.0；
2）The activation of Procalcitonin
Following chemicals is added to stirring and dissolving in beaker：10-30 mg Procalcitonins, 10-40 mg1- ethyl -3- (- 3-
Dimethylaminopropyl) carbodiimide, 2-10 mg N- hydroxy thiosuccinimides, be dissolved in 2- (N- morpholines) ethyl sulfonic acid
（MES）In solution, stirring and dissolving, reacts 15-60 minutes at room temperature；
3）The coupling of GDH and Procalcitonin
A. the Procalcitonin solution of above-mentioned activation is added dropwise in the GDH solution in above-mentioned stirring；
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-Procalcitonin conjugate, in 2-
Stored at 8 DEG C.
A kind of 6. preparation method of Procalcitonin immunologic function test reagent according to claim 5, it is characterised 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 0.5-2L of 2-5 g oxidation state
Homogeneous zymolyte is made in phosphate buffer dissolving；Procalcitonin 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-Procalcitonin conjugate is added in phosphate buffer, above-mentioned idol
The volume ratio for joining thing and phosphate buffer is 1:100~1:10000.
7. using the detection method of the Procalcitonin immunologic function test reagent described in 4 any one of Claims 1-4, its feature exists
In comprising the following steps：
1）Sample to be tested is contacted with Procalcitonin antibody；
2）According to the combination situation of Procalcitonin in sample to be tested and Procalcitonin antibody, using being dropped in indicator judgement sample
The former content of calcium element；The sample to be tested is serum, blood plasma, saliva or urine.
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Citations (1)
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| CN101048660A (en) * | 2004-08-27 | 2007-10-03 | 灵芝国际股份有限公司 | Homogeneous enzyme immunoassay for oral fluid |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101048660A (en) * | 2004-08-27 | 2007-10-03 | 灵芝国际股份有限公司 | Homogeneous enzyme immunoassay for oral fluid |
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