CN105115945B - The detection method of gamma Globulin - Google Patents
The detection method of gamma Globulin Download PDFInfo
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- CN105115945B CN105115945B CN201510367483.3A CN201510367483A CN105115945B CN 105115945 B CN105115945 B CN 105115945B CN 201510367483 A CN201510367483 A CN 201510367483A CN 105115945 B CN105115945 B CN 105115945B
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Abstract
The invention discloses a kind of detection method of gamma Globulin, the detection method includes：Will as shown in formula (I) structure polymer P PEASO3Obtained solvent soluble in water；The gamma Globulin standard liquid of various concentrations is placed in solvent obtained above, and adds water and cushioning liquid constant volume that the solution to be measured of various concentrations is made, determines the maximum fluorescence intensity of each solution to be measured；Using the ratio of the fluorescence intensity of the fluorescence emission peak of solvent and the fluorescence intensity of the fluorescence emission peak of solution to be measured as ordinate, the concentration of gamma Globulin solution establishes the equation of the fluorescent absorption curve of spectrum for abscissa；The maximum fluorescence intensity of the gamma Globulin solution of concentration to be measured is detected, the concentration of gamma Globulin in the gamma Globulin solution of concentration to be measured is then calculated according to the equation of the fluorescent absorption curve of spectrum；Wherein, n is positive integer.Realize that Concentration Testing high sensitivity, the stability of gamma Globulin are good and repeatable high, simple to operate, the effect that can quickly determine.
Description
Technical field
The present invention relates to the detection method of biological vivo protein, in particular it relates to a kind of detection side of gamma globulin
Method.
Background technology
Serum globulins are synthesized by human monocytic-mononuclear phagocyte system, and it can be divided into α 1-, α 2-, β by protein electrophoresis
1-, β 2- and gamma globulin.It is with antibody activity and can be special with corresponding antigens and gamma globulin is also referred to as Class C globulin
A kind of globulin that strange land combines, an and important indicator in liver function test, gamma globulin it is higher almost see it is all
Disease in the liver and gallbladder.When patient is virus hepatitis, gamma globulin meeting moderate increases；When patient is heavy type hepatitis, γ-ball egg
In vain can be significantly raised；When patient is hepatic sclerosis, gamma globulin generally increases, especially late or progressive decompensation liver is hard
Change, gamma globulin can extremely increase.The current common disease triggered extremely by immunoglobulin has PID
With variability immunodeficiency symptoms.Therefore can quickly show that liver function has according to the changes of contents of internal gamma globulin not hindering
Hinder, reduce risk caused by disease in the liver and gallbladder as far as possible.
It has been suggested currently for the assay method of gamma globulin, among these including Coomassie Brilliant Blue and bromine phenol
Blue laws.In these methods, because susceptibility, multiplicity etc. are not high enough, therefore in practical application with certain limitation
And unstability.In general fluorescence spectrophotometry is in actual test due to meetings such as the similitudes of different kinds of proteins architectural characteristic
Inevitably there are many interference, and short wavelength emissions also result in many unnecessary signal interferences.
Therefore it provides a kind of high sensitivity, stability are good and with preferably repeatability, and simple to operate, can be fast
The problem of detection method that speed measures the gamma globulin of the concentration of gamma globulin is urgent need to resolve of the present invention.
The content of the invention
For above-mentioned prior art, it is an object of the invention to overcome in the prior art gamma globulin in continuous mode,
Often because the similitude of different kinds of proteins architectural characteristic is so as to inevitably occur much disturbing, while short wavelength emissions
It is the problems such as many unnecessary signal interferences can be caused, good and have and preferably may be used so as to provide a kind of high sensitivity, stability
Repeatability, and it is simple to operate, it can quickly measure the detection method of the gamma globulin of the concentration of gamma globulin.
To achieve these goals, the invention provides a kind of detection method of gamma globulin, wherein, the detection side
Method includes：
(1) will as shown in formula (I) structure polymer P PEASO3Obtained solvent soluble in water；
(2) the gamma globulin standard liquid of various concentrations is placed in solvent made from step (1), and adds water and buffering
The solution to be measured of various concentrations is made in solution constant volume, determines the maximum fluorescence intensity of each solution to be measured；
(3) with the ratio of the fluorescence intensity of the fluorescence emission peak of solvent and the fluorescence intensity of the fluorescence emission peak of solution to be measured
For ordinate, the concentration of gamma globulin solution establishes the equation of the fluorescent absorption curve of spectrum for abscissa；
(4) maximum fluorescence intensity of the gamma globulin solution of concentration to be measured is detected, then according to the fluorescent absorption curve of spectrum
Equation calculate the concentration of gamma globulin in the gamma globulin solution of concentration to be measured；
Wherein, n is positive integer.
Pass through above-mentioned technical proposal, it is of the invention by polymer P PEASO3Obtained solvent soluble in water, then passes through the solvent
And cushioning liquid mixes with the gamma globulin standard liquid of various concentrations, then solution to be measured is made in constant volume, and determines above-mentioned to be measured
The maximum fluorescence intensity of solution, and with the fluorescence intensity of the fluorescence emission peak of solvent and the fluorescence of the fluorescence emission peak of solution to be measured
The ratio of intensity is ordinate, and the concentration of gamma globulin solution is established fluorescent absorption curve of spectrum equation for abscissa, finally surveyed
The maximum fluorescence intensity of gamma globulin solution detected is needed calmly, and is calculated according to above-mentioned fluorescent absorption curve of spectrum equation
To the concentration of gamma globulin, so as in this way, after fluorescent absorption curve of spectrum establishing equation is come out, then only need
Determine a maximum fluorescence intensity, you can obtain needing the concentration of gamma globulin solution detected, realize high sensitivity, steady
It is qualitative to get well and with preferably repeatability, and simple to operate, quickly determine the effect of the concentration of gamma globulin solution.
Other features and advantages of the present invention will be described in detail in subsequent specific embodiment part.
Brief description of the drawings
Accompanying drawing is for providing a further understanding of the present invention, and a part for constitution instruction, with following tool
Body embodiment is used to explain the present invention together, but is not construed as limiting the invention.In the accompanying drawings：
Fig. 1 is the fluorescence intensity collection of illustrative plates of the gamma globulin solution of each concentration provided in embodiment；
Fig. 2 is a kind of equation of fluorescent absorption curve of spectrum provided in embodiment；
Fig. 3 is the equation of the embodiment obtained fluorescent absorption curve of spectrum at different temperatures；
Fig. 4 is PPEASO in embodiment3Fluorescence decay situation of the solution in the presence of the gamma globulin under various concentrations.
Embodiment
The embodiment of the present invention is described in detail below.It is it should be appreciated that described herein specific
Embodiment is merely to illustrate and explain the present invention, and is not intended to limit the invention.
The invention provides a kind of detection method of gamma globulin, wherein, the detection method includes：
(1) will as shown in formula (I) structure polymer P PEASO3Obtained solvent soluble in water；
(2) the gamma globulin standard liquid of various concentrations is placed in solvent made from step (1), and adds water and buffering
The solution to be measured of various concentrations is made in solution constant volume, determines the maximum fluorescence intensity of each solution to be measured；
(3) with the ratio of the fluorescence intensity of the fluorescence emission peak of solvent and the fluorescence intensity of the fluorescence emission peak of solution to be measured
For ordinate, the concentration of gamma globulin solution establishes the equation of the fluorescent absorption curve of spectrum for abscissa；
(4) maximum fluorescence intensity of the gamma globulin solution of concentration to be measured is detected, then according to the fluorescent absorption curve of spectrum
Equation calculate the concentration of gamma globulin in the gamma globulin solution of concentration to be measured；
Wherein, n is positive integer.
Above-mentioned design is by by polymer P PEASO3Obtained solvent soluble in water, then passes through the solvent and cushioning liquid
Mixed with the gamma globulin standard liquid of various concentrations, then solution to be measured is made in constant volume, and determine the maximum of above-mentioned solution to be measured
Fluorescence intensity, and with the ratio of the fluorescence intensity of the fluorescence emission peak of solvent and the fluorescence intensity of the fluorescence emission peak of solution to be measured
For ordinate, the concentration of gamma globulin solution establishes fluorescent absorption curve of spectrum equation for abscissa, and finally measure needs to detect
Gamma globulin solution maximum fluorescence intensity, and gamma globulin is calculated according to above-mentioned fluorescent absorption curve of spectrum equation
Concentration, so as in this way, after fluorescent absorption curve of spectrum establishing equation is come out, then only need measure once maximum
Fluorescence intensity, you can obtain needing the concentration of gamma globulin solution detected, it is good and have to realize high sensitivity, stability
Preferably repeatability, and it is simple to operate, it can quickly determine the effect of the concentration of gamma globulin solution.
The polymer P PEASO3Ratio between the cushioning liquid can not be further qualified, still, in order to
The detection data for making to obtain are more accurate, and then improve the accuracy of detection, while save cost as far as possible, the one of the present invention
In kind preferred embodiment, relative to the 1mg polymer P PEASO3, the dosage of the cushioning liquid is 0.01-0.2 μ
mol。
The cushioning liquid can be buffer species commonly used in the art, for example, in a kind of excellent of the present invention
In the embodiment of choosing, in order that the buffering effect of cushioning liquid is more preferable, the cushioning liquid can be defined to disodium hydrogen phosphate-
Potassium dihydrogen phosphate or three (methylol) aminomethane-hydrochloric acid buffer solutions.Certainly, select which kind of cushioning liquid is mainly here
Selection is carried out according to specific pH value in experiment, those skilled in the art can be directly selected accordingly, herein not one by one
Repeat.
Certainly, in order in whole system pH value it is more suitable, so as to farthest make it that the result that measures is accurate, at this
In a kind of embodiment being more highly preferred to of invention, the disodium hydrogen phosphate-potassium dihydrogen phosphate and three (methylol) amino first
The pH value of alkane-hydrochloric acid buffer solution can be further defined to 5-9.
The polymer P PEASO3Can be conventional use of type, for example, can be pure substance, certainly, in order to the greatest extent may be used
It can be easy to preserve in operation and use, in a kind of preferred embodiment of the present invention, polymer described in step (1)
PPEASO3Can be by polymer P PEASO3Solution provides, and the solvent in solution can be type of solvent commonly used in the art,
For example, in a kind of embodiment being more highly preferred to of the present invention, the polymer P PEASO3The solvent of solution can be water.
Polymer P PEASO3Polymer P PEASO in solution3Concentration can not be further qualified, certainly, in order that
It is easy to the use that preserves and can meet the present invention when in use, described poly- in a kind of preferred embodiment of the present invention
Compound PPEASO3Polymer P PEASO in solution3Concentration can be 7.5-22.5 μ g/mL.
In order that it is more accurate to detect the fluorescence intensity data obtained during solution to be measured, can be right in whole system
The pH of solution to be measured is further qualified, for example, in a kind of preferred embodiment of the present invention, in order to allow whole system
Condition can make the fluorescence intensity of detection more accurate and clear, and the pH value of the solution to be measured can be further defined to 5-9.
In order to allow each material in system fully to be reacted, be further ensured that detection fluorescence intensity it is accurate, in this hair
In a kind of bright preferred embodiment, the detection method can also include determining it after solution to be measured is placed into 1-80min
Fluorescence intensity.
The detection of fluorescence intensity can be operated according to this area common detection methods, meanwhile, in one kind of the present invention
In preferred embodiment, in order to obtain maximum fluorescence intensity, in step (2), the maximum fluorescence for detecting each solution to be measured is strong
Degree wave-length coverage is 590-820nm.
Certainly, in order that the linear correlation of the equation of the obtained fluorescent absorption curve of spectrum levels off to 1 as far as possible, at this
In a kind of preferred embodiment of invention, it can also be further defined to determine maximum under conditions of temperature is 293-320K
Fluorescence intensity.
The present invention will be described in detail by way of examples below.In following examples, the polymer P PEASO3For
It is made according to following preparation methods, the gamma globulin is the commercially available product of Shanghai Jing Chun biochemical technologies limited company, described
Disodium hydrogen phosphate-potassium dihydrogen phosphate buffer solution is to be made according to following preparation methods.
Preparation example 1
According to《Microchim.Acta》Polymer is made in method described in the 357-364 pages of the phase of periodical the 177th
PPEASO3。
Preparation example 2
3.561g disodium hydrogen phosphates are dissolved in 100mL water, 0.2mol/L disodium phosphate soln are made, by 3.06g
Potassium dihydrogen phosphate is dissolved in 100mL water, 0.2mol/L potassium dihydrogen phosphate is made, then by above-mentioned 31.5mL phosphoric acid hydrogen
Two sodium solutions are added drop-wise in 68.5mL potassium dihydrogen phosphate, regulation pH value to 6.5, so as to which disodium hydrogen phosphate-phosphoric acid be made
Potassium dihydrogen cushioning liquid.
Embodiment 1
By the polymer P PEASO that 400 μ L concentration are 0.5mg/mL3It is dissolved in the above-mentioned phosphoric acid hydrogen two that concentration is 0.2mol/L
Solvent is made in sodium-potassium dihydrogen phosphate buffer solution；10 parts of above-mentioned solvents are taken, and it is 5 μ to be separately added into concentration in every part of solvent
G/mL gamma globulin standard liquid 0 μ l, 10 μ l, 20 μ l, 100 μ l, 200 μ l, 500 μ l, 700 μ l, 1000 μ l, 1500 μ l and
2000 μ l, and add water constant volume that the solution to be measured of various concentrations is made, place the fluorescence for determining above-mentioned solution to be measured after 40min respectively
Intensity, as shown in figure 1, the maximum fluorescence intensity (nm) of each solution to be measured of measure is respectively 534nm, 508nm, 488.7nm,
425.8nm, 396.4nm, 332.2nm, 301.9nm, 247nm, 205.3nm and 182.5nm, wherein, gamma globulin mark in Fig. 1
The concentration of quasi- solution is followed successively by 0 μ g/L, 5 μ g/L, 10 μ g/L, 50 μ g/L, 100 μ g/L, 250 μ g/L, 350 μ g/L from top to bottom,
500 μ g/L, 750 μ g/L and 1000 μ g/L；As shown in Fig. 2 using the fluorescence intensity of the fluorescence emission peak of solvent (fluorescence intensity level as
534nm) with the ratio of the fluorescence intensity of the fluorescence emission peak of solution to be measured it is ordinate, the concentration of gamma globulin solution is horizontal stroke
Coordinate establishes the equation of the fluorescent absorption curve of spectrum, and the equation for obtaining the fluorescent absorption curve of spectrum under temperature is 293K is：Y=
1.11246+0.0019x wherein linear correlation is R=0.99789.
As shown in figure 3, the fluorescent absorption curve of spectrum in the case where temperature is 310K and 320K is made respectively according to the method described above
Figure.As can be seen that the quenching constant highest under conditions of temperature is 293K.
As shown in figure 4, take 4 parts of PPEASO3Solution, the 1st part of PPEASO3Gamma globulin, 2-4 parts are added without in solution
PPEASO3The concentration that same volume (10 μ L) is separately added into solution is 100ng/mL, 250ng/mL, 500ng/mL γ-ball
Albumen, its fluorescence decay time is determined, wherein, the fluorescence decay time for not adding gamma globulin is 0.781ns, adds concentration
Fluorescence decay time for 100ng/mL gamma globulin is 0.787ns, and the fluorescence for adding 250ng/mL gamma globulin declines
The fluorescence decay time for subtracting the gamma globulin that the time is 0.793ns, 500ng/mL is 0.794ns, it is possible thereby to clearly see
Go out, fluorescence decay time is almost unchanged.Therefore, it further determined that gamma globulin to PPEASO3The quenching of polymer is static
Quenching.
Application examples 1
Operated according to the method for embodiment, unlike, the concentration of the gamma globulin solution is 0.2 μ g/ml,
The maximum fluorescence intensity measured is 340.8nm, and the concentration that gamma globulin solution is calculated according to above-mentioned equation is A1=
0.196μg/ml。
Application examples 2
Operated according to the method for embodiment, unlike, the concentration of the gamma globulin solution is 0.4 μ g/ml,
The maximum fluorescence intensity measured is 289nm, and the concentration that gamma globulin solution is calculated according to above-mentioned equation is A2=0.408 μ
g/ml。
Application examples 3
Operated according to the method for embodiment, unlike, the concentration of the gamma globulin solution is 0.25 μ g/ml,
The maximum fluorescence intensity measured is 343.6nm, and the concentration that gamma globulin solution is calculated according to above-mentioned equation is A3=
0.257μg/ml。
Application examples 4
Operated according to the method for embodiment, unlike, the concentration of the gamma globulin solution is 0.5 μ g/ml,
The maximum fluorescence intensity measured is 284.3nm, and the concentration that gamma globulin solution is calculated according to above-mentioned equation is A4=
0.516μg/ml。
By above-mentioned as can be seen that the concentration and gamma globulin of the gamma globulin solution determined by the method for the present invention
The actual concentrations value difference of solution is away from very little, thus when detecting the concentration of gamma globulin solution with higher sensitivity, and grasps
Make having good stability for method, repeatability is high, and detects the concentration operating method of gamma globulin solution in this way
Simply, the concentration of gamma globulin solution can be quickly measured, greatly reduces interference of other protein to detection.
The preferred embodiment of the present invention described in detail above, still, the present invention are not limited in above-mentioned embodiment
Detail, in the range of the technology design of the present invention, a variety of simple variants can be carried out to technical scheme, this
A little simple variants belong to protection scope of the present invention.
It is further to note that each particular technique feature described in above-mentioned embodiment, in not lance
In the case of shield, can be combined by any suitable means, in order to avoid unnecessary repetition, the present invention to it is various can
The combination of energy no longer separately illustrates.
In addition, various embodiments of the present invention can be combined randomly, as long as it is without prejudice to originally
The thought of invention, it should equally be considered as content disclosed in this invention.
Claims (10)
1. a kind of detection method of gamma globulin, it is characterised in that the detection method includes：
(1) will as shown in formula (I) structure polymer P PEASO3Obtained solvent soluble in water；
(2) the gamma globulin standard liquid of various concentrations is placed in solvent made from step (1), and adds water and cushioning liquid
The solution to be measured of various concentrations is made in constant volume, determines the maximum fluorescence intensity of each solution to be measured；
(3) it is vertical using the ratio of the fluorescence intensity of the fluorescence emission peak of solvent and the fluorescence intensity of the fluorescence emission peak of solution to be measured
Coordinate, the concentration of gamma globulin solution establish the equation of the fluorescent absorption curve of spectrum for abscissa；
(4) maximum fluorescence intensity of the gamma globulin solution of concentration to be measured is detected, then according to the side of the fluorescent absorption curve of spectrum
The concentration of gamma globulin in the gamma globulin solution of journey calculating concentration to be measured；
Wherein, n is positive integer.
2. detection method according to claim 1, wherein, relative to the 1mg polymer P PEASO3, it is described buffering it is molten
The dosage of liquid is 0.01-0.2 μm of ol.
3. detection method according to claim 1 or 2, wherein, the cushioning liquid is disodium hydrogen phosphate-potassium dihydrogen phosphate
Or three (methylol) aminomethane-hydrochloric acid buffer solution.
4. detection method according to claim 3, wherein, the disodium hydrogen phosphate-potassium dihydrogen phosphate and three (methylols)
The pH value of aminomethane-hydrochloric acid buffer solution is 5-9.
5. detection method according to claim 1 or 2, wherein, polymer P PEASO described in step (1)3By polymer
PPEASO3Solution provides, and the polymer P PEASO3The solvent of solution is water.
6. detection method according to claim 5, wherein, the polymer P PEASO3Polymer P PEASO in solution3's
Concentration is 7.5-22.5 μ g/mL.
7. detection method according to claim 1 or 2, wherein, the pH value of the solution to be measured is 5-9.
8. detection method according to claim 1 or 2, wherein, the detection method also includes solution to be measured placing 1-
Its fluorescence intensity is determined after 80min.
9. detection method according to claim 1 or 2, wherein, in step (2), detect the maximum glimmering of each solution to be measured
Luminous intensity wave-length coverage is 590-820nm.
10. detection method according to claim 1 or 2, wherein, determined under conditions of temperature is 293-320K maximum glimmering
Luminous intensity.
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| CN1712940A (en) * | 2005-08-02 | 2005-12-28 | 武汉大学 | Production of fluorescent sensor based on soluble conjugated polymer |
| CN101671427A (en) * | 2009-10-22 | 2010-03-17 | 湖南师范大学 | Water soluble fluorescent conjugated polymer and synthesis method thereof |
| CN103969446A (en) * | 2014-05-11 | 2014-08-06 | 桂林理工大学 | Method for detecting concentration of trace immunoglobulin G in human serum |
| CN104090113A (en) * | 2013-06-27 | 2014-10-08 | 桂林电子科技大学 | Method for detecting human immune globulin E with concentration of 0.5-10[mu]g/mL |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP4891363B2 (en) * | 2009-05-18 | 2012-03-07 | 株式会社日立ハイテクノロジーズ | Fluorescence detection method and fluorescence detection apparatus |
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2015
- 2015-06-26 CN CN201510367483.3A patent/CN105115945B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1712940A (en) * | 2005-08-02 | 2005-12-28 | 武汉大学 | Production of fluorescent sensor based on soluble conjugated polymer |
| CN101671427A (en) * | 2009-10-22 | 2010-03-17 | 湖南师范大学 | Water soluble fluorescent conjugated polymer and synthesis method thereof |
| CN104090113A (en) * | 2013-06-27 | 2014-10-08 | 桂林电子科技大学 | Method for detecting human immune globulin E with concentration of 0.5-10[mu]g/mL |
| CN103969446A (en) * | 2014-05-11 | 2014-08-06 | 桂林理工大学 | Method for detecting concentration of trace immunoglobulin G in human serum |
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| Sun Lilin et al..Highly sensitive fluorescent sensor for copper (II) based on amplified fluorescence quenching of a water-soluble NIR emitting conjugated polymer.《Microchim Acta》.2012,第177卷第359页左栏第4段、第360页右栏第3段-第361页左栏第1段及结构图1、图3及表3. * |
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