CN103969446A - Method for detecting concentration of trace immunoglobulin G in human serum - Google Patents
Method for detecting concentration of trace immunoglobulin G in human serum Download PDFInfo
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- CN103969446A CN103969446A CN201410195006.9A CN201410195006A CN103969446A CN 103969446 A CN103969446 A CN 103969446A CN 201410195006 A CN201410195006 A CN 201410195006A CN 103969446 A CN103969446 A CN 103969446A
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
- G01N33/542—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with steric inhibition or signal modification, e.g. fluorescent quenching
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Abstract
The invention discloses a method for detecting the concentration of trace immunoglobulin G in human serum. An energy transferring system with stable performance consists of goat anti human immunoglobulin G antibody-labeled fluorescein isothiocyanate serving as an energy transferring supplier and nano gold serving as a receptor; the goat anti human immunoglobulin G antibody-labeled fluorescein isothiocyanate transmits energy to the nano gold, so that fluorescence of the fluorescein isothiocyanate is quenched; however, after immunoglobulin G is added, the fluorescence of the fluorescein isothiocyanate is recovered; furthermore, the fluorescence recovery value and the concentration of the immunoglobulin G are in good linear relation within a range of 4.0-220.0 nanograms per milliliter, so that a novel method for detecting the immunoglobulin G is built. According to the method disclosed by the invention, the shortcomings of low sensitivity, complicated operation and the like during detection in the prior art are overcome, and the immunoglobulin G in the human serum can be conveniently and quickly detected.
Description
Technical field
The present invention relates to a kind of method of utilizing different sulphur cyanines acid fluorescein and nm of gold energy transfer technique speed to detect trace immunoglobulin G concentration in human serum.
Background technology
Immunoglobulin G is the highest a kind of immune protein of content in serum, account for 70% ~ 80% of sero-immunity albumen, after body secondary immune response, to form the key component of antibody, in body defense mechanism, play an important role, clinically as the important indicator of various diseases diagnosis and therapeutic evaluation, so its qualitative and quantitative analysis is very important.At present, the main method that detects immunoglobulin G while both at home and abroad has euzymelinked immunosorbent assay (ELISA), voltage immune sensing technology, electrochemiluminescence immunological technique, fluorescence sense technology, ultra-violet absorption spectrometry etc.The complicated operation that above method has, some sensitivity is not high.Therefore, seek a kind of fast, accurately, the method for high selectivity detects immunoglobulin G in human serum and has great importance.Energy transfer technique is a kind of novel fluorescence detection technique, has highly sensitively, compares be subject to Rayleigh scattering light to disturb the features such as little with conventional fluorescence method with Resonance Light Scattering Method.Meanwhile, utilize immunoglobulin G and the nm of gold Competition to the different sulphur cyanines acid fluorescein of goat anti-human immunoglobulin G mark herein, only need mark fluorescent to body, and medicine has been saved in the operation of having save labeled receptor.This method is utilized the detection characteristic of its uniqueness, thereby sets up quick, sensitive, to detect accurately immunoglobulin G new method.The different sulphur cyanines acid fluorescein of domestic and international application and nm of gold energy transfer method there is not yet report in the detection of immunoglobulin G at present.
Summary of the invention
The object of this invention is to provide a kind of method simple, highly sensitive, selectivity is good, detects easily and fast the method for trace immunoglobulin G concentration in human serum.
Thinking of the present invention: the different sulphur cyanines acid fluorescein of goat anti-human immunoglobulin G antibody labeling of usining as energy, shift to body, nm of gold is as acceptor, form the energy transfer system of stable performance, the different sulphur cyanines acid fluorescein of goat anti-human immunoglobulin G antibody labeling passes to nm of gold by energy, makes its fluorescence generation quencher.And immunoglobulin G makes the fluorescence of different sulphur cyanines acid fluorescein recover after adding, and the concentration of its fluorescence recovery value and immunoglobulin G is good linear relationship within the scope of 4.0 ~ 220.0 nanograms/milliliter, thereby set up the new method that detects immunoglobulin G while.
Concrete mechanism of the present invention: different sulphur cyanines acid fluorescein and the nm of gold generation energy of goat anti-human immunoglobulin G antibody labeling shift the fluorescence generation quencher that makes himself.Energy transfer rat with give body, distance dependent between acceptor, immunoglobulin G add can with different sulphur cyanines acid fluorescein on the immune response of goat anti-human immunoglobulin G antibody generation antigen and antibody specific, make the distance between nm of gold and different sulphur cyanines acid fluorescein farther, energy transfer phenomenon is suppressed, and makes the regular recovery of fluorescence of the different sulphur cyanines acid fluorescein of goat anti-human immunoglobulin G antibody labeling.
Concrete steps are:
(1) preparation of nano-Au solution:
5 milliliters of 5 mM/ls of gold chlorides are joined in 100 milliliters of sub-boiling water, under constantly stirring, be heated to boil, then add fast the citric acid solution that 5 milliliters of mass body volume concentrations are 0.1%, keep boiling 10 minutes, the color of solution becomes claret gradually, stop heating, under 10000 revs/min of rotating speeds centrifugal 10 minutes, remove excessive citric acid, make concentration and be 50 nanomoles/liter nano-Au solution, under the condition of 4 ℃, preserve, standby.
(2) preparation of the different sulphur cyanines acid of goat anti-human immunoglobulin G antibody labeling luciferin solution:
By 20 milliliter 1.0 * 10
-5the different sulphur cyanines acid of mol/L fluorescein, the N-maloyl imines of the 1-of 100 microlitre 20 mg/ml (3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride and 100 microlitre 10 mg/ml is mix and blend 0.5 hour at room temperature, add again 100 microlitre 1 mg/ml goat anti-human immunoglobulin G antibody, at room temperature hatch 2 hours, last under the rotating speed of 10000 revs/min centrifugal 30 minutes, remove unnecessary antibody, make the different sulphur cyanines acid of goat anti-human immunoglobulin G antibody labeling luciferin solution, under the condition of 4 ℃, preserve, standby.
(3) obtain solution:
The different sulphur cyanines acid of the goat anti-human immunoglobulin G antibody labeling luciferin solution of respectively being prepared by 50 microlitre steps (2), nano-Au solution and 0,2.0,5.0,10.0,20.0,30.0,50.0,70.0 prepared by 800 microlitre steps (1), 110.0 microlitres 1.0 * 10
-5the immunoglobulin G of grams per milliliter joins in 95 milliliters of colour tubes, with the buffer solution of pH=7.4, is settled to scale, reacts 10 minutes; The sodium dihydrogen phosphate of the sodium hydrogen phosphate that the buffer solution of described pH=7.4 contains 0.05 mol/L and 0.05 mol/L.
(4) drafting of working curve:
The solution that step (3) is made carries out fluorescence intensity detection with RF-5301 PC fluorophotometer respectively, and excitation wavelength is 480 nanometers, excites and launch slit width to be 5 nanometers; According to measurement result, the concentration c of immunoglobulin G within the scope of 4.0 ~ 220 nanograms/milliliter with fluorescence recovery value Δ I
fbe good linear relationship, its equation of linear regression is: Δ I
f=9.52+1.68c, linearly dependent coefficient r=0.9987.
(5) detection of immunoglobulin G in human serum:
A. human serum sample to be measured is diluted to 1000 times as solution to be measured with the phosphate buffer of 50 mM/ls.
B. the different sulphur cyanines acid of the goat anti-human immunoglobulin G antibody labeling luciferin solution of respectively being prepared by 50 microlitre steps (2), the solution to be measured of nano-Au solution prepared by 800 microlitre steps (1) and step (5) a step preparations adds in 5 milliliters of colour tubes, with the buffer solution of pH=7.4, be settled to scale, react 10 minutes; With RF-5301 PC fluorophotometer, in excitation wavelength, be 480 nanometers, excite and launch slit width to be under the condition of 5 nanometers and to carry out fluorescence intensity detection, record fluorescence amount of recovery Δ I
f, according to the equation of linear regression of step (4) gained, calculate the concentration c of immunoglobulin G; The sodium dihydrogen phosphate of the sodium hydrogen phosphate that the buffer solution of described pH=7.4 contains 0.05 mol/L and 0.05 mol/L.
The inventive method overcome prior art when detecting, exist reaction time length, complex operation, sensitivity low, etc. shortcoming, improved better sensitivity and selectivity, for the more accurate fast and easy of detection of immunoglobulin G in human serum.
Accompanying drawing explanation
Fig. 1 is that the immunoglobulin G of variable concentrations in the present invention recovers spectrogram to the fluorescence of different sulphur cyanines acid fluorescein.
In figure, a is respectively 0,2.0,5.0,10.0,20.0,30.0,50.0,70.0,110.0 microlitres 1.0 * 10 to j
-5after the immunoglobulin G of grams per milliliter is settled to scale in 5 milliliters of color comparison tubes, the fluorescence of different sulphur cyanines acid fluorescein is recovered to spectrogram.
Fig. 2 is the graph of a relation of the fluorescence amount of recovery of different sulphur cyanines acid fluorescein in the concentration c of immunoglobulin G in the present invention and different sulphur cyanines acid fluorescein-nm of gold energy transfer system.
Embodiment
embodiment:
(1) preparation of nano-Au solution:
5 milliliters of 5 mM/ls of gold chlorides are joined in 100 milliliters of sub-boiling water, under constantly stirring, be heated to boil, then add fast the citric acid solution that 5 milliliters of mass body volume concentrations are 0.1%, keep boiling 10 minutes, the color of solution becomes claret gradually, stop heating, under 10000 revs/min of rotating speeds centrifugal 10 minutes, remove excessive citric acid, make concentration and be 50 nanomoles/liter nano-Au solution, under the condition of 4 ℃, preserve, standby.
(2) preparation of the different sulphur cyanines acid of goat anti-human immunoglobulin G antibody labeling luciferin solution:
By 20 milliliter 1.0 * 10
-5the different sulphur cyanines acid of mol/L fluorescein, the N-maloyl imines of the 1-of 100 microlitre 20 mg/ml (3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride and 100 microlitre 10 mg/ml is mix and blend 0.5 hour at room temperature, add again 100 microlitre 1 mg/ml goat anti-human immunoglobulin G antibody, at room temperature hatch 2 hours, last under the rotating speed of 10000 revs/min centrifugal 30 minutes, remove unnecessary antibody, make the different sulphur cyanines acid of goat anti-human immunoglobulin G antibody labeling luciferin solution, under the condition of 4 ℃, preserve, standby.
(3) obtain solution:
The different sulphur cyanines acid of the goat anti-human immunoglobulin G antibody labeling luciferin solution of respectively being prepared by 50 microlitre steps (2), nano-Au solution and 0,2.0,5.0,10.0,20.0,30.0,50.0,70.0 prepared by 800 microlitre steps (1), 110.0 microlitres 1.0 * 10
-5the immunoglobulin G of grams per milliliter joins in 95 milliliters of colour tubes, with the buffer solution of pH=7.4, is settled to scale, reacts 10 minutes; The sodium dihydrogen phosphate of the sodium hydrogen phosphate that the buffer solution of described pH=7.4 contains 0.05 mol/L and 0.05 mol/L.
(4) drafting of working curve:
The solution that step (3) is made carries out fluorescence intensity detection with RF-5301 PC fluorophotometer respectively, and excitation wavelength is 480 nanometers, excites and launch slit width to be 5 nanometers; According to measurement result, the concentration c of immunoglobulin G within the scope of 4.0 ~ 220 nanograms/milliliter with fluorescence recovery value Δ I
fbe good linear relationship, its equation of linear regression is: Δ I
f=9.52+1.68c, linearly dependent coefficient r=0.9987.
(5) detection of immunoglobulin G content in human serum:
The human serum sample to be measured that Hospital of a.Jiang Cong Guilin University of Technology obtains dilutes 1000 times as solution to be measured with the phosphate buffer of 50 mM/ls.
B. the solution to be measured step (5) a steps being made is by method constant volume of the present invention and measure, carry out standard simultaneously and add recovery test, result is as shown in table 1, its RSD≤2.86% (n=6), recovery of standard addition, 98.7% ~ 105.0%, illustrates that the inventive method has higher accuracy and good precision.
Table 1: sample determination and mark-on recovery test data
| Sample | Measured value (n=6) ng/mL | RSD(n=6)% | Add scalar ng/mL | Record total amount ng/mL | Recovery % |
| Serum 1 | 83.8 | 3.32 | 100.00 | 188.8 | 105.0 |
| Serum 2 | 116.8 | 2.07 | 100.00 | 217.8 | 101.0 |
| Serum 3 | 106.1 | 2.26 | 100.00 | 204.8 | 98.7 |
Claims (1)
1. detect a method for trace immunoglobulin G concentration in human serum, it is characterized in that concrete steps are:
(1) preparation of nano-Au solution:
5 milliliters of 5 mM/ls of gold chlorides are joined in 100 milliliters of sub-boiling water, under constantly stirring, be heated to boil, then add fast the citric acid solution that 5 milliliters of mass body volume concentrations are 0.1%, keep boiling 10 minutes, the color of solution becomes claret gradually, stop heating, under 10000 revs/min of rotating speeds centrifugal 10 minutes, remove excessive citric acid, make concentration and be 50 nanomoles/liter nano-Au solution, under the condition of 4 ℃, preserve, standby;
(2) preparation of the different sulphur cyanines acid of goat anti-human immunoglobulin G antibody labeling luciferin solution:
By 20 milliliter 1.0 * 10
-5the different sulphur cyanines acid of mol/L fluorescein, the N-maloyl imines of the 1-of 100 microlitre 20 mg/ml (3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride and 100 microlitre 10 mg/ml is mix and blend 0.5 hour at room temperature, add again 100 microlitre 1 mg/ml goat anti-human immunoglobulin G antibody, at room temperature hatch 2 hours, last under the rotating speed of 10000 revs/min centrifugal 30 minutes, remove unnecessary antibody, make the different sulphur cyanines acid of goat anti-human immunoglobulin G antibody labeling luciferin solution, under the condition of 4 ℃, preserve, standby;
(3) obtain solution:
The different sulphur cyanines acid of the goat anti-human immunoglobulin G antibody labeling luciferin solution of respectively being prepared by 50 microlitre steps (2), nano-Au solution and 0,2.0,5.0,10.0,20.0,30.0,50.0,70.0 prepared by 800 microlitre steps (1), 110.0 microlitres 1.0 * 10
-5the immunoglobulin G of grams per milliliter joins in 95 milliliters of colour tubes, with the buffer solution of pH=7.4, is settled to scale, reacts 10 minutes; The sodium dihydrogen phosphate of the sodium hydrogen phosphate that the buffer solution of described pH=7.4 contains 0.05 mol/L and 0.05 mol/L;
(4) drafting of working curve:
The solution that step (3) is made carries out fluorescence intensity detection with RF-5301 PC fluorophotometer respectively, and excitation wavelength is 480 nanometers, excites and launch slit width to be 5 nanometers; According to measurement result, the concentration c of immunoglobulin G within the scope of 4.0 ~ 220 nanograms/milliliter with fluorescence recovery value Δ I
fbe good linear relationship, its equation of linear regression is: Δ I
f=9.52+1.68c, linearly dependent coefficient r=0.9987;
(5) detection of immunoglobulin G in human serum:
A. human serum sample to be measured is diluted to 1000 times as solution to be measured with the phosphate buffer of 50 mM/ls;
B. the different sulphur cyanines acid of the goat anti-human immunoglobulin G antibody labeling luciferin solution of respectively being prepared by 50 microlitre steps (2), the solution to be measured of nano-Au solution prepared by 800 microlitre steps (1) and step (5) a step preparations adds in 5 milliliters of colour tubes, with the buffer solution of pH=7.4, be settled to scale, react 10 minutes; With RF-5301 PC fluorophotometer, in excitation wavelength, be 480 nanometers, excite and launch slit width to be under the condition of 5 nanometers and to carry out fluorescence intensity detection, record fluorescence amount of recovery Δ I
f, according to the equation of linear regression of step (4) gained, calculate the concentration c of immunoglobulin G; The sodium dihydrogen phosphate of the sodium hydrogen phosphate that the buffer solution of described pH=7.4 contains 0.05 mol/L and 0.05 mol/L.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201410195006.9A CN103969446A (en) | 2014-05-11 | 2014-05-11 | Method for detecting concentration of trace immunoglobulin G in human serum |
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| CN201410195006.9A CN103969446A (en) | 2014-05-11 | 2014-05-11 | Method for detecting concentration of trace immunoglobulin G in human serum |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105115945A (en) * | 2015-06-26 | 2015-12-02 | 安徽师范大学 | gamma-globulin detection method |
| CN113804659A (en) * | 2020-06-12 | 2021-12-17 | 武汉戴安生物技术有限公司 | Method for detecting concentration of trace immunoglobulin G in human serum |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101881767A (en) * | 2009-05-06 | 2010-11-10 | 北京现代高达生物技术有限责任公司 | Special specimen dilution for detecting kit by using IgG (Immunoglobulin G) compatibility index method |
| CN102507932A (en) * | 2011-12-02 | 2012-06-20 | 无锡博慧斯生物医药科技有限公司 | IgM (immunoglobulin M) antibody detection test strip |
| CN103575875A (en) * | 2013-11-12 | 2014-02-12 | 镇江市第一人民医院 | Novel functionalized gold nanorod immune probe as well as preparation method and application of gold nanorod biological chip |
-
2014
- 2014-05-11 CN CN201410195006.9A patent/CN103969446A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101881767A (en) * | 2009-05-06 | 2010-11-10 | 北京现代高达生物技术有限责任公司 | Special specimen dilution for detecting kit by using IgG (Immunoglobulin G) compatibility index method |
| CN102507932A (en) * | 2011-12-02 | 2012-06-20 | 无锡博慧斯生物医药科技有限公司 | IgM (immunoglobulin M) antibody detection test strip |
| CN103575875A (en) * | 2013-11-12 | 2014-02-12 | 镇江市第一人民医院 | Novel functionalized gold nanorod immune probe as well as preparation method and application of gold nanorod biological chip |
Non-Patent Citations (1)
| Title |
|---|
| HUILIN TAO ET AL: "Detection of immunoglobulin G based on nanoparticle surface energy transfers from fluorescein isothiocyanate to gold nanoparticles", 《ANALYTICAL METHODS》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105115945A (en) * | 2015-06-26 | 2015-12-02 | 安徽师范大学 | gamma-globulin detection method |
| CN105115945B (en) * | 2015-06-26 | 2018-01-19 | 安徽师范大学 | The detection method of gamma Globulin |
| CN113804659A (en) * | 2020-06-12 | 2021-12-17 | 武汉戴安生物技术有限公司 | Method for detecting concentration of trace immunoglobulin G in human serum |
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Application publication date: 20140806 |