CN106526197A - Method for detecting human IgG concentration - Google Patents
Method for detecting human IgG concentration Download PDFInfo
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- CN106526197A CN106526197A CN201610883454.7A CN201610883454A CN106526197A CN 106526197 A CN106526197 A CN 106526197A CN 201610883454 A CN201610883454 A CN 201610883454A CN 106526197 A CN106526197 A CN 106526197A
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- igg
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- elisa plate
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- concentration
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
- G01N33/6806—Determination of free amino acids
- G01N33/6812—Assays for specific amino acids
Abstract
The invention discloses a method for detecting human IgG concentration. The method includes: using mesoporous NH2-silicon dioxide nano particles as the carrier to build an anti-IgG-(secondary antibody-MSN-enzyme compound) three-layer sandwiched structure; using the three-layer sandwiched structure to catalyze the oxygenolysis of glucose to generate hydrogen peroxide, and using the principle that the hydrogen peroxide oxidizes Fe<2+> to generate Fe<3+> and orthophenanthroline and the Fe<3+> and orthophenanthroline form orange complex to detect the IgG concentration. The method is good in selectivity, high in sensitivity, low in detection limit, wide in detection range, and the like.
Description
Technical field
The present invention relates to a kind of detection method of IgG concentration, more particularly to an a kind of anti-igg (two resist
MSN multienzyme complexs) three-layer sandwich type structure structure and its for the detection method of human immunoglobulin IgG concentration；Category
In biosensor technique field.
Background technology
IgG is a kind of immunoglobulin G (Immunoglobulin G, IgG), is the main antibody component of serum, accounts for
The 75% of serum I g.The function of IgG mainly shields in immunity of organism, most of antibacterials, antiviral, can be effective
The corresponding infectious diseases of ground prevention.For diagnosis, some diseases have greater significance to its index.
As IgG is more easy to be diffused in EV gap compared with other classes Ig, thus in conjugated complement, enhancing immunocyte
Phagocytosis pathogenic microorganism and the bacteriotoxic ability side of neutralization have important function, can be effective against infection.Traditional measure IgG
Method have fluoroimmunoassay, radioimmunology, electrochemical assay etc..Although the spirit to a certain extent of these detection methods
It is quick effective but high to technical requirements, take time and effort, and have certain requirement to instrument, each with certain limitation.
The content of the invention
For the deficiency that the method for existing traditional detection IgG is present, it is to provide a kind of that the purpose of the present invention is
The method of the detection human immunoglobulin IgG concentration that selective good, sensitivity is high, test limit is low, detection range is wide.
In order to realize above-mentioned technical purpose, the invention provides a kind of detection method of IgG concentration, the method includes
Following steps：
1) in mesoporous NH2Modify glucose oxidase and two anti-Ab on Nano particles of silicon dioxide (MSN) carrier simultaneously2,
Obtain two anti-MSN multienzyme complexs；
2) by an anti-Ab1Modification is in each hole of ELISA Plate, then ELISA Plate is closed with ethanolamine solutions, obtains one
Anti- Ab1The ELISA Plate of modification；
3) a series of standard IgG solution for taking variable concentrations is added separately to described one anti-Ab1The ELISA Plate of modification it is each
An IgG and anti-Ab is carried out in hole1Between specific binding reaction, obtain combining the ELISA Plate of IgG；
4) adding described two anti-MSN multienzyme complexs carries out IgG and two to each hole of the ELISA Plate for combining IgG
Anti- Ab1Between specific binding reaction, obtain containing an anti-igg (two anti-MSN multienzyme complexs) three-layer sandwich type tie
The ELISA Plate of structure；
5) into each hole of the ELISA Plate containing an anti-igg (two anti-MSN multienzyme complexs) three-layer sandwich type structure
Add glucose/Fe2+After mixed solution is reacted, Phen developer is added, and detects the extinction of solution in each hole
Angle value；
6) standard is set up with corresponding standard IgG solution concentration according to solution absorbance value in each hole for obtaining of detection bent
Line；
7) take IgG solution to be measured replace standard IgG solution by 3), 4) He 5) the step of operated, detect and obtain extinction
Angle value, establishing criteria curve obtain final product the concentration of IgG solution to be measured.
Preferred scheme, in mesoporous NH2Glutaraldehyde solution is added in Nano particles of silicon dioxide carrier solution in room temperature
Lower reaction, adds glucose oxidase and two anti-Ab2, reacted at a temperature of 3～5 DEG C, obtained final product two anti-MSN enzymes multiple
Compound.The effect of glutaraldehyde is by glucose oxidase and two anti-Ab2It is fixed on mesoporous NH2Nano particles of silicon dioxide carrier
On.
Preferred scheme, first by an anti-Ab1Solution is added to each hole of ELISA Plate at a temperature of 3～5 DEG C overnight, then will
Ethanolamine solutions are added to each hole of ELISA Plate and are closed at room temperature, obtain final product an anti-Ab1The ELISA Plate of modification.More preferably
Scheme, by 100 μ L, an anti-Ab of 10 μ g/mL1Solution is added at 4 DEG C of orifice plate overnight, with PBST as cleaning solution board-washing 2 times,
150 μ L, 1M ethanolamine solutions are added to be closed per hole, board-washing 2 times after 2～3h.
More preferably scheme, two anti-Ab2For goat anti-human IgG antibodies.
More preferably scheme, an anti-Ab1For mouse anti human IgG antibody.
More preferably scheme, the mesoporous NH2Nano particles of silicon dioxide is prepared via a method which to obtain：By 16
After alkyl trimethyl ammonium bromide is dissolved in sodium hydroxide solution, adds tetraethyl orthosilicate to be hydrolyzed reaction, obtain silica
Nano-particle is precipitated；After Nano particles of silicon dioxide precipitation is suspended in toluene, exist with aminopropyl triethoxysilane
Under nitrogen protection, 11～12h, reaction products therefrom and NH is reacted at a temperature of 75～80 DEG C4NO3Ethanol solution 55～60
2～3h is reacted at a temperature of DEG C, is obtained final product.
Preferred scheme, 3) or 4) in the condition of specific binding reaction be：Temperature be 36～37 DEG C, the time be 3～
5h。
Preferred scheme, by concentration be respectively 1pg/mL, 5pg/mL, 10pg/mL, 100pg/mL, 1ng/mL, 10ng/mL,
The series of standards IgG solution of 100ng/mL, 1 μ g/mL and 10 μ g/mL is separately added into an anti-Ab1Each hole of the ELISA Plate of modification
It is interior, carry out specific binding reaction.
Preferred scheme, glucose/Fe2+Mixed solution ferrous ions concentration is 0.15～0.16mM, concentration of glucose
For 16～17mg/mL.
Preferred scheme, the Detection wavelength of absorbance is 490nm.
Preferred scheme, ELISA Plate are 96 hole polystyrene ELISA Plates.
Preferred scheme, glucose/Fe2+Mixed solution is comprising glucose and Fe2+Hac buffer, acetic acid is molten
Liquid concentration is 10mM.Glucose is decomposed under glucose oxidase catalytic action, produces H2O2With the Fe in solution2+Carry out
Redox reaction, Fe2+It is oxidized to Fe3+。
In the solution of the present invention, Phen developer with not by the Fe of hydrogen peroxide oxidation2+Reaction forms orange red network
Compound, makes solution develop the color, and is conducive to being measured absorbance by ELIASA.
Preferred scheme, 2) in, by an anti-Ab1Modification, an anti-Ab are added to each hole of ELISA Plate1In the form of a solution plus
Enter, containing an anti-Ab1The volume of solution is 100 μ L, and concentration is 10 μ g/mL.
Preferred scheme, 5) in reaction condition be：3～5h is reacted at a temperature of 37 DEG C.
The method of the detection IgG concentration of the present invention is anti-by vector construction one of amino mesoporous silicon sphere nano-particle
IgG (two anti-MSN multienzyme complexs) three-layer sandwich type structure, uses it for detecting IgG concentration, with selectivity
Good, sensitivity is high, test limit is low, and the advantages of detection range width.Technical scheme, carries out amino mesoporous silicon sphere first
Synthesis, the silicon ball nano-particle of synthesis is due to band-NH2Group, can be used for binding antibody and enzyme；ELISA Plate is resisted with one to be carried out
Bed board, is closed to orifice surface with monoethanolamine, prevents that non-specific adsorption occurs in orifice surface；Then add in plate hole
Enter IgG to be reacted, bed board one is anti-to carry out specific recognition with IgG and combine；To in orifice plate, add two anti-MSN- enzymes to be combined
Thing, can be with two anti-generation specific bindings in MSN by the IgG of an anti-capture；Add glucose/Fe2+Solution, makes the Portugal on MSN
Grape carbohydrate oxidase is catalyzed breakdown of glucose, produces H2O2By the Fe in solution2+It is oxidized to Fe3+, Phen and residual F e2+Formed
Orange red complex compound (Fe (phen)3)2+；It is 490nm to arrange ELIASA Detection wavelength, and each hole absorbance of ELISA Plate is examined
Survey, absorbance is in inverse ratio with the concentration of IgG, so as to realize the detection of IgG solution concentrations.And in traditional ELISA method, by
In signaling molecule, enzyme number of links not enough, generally causes detection sensitivity and test limit limited.And synthesize in the present invention
There is abundant-NH on mesoporous silicon sphere surface2, can be covalently attached with up to a hundred enzyme molecules so as to improving MSN surface enzymes as carrier
And antibody levels, and then signal is amplified, sensitivity is improved, is reduced test limit.Surveyed by adding variable concentrations IgG Jing ELIASAs
Calibration curve is obtained after amount process, the calibration curve is straight line (such as Fig. 4), and the bigger ELIASA of IgG concentration measures OD values more
It is little；Add unknown concentration IgG and form sandwich type structural measurement OD values, the OD values are compared with calibration curve and obtain unknown
The concentration of concentration IgG.Technical scheme utilizes mesoporous NH2A large amount of-NH that Nano particles of silicon dioxide contains2, can tie
A large amount of enzymes and antibody molecule is closed, is promoted signal to amplify, is enhanced sensitivity, reduce test limit.
Compared with prior art, the advantage of technical solution of the present invention is：
(1) with amino mesoporous silicon sphere nano-particle, as one anti-igg of vector construction, (two resist technical scheme
MSN multienzyme complexs) three-layer sandwich type structure, antibody and enzyme carrying capacity is considerably increased, and by specificity knot between IgG
Close, promote signal to amplify, and then improve detection sensitivity, reduce test limit, detection range is wide.
(2) technical scheme can survey the IgG that concentration is 1pg/mL-10 μ g/mL, and IgG in normal human cells
Concentration be 9.5～12.5mg/mL, when suffer from allergic disease,Autoimmune disease, it is various infection andMultiple bone MyelomaDeng when, immunoglobulin (Ig) can increase extremely.Immunoglobulin (Ig) when suffering from the immunodeficiencies such as acquired immunodeficiency syndrome disease
Can reduce extremely, there is enough detection sensitivities by technical scheme to patient's serum after dilution.
(3) technical method detection range width of the invention, without precision instrument and equipment, and may extend to other biographies
Sensor, for detecting to the concentration of different albumen or small molecule.
Description of the drawings
【Fig. 1】For the principle schematic of the detection method of the present invention；
【Fig. 2】For mesoporous NH2The TEM diagrams of Nano particles of silicon dioxide；
【Fig. 3】The colour developing figure of signal is produced for variable concentrations IgG in embodiment 1；
【Fig. 4】For the canonical plotting of variable concentrations IgG correspondences OD values in embodiment 1.
Specific embodiment
For the ease of understanding the present invention, more complete is made to the present invention below in conjunction with Figure of description and preferred embodiment
Face, meticulously describe, but protection scope of the present invention is not limited to embodiment in detail below.
Unless otherwise defined, the implication that all technical terms used hereinafter are generally understood that with those skilled in the art
It is identical.Technical term used herein is intended merely to describe the purpose of specific embodiment, is not intended to limit the present invention
Protection domain.
Unless otherwise specified, the various raw material used in the present invention, reagent, instrument and equipment etc. can pass through city
Field is commercially available or can be prepared by existing method.
Embodiment 1
A kind of embodiment of the detection method of human IgG of the present invention, its principle schematic are as shown in Figure 1.The detection method has
Body is comprised the following steps：
(1) mesoporous NH2The synthesis of Nano particles of silicon dioxide (MSN)：First by 1g cetyl trimethylammonium bromides
(CTAB) it is dissolved in 480mL water and NaOH (2.00mol/L, 3.5mL) mixed liquor, stirring 30min under the conditions of 80 DEG C makes CTAB
Dissolving, rapidly joins tetraethyl orthosilicate (TEOS) (4.75g, 22.83mmol), and the precipitation obtained after reacting 2h is suspended in 200mL
In dry toluene, 12h, gained is refluxed for 80 DEG C under nitrogen protection with 0.17mL aminopropyl triethoxysilanes (APTES)
Product and NH4NO30.6% 60 DEG C of backflow 2h of ethanol solution, obtain carrier NH2—MSN。
(2) to NH2MSN carries out modification two and resists and enzyme：50μL NH2During MSN solution adds 2950 μ L, 25% glutaraldehyde
3h is reacted under room temperature, and centrifuge washing removes unreacted glutaraldehyde, then is added glucose oxidase (GOX) anti-with two simultaneously,
(wherein m bis- resists：M GOX=1:75) to react at 4 DEG C overnight, unreacted enzyme and antibody are removed in next day centrifugation, obtain two
Anti- MSN enzymes.
(3) bed board one is anti-and closes：The one anti-solution of 100 μ L, 10 μ g/mL is added at 4 DEG C of orifice plate overnight, is made with PBST
For cleaning solution board-washing 2 times, 150 μ L, 1M ethanolamine solutions are added to be closed per hole, board-washing 2 times after 2h obtain modified one and resist
ELISA Plate.
(4) variable concentrations human IgG specific binding：By concentration be 1pg/mL, 5pg/mL, 10pg/mL, 100pg/mL,
1ng/mL, 10ng/mL, 100ng/mL, 1 μ g/mL, the human IgG solution of 10 μ g/mL are separately added in ELISA Plate plate hole, 37 DEG C of water
Bath reaction 4h, the IgG that PBST board-washings are removed on uncombined for 3 times, obtain combining the ELISA Plate hole of variable concentrations IgG.
(5) IgG and MSN specifically binds：The two anti-MSN- enzyme solutions that 100 μ L dilute 4 times are added in ELISA Plate,
4h is reacted under 37 DEG C of water bath conditions, with the NaAc_HAc buffer solution board-washing 3 times of 10mM, remove not reacted two anti-
MSN- enzymes, obtain an anti-igg (two anti-MSN enzymes) three-layer sandwich type structure.
(6) glucose/Fe2+Solution is reacted with MSN：Add in the ELISA Plate hole for forming three-layer sandwich type structure and contain
Fe2+Glucose solution reacted, wherein solution ferrous ion concentration be 0.15mM, concentration of glucose be 16.8mg/mL, 37
DEG C water-bath 10min.
(7) Phen colour developing：20 μ L, 15mM Phens solution are added to develop the color in ELISA Plate, with residual F e2+Form orange
Red complex, surveys the OD values that absorbance obtains each hole with ELIASA at the 490nm wavelength, then by the OD values in each hole with it is corresponding
IgG concentration make calibration curve, obtain measure IgG concentration calibration curve.
(8) measurement of actual sample：The serum solution of 5pg/mL, 500pg/mL, 5ng/mL, 50ng/mL IgG is taken,
Addition is modified with an anti-ELISA Plate, has reacted board-washing, and the two anti-MSN- enzyme solutions that 100 μ L dilute 4 times are added enzyme then
In target, 4h is reacted under 37 DEG C of water bath conditions, with the NaAc_HAc buffer solution board-washing 3 times of 10mM, remove not reacted
MSN, obtain an anti-igg (two anti-MSN enzymes) three-layer sandwich type structure.Add glucose/Fe2+Solution, 37 DEG C of bars
10min is reacted under part, adds 20 μ L, 15mM Phens solution to develop the color, with residual F e in ELISA Plate2+Form orange red complexing
Thing, at 490nm wavelength is surveyed absorbance with ELIASA and obtains OD values, compared with calibration curve and obtain measuring concentration value, measured dense
Angle value is recycled rate for 96%～103% with the contrast of spiked levels value.
(9) glucose/Fe2+Solution is reacted with MSN:Add in the ELISA Plate hole for forming three-layer sandwich type structure and contain
Fe2+Glucose solution reacted, wherein solution ferrous ion concentration be 0.15mM, concentration of glucose be 16.8mg/mL, 37
DEG C water-bath 10min.
(10) Phen colour developing：20 μ L, 15mM Phens solution are added to develop the color in ELISA Plate hole, with residual F e2+Shape
Into orange red complex compound, the OD values that absorbance obtains each hole are surveyed at 490nm wavelength, then the OD values and calibration curve are carried out
Control, that is, measure the concentration of the IgG of the unknown concentration, the OD that the concentration of the IgG to be measured is measured in being calibration curve
Corresponding abscissa concentration value.
The preferred embodiments of the present invention are the foregoing is only, the present invention is not limited to, for the skill of this area
For art personnel, the present invention can have various modifications and variations.It is all within the spirit and principles in the present invention, made any repair
Change, equivalent, improvement etc., should be included within the scope of the present invention.
Claims (9)
1. a kind of detection method of IgG concentration, it is characterised in that：Comprise the following steps：
1) in mesoporous NH2Modify glucose oxidase and two anti-Ab on Nano particles of silicon dioxide carrier simultaneously2, obtain two
Anti- MSN multienzyme complexs；
2) by an anti-Ab1Modification is in each hole of ELISA Plate, then ELISA Plate is closed with ethanolamine solutions, obtains an anti-Ab1
The ELISA Plate of modification；
3) a series of standard IgG solution for taking variable concentrations is added separately to described one anti-Ab1Enter in each hole of the ELISA Plate of modification
A row IgG and anti-Ab1Between specific binding reaction, obtain combining the ELISA Plate of IgG；
4) adding described two anti-MSN multienzyme complexs to each hole of the ELISA Plate for combining IgG carries out IgG and two anti-Ab2
Between specific binding reaction, obtain the enzyme containing an anti-igg (two anti-MSN multienzyme complexs) three-layer sandwich type structure
Target；
5) add into each hole of the ELISA Plate containing an anti-igg (two anti-MSN multienzyme complexs) three-layer sandwich type structure
Glucose/Fe2+After mixed solution is reacted, Phen colour developing is added, and detects the absorbance of solution in each hole；
6) calibration curve is set up with corresponding standard IgG solution concentration according to solution absorbance value in each hole for obtaining of detection；
7) take IgG solution to be measured replace standard IgG solution by 3), 4) He 5) the step of operated, detect and obtain absorbance,
Establishing criteria curve, obtains final product the concentration of IgG solution to be measured.
2. the detection method of IgG concentration according to claim 1, it is characterised in that：In mesoporous NH2Silica nanometer
Add glutaraldehyde solution to react in particle carrier solution at room temperature, add glucose oxidase and two anti-Ab2, at 3～5 DEG C
At a temperature of reacted, obtain final product two anti-MSN multienzyme complexs.
3. the detection method of IgG concentration according to claim 1, it is characterised in that：First by an anti-Ab1Solution is added to enzyme
Modified at a temperature of 3～5 DEG C in each hole of target, then ethanolamine solutions are added to each hole of ELISA Plate at room temperature
Closed, obtained final product an anti-Ab1The ELISA Plate of modification.
4. the detection method of the IgG concentration according to any one of claims 1 to 3, it is characterised in that：Two described anti-Ab2For
Goat anti-human IgG antibodies；A described anti-Ab1For mouse anti human IgG antibody.
5. the detection method of IgG concentration according to claim 3, it is characterised in that：The mesoporous NH2Silica is received
Rice corpuscles is prepared via a method which to obtain：After cetyl trimethylammonium bromide is dissolved in sodium hydroxide solution, just add
Silester is hydrolyzed reaction, obtains Nano particles of silicon dioxide；The Nano particles of silicon dioxide is suspended in toluene
Afterwards, with aminopropyl triethoxysilane under nitrogen protection, 11～12h is reacted at a temperature of 75～80 DEG C, react products therefrom
With NH4NO3Ethanol solution react 2～3h at a temperature of 55～60 DEG C, obtain final product.
6. the detection method of IgG concentration according to claim 1, it is characterised in that：Or 4) 3) specific binding in is anti-
The condition answered is：Temperature is 36～37 DEG C, and the time is 3～5h.
7. the detection method of IgG concentration according to claim 1, it is characterised in that：Concentration is respectively into 1pg/mL, 5pg/
Series of standards IgG of mL, 10pg/mL, 100pg/mL, 1ng/mL, 10ng/mL, 100ng/mL, 1 μ g/mL and 10 μ g/mL is molten
Liquid is separately added into an anti-Ab1In each hole of the ELISA Plate of modification, specific binding reaction is carried out.
8. the detection method of IgG concentration according to claim 1, it is characterised in that：Described glucose/Fe2+Mixing is molten
Liquid ferrous ions concentration is 0.15～0.16mM, concentration of glucose is 16～17mg/mL.
9. the detection method of class IgG concentration according to claim 1, it is characterised in that：The Detection wavelength of absorbance is
490nm。
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Cited By (1)
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| CN108362872A (en) * | 2018-01-10 | 2018-08-03 | 浙江工商大学 | A kind of quantitative detecting method of the white diarrhea and avian infectious bronchitis nephritis virus of non-diagnostic purpose |
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Application publication date: 20170322 |