CN105137070B - The methods and applications of 1 high-density lipoprotein of β before improvement detected through gel electrophoresis serum - Google Patents
The methods and applications of 1 high-density lipoprotein of β before improvement detected through gel electrophoresis serum Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/24—Extraction; Separation; Purification by electrochemical means
- C07K1/26—Electrophoresis
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Abstract
The present invention relates to a kind of methods using nonlinear gradient gel electrophoresis accurate separation and 1 HDL of quantitative analysis serum pre β.The invention has the advantages that：The preparation process that the non-linear concentration gradient of the present invention is independently layered is simple and practicable, does not need Special Mixed device；Implementing electrophoresis only needs≤3.5h；Quantitative approach is not necessarily to radioactive substance.The nonlinear gradient gel electrophoresis that the present invention establishes can precisely detect 1 HDL of pre β, have apparent superiority in performance, and be currently the only feasible method.
Description
Technical field
The present invention relates to biotechnologies, specifically, being 1 high density lipoprotein levels of β before improvement detected through gel electrophoresis serum
White methods and applications.
Background technology
1 high-density lipoprotein of β (pre β 1-HDL) is a kind of HDL of new life before serum, is sent out during reverse cholesterol transport
Critical function is waved, has the function of antiatherosclerosis.But it is puzzling, clinical data shows pre β 1-HDL
Content in patients with coronary heart disease significantly increases, rather than negatively correlated relationship.The key of problem is methodology existing defects,
The gradient gel electrophoresis (GGE) applied in the past is as a result to cannot distinguish between free apolipoprotein based on linear concentration gradient
Apo A1, therefore cannot accurately detect pre β 1-HDL.
Previous linear GGE is applied to two-dimensional gel electrophoresis, and combined immunization engram technology detects pre β 1-HDL.U.S. boss
The method that the Asztalos of Tufts universities is established is published in 1993, widely available later.Through Boston Heart
The exploitation of Diagnostics companies is used for Boston Heart HDLCore technology.This technology answers over more than 20 years
With bringing economic benefit, while understanding of this field academia to pre β 1-HDL is influenced, so that as the warp generally acknowledged in the industry
Allusion quotation.However, the pre β 1-HDL of Miyazaki report bidimensional electrophoretic techniques detections in 2014 are substantially that a kind of free Apo A1 are mono-
Body.It is clear that because free Apo A1 cannot be distinguished, there are critical defects for this technology, for a long time for pre β 1-
The understanding of HDL always exists conceptual confusion, and the content for leading to be mistakenly considered the pre β 1-HDL with protective effect is being preced with
It is significantly increased instead in cardiaopath.In addition, the gel (such as 2%-36%) of linear gradient needs special mixing in preparation
Device, stability are also not so good as the setting of nonlinear gradient.Implementing electrophoresis time often will be for 24 hours.Quantitative approach application125I needs
Stringent radioactivity experiment condition.It can be seen that complex procedures, operation takes, and can not avoid radiation sex chromosome mosaicism, becomes the method
The shortcomings that being difficult to overcome.With reference to：http://www.bostonheartdiagnostics.com/science_portfolio_
map_test.php。
Invention content
The purpose of the present invention is being directed to deficiency in the prior art, nonlinear gradient gel and/or sudan black B stain are provided
Application of the liquid in accurate separation and quantitative analysis serum pre β 1-HDL.
Second object of the present invention is to provide a kind of method of accurate separation and quantitative analysis serum pre β 1-HDL.
To achieve the above object, the technical solution adopted by the present invention is that：Nonlinear gradient gel and/or sudan black B stain
Application of the liquid in accurate separation and quantitative analysis serum pre β 1-HDL, the nonlinear gradient gel is by three sections of concentration
It is followed successively by 3.0%, 3.6% and 7.0% polyacrylamide gel to collectively constitute, Sudan black B in the sudan black B stain liquid
A concentration of 0.5-1.0w/v%, the volume ratio of solvent isopropanol and ethylene glycol is 4:1.
The nonlinear gradient gel is respectively as one-dimensional nonlinear gradient tubulose gel rubber system and two-dimension non linearity ladder
Spend the electrophoretic medium of slab gel system separation serum pre β 1-HDL, the one-dimensional nonlinear gradient tubulose gel rubber system point
Serum pre β 1-HDL prestained from Sudan black B.
To realize above-mentioned second purpose, the technical solution adopted by the present invention is that：A kind of precisely separation and quantitative analysis blood
The method of clear pre β 1-HDL, using the accurate separation of nonlinear gradient gel and quantitative analysis serum pre β 1-HDL, wherein non-thread
Property gradient gel is to be followed successively by 3.0%, 3.6% and 7.0% polyacrylamide gel by three sections of concentration to collectively constitute.
The nonlinear gradient gel is respectively as one-dimensional nonlinear gradient tubulose gel rubber system and two-dimension non linearity ladder
Spend the electrophoretic medium of slab gel system separation serum pre β 1-HDL.
It is described using the prestained serum pre β 1-HDL of one-dimensional nonlinear gradient tubulose gel rubber system separation Sudan black B
The volume ratio of a concentration of 0.5-1.0w/v% of Sudan black B in sudan black B stain liquid, solvent isopropanol and ethylene glycol is 4:1.
Three sections of concentration are followed successively by 3.0%, 3.6% and 7.0% polyacrylamide gel as separation gel, corresponding
Glue-line height be respectively 7mm, 40mm and 45mm, error range is ± 3mm, and the total height of three layer separation glue is 82mm, error
Range ± 5mm.
Specification of the nonlinear gradient gel in one-dimensional tubulose gel rubber system be internal diameter 6-8mm, outer diameter 8-10mm,
Specification of the nonlinear gradient gel in two-dimensional flat plate gel rubber system is that thickness is 2-3mm, width 60-80mm.
The one-dimensional nonlinear gradient tubulose gel rubber system detaches the prestained serum pre β 1-HDL of Sudan black B, electrophoresis
Buffer solution is 5mM Tris and 38.4mM glycine, deposition condition 100V, 2.5h, by migration distance origin 65- after electrophoresis
The dyeing zone isolated at 75mm is defined as pre β 1-HDL；The two-dimension non linearity gradient plate gel rubber system detaches serum
Pre β 1-HDL, electrophoretic buffer are 5mM Tris and 38.4mM glycine, deposition condition 100V, 3.5h, by pre β after electrophoresis
Trace zone is defined as pre β 1-HDL and free Apo A1 polymers at migration distance origin 65-75mm on swimming lane, migration away from
Free Apo A1 monomers are defined as from trace zone at origin 80-90mm.
The two-dimension non linearity gradient plate gel rubber system detaches serum pre β 1-HDL, is added in sample application zone before electrophoresis
A small amount of phenol red solution is as electrophoresis indicator.
The one-dimensional nonlinear gradient tubulose gel rubber system detaches the prestained serum pre β 1-HDL of Sudan black B, uses
Densitometric scan is quantitative, is unified reference with Blank gel scan values, calculates pre β 1-HDL and accounts for the percentage of blood fat totality and contains
Amount；Two-dimension non linearity gradient plate gel rubber system detaches serum pre β 1-HDL, free Apo A1 monomers and polymer, uses
Immunoblotting, chemiluminescence and Film technique progress are qualitative, in same a collection of internal ratio compared with its relative amount.
The invention has the advantages that：
1, the method that the present invention establishes one-dimensional nonlinear GGE, is respectively 3.0%, 3.6% and using three sections of concentration
For 7.0% polyacrylamide gel as electrophoretic medium, it is a kind of special clearly to isolate the serum of sudan black B stain
HDL subclass, i.e. pre β 1-HDL；
2, the method that the present invention has also set up two-dimension non linearity GGE, can be very clearly by pre β 1-HDL and free
Apo A1 are separated；
3, the preparation process that non-linear concentration gradient of the invention is independently layered is simple and practicable, does not need Special Mixed dress
It sets；Implementing electrophoresis only needs≤3.5h；Quantitative approach is not necessarily to radioactive substance.The non-linear GGE that the present invention establishes can be examined precisely
Pre β 1-HDL are surveyed, there is apparent superiority in performance, and be currently the only feasible method.
Description of the drawings
Fig. 1 one-dimensional nonlinear GGE- lipophilicitys SBB dyes collection of illustrative plates.A:The schematic diagram of one-dimensional nonlinear gradient tubulose gel.
B:Repeated experiment result.The electrophoretic migration of pre β 1-HDL is presented clearly SBB and dyes zone apart from origin 70mm.Identical blood
The coefficient of variation that sample detects pre β 1-HDL is 2.31%, illustrates that the method has good repeatability.C:Clinical blood sample one-dimensional nonlinear
GGE- lipophilicitys SBB dyes collection of illustrative plates.Pre β 1-HDL rich contents in umbilical cord blood, and in acute myocardial infarction patient and
Content in CETP variation persons is less than health adult.D:Electrophoresis scanning spectra.
Fig. 2 two-dimension non linearity GGE- immunoblotting collection of illustrative plates.A:Two-dimension non linearity gradient plate gel prepares schematic diagram.
B:Immunoblotting collection of illustrative plates after two dimensional electrophoresis containing Apo A1 particles in serum.The electrophoretic migration about 70mm on pre β swimming lanes
Zone is pre β 1-HDL (including Apo A1 polymers), and the zone of electrophoretic migration about 85mm is the Apo A1 monomers to dissociate, clearly
Clear visible isolated trace zone.The collection of illustrative plates performance of newborn's blood sample is relatively grown up complicated.C:Two-dimension non linearity GGE- immunoblottings
Schematic diagram.Arrow indicates one to (1D) and two to the electrophoresis direction of (2D).
The experimental demonstration of Fig. 3 .pre β 1-HDL：Grain density and external development.A:Ultracentrifugation detaches serum hdl.B:From
Different densities range samples after ultracentrifugation carry out one-dimensional nonlinear GGE after SBB is dyed.Pre β 1-HDL are appeared in
In the density fluid of 1.210g/ml, electrophoretic migration 70mm.C:Two-dimension non linearity GGE- immunoblotting collection of illustrative plates, pre β 1-HDL equally go out
In the density fluid of present 1.210g/ml, migration position is equally at 70mm.D:DTNB body outer suppressioning experiments.Normal serum is external
After 37 DEG C of water-bath 6h, the content of pre β 1-HDL gradually decreases, and the development of this particle can be completely inhibited by DTNB.Because
DTNB is the inhibitor of lecithin cholesterol acyltransferase, and newborn pre β 1-HDL can effectively be inhibited to spread out to bulky grain HDL
Become, so result further illustrates, this electrophoretic migration 70mm and the isolated Zone electophoresis band essence that can be dyed by lipophilicity SBB
On be pre β 1-HDL.
The experimental demonstration of Fig. 4 .pre β 1-HDL：Granular size and chemical component.A:The electron microscopic collection of illustrative plates of serum hdl.Arrow
Signified head is pre β 1-HDL.B:The Apo A1 of serum hdl and the relative weight ratio of cholesterol, wherein in the ratio of pre β 1-HDL
Highest.
The experimental demonstration of Fig. 5 .pre β 1-HDL：Clinical case compares collection of illustrative plates.A-B:Acute myocardial infarction patient and normal healthy controls blood
The non-linear GGE- immunoblottings collection of illustrative plates of clear exemplary two dimensional.The content of acute myocardial infarction patient pre β 1-HDL is apparent compared with normal healthy controls
It reduces.Because pre β 1-HDL have antiatherosclerosis protective effect, such result natural.C:Tangier
Patient and the one-dimensional nonlinear GGE- lipophilicitys SBB of normal healthy controls serum dye collection of illustrative plates.Due to ABCA1 gene defects,
Tangier patients cannot generate pre β 1-HDL, cause to lack completely in collection of illustrative plates.D:Tangier patient's serum two dimensions are non-
Linear GGE- immunoblotting collection of illustrative plates.The high-visible free Apo A1 monomers at migration position 85mm, while in migration position
Still visible micro trace zone at 70mm, although position is identical with pre β 1-HDL, substantial not instead of pre β 1-HDL,
Free Apo A1 polymers.D:Free Apo A1 standard items (0.2 μ g, Sigma) two-dimension non linearity GGE- immunoblotting collection of illustrative plates.
Trace zone is concentrated mainly at migration position 85mm, this is the Apo A1 monomers to dissociate, but at migration position 70mm still
Right visible a small amount of trace zone, this is Apo A1 polymers.It can so explain, the Apo A1 of Tangier patient's serum are more
Aggressiveness can appear in two-dimentional GGE- immunoblottings collection of illustrative plates, but because this part is free of lipid, in one-dimensional nonlinear GGE-
Lipophilicity SBB dyeing collection of illustrative plates can not be then detected.Therefore, only one-dimensional nonlinear GGE could be used to precisely detect serum pre
β1-HDL。
The two-dimensional linear GGE- immunoblotting collection of illustrative plates of the China Fig. 6 application.A:One-dimensional agarose electrophoresis；B:Two-dimensional linear
GGE。
The linear GGE- immunoblottings collection of illustrative plates of Fig. 7 conventional two-dimensionals.
Specific implementation mode
It elaborates below in conjunction with the accompanying drawings to specific implementation mode provided by the invention.
Embodiment 1
One, one-dimensional nonlinear GGE
1, reagent
(1) storage liquid A：Acrylamide (Acr) 9.60g and methylene diacrylamide (Bis) 0.25g is dissolved in deionization steaming
Distilled water (dH2O) to 100ml, 4 DEG C are stored in brown bottle, term of validity March.
(2) storage liquid B：Trishydroxymethylaminomethane (Tris) 18.3g and 1N hydrochloric acid 24ml, are dissolved in dH2O to 100ml is used
Brown bottle is stored in 4 DEG C, term of validity March.
(3) storage liquid C：Acr 19.6g and Bis 0.4g, are dissolved in dH2O to 100ml is stored in 4 DEG C, the term of validity with brown bottle
March.
(4) storage liquid D：Tris 6.06g and disodium ethylene diamine tetraacetate (EDTA-Na2) 1.17g, it is dissolved in dH2O is extremely
100ml is stored in 4 DEG C with brown bottle, term of validity March.
(5) initiator：Ammonium persulfate (APS) 1.0g is dissolved in dH2O to 10ml is stored in -80 DEG C after packing.
(6) dyeing liquor：Sudan black (SBB) 0.125g is dissolved in isopropanol 20ml and ethylene glycol 5ml, and 37 DEG C of water-baths are stayed overnight, room
It is stored in the brown bottle of sealing under temperature, term of validity June.
(7) buffer solution H：Tris 6.0g and glycine 28.8g are dissolved in dH2O to 1000ml, is stored in 4 DEG C, and when use is used
dH2O dilutes 10 times.
2, sample collection limosis vein blood isolates serum in 3 hours.It takes 100 μ l of serum to add 10 μ l of SBB dyeing liquors, shakes
37 DEG C of water-bath 45min of mixing postposition are swung, 3000rpm, 10min are then centrifuged for.
3, the specification of tubulose gel glass pipe is internal diameter 6mm, outer diameter 8mm, length 10cm.Sealed glass tube one end, by it
It is vertically fixed on glue holder.Three layers of various concentration that height is respectively 45mm, 40mm and 7mm are gradually prepared according to table 1
The gel of (7.0%, 3.6% and 3.0%).Every layer of gel gently injects 100 μ l dH immediately after encapsulating2O covers its surface,
Keep gel surface polymerisation smooth.Remove water layer, lower layer of coagulant liquid of Reperfu- sion after standing 30min gel polymerisations.
4, the gel tube prepared is vertically placed in disc electrophoresis slot (III 27A of DYY-, 61 electrophoresis apparatus of Beijing by pipe gel electrophoresis
Device factory).Electrophoretic buffer (positive slot 600ml, cathode slot 400ml) is injected into positive and negative anodes electrophoresis tank respectively.By pre-dyed serum
(50 μ l/ pipes) is slowly added into gel tube, is covered in the surface of separation gel.Connection regulated power supply (Model 1000/500,
Bio-Rad), electrophoresis 100V, 2.5h.
4, scan and quantify electrophoresis terminate to remove gel tube immediately, with two waveband flying spot scanner (CS-9000,
Shimadzu) gel tube is scanned and is imaged respectively with optical scanner.The a length of 604nm of flying-spot scanner monochromatic optical wave.It sweeps
Along the center line of gel tube when retouching, carried out from clear area to the direction of origin.With Blank gel to unify reference when analysis, with adjacent
Inflection point between electrophoresis peak calculates the percentage composition that area under each electrophoresis peak accounts for serum lipoprotein entirety as segmentation marker.
The gel solution of 1. one-dimensional nonlinear GGE of table configures
T:Concentration；C:The degree of cross linking.dH2O:Deionized-distilled water；TEMED:Tetramethylethylenediamine；APS:Ammonium persulfate is molten
Liquid.
Two, two-dimension non linearity GGE
1, glue reagent is same as above.Agarose gel electrophoresis buffer solution is with 25mM Tris-Tricine (pH 8.6).Two-dimentional electricity
Swimming buffer solution is 5mM Tris and 38.4mM glycine (pH 8.4).0.3% glutaraldehyde fixer.1 anti-Apo A1 antibody is purchased from
Abcam.2 anti-igg are purchased from Novus Biologicals.
2, slab gel glass plate specification 10cm × 10cm, thickness 2mm, gasket specification 10cm × 1cm, thickness 2mm.Gasket
It is placed between two pieces of glass plate both sides, is vertically fixed on glue holder, bottom end seal.According to one-dimensional nonlinear gradient gel
Encapsulating step successively prepares slab gel.
3,4 μ l serum are carried out 0.75% agarose gel electrophoresis separation (100V, 1.5h) by plate gel electrophoresis, SBB dyeing
4 μ l of serum are as sync mark.Agarose gel electrophoresis slot comes from GT Cell apparatus(Bio-
Rad).The position migrated from β to α along swimming lane after electrophoresis cuts out the adhesive tape (thickness 2mm) of 40mm long, and adhesive tape is moved into tablet
Two pieces of adhesive tape can be added in the upper surface of gel simultaneously.Slab gel is moved into (XCell SureLock in two dimensional electrophoresis slotTM
Mini-Cell apparatus, Life Technologies), two dimensional electrophoresis buffer solution is added, powers on, electrophoresis 100V,
3.5h.A little phenol red solution is added between adhesive tape as electrophoresis indicator, electrophoresis is terminated after phenol red whole swims out gel,
Electrophoresis time can be adjusted according to lipoprotein migration position.It, can be in the glass plate of contact to prevent buffer solution from overflowing positive electrophoresis tank
It is upper to apply a little vaseline isolation water layer.It takes out slab gel after two dimensional electrophoresis, carries out transferring film electrophoresis 40V, 1.5h, lipoprotein
It is transferred on vinegar fibre film.
Three, immunoblotting
After two-dimension non linearity GGE and transferring film electrophoresis, the lipoprotein on vinegar fibre film is fixed with 0.3% glutaraldehyde solution
10min carries out immunoblotting after clear water rinsing.5% degreasing milk-TBST and 5% balf serum albumin-TBST is successively added
Solution closes off 8h or so in cold house.Then 1 anti-Apo A1 antibody (cold room overnight) is added.It is used on room temperature shaker
Unbonded 1 anti-of TBST solution washes aways.It is subsequently added into 2 anti-igg (1h at room temperature).2 that TBST solution washes aways are not associated with are used again
It is anti-.Chemiluminescence reaction is finally carried out, and carries out film imaging in darkroom.
Four, ultracentrifugation
Human serum 6ml is acquired, is 1.30g/ml (potassium bromide 2.93g is added) using formula tune density.It is super to be added to 28ml
The bottom of fast centrifuge tube, upper layer are covered with the density fluid of 1.063g/ml.Ultracentrifuge model Beckman L8-M, rotor are
50.2Ti, centrifugal condition are 40K rpm, 12h, 10 DEG C.It is slowly inhaled successively since bottom with long syringe needle after ultracentrifugation
Go out the separating liquid of isometric 1ml different densities range.800 μ l are quantified for the different densities liquid after separation, directly with accurate day
Scale claims its weight, calculates density.Finally use super filter tube (30KD, Millipore) by the volume of different densities range separating liquid
It is concentrated to 500 μ l.Then it samples and carries out a peacekeeping two dimension GGE respectively.
Five, electron microscopic
After one-dimensional GGE, the Zone electophoresis band of serum hdl subclass is cut and shredded from separating gel respectively, is put into
PBS solution containing antibiotic free diffusing 3-5 days in cold house.HDL solution is collected by super filter tube dialysis and concentration.
0.4% sodium bicarbonate solution of dialyzate.Treated 10 μ l of HDL solution and 2% isometric Salkowski's solution mixing, draw
It is a little to carry out negative staining Transmission electron microscopy.Transmission electron microscope is daily output JEM-1200EX, and electronic intensity 80KV is put
Big multiple 100K.Electronic Speculum imaging after with image analysis software Image-Pro Plus 6.0 (Media Cybernetics,
Inc.USA grain size analysis) is carried out to HDL particles, measures its granular size.
Six, biochemistry detection
Various HDL solution are collected, with the detection of daily output 911 automatic clinical chemistry analyzers of Hitachi wherein cholesterol and Apo
The content of A1 calculates their content ratio.The detection method of cholesterol uses enzyme process, and the detection method of Apo A1 is using immune
Turbidimetry, detection reagent come from Roche Diagnostics.
Seven, result and conclusion
The blood lipid level and lipoprotein of 2. healthy newborn of table, adult and acute myocardial infarction patient are composed
With<Health adult compares within 60 years old,With>Health adult compares within 60 years old,#P<0.05,*P
<0.01,△P<0.001.TG:Triglycerides；TC:Total cholesterol；LDL-C:Low density lipoprotein cholesterol；HDL-C:High density
Lipoprotein cholesterol；IDL:Intermediated-density lipoprotein；Lp(a):Lipoprotein (a).
Experimental diagrams show that the one-dimensional nonlinear GGE that the present invention establishes can be clearly from the prestained serum of Sudan black B
In isolate a kind of special HDL subclass, i.e. pre β 1-HDL, also known as newborn HDL.Meanwhile the present invention is using newly-established
Two-dimension non linearity GGE- immunoblot assays, the pre β 1- for finding this HDL subclass and initially being defined by the laboratories Fielding
HDL has consistent feature.Compare with other HDL subclass, pre β 1-HDL have high grain density (>1.20g/ml), and
Wherein the content of aPoA po A1 and cholesterol compares highest.Experiment in vitro shows that 2- nitrobenzoic acids (DTNB) can be complete
It is complete that pre β 1-HDL is inhibited to develop to bulky grain HDL.Pre β 1-HDL rich contents in umbilical cord blood, and in acute myocardial infarction
Content in patient and CETP variation persons is less than health adult, can not then be detected in Tangier patients.Result of study table
Bright, bidimensional gel electrophoresis-Western blot cannot distinguish between pre β 1-HDL and free Apo A1, therefore method detection pre β 1-
HDL is inaccurate.The non-linear GGE technologies that only we establish can precisely detect pre β 1-HDL, have good application
Foreground.
China is in 1999 by Huaxi Medical Univ's apolipoprotein research department Wu Xin at that time is big and Fu Mingde etc. uses agar
Sugar and gradient polyacrylamide gels dielectrophoresis, western blot test and spot scanning analysis, establish serum hdl subclass
Detection method, Fig. 6.This is the domestic relatively early research for carrying out detection pre β 1-HDL, had successively much apply document report later.
The method is using the linear GGE of 2%-30% as two dimensional electrophoresis, and similar with Asztalos methods, the two divides pre β 1-HDL
Distinguish that effect is similar.Although the method improves to some extent in quantitative approach, but still that apply is linear GGE, equally exists technology and lacks
It falls into, pre β 1-HDL and free Apo A1 cannot be distinguished.Regrettably, this problem is not realized for a long time.
Fig. 7 is that linear GGE is applied to two-dimensional gel electrophoresis, and combined immunization engram technology detects pre β 1-HDL.The method is by U.S.
The Asztalos of Boston Tufts universities of state is published in 1993, widely available later.Through Boston Heart
The exploitation of Diagnostics companies is used for Boston Heart HDLCore pre β 1-HDL understanding so that as this
Classics recognized within the industry.However, the pre β 1-HDL of Miyazaki report bidimensional electrophoretic techniques detections in 2014 are substantially a kind of
Free Apo A1 monomers.It is clear that because free Apo A1 cannot be distinguished, there are critical defects for this technology, for a long time
Since conceptual confusion is always existed for the understanding of pre β 1-HDL, lead to be mistakenly considered the pre β with protective effect
The content of 1-HDL significantly increases instead in patients with coronary heart disease.In addition, the gel (such as 2%-36%) of linear gradient is in preparation
Special mixing arrangement, stability is needed also to be not so good as the setting of nonlinear gradient.Implementing electrophoresis time often will be for 24 hours.Quantitative square
Method application125I needs stringent radioactivity experiment condition.It can be seen that complex procedures, operation takes, and can not avoid radiating
Sex chromosome mosaicism, becomes this and is considered as classical method and be difficult to the technology restriction overcome.
In contrast, we establish non-linear GGE technologies (Fig. 1-2) can then cleanly separate out pre β 1-HDL and
Free Apo A1.Clinical data shows that the content of coronary heart disease acute myocardial infarction patients serum pre β 1-HDL significantly reduces, this makes
The protective effect of pre β 1-HDL is easier to understand with its clinical correlation.And the preparation that non-linear concentration gradient is independently layered
Journey is simple and practicable, does not need Special Mixed device；Implementing electrophoresis only needs≤3.5h；Quantitative approach is not necessarily to radioactive substance.Therefore,
The non-linear GGE that we establish has apparent technical advantage, and is that precisely the unique of detection serum pre β 1-HDL can at present
Capable method.
The Technical comparing of the non-linear GGE that 3. present invention of table establishes and classical linear GGE
In conclusion the present invention establishes the method (Fig. 1) of one-dimensional nonlinear GGE.The method is respectively using three sections of concentration
3.0%, 3.6% and 7.0% polyacrylamide gel clearly detaches the serum of sudan black B stain as electrophoretic medium
Go out a kind of special HDL subclass, i.e. pre β 1-HDL, also known as newborn HDL.And then we are set using same three kinds of concentration
It sets, original tubulose gel is changed to slab gel (Fig. 2), and this plate glue is applied in bidimensional gel electrophoresis system, it is right
The HDL of agarose gel electrophoresis separation carries out two dimensional electrophoresis and detaches again, in conjunction with immunoblot assay, as a result, it has been found that this HDL is sub-
The Electrophoretic Characterization of class and the pre β 1-HDL (CastroG.R.and Fielding C.J., 1988) initially defined are completely the same.
Then this special HDL subclass designations are by convention pre β 1-HDL by we.Meanwhile the two-dimension non linearity of our foundation
GGE technologies can clearly separate pre β 1-HDL and free Apo A1 very much.Clinical data shows that coronary heart disease is acute
The content of heart infarction patients serum pre β 1-HDL significantly reduces, this makes the protective effect of pre β 1-HDL more hold with its clinical correlation
It is readily understood.The preparation process that non-linear concentration gradient is independently layered is simple and practicable, does not need Special Mixed device；Implement electrophoresis only
Need≤3.5h；Quantitative approach is not necessarily to radioactive substance.Therefore, the non-linear GGE that we establish can precisely detect pre β 1-
HDL has apparent superiority in performance, and is currently the only feasible method.
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
Member, under the premise of not departing from the method for the present invention, can also make several improvement and supplement, these are improved and supplement also should be regarded as
Protection scope of the present invention.
Claims (10)
1. the application of nonlinear gradient gel and sudan black B stain liquid in accurate separation and quantitative analysis serum pre β 1-HDL,
The nonlinear gradient gel be followed successively by by three sections of concentration 3.0%, 3.6% and 7.0% polyacrylamide gel it is common
It forms, a concentration of 0.5-1.0w/v% of Sudan black B, the body of solvent isopropanol and ethylene glycol in the sudan black B stain liquid
Product is than being 4:1.
2. application according to claim 1, which is characterized in that the nonlinear gradient gel is respectively as one-dimensional non-thread
Property gradient tubulose gel rubber system and two-dimension non linearity gradient plate gel rubber system separation serum pre β 1-HDL electrophoretic medium, institute
The prestained serum pre β 1-HDL of one-dimensional nonlinear gradient tubulose gel rubber system separation Sudan black B stated.
3. a kind of method of accurate separation and quantitative analysis serum pre β 1-HDL, which is characterized in that use nonlinear gradient gel
With the accurate separation of sudan black B stain liquid and quantitative analysis serum pre β 1-HDL, wherein nonlinear gradient gel is by three sections of concentration
It is followed successively by 3.0%, 3.6% and 7.0% polyacrylamide gel to collectively constitute, Sudan black B in the sudan black B stain liquid
A concentration of 0.5-1.0w/v%, the volume ratio of solvent isopropanol and ethylene glycol is 4:1.
4. according to the method described in claim 3, it is characterized in that, the nonlinear gradient gel is respectively as one-dimensional non-thread
Property gradient tubulose gel rubber system and two-dimension non linearity gradient plate gel rubber system separation serum pre β 1-HDL electrophoretic medium.
5. according to the method described in claim 4, reviving it is characterized in that, being detached using one-dimensional nonlinear gradient tubulose gel rubber system
The red black prestained serum pre β 1-HDL of B, a concentration of 0.5-1.0w/v% of Sudan black B in the sudan black B stain liquid,
The volume ratio of solvent isopropanol and ethylene glycol is 4:1.
6. according to the method described in claim 3, it is characterized in that, three sections of concentration are followed successively by 3.0%, 3.6% and
7.0% polyacrylamide gel is as separation gel, and corresponding glue-line height is respectively 7mm, 40mm and 45mm, and error range is
The total height of ± 3mm, three layer separation glue are 82mm, error range ± 5mm.
7. according to the method described in claim 4, it is characterized in that, the nonlinear gradient gel is in one-dimensional tubulose gelinite
Specification in system is internal diameter 6-8mm, and outer diameter 8-10mm, specification of the nonlinear gradient gel in two-dimensional flat plate gel rubber system is thickness
Degree is 2-3mm, width 60-80mm.
8. according to the method described in claim 4, it is characterized in that, the one-dimensional nonlinear gradient tubulose gel rubber system separation
The prestained serum pre β 1-HDL of Sudan black B, electrophoretic buffer are 5mM Tris and 38.4mM glycine, and deposition condition is
The dyeing zone isolated at migration distance origin 65-75mm is defined as pre β 1-HDL after electrophoresis by 100V, 2.5h；Described two
It ties up nonlinear gradient slab gel system and detaches serum pre β 1-HDL, electrophoretic buffer is 5mM Tris and 38.4mM glycine,
Deposition condition is 100V, 3.5h, by trace zone is defined as pre β at migration distance origin 65-75mm on pre β swimming lanes after electrophoresis
1-HDL and free Apo A1 polymers, it is mono- to be defined as free Apo A1 for trace zone at migration distance origin 80-90mm
Body.
9. according to the method described in claim 8, it is characterized in that, the two-dimension non linearity gradient plate gel rubber system separation
A small amount of phenol red solution is added as electrophoresis indicator in sample application zone before electrophoresis in serum pre β 1-HDL.
10. according to the method described in claim 8, it is characterized in that, the one-dimensional nonlinear gradient tubulose gel rubber system point
Serum pre β 1-HDL prestained from Sudan black B, it is quantitative using densitometric scan, it is unified reference with Blank gel scan values,
Calculate the percentage composition that pre β 1-HDL account for blood fat totality；Two-dimension non linearity gradient plate gel rubber system detaches serum pre β 1-
HDL, free Apo A1 monomers and polymer, it is qualitative using the progress of immunoblotting, chemiluminescence and Film technique, same
Compare its relative amount in a collection of.
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| CN105527449A (en) * | 2015-12-24 | 2016-04-27 | 山东博科生物产业有限公司 | Apolipoprotein A1 immunoturbidimetry detection kit |
| CN108387630B (en) * | 2018-01-29 | 2019-09-03 | 温州科技职业学院 | A kind of improvement SDS- polyacrylamide gel electrophoresis technique |
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