CN105158460A - Electrophoresis flowing type ELISA method based on polyelectrolyte multilayer (PEM) substrate - Google Patents
Electrophoresis flowing type ELISA method based on polyelectrolyte multilayer (PEM) substrate Download PDFInfo
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- CN105158460A CN105158460A CN201510316659.2A CN201510316659A CN105158460A CN 105158460 A CN105158460 A CN 105158460A CN 201510316659 A CN201510316659 A CN 201510316659A CN 105158460 A CN105158460 A CN 105158460A
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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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- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/5302—Apparatus specially adapted for immunological test procedures
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Abstract
The invention discloses an electrophoresis flowing type ELISA method based on a PEM substrate, belonging to the field of biomaterials. The method comprises the following steps: allowing a first antibody to be adsorbed on the surface of a porous membrane and then carrying out blocking agent protein adsorption; carrying out antigen-first antibody reaction under the driving of electrophoresis; carrying out second antibody-antigen reaction and carrying out color development and quantification on the enzyme-labeled second antibody, i.e., after the antigen-first antibody reaction under the driving of electrophoresis, placing the porous membrane substrate having undergone electrophoresis in a second antibody solution, carrying out a reaction, then carrying out cleaning, placing the obtained porous membrane substrate in a substrate solution of second antibody identification enzyme and carrying out color development; and carrying out quantitative analysis on antigen by detecting absorbance. Preferably, an electropositive PDDA/PSS modified cellulose acetate membrane PEMs-CA is used. According to the invention, through modification of PEMs, non-specific adsorption is reduced; electrophoresis driving force enables antigen molecules to be locally concentrated around the first antibody in a short period of time; so rapid and sensitive detection is realized.
Description
Technical field
The invention belongs to technical field of biological material, particularly the preparation of polyelectrolyte multilayer film (polyelectrolytemultilayers:PEMs), the method for the enzyme linked immunosorbent assay (ELISA) under electrophoresis drives.
Background technology
In recent years, since immunization method is established, immunosensor, as emerging biology sensor, is favored with its qualification material high degree of specificity, sensitivity and stability.ELISA is a small amount of protein detection and quantitative test method, is widely used in the fields such as food, industry, environmental monitoring and clinical medicine.There is following problem in the static incubation reaction of ELISA on polystyrene substrate (polystyrene:PS) substrate of routine: the easy sex change of (1) first antibody on PS, and be not easy exposed at substrate surface with antigen reactive position; (2) sealing effect is not good, causes the non-specific adsorption of antigen and second antibody, have impact on the sensitivity of ELISA system; (3) in antigen-antibody reaction process, antigen needs long free diffusing, and long incubation reaction becomes rate-limiting step, causes slowly in conjunction with speed, have impact on sensitivity and the dynamic range of analysis.Much prepare by nano material the research improving ELISA system sensitivity, its complicated operation and high cost although have, cause being difficult to practical application.In addition, Microfabricated chips board group device, micro-array chip, micro-fluidic technologies, but because the drawbacks such as the shortcoming of its surface modification technology, the complicacy of operation can not be widely used in highly sensitive clinical detection.In addition, the local concentration of antigen near first antibody is improved by external force, the while of can carrying highly sensitive, can also Reaction time shorten greatly.Punyadeera [Lieshout, R.M.L.van; Domburg, T.van; Saalmink, M.; Verbeek, R.; Wimberger-Friedl, R.; Dieijen-Visser, M.P.van; Punyadeera, C.Anal.Chem.2009,81,5165 – 5171], Bruening [Dai, J.; Baker, G.L.; Bruening, M.L.Anal.Chem.2009,78,135 – 140] etc. will detect sample flow by pressure mode and carry out antigen-antibody reaction while the perforated membrane fixed through first antibody, effectively improve detection efficiency and sensitivity, but pressure type flow system does not have preliminary screening process for the detection of biased sample (serum, whole blood), causes perforated membrane easily to block, and limits the raising of detection efficiency and sensitivity.For the detection drawback of above ELISA system, therefore, find one and " quick-sensitive-easy " detection ELISA system can seem particularly important.The chemical modification of polymeric membrane and electrophoretic techniques are organically blended in ELISA system, solves the problems such as conventional ELISA system non-specific adsorption is high, reaction efficiency is low, biased sample detection difficult, have great importance.
Summary of the invention
The object of the invention is to solve sample detection low efficiency problem in ELISA system and solve the problem of muting sensitivity further and a kind of method of electrophoresis flow model ELISA is provided, especially a kind of method of polyelectrolyte multilayer film substrate electrophoresis flow model ELISA is provided, to solve Problems existing in background technology.In polyelectrolyte multilayer film substrate electrophoresis flow model ELISA method provided by the present invention, electropositivity PEMs modifies perforated membrane, increases substantially the adsorbance of sealer albumen, effectively suppresses the non-specific adsorption of antigen and second antibody.Secondly electrophoretic techniques is as the type of drive of antigen flow detection in ELISA system, substantially reduces detection time, improves detection efficiency.
Electrophoresis flow model ELISA method provided by the present invention, the technical scheme adopted is:
(1) first antibody is adsorbed onto on perforated membrane substrate;
(2) the perforated membrane substrate adsorbing first antibody carries out sealer protein adsorption again;
(3) electrophoresis drives antigen-first antibody reaction: be fixed in electrophoretic apparatus by the perforated membrane substrate after step (2) first antibody and sealer protein adsorption, in electrophoretic apparatus, antigenic solution (i.e. electrophoresis solution) is injected in perforated membrane substrate side, perforated membrane substrate opposite side injects phosphate buffered solution, if antigen molecule is negative charge, negative electrode is inserted in antigenic solution side, opposite side inserts positive electrode, otherwise then anti-.Electrode making alive, carries out antigen-first antibody reaction.
(4) second antibody-antigen-reactive, colour developing is carried out to enzyme mark second antibody quantitative: after step (3) electrophoresis drives antigen-first antibody reaction, perforated membrane substrate after electrophoresis is put in second antibody solution, react, then clean, gained perforated membrane substrate is put into the substrate solution of second antibody mark enzyme, develop the color; By detecting absorbance, carry out antigen quantify analysis.
Preferred above-mentioned perforated membrane substrate step (1) perforated membrane substrate is the CA perforated membrane that cellulose acetate perforated membrane (CA) or polyelectrolyte multilayer film are modified, the CA perforated membrane that polyelectrolyte multilayer film is modified is the CA perforated membrane utilizing poly-(diallyldimethylammonium chloride) and the cross layered modification of polystyrene sulphuric acid sodium successively, and the outermost layer modified is poly-(diallyldimethylammonium chloride), obtain electropositive PDDA/PSS and modify CA, i.e. PEMs-CA (in preferred PEMs-CA poly-(diallyldimethylammonium chloride) and the cross layered modification of polystyrene sulphuric acid sodium 7 layers), the adsorbance of first antibody and sealer (ovalbumin ovalbumin:OVA) is improved by its surface charge, improve the density (signal) of antigen and antibody response, suppress the non-specific adsorption (noise) of antigen and second antibody, and then increase substantially sensitivity (signal and noise ratio).
Further preferred steps (3) electrophoresis drives electrophoretic voltage and the electrophoresis time of antigen-first antibody reaction, and the voltage swing of electrophoresis determines the translational speed of protein; The length of electrophoresis time affects the efficiency of antigen and first antibody combination and the stability of self-assembled film.Regulate voltage and the time of electrophoresis, improve detection sensitivity.Above-mentioned perforated membrane substrate can choose cellulose acetate (celluloseacetate, CA) perforated membrane as detection solution through substrate; The cellulose acetate membrane that PDDA/PSS modifies is PEMs-CA.
CA and PEMs-CA electrophoretic voltage and time-optimized scope are 15-25V and 2-8min, and preferably CA electrophoretic voltage and time are 15v, 2min further; PEMs-CA electrophoretic voltage and time are 25v, 2min.
In this electrophoresis flow model ELISA system, by orthogonal test, optimization is carried out to second antibody concentration, determine that the optium concentration of the second antibody of CA and PEMs-CA is respectively 0.5,1 μ g/mL.Utilize the quantitation curves of this second antibody Concentration Testing antigen, the detection limit concentration obtaining PEMs-CA is 5ng/mL.While reaching traditional E LISA detection level, detection speed improves 30 times.
The present invention has following beneficial effect:
1) polyelectrolyte multilayer film substrate electrophoresis flow model ELISA method provided by the present invention, efficiently can suppress the PEMs of ELISA non-specific adsorption in initiative electrophoresis environment.
2) polyelectrolyte multilayer film substrate electrophoresis flow model ELISA method provided by the present invention, formulates sensitive, quick, easy electrophoresis flow model ELISA system.The driving flowing of electrophoresis effectively controls the different protein of isoelectric point and flows to the electrode direction of opposite-sign, pass through perforated membrane, so make near antigen localized concentrations to perforated membrane (i.e. first antibody) in the flow process of short time, in few interference environment, complete sensitive, antibody-antigene reaction fast, significantly shorten the reaction time compared with traditional E LISA.
Accompanying drawing explanation
Fig. 1 embodiment of the present invention electrophoretic apparatus schematic diagram used.
The electrophoresis flow model ELISA detection curve of Fig. 2 embodiment 1 and 2.
Embodiment
Below in conjunction with embodiment, the present invention will be further described, but the present invention is not limited to following examples.Fig. 1 is shown in by the electrophoretic apparatus that the embodiment of the present invention adopts.Electrophoretic apparatus is one keep flat the drum structure sent out, and perforated membrane is positioned at and middle is divided into two by drum, and the side of perforated membrane is antigenic solution, and end face is provided with solution inlet port and electrode; Opposite side is PB damping fluid, and end face is provided with solution inlet port and electrode.
Embodiment 1
CA perforated membrane substrate electrophoresis ELISA
1) configuration of solution:
A) configuration of 50mMTris-HCl0.15MNaCl solution: 1.15175gTris-base solid is dissolved in 125mL ultrapure water, get 0.84mLHCl and to be dissolved in 100mL ultrapure water obtaining 0.1MHCl solution, after to be dissolved, HCl solution is added dropwise to till adjusting pH=7.4 in Tris-base solution, be settled to 250mL, weigh 2.208gNaCl solid and add in Tris-HCl.
B) pH=7.4 concentration is the configuration of 0.15M phosphate buffer (PB): take 10.74gNa
2hPO
412H
2o solid joins in 200mL ultrapure water, takes 4.68gNaH
2pO
42H
2o solid joins in 200mL ultrapure water, by NaH after to be dissolved
2pO
4solution joins Na
2hPO
4in solution, adjust pH=7.4.
C) 0.05%Tween200.03MPB configuration: get 250 μ LTween-20 and join in 500mL0.03MPB damping fluid.
D) 2MH
2sO
4configuration: get 8mL ultrapure water and add the dense H of 1mL
2sO
4(98%).
2) first antibody adsorption experiment.The CA film soaked with ultrapure water, be immersed in 60 μ g/mL rabbit anti-mouse igg antibody (rabbitanti-mouseIgG0.03MPB) solution, after absorption at room temperature 2h, CA film is cleaned each 30s through 0.05%Tween200.03MPB solution, ultrapure water successively.
3) sealer albumen (ovalbumin, OVA) adsorption experiment.The CA film of first antibody absorption puts into 1mg/mLOVA solution, after adsorbing 1h, CA film is cleaned each 30s through 0.05%Tween200.03MPB solution, 0.03MPB solution, ultrapure water successively at 37 DEG C.
4) optimize electrophoretic voltage and time, electrophoresis drives antigen-first antibody reaction.First antibody and the good CA film of sealer protein adsorption are fixed on glass disengagement chamber central authorities (Fig. 1), disengagement chamber entrance side injects antigenic solution (1mL5 μ g/mL mouse-anti body mouseIgG0.03MPB), and disengagement chamber exit adds 0.03MPB solution.Entrance side access power cathode, outlet side access positive source.Regulation voltage is 15V, 20V, 25V respectively, and the time is 2min, 5min, 8min, carries out antigen-first antibody reaction.By the CA film after electrophoresis, successively through 0.05%Tween200.03MPB solution, 0.03MPB solution, ultrapure water cleaning 30s.
5) second antibody-antigen-reactive, the colour developing of enzyme mark quantitatively.After antigen-reactive, CA film is put into 0.5 μ g/mL second antibody (sheep anti-mouse igg antibody-horseradish peroxidase mark (goatanti-mouseIgG-horseradishperoxidaseconjugatedIgG)), after 37 DEG C of reaction 1h, CA film is respectively cleaned 30s through 0.05%Tween200.03MPB solution, 0.03MPB solution, ultrapure water successively.CA film is joined the tetramethyl biphenyl amine aqueous solution (TMB) of 0.4mL, under room temperature after lucifuge reaction 30min, then add 0.4mL2MH
2sO
4solution, cessation reaction.By detecting 450nm place absorbance, carry out antigen quantify.
6) CA substrate electrophoresis flow model ELISA detection curve measures.By measuring under different voltage and time detection sensitivity (snr value (S/N)), (signal S is second antibody reaction signal when antigen exists; When noise N is without antigen, the signal of second antibody non-specific adsorption), the voltage and the time conditions that obtain optimum are 15v, 2min.Again under this condition, detect the signal of variable concentrations antigen, obtain CA substrate electrophoresis flow model ELISA detection curve (Fig. 2).
Embodiment 2
1, PEMs modifies the preparation of CA perforated membrane
1) PDDA and PSS solution is prepared.Take each 2mg of PDDA and PSS solid respectively, be dissolved in respectively in the solution of 10mL50mMTris-HCl0.15MNaClpH=7.4, obtaining PDDA and PSS concentration is 0.2mg/mL.
2) PEMs modifies the preparation of CA perforated membrane.The CA film that ultrapure water soaks puts into PDDA solution, floods 2min under room temperature, cleans 30s afterwards with ultrapure water, washes away the polyelectrolyte solution do not adsorbed.Then put into PSS solution, flood 2min under room temperature, clean 30s afterwards with ultrapure water, above PDDA and PSS hockets, and prepares 7 layers of electropositive self-assembled multilayer film PEMs ((PDDA/PSS)
3pDDA).2, PEMs-CA perforated membrane substrate electrophoresis ELISA
1) with embodiment 1 step 1)
2) with embodiment 1 step 2), PEMs-CA replaces CA.
3) with embodiment 1 step 3), PEMs-CA replaces CA.
4) with embodiment 1 step 4), PEMs-CA replaces CA.
5) with embodiment 1 step 5), PEMs-CA replaces CA, and the concentration of second antibody solution is 1 μ g/mL.
6) PEMs-CA substrate electrophoresis flow model ELISA detection curve measures.By measuring under different voltage and time detection sensitivity (snr value (S/N)), (signal S is second antibody reaction signal when antigen exists; When noise N is without antigen, the signal of second antibody non-specific adsorption), the voltage and the time conditions that obtain optimum are 25v, 2min.Again under this condition, detect the signal of variable concentrations antigen, obtain PEMs-CA substrate electrophoresis flow model ELISA detection curve (Fig. 2).
Claims (6)
1. electrophoresis flow model ELISA method, is characterized in that, comprises the following steps:
(1) first antibody is adsorbed onto on perforated membrane substrate;
(2) the perforated membrane substrate adsorbing first antibody carries out sealer protein adsorption again;
(3) electrophoresis drives antigen-first antibody reaction: be fixed in electrophoretic apparatus by the perforated membrane substrate after step (2) first antibody and sealer protein adsorption, in electrophoretic apparatus, antigenic solution is injected in perforated membrane substrate side, perforated membrane substrate opposite side injects phosphate buffered solution, if antigen molecule is negative charge, negative electrode is inserted in antigenic solution side, opposite side inserts positive electrode, otherwise then anti-; Electrode making alive, carries out antigen-first antibody reaction;
(4) second antibody-antigen-reactive, colour developing is carried out to enzyme mark second antibody quantitative: after step (3) electrophoresis drives antigen-first antibody reaction, perforated membrane substrate after electrophoresis is put in second antibody solution, react, then clean, gained perforated membrane substrate is put into the substrate solution of second antibody mark enzyme, develop the color; By detecting absorbance, carry out antigen quantify analysis.
2. according to the method for claim 1, it is characterized in that, step (1) perforated membrane substrate is the CA perforated membrane that cellulose acetate perforated membrane (CA) or polyelectrolyte multilayer film are modified, the CA perforated membrane that polyelectrolyte multilayer film is modified is the CA perforated membrane utilizing poly-(diallyldimethylammonium chloride) and the cross layered modification of polystyrene sulphuric acid sodium successively, and the outermost layer modified is poly-(diallyldimethylammonium chloride), obtain electropositive PDDA/PSS and modify CA, i.e. PEMs-CA.
3. according to the method for claim 2, it is characterized in that, poly-(diallyldimethylammonium chloride) and the cross layered modification of polystyrene sulphuric acid sodium 7 layers in PEMs-CA.
4. according to the method for claim 1, it is characterized in that, step (3) electrophoresis drives electrophoretic voltage and the electrophoresis time of antigen-first antibody reaction, and the voltage swing of electrophoresis determines the translational speed of protein; The length of electrophoresis time affects the efficiency of antigen and first antibody combination and the stability of self-assembled film.
5. according to the method for claim 1 or 2, it is characterized in that, CA and PEMs-CA electrophoretic voltage and time range are 15-25V and 2-8min.
6. according to the method for claim 5, it is characterized in that, CA electrophoretic voltage and time are 15v, 2min; PEMs-CA electrophoretic voltage and time are 25v, 2min.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105891306B (en) * | 2016-06-24 | 2019-04-26 | 北京化工大学 | A kind of method of antibody modification nanosphere electrophoresis flow model ELISA |
| CN111007242A (en) * | 2019-12-20 | 2020-04-14 | 苏州和迈精密仪器有限公司 | Fluorescence immunoassay method and device based on multilayer high molecular porous membrane and application |
-
2015
- 2015-06-10 CN CN201510316659.2A patent/CN105158460A/en active Pending
Non-Patent Citations (2)
| Title |
|---|
| HEYUN SHEN, ET AL.: "Polyelectrolyte multilayers-modified membrane filter for rapid immunoassay: protein condensation by centrifugal permeation.", 《POLYMER JOURNAL》 * |
| HEYUN SHEN, ET AL.: "Polyelectrolyte multilayers-Modified Polystyrene Plate Improves Conventional Immunoassay: Full Covering of hte Blocking Reagent.", 《ANAL.CHEM.》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105891306B (en) * | 2016-06-24 | 2019-04-26 | 北京化工大学 | A kind of method of antibody modification nanosphere electrophoresis flow model ELISA |
| CN111007242A (en) * | 2019-12-20 | 2020-04-14 | 苏州和迈精密仪器有限公司 | Fluorescence immunoassay method and device based on multilayer high molecular porous membrane and application |
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Application publication date: 20151216 |