CN109406772A - The method of chemiluminescent enzyme-linked immunosorbent immune detection DNMT1 based on Magnetic Isolation - Google Patents
The method of chemiluminescent enzyme-linked immunosorbent immune detection DNMT1 based on Magnetic Isolation Download PDFInfo
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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
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
- G01N33/54326—Magnetic particles
- G01N33/54333—Modification of conditions of immunological binding reaction, e.g. use of more than one type of particle, use of chemical agents to improve binding, choice of incubation time or application of magnetic field during binding reaction
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/76—Chemiluminescence; Bioluminescence
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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
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/535—Production of labelled immunochemicals with enzyme label or co-enzymes, co-factors, enzyme inhibitors or enzyme substrates
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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
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
- G01N33/537—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with separation of immune complex from unbound antigen or antibody
- G01N33/539—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with separation of immune complex from unbound antigen or antibody involving precipitating reagent, e.g. ammonium sulfate
- G01N33/541—Double or second antibody, i.e. precipitating antibody
Abstract
The method of the present invention provides a kind of chemiluminescent enzyme-linked immunosorbent immune detection DNMT1 based on Magnetic Isolation, comprising: closing pretreatment plus magnetic monoclonal antibody plus antigen add clonal antibody plus ELIAS secondary antibody plus chemiluminescence bottom liquid, shine and detection and establish regression equation.The above method provided by the invention constructs a kind of novel Chemiluminescence System HRP-Luminol-H2O2- BIP, using immunomagnetic beads as solid phase carrier, the advantages of uniformity and magnetic bead Magneto separate for being detected simultaneously in conjunction with polystyrene micropore plate multisample, the efficient specificity three of chemiluminescent high sensitivity, the high specific of antigen-antibody reaction and enzymatic is combined, double-antibody method is established and DNMT1 is measured.
Description
Technical field
The present invention relates to detection technique fields, specifically, relate to a kind of chemiluminescent enzyme-linked immunosorbent based on Magnetic Isolation
The method of immune detection DNMT1.
Background technique
In detection field, it is often necessary to qualitatively or quantitatively be detected to all kinds of macromolecular antigens or antibody.Existing skill
In art, panimmunity response analysis method is derived based on " double antibodies sandwich ", such as: radioimmunology, enzyme linked immunological
Method, chemoluminescence method, time-resolved fluorescence method and fluorescent immune method etc. can be used for determining pathogenic microorganism, to the special of human body
Property protein quantification detection to carry out auxiliary diagnosis or monitoring etc. to disease, purposes is very extensive.But use double-antibody method
When measuring macromolecular antigen, enzyme labelled antibody is needed from the disadvantages of mark, time and effort consuming is uneconomical, and at high cost sometimes.
In order to solve the above problems, people are seeking always a kind of ideal technical solution.
Summary of the invention
In view of this, the present invention is it is necessory to provide a kind of chemiluminescent enzyme-linked immunosorbent immune detection based on Magnetic Isolation
DNMT1 method, to solve the above problems.
To solve the above-mentioned problems, the technical scheme adopted by the invention is that: a kind of chemiluminescence based on Magnetic Isolation
The method of enzyme linked immunosorbent detection DNMT1, specific steps include:
Confining liquid is added in polystyrene micropore plate by closing pretreatment successively to be carried out incubating and the processing of first stage board-washing；
Add magnetic monoclonal antibody that Fe is added into the polystyrene micropore plate handled by first stage board-washing3O4@McAbDNMT1
Dilution simultaneously carries out Magneto separate and the processing of second stage board-washing, so that the polystyrene micropore plate is suspended with the first compound,
First compound has Fe3O4@McAbDNMT1Structure, and the Fe3O4@McAbDNMT1It is surface by DNMT1 mouse anti human list
The Fe of clonal antibody covalent modification3O4Nano immune magnetic bead；
Add antigen that DNMT1 standard antigen solution or to be measured is added into the polystyrene micropore plate for being suspended with first compound
Antigenic solution, and successively incubated, the processing of Magneto separate and phase III board-washing, so that the polystyrene micropore plate is suspended with
Second compound, second compound have Fe3O4@McAbDNMT1@AgDNMT1Structure；
It adds clonal antibody and rabbit anti-human polyclonal antibody is added into the polystyrene micropore plate for being suspended with second compound
PAbDNMT1Dilution, and successively incubated, the processing of Magneto separate and fourth stage board-washing, so that the polystyrene micropore plate is outstanding
Floating to have third compound, which has Fe3O4@McAbDNMT1@AgDNMT1@PAbDNMT1Structure；
ELIAS secondary antibody is added to be added ELIAS secondary antibody dilution into the polystyrene micropore plate for being suspended with the third compound, and according to
It is secondary incubated, the processing of Magneto separate and the 5th stage board-washing, and the 4th compound is suspended in the polystyrene micropore plate, should
4th compound has Fe3O4@McAbDNMT1@AgDNMT1@PAbDNMT1@ELIAS secondary antibody structure；
Add chemiluminescence bottom liquid sequentially added into the polystyrene micropore plate for being suspended with the 4th compound luminous bottom liquid A and
Hydrogenperoxide steam generator carries out chemiluminescence processing, forms chemiluminescent solution, wherein includes luminol in the luminous bottom liquid A
And xenol；
It shines and detects and establish the luminous intensity that regression equation detects the chemiluminescent solution using chemiluminescence detector, root
According to the relationship between the luminous intensity and the DNMT1 standard antigen solution or determined antigen solution concentration of detection, establish
Detect the regression equation of DNMT1.
Based on above-mentioned, the pretreated step of closing include: by concentration be 0.5% BSA/PBS confining liquid with every hole
250~350 μ L are added in the polystyrene micropore plate, then are placed in 60~120 min of incubation in 37 DEG C of constant incubators,
Then it is handled, and patted dry with the board-washing that PBST solution carries out the first stage again.
Based on above-mentioned, the step of addition monoclonal antibody includes: with 0.008~0.012 mmol/L pH 7~7.5
PBS solution with 1: 35~45 thinner ratio dilute Fe3O4@McAbDNMT1The Fe is made in stoste3O4@McAbDNMT1Dilution
Liquid, and by the Fe3O4@McAbDNMT1Dilution is added in the polystyrene micropore plate with every 100 μ L of hole, then successively
Magneto separate processing is carried out and carries out the second stage board-washing using PBST solution to handle, so that in the polystyrene micropore plate
It is suspended with first compound.
Based on above-mentioned, the Fe3O4@McAbDNMT1The preparation method of stoste the following steps are included:
Cleaning: by carboxylated Fe3O4Nano granule suspension used after ultrasound, Magneto separate abandon supernatant processing concentration for 0.01~
The MES buffer that 0.015 mol/L, pH is 6.0 balances 2~5 min to it, and then it is heavy to obtain first for progress Magneto separate abandoning supernatant
Starch；
Activation: it is 8~11 that EDC solution that concentration is 8~10 mg/mL and concentration are sequentially added in the first sediment of Xiang Suoshu
The NHS solution of mg/mL carries out 10~15 min of reaction that are vortexed, and Magneto separate washs it using MES buffer after abandoning supernatant processing
3 times, Magneto separate is then carried out again, abandoning supernatant handles to obtain the second sediment；
Buffer system conversion: second sediment is washed 3~4 times using PBS buffer solution, Magneto separate obtains third precipitating
Object；
Coupling: being added DNMT1 monoclonal antibody in Xiang Suoshu third sediment and PBS buffer solution carries out the reaction 1.5~2 that is vortexed
H through Magneto separate, is abandoned after supernatant is handled and is cleaned to it 2 times using PBS buffer solution, and Magneto separate, abandoning supernatant obtain the 4th sediment；
Closing: being added BSA confining liquid in the 4th sediment of Xiang Suoshu, carries out 0.5~1 h of reaction that is vortexed, and Magneto separate abandons supernatant
After processing, using PBST buffer solution for cleaning 3~4 times, Magneto separate, abandoning supernatant obtain the 4th i.e. Fe of sediment3O4@McAbDNMT1；Its
Middle BSA confining liquid represents bovine serum albumin(BSA) confining liquid；
It saves: in coupled product Fe3O4@McAbDNMT1Middle addition PBS buffer solution, light shake shake up, and are made the Fe3O4@
McAbDNMT1Stoste.
Based on above-mentioned, the step of described plus antigen includes: the PBS with 0.008~0.012 mmol/L pH 7~7.5
DNMT1 antigen standard is diluted to the DNMT1 standard antigen solution of multiple and different concentration by buffer solution, with identical pH value
PBS buffer solution is placebo solution；The DNMT1 standard antigen solution and placebo solution are added with every 100 μ L of hole
Enter to being suspended in the polystyrene micropore plate of first compound, be placed in 37 DEG C of constant incubators incubate 60~
Then 120 min successively carry out Magneto separate processing and carry out the phase III board-washing using PBST solution to handle, so that described
Second compound is suspended in polystyrene micropore plate.
Based on above-mentioned, it by concentration is 1 that described the step of adding clonal antibody, which includes: with containing 0.5% BSA/PBS buffer solution,
The rabbit anti-human polyclonal antibody PAb of mg/mLDNMT1Solution dilutes 1500~2500 times of obtained rabbit anti-human polyclonal antibody PAbDNMT1
Dilution, then by the rabbit anti-human polyclonal antibody PAbDNMT1Dilution is added to every 100 μ L of hole and is suspended with described second
In the polystyrene micropore plate of compound, 60~120 min of incubation in 37 DEG C of constant incubators are placed in, are then successively carried out
Magneto separate handles and carries out the fourth stage board-washing using PBST solution and handles, so that suspending in the polystyrene micropore plate
There is the third compound.
Based on above-mentioned, it by concentration is 2 that the step of described plus ELIAS secondary antibody, which includes: with containing 0.5% BSA/PBS buffer solution,
The ELIAS secondary antibody solution of mg/mL dilutes 3500~4500 times of obtained ELIAS secondary antibody dilutions, by the ELIAS secondary antibody dilution with
Every 100 μ L of hole, which is added to, to be adsorbed in polystyrene micropore plate described in the third compound, is placed in 37 DEG C of constant incubators
60~120 min are incubated, then successively carry out Magneto separate processing and carry out the 5th stage board-washing using PBST solution to handle,
So that being suspended with the 4th compound in the polystyrene micropore plate.
Based on above-mentioned, the step of described plus chemiluminescence bottom liquid includes: that the luminous bottom liquid A and the hydrogen peroxide is molten
Liquid is added in the polystyrene micropore plate for being suspended with the 4th compound with every 100 μ L of hole respectively, in the polystyrene
The chemiluminescent solution is formed in microwell plate, wherein the luminous bottom liquid A include concentration be 0.001mol/L luminol and
5×10-5The luminescence enhancer xenol of mol/L.
Based on above-mentioned, the luminous detection and the step of establishing regression equation include: first to be examined using chemiluminescence detector
Survey the RLU value or RLU of the chemiluminescent solution in each hole of the polystyrene micropore plate0, then antigen concentration be 0.5 ~
In the range of 128 ng/mL, the equation of linear regression of detection DNMT1 is established:y=0.50143x+ 1.76886, whereinyFor RLU/
RLU0,xFor logCDNMT1, related coefficientRThe luminous intensity values of DNMT1 determined antigen, RLU are represented for 0.9907, RLU0Represent sky
The luminous intensity values of white test measurement, CDNMT1Represent DNMT1 determined antigen concentration.It is described based on Magnetic Isolation based on above-mentioned
Chemiluminescent enzyme-linked immunosorbent immune detection DNMT1 method, the lowest detection of the DNMT1 determined antigen concentration is limited to 0.01 ng/
mL。
It should be noted that in above steps, McAbDNMT1Represent DNMT1 mouse monoclonal anti-human antibody, PcAbDNMT1
Represent DNMT1 rabbit anti-human polyclonal antibody, HRP-Second-Ab represents goat-anti rabbit ELIAS secondary antibody.CB represent carbonate buffer solution,
PBS represents phosphate buffer solution, PBST represents the phosphate buffer solution of addition Tween-20 nonionic surfactant.
The present invention has substantive distinguishing features outstanding and significant progress compared with the prior art, and specifically, the present invention mentions
The method of the above-mentioned chemiluminescence enzyme immune detection DNMT1 based on Magnetic Isolation supplied constructs a kind of novel new body of chemiluminescence
It is HRP-Luminol-H2O2- BIP is detected using immunomagnetic beads as solid phase carrier in conjunction with polystyrene micropore plate multisample simultaneously
Uniformity and the advantages of magnetic bead Magneto separate, chemiluminescent high sensitivity, the high specific of antigen-antibody reaction and enzyme are urged
The efficient specificity three changed combines, and establishes double-antibody method and is measured to DNMT1, anti-without carrying out enzyme label
Body, it is time saving and energy saving, it is economical, it is at low cost.
Further, the method for above-mentioned detection DNMT1 provided by the invention, can according to the luminous intensity of detection with it is described
Relationship between DNMT1 standard sample or target to be measured concentration establishes the regression equation of detection DNMT1, to utilize this method
Calculate the content of DNMT1 in sample, the linear model that method and step simply and by the regression equation that this method is established detects DNMT1
Wide, detection is enclosed to limit low, precision, accuracy height and there is good specificity.
Detailed description of the invention
Fig. 1 is the principle signal of the chemiluminescence magnetic enzyme linked immunosorbent detection DNMT1 provided by the invention based on Magnetic Isolation
Figure.
Fig. 2 is HRP-Luminol-H in the present invention2O2The relational graph of the concentration of HRP and RLU in-BIP luminescence system.
Fig. 3 is HRP-Luminol-H in the present invention2O2The relational graph of the concentration of Luminol and RLU in-BIP luminescence system.
Fig. 4 is HRP-Luminol-H in the present invention2O2H in-BIP luminescence system2O2Concentration and RLU relational graph.
Fig. 5 is HRP-Luminol-H in the present invention2O2- BIP luminescence system and HRP-Luminol-H2O2Luminescence system
Kinetic curve comparison diagram.
Fig. 6 is HRP-Luminol-H in the present invention2O2The relational graph of the concentration of BIP and RLU in-BIP luminescence system.
Fig. 7 is HRP-Luminol-H in the present invention2O2Different buffer solutions are as dilution and RLU in-BIP luminescence system
Relational graph.
Fig. 8 is the relational graph of the RLU in the extension rate and chemical luminous system of immunomagnetic beads provided by the invention.
Fig. 9 is provided by the invention when the thinner ratio difference of immunomagnetic beads, the comparison diagram of the RLU value detected.
Figure 10 is the relational graph of the RLU of solid phase antibody capture antigenic action time provided by the invention and reaction system.
Figure 11 is provided by the invention as polyclonal antibody PcAbDNMT1Thinner ratio difference when, pair of the RLU value detected
Than figure.
Figure 12 is the RLU value provided by the invention when the thinner ratio difference of ELIAS secondary antibody HRP-Second-Ab, detected
Comparison diagram.
Figure 13 is the DNMT1 Concentration Testing standard curve schematic diagram that the present invention establishes.
Specific embodiment
Below by specific embodiment, technical scheme of the present invention will be described in further detail.
Reagent used in the present invention and instrument type are as follows: source of people DNMT1(Abcam, OriGene)；DNMTI mouse is anti-
Human monoclonal antibodies (McAbDNMT1, Abcam)；DNMT1 rabbit anti-human polyclonal antibody (PcAbDNMT1, Abcam)；Goat-anti rabbit enzyme mark
Secondary antibody (HRP-Second-Ab, Abcam)；30% hydrogen peroxide (Hao Tian reagent Chemical Co., Ltd.)；Luminol (English Luminol)
(>=98%, Sigma)；Ethyl alcohol (Tianjin Bo Di Chemical Co., Ltd.)；Three (methylol) aminomethanes (Tris, Suo Laibao science and technology
Co., Ltd)；People's DNA methylation transferase 3a ELISA kit；People DNA methylation transferase 3b ELISA kit (on
The bright Biotechnology Co., Ltd in Haikang)；Fe3O4Nano particle (self-control)；(BSA, Beijing Suo Laibao science and technology have bovine serum albumin(BSA)
Limit company)；Other reagents are that analysis is pure.Such as no special explanation, experimental water is that (resistivity is greater than Milli-Q ultrapure water
18.2 M Ω cm).
Instrument used in the present invention is as follows: board-like chemiluminescence detector (LB960, German Berthold company)；96
Hole polystyrene white board (Jin Canhua Industrial Co., Ltd., Shenzhen)；Multifunctional polystyrene microplate reader
(SpectraMax M2e, the U.S.), electro-heating standing-temperature cultivator (DHG-9080 type, the upper macro experimental facilities Co., Ltd of Nereid), number
It controls ultrasonic washing instrument (KQ-300DE type, Kunshan Ultrasonic Instruments Co., Ltd.), adjustable micro sample adding appliance (Eppendorf, moral
State), XH-C type vortex blending instrument (Guo Wang laboratory apparatus factory, Jintan City), refrigerated centrifuge (5424 R of Centrifuge,
Eppendorf).
The preparation of various solution
(1) 0.1 mol/L Tris-HCl(pH 8.5) buffer solution: 3.0285 g tri- (methylol) aminomethanes are accurately weighed,
It is to be transferred in clean bomb before 8.5(is used with pH is adjusted with 1.0 mol/L HCl after the dissolution of MilliQ water, through high temperature
After high pressure sterilization processing, being cooled to room temperature rear be can be used).
(2) 0.01 mol/L Luminol stock solutions: accurately weighing 0.1772 g of Luminol, fixed with 1 mol/L NaOH
Be dissolved in 100 mL brown volumetric flasks, be protected from light 4 DEG C place 3 ~ 5 days after use, with 0.1 mol/L Tris-HCl(pH when use
8.5) buffer solution is diluted to required concentration.
(3) BIP(parazon) stock solution: 0.0596 g BIP is accurately weighed, with n,N-Dimethylformamide (DMF)
Constant volume is dissolved in 100 mL brown volumetric flasks, is made into 3.5 × 10-3The stock solution of mol/L, 4 DEG C save backup.
(4) 1 mL Luminol stock solution, 143 μ L 3.5 × 10 chemiluminescence bottom liquid A: are taken-3The BIP solution of mol/L,
It is settled in 10 mL brown volumetric flasks with the buffer solution dilution of 1 mol/L Tris-HCl(pH 8.5), it is ready-to-use.
(5) chemiluminescence bottom liquid B: 30% H is taken2O215.3 μ L of solution is dissolved with Milli-Q ultrapure water, and constant volume is in 25
It is ready-to-use in mL volumetric flask.
(6) Fe3O4@McAbDNMT1The preparation method of stoste the following steps are included:
1) it cleans: by carboxylated Fe3O4Nano granule suspension uses concentration for 0.01 after ultrasound, Magneto separate abandon supernatant processing
The MES buffer that~0.015 mol/L, pH is 6.0 balances the min of 2 min~5 to it, then carries out Magneto separate abandoning supernatant and obtains
First sediment；Wherein MES represents morpholino b acid buffer；
2) activate: it is 8~11 that EDC solution that concentration is 8~10 mg/mL and concentration are sequentially added in the first sediment of Xiang Suoshu
The NHS solution of mg/mL carries out 10~15 min of reaction that are vortexed, and Magneto separate washs it using MES buffer after abandoning supernatant processing
3 times, Magneto separate is then carried out again, abandoning supernatant handles to obtain the second sediment；Wherein, EDC represents (1- (3- dimethylamino third
Base) that -3- ethyl-carbodiimide hydrochloride solution, NHS represent n-hydroxysuccinimide solution, MES represents morpholino b acid is slow
Fliud flushing, mg/mL represent every milliliter of milligram；
3) buffer system is converted: being washed 3~4 times using PBS buffer solution to second sediment, Magneto separate obtains third precipitating
Object；
4) be coupled: be added in Xiang Suoshu third sediment DNMT1 monoclonal antibody and PBS buffer solution be vortexed reaction 1.5~
2h through Magneto separate, is abandoned after supernatant is handled and is cleaned to it 2 times using PBS buffer solution, and Magneto separate, abandoning supernatant obtain the 4th sediment；
5) it closes: BSA confining liquid being added in the 4th sediment of Xiang Suoshu, 0.5~1 h of reaction that is vortexed is carried out, on Magneto separate, abandoning
After clear processing, using PBST buffer solution for cleaning 3~4 times, Magneto separate, abandoning supernatant obtain the 4th i.e. Fe of sediment3O4@McAbDNMT1；
Wherein BSA confining liquid represents bovine serum albumin(BSA) confining liquid；
6) it saves: in coupled product Fe3O4@McAbDNMT1Middle addition PBS buffer solution, light shake shake up, and are made the Fe3O4@
McAbDNMT1Stoste, 4 DEG C save backup.
The present invention provides the method for chemiluminescent enzyme-linked immunosorbent immune detection DNMT1 based on Magnetic Isolation a kind of, the detection
The corresponding testing principle of DNMT1 method is as shown in Figure 1.The method specific steps of detection DNMT1 include: to close pretreatment plus magnetic
Property monoclonal antibody, plus antigen, add clonal antibody, plus ELIAS secondary antibody, plus chemiluminescence bottom liquid, shine detection and establish return
Equation and etc..Wherein, each step particular content is as follows:
Confining liquid is added in polystyrene micropore plate successively to carry out incubating and handle with first stage board-washing by closing pretreatment, specifically
Ground, Xiang Suoshu polystyrene micropore plate --- 300 μ L confining liquids (0.5% are added in every hole in 96 hole polystyrene white boards
BSA/PBS solution), it is placed in 37 DEG C of constant incubators and incubates 90 min, PBST board-washing 3 times, pat dry, uneven to avoid coating
Problem, while solving non-specific adsorption.
Add magnetic monoclonal antibody that Fe is added into the polystyrene micropore plate handled by first stage board-washing3O4@
McAbDNMT1Dilution simultaneously carries out Magneto separate and the processing of second stage board-washing, so that the polystyrene micropore plate is suspended with first
Compound, first compound have Fe3O4@McAbDNMT1Structure, wherein the Fe3O4@McAbDNMT1It is small by DNMT1 for surface
The Fe of mouse anti-human monoclonal's antibody covalent modification3O4Nano immune magnetic bead；Specifically, with the PBS of 0.01 mmol/L pH 7.4 with
The thinner ratio of 1:40 dilutes Fe3O4@McAbDNMT1 stoste, every hole add 100 μ L, Magneto separate, and PBST board-washing 3 times, in the polyphenyl
First compound is formed in each hole of ethylene microwell plate.
Add antigen be added into the polystyrene micropore plate for being suspended with first compound DNMT1 standard antigen solution or
Determined antigen solution, and successively incubated, the processing of Magneto separate and phase III board-washing, so that the polystyrene micropore plate is outstanding
Floating to have the second compound, which has Fe3O4@McAbDNMT1@AgDNMT1Structure；Specifically, with 0.01 mmol/L
DNMT1 antigen standard is diluted to 0.001 by the buffer solution of PBS pH 7.4,0.005,0.01,0.05,0.1,0.5,1,2,
4,15 various concentrations such as 8,16,32,64,128 ng/mL, using 7.4 PBS buffer solution of pH as blank control, every hole is added
100 μ L are placed in 37 DEG C of constant incubators and incubate 90 min, Magneto separate, with PBST board-washing 3 times, in the polystyrene micropore
Second compound is formed in each hole of plate.
Adding clonal antibody, that rabbit-anti people is added into the polystyrene micropore plate for being suspended with second compound is polyclonal
Antibody PAbDNMT1Dilution, and successively incubated, the processing of Magneto separate and fourth stage board-washing, so that the polystyrene micropore
Plate is suspended with third compound, which has Fe3O4@McAbDNMT1@AgDNMT1@PAbDNMT1Structure；Specifically, with containing
The rabbit anti-human polyclonal antibody PAbDNMT1 solution that concentration is 1 mg/mL is diluted 2000 times by 0.5% BSA/PBS buffer solution,
100 μ L are added in every hole, are placed in 37 DEG C of constant incubators and incubate 90 min, Magneto separate, with PBST board-washing 3 times, in the polyphenyl
The third compound is formed in each hole of ethylene microwell plate.
Add ELIAS secondary antibody that ELIAS secondary antibody dilution is added into the polystyrene micropore plate for being suspended with the third compound,
And it is successively incubated, the processing of Magneto separate and the 5th stage board-washing, and it is compound to be suspended with the 4th in the polystyrene micropore plate
Object, the 4th compound have Fe3O4@McAbDNMT1@AgDNMT1@PAbDNMT1@ELIAS secondary antibody structure；Specifically, with containing 0.5%
The HRP-Second-Ab solution that concentration is 2 mg/mL is diluted 4000 times by the PBS solution of BSA, and 100 μ L are added in every hole, are placed in
90 min, Magneto separate, with PBST board-washing 5 times, in each control of the polystyrene micropore plate are incubated in 37 DEG C of constant incubators
Form the 4th compound.
Chemiluminescence bottom liquid is added to sequentially add luminous bottom into the polystyrene micropore plate for being suspended with the 4th compound
Liquid and hydrogenperoxide steam generator carry out chemiluminescence processing, form chemiluminescent solution, wherein include Rumi in the luminous bottom liquid
Promise and xenol；Specifically, chemiluminescence bottom liquid A and describedization described in 100 μ L is added in every hole on polystyrene micropore plate
The bottom liquid B that shines is learned, the chemiluminescent solution is formed in each hole of the polystyrene micropore plate, wherein the chemistry hair
Light bottom liquid A includes the luminol and 5 × 10 that concentration is 0.001mol/L-5The luminescence enhancer xenol of mol/L.
Shining, it is each on the polystyrene micropore plate using detecting on chemiluminescence detector to detect and establish regression equation
The luminous intensity values of chemiluminescent solution in hole, parallel 6 holes of the standard antigen solution or determined antigen solution of each concentration,
According to the relationship between the luminous intensity of detection and the DNMT1 standard antigen solution or determined antigen solution concentration, build
The regression equation of vertical detection DNMT1.
It can be seen that the above-mentioned chemiluminescent enzyme-linked immunosorbent immune detection DNMT1 based on Magnetic Isolation of one kind provided by the invention
Method relate generally to chemical luminous system HRP-Luminol-H2O2- BIP system and immune response system, two systems pair
Testing result has important influence.This is further described in influence with regard to the influence factor of the two systems to testing result below
The method that the above-mentioned chemiluminescence enzyme immune detection DNMT1 based on Magnetic Isolation provided is provided.
The influence factor of chemical luminous system
For a chemical luminous system, chemiluminescence intensity is vulnerable to chemical reaction condition, such as pH, ionic strength, molten
Liquid composition, temperature etc. influence.Usual Luminol and H2O2Chemical reaction rate it is relatively slow, but under the catalytic action of HRP,
Reaction rate greatly improves.HRP activity is high, and stability is good, is most common enzyme in CLEIA method.Utilize Luminol, H2O2Make
For chemiluminescent substrate, luminous intensity depends on the concentration of enzyme in immune response, therefore investigates to HRP, verifies HRP-
Luminol-H2O2Feasibility of the luminescence system as reaction system.In alkaline aqueous solution, Luminol can be oxidized generation
The intermediate of excitation state, the intermediate return to ground state by emitting light radiation transition；H2O2Reaction as chemical luminous system
Luminol is oxidized to the oxidant of excitation state, and is catalyzed by HRP specificity by object, increases chemiluminescence intensity.Therefore, it needs
The two is investigated.Phenol type substances are as Luminol-H2O2The reinforcing agent of-HRP system, chemiluminescence intensity after addition
Enhancing, fluorescent lifetime is more longlasting, keeps stablizing in a short time, so, BIP generates shadow to the luminescence system as reinforcing agent
It rings；The luminous pH of the optimization of Luminol is 11.5, but the optimal pH of HRP is 7.0, and at room temperature, holding is stablized in several weeks,
The optimum pH of the two is different, so different pH has a certain impact to RLU, therefore, it is also desirable to the luminescence system
The dilution of pH(, that is, Luminol) it is investigated.
So below with regard to HRP concentration, Luminol concentration, H2O2Concentration, the concentration of BIP and dilution of Luminol etc.
Influence factor, to HRP-Luminol-H2O2The influence of-BIP luminescence system is further analysed.
1.1 HRP concentration are to HRP-Luminol-H2O2The catalytic action of luminescence system
In the identical situation of other conditions, to concentration range 1 × 10-6Mol/L~15 × 10-6HRP between mol/L
To HRP-Luminol-H2O2The catalytic affect of luminescence system is detected, and testing result is as shown in Figure 2: with the increasing of HRP concentration
Greatly, HRP-Luminol-H2O2The RLU value of system first increases, after tend to be steady, present positive correlation.Therefore, HRP- is used
Luminol-H2O2System has feasibility as reaction system.
1.2 Luminol concentration are to HRP-Luminol-H2O2The influence of luminescence system
In the identical situation of other conditions, to the Luminol that concentration range is the mmol/L of 0.4 mmol/L~1.4 to HRP-
Luminol- H2O2The influence of luminescence system is detected, and testing result is as shown in Figure 3: RLU value with Luminol concentration increasing
Add and increase, Luminol concentration RLU at 1.4 mmol/L reaches maximum, but considers actual conditions, selects speedup most fast
1.0 mmol/L are the optimal concentration of Luminol.
1.3 H2O2Concentration is to HRP-Luminol-H2O2The influence of luminescence system
In the identical situation of other conditions, the H for being the mmol/L of 0.5 mmol/L ~ 12 to concentration range2O2To HRP-
Luminol-H2O2The influence of luminescence system is detected, and testing result is as shown in Figure 4: H2O2RLU value when concentration is 6 mmol/L
Relatively high and precision is good, and RLU value amplification is unobvious when concentration is 12 mmol/L, therefore selects 6 mmol/L for H2O2Most
Excellent concentration.
1.4 BIP concentration are to HRP-Luminol-H2O2The influence of-BIP luminescence system
In the identical situation of other conditions, with BIP concentration for 5 × 10-5The HRP-Luminol-H of mol/L2O2- BIP shines
System is as measurement experiment group (adding BIP), with HRP-Luminol-H2O2As a control group (BIP is not added) in luminescence system, when measurement
Between 1 s, to measure humidification of the relationship of dynamics time and RLU value to investigate BIP to luminescence system, measurement result is as schemed
Shown in 5.The kinetic curve of Fig. 5 shows that, relative to control group chemiluminescence intensity, the addition of BIP significantly increases RLU value,
It can be seen that BIP is obvious to chemical luminous system reinforcing effect.
In the identical situation of other conditions, concentration range is 0.5 × 10-5Mol/L~10 × 10-5The BIP of mol/L
To HRP-Luminol-H2O2The influence of-BIP luminescence system is detected, and testing result is as shown in Figure 6: in optimal L uminol and
H2O2Under concentration conditions, when the concentration of BIP is 5 × 10-5When mol/L, RLU value reaches maximum, and concentration is bigger, inhibits instead
Chemiluminescence, therefore selecting BIP concentration is 5 × 10-5 mol/L。
1.5 pH value are to HRP-Luminol-H2O2The influence of-BIP luminescence system
It is respectively adopted the common 4 kinds of buffer solutions in laboratory: the CB of Tris-HCl, pH 9.6 of PBS, pH 8.5 of pH 7.4,
The Tris-HCl of pH 10.2 dilutes HRP-Luminol-H2O2- BIP luminescence system investigates pH value to HRP-Luminol-
H2O2The influence of-BIP luminescence system.It is as shown in Figure 7 to investigate result.As can be seen from Figure 7: the Tris-HCl with pH 8.5 is dilute
Luminol is released, RLU value is maximum；Therefore select the Tris-HCl of pH 8.5 as HRP-Luminol-H2O2- BIP luminescence system
Dilution.
The influence factor of immune response system
Each factor of immune response system influences RLU value very big.Immune response system in detection method provided by the invention
Immunomagnetic beads are introduced as solid phase carrier, luminous intensity may be had an impact；Immunoreagent interaction time be
Determine one of the key parameter of detection sensitivity, solid phase antibody and antigenic action time also affect capture rate, and then influence
Detection sensitivity；Polyclonal antibody, ELIAS secondary antibody are to influence two key factors of sensitivity and linear measurement range；Therefore it needs below
Will concentration to immunomagnetic beads to the effect of chemical luminous system and thinner ratio, the immune response time, polyclonal antibody thinner ratio,
The factors such as ELIAS secondary antibody thinner ratio.
2.1 immunomagnetic beads Fe3O4@McAbDNMT1To HRP-Luminol-H2O2The influence of-BIP chemical luminous system and dilute
It releases than optimization
A series of carboxylated immunomagnetic beads are prepared according to the ratio of 1:320,1:160,1:80,1:40,1:20,1:10,1:5,
Luminol、H2O2, BIP be measured according to the optimization concentration in above-mentioned " influence factors of 1. chemical luminous systems ", it is each dilute
Release parallel 3 holes of concentration, under the immunomagnetic beads for then measuring different thinner ratios, the RLU value of chemical luminous system.Measurement result figure
Shown in 8: with the continuous increase of immunomagnetic beads concentration, RLU is successively reduced, and the reduction of background value is conducive to the spirit of improvement method
Sensitivity；But immunomagnetic beads be not it is The more the better, it is more more to reduce chemiluminescence intensity instead.It may be due to Fe3O4With certain
Partial size and also Fe3O4Color is slightly deep, (main reason is that light is by the way that after solid, intensity can subtract caused by transmitting light absorption
It is weak, be because a part of light energy is by solid sorbent)；Or the light quantum effect generated to chemiluminescence has and certain work is quenched
With, or having certain shadowing effect to light, the mechanism for reducing chemiluminescence intensity is still not clear.Therefore, it is necessary to immune
The concentration of magnetic bead optimizes.
It is 0 ng/mL and 100 ng/mL in DNMT1 antigen concentration, and in the identical situation of other conditions, measurement is different dilute
Release the immunomagnetic beads Fe of multiple 1:5,1:10,1:20,1:40,1:803O4@McAbDNMT1Influence to chemiluminescence intensity, measurement
As a result as shown in Figure 9.Theoretically, thinner ratio is smaller, and the amount of antibody of load is more, and RLU should be bigger, still, can be with from Fig. 9
Find out: when thinner ratio is low, Fe3O4Amount it is more, RLU is lower instead, in certain negative correlation.Fe3O4@McAbDNMT1Dilution
When than for 1:80, the McAb of loadDNMT1Amount is few, and RLU equally weakens.Fe3O4@McAbDNMT1Thinner ratio be 1:40 when, RLU reaches
To maximum, and it is also maximum with the difference of background, therefore select 1:40 as Fe3O4@McAbDNMT1Optimal thinner ratio.
The optimization of 2.2 immune response times
DNMT1 antigen liquid concentration be 100 ng/mL and the identical situation of other conditions under, respectively to immune response act on when
Between 30 min, 60 min, 90 min, 120 min when RUL value be determined, measurement result is as shown in Figure 10.From Figure 10
It can be seen that RLU value is relatively high when the reaction time of solid phase antibody and determined antigen DNMT1 is 90 min, 120 min, but
The variation of RLU value is unobvious after 90 min, shows that the combination of antigen-antibody complex has reached balance, it is contemplated that time cost,
Therefore the reaction time selects 90 min.Meanwhile subsequent and PcAbDNMT1, HRP-Second-Ab action time be selected as 90
min。
2.3 polyclonal antibody PcAbDNMT1The optimization of thinner ratio
With 0.5% BSA/PBS of pH 7.4 by PcAbDNMT1Dilute 5 different multiples, respectively 500,1000,2000,
3000,4000, the values of chemiluminescence RLU that DNMT1 antigen concentration under different extension rates is 100 ng/mL is then measured respectively；
It is simultaneously 0 ng/mL as blank control using DNMT1 antigen concentration, measures RLU0；Then with the two difference DELTA RLU mapping, such as scheme
Shown in 11.As can be seen from Figure 11: as the thinner ratio 1:2000 of polyclonal antibody, the difference DELTA RLU of the two is maximum, because
This, the best thinner ratio of polyclonal antibody is 1:2000.
The optimization of 2.4 ELIAS secondary antibody HRP-Second-Ab thinner ratios
HRP-Second-Ab is diluted into 5 different multiples with 0.5% BSA/PBS of pH 7.4, respectively 1000,2000,
4000,6000,8000, the values of chemiluminescence RLU that DNMT1 concentration under different extension rates is 100 ng/mL is then measured respectively；
It is simultaneously 0 ng/mL as blank control using DNMT1 concentration, measures RLU0；Again with the two difference DELTA RLU mapping, as shown in figure 12.
As can be seen from Figure 12: as the thinner ratio 1:4000 of HRP-Second-Ab, the difference DELTA RLU of the two is maximum, therefore, enzyme
The best thinner ratio of labeling antibody is 1:4000.
The foundation of standard curve
DNMT1 standard items are diluted to 9 various concentrations, respectively 0.5 ng/mL, 1.0 with the PBS buffer solution of pH 7.4
ng/mL,2.0 ng/mL,4.0 ng/mL,8.0 ng/mL,16 ng/mL,32 ng/mL,64 ng/mL,128 ng/mL；Simultaneously
The DNMT1 of 5 low concentrations is determined, concentration is respectively as follows: 0.001 ng/mL, 0.005 ng/mL, 0.01 ng/mL, 0.05 ng/
mL,0.1 ng/mL；Then by above-mentioned " influence factors of 1. chemical luminous systems " and " 2. be immunoreacted systems influence because
Experiment condition in element " after optimization is measured, and each concentration is 6 times parallel, and measurement result is as shown in table 1 below:
The MCLEIA data (n=6) of 1 DNMT1 of table
Antigen DNMT1 concentration is within the scope of 0.001~128 ng/mL, with CDNMT1For abscissa, RLU is that ordinate draws curve
As shown in figure 13.According to the data that table 1 is measured, by antigen DNMT1 concentration within the scope of 0.5~128 ng/mL and its institute it is right
The RLU/RLU answered0, linear fit is carried out with four kinds of different forms respectively, obtains different linear equations and its related coefficientR, chooseRClosest to 1 linearity curve as final standard curve.The standard curve and its correspondence of four kinds of distinct methods fittings
Related coefficientR, as shown in table 2 below.In 0.5~128 ng/mL concentration range, RLU/RLU0With lgC in good linear
Relationship, linear equation arey=0.50143x+ 1.76886(yFor RLU/RLU0,xFor logCDNMT1), related coefficientRIt is 0.9907.
The standard curve and related coefficient of 2 four kinds of different forms of table fitting
4. the method validation test of detection DNMT1 provided by the invention
4.1 sensitivity
The RLU value of the DNMT1 antigen samples of 6 low concentrations is measured, testing result is shown in Table 3, while detecting 6 DNMT1 blank samples
Product are 74887.67,SDIt is 6479.979, finds out+3SDValue be 94327.607, RLU(0.01)> 94327.607, therefore the party
The detection of method is limited to 0.01 ng/mL.
The MCLEIA testing result of 3 low concentration DNMT1 of table
Note: MCLEIA is the chemiluminescence enzyme linked immune analysis based on Magnetic Isolation, i.e., detection method provided by the invention
4.2 preci-sion and accuracy
It is 0.5 ng/mL, 4.0 ng/mL, 32.0 ng/mL tri- that 100 μ L concentration are separately added into 96 polystyrene micropore plates
The DNMT1 standard sample of a concentration, each concentration are 6 times parallel；Separately 96 polystyrene micropore plates is taken equally to be separately added into 100
The DNMT1 standard sample of basic, normal, high three concentration of μ L, concentration are respectively 0.5 ng/mL, 4.0 ng/mL, 32.0 ng/mL, often
A concentration is 6 times parallel, detects their RLU value respectively, then calculates separately in plate between plateRSDAnd the rate of recovery, as a result such as
Shown in table 4.In the plate of the method for detection DNMT1 between plateRSDRange is respectively 14.3%~18.1% and 15.8%~16.9%
(n=6), precision is good；Rate of recovery range is respectively 70.0%~106.2%, 78.4%~116.8%(n=6 between plate in plate),
The rate of recovery of concentration middle and high concentration when being 4.0 ng/mL and 32.0 ng/mL is preferable, and the rate of recovery of low concentration is relatively low.
Precision and accuracy (n=6) between plate between 4 plate of table
4.3 specific
Chemiluminescence enzyme linked immune analysis (MCLEIA) method based on Magnetic Isolation established will be used for DNMT1 in blood serum sample
Detection, due to the complicated component in serum, it is desirable that the MCLEIA method of foundation is to DNMT1 specificity with higher, therefore
Selected two kinds it is similar with DNMT1 structure, may with DNMT1 antibody occur specific reaction DNMTs of the same clan, such as DNMT3A
And DNMT3B, DNMT1, DNMT3A, the DNMT3B of 64 ng/mL, verifying specificity are prepared respectively；The RLU/RLU that will be measured0Value
It is brought into standard curvey=0.50143xIn+1.76886, cross reacting rate is calculated, the results are shown in Table 5.
From the data of table 5 it can be seen that DNMT3A, DNMT3B and the DNMT1 cross reacting rate of 64 ng/mL is respectively
0.31% and 0.34%, cross reacting rate is low, illustrates McAbDNMT1To other DNMTs without specific reaction, therefore the MCLEIA established
Method measures DNMT1 specificity well, can be used for the detection of actual sample.
The specificity (n=6) of 5 MCLEIA of table measurement DNMT1
Note: MCLEIA is the chemiluminescence enzyme linked immune analysis based on Magnetic Isolation, i.e., detection method provided by the invention
The recovery of standard addition of 4.4 blood serum samples
Since the content of DNMT1 in serum is lower, in addition the otherness of individual, can not carry out quantitative analysis to it sometimes, so
When selecting the background sample of recovery of standard addition, low value serum that this experiment uses kit to detect: DNMT1 concentration is 0.24
The DNMT1 standard sample of basic, normal, high three concentration, concentration point is added as Background control in ng/mL in low value serum respectively
Not Wei 1 ng/mL, 50 ng/mL, 100 ng/mL, measure mark-on serum RLU, each concentration samples are 3 times parallel, according to following
Formula (1) calculate blood serum sample in recovery of standard addition, the results are shown in Table 6, and the recovery of standard addition range of MCLEIA is
Between 66.8%~118.8%, with recovery of standard addition 78.4% between recovery of standard addition 70.0%~106.2%, plate in the plate of serum-free~
116.8% compares, and is all that low concentration sample recovery of standard addition is low, the rate of recovery of middle and high concentration is preferable.
Recovery of standard addition (n=3) in 6 MCLEIA serum sample of table
Note: MCLEIA is the chemiluminescence enzyme linked immune analysis based on Magnetic Isolation, i.e., detection method provided by the invention
From various tests above it can be seen that the method for detection DNMT1 provided in an embodiment of the present invention construct it is a kind of novel
Chemiluminescence System HRP-Luminol-H2O2- BIP, using immunomagnetic beads as solid phase carrier, in conjunction with microwell plate polystyrene
The advantages of uniformity and magnetic bead Magneto separate that microwell plate multisample detects simultaneously, by chemiluminescent high sensitivity, antigen-antibody
The high specific of reaction and the efficient specificity three of enzymatic combine, and establish double-antibody method and carry out to DNMT1
Measurement.
It is optimized by the every factor to shine to influence system, optimum reaction condition are as follows: Luminol concentration is 1.0
Mmol/L, H2O2Concentration be 6 mmol/L, the concentration of BIP is 5 × 10-5Mol/L, reaction system buffer are that pH is 8.5
Tris-HCl；Every factor of immune response system is optimized simultaneously, optimum reaction condition are as follows: Fe3O4@McAbDNMT1
Thinner ratio is 1:40, incubation time 90 min, PcAbDNMT1Thinner ratio is 1:2000, and HRP-Second-Ab thinner ratio is 1:
4000.Under the conditions of after optimization, DNMT1 is in 0.5~128 ng/mL concentration range, RLU/RLU0With lgC in good linear
Relationship, linear equation arey=0.50143x+ 1.76886(yFor RLU/RLU0,xFor logCDNMT1), related coefficientRIt is 0.9907,
Detection is limited to 0.01 ng/mL；Precision is respectively 14.3%~18.1% and 15.8%~16.9%(n=6 between plate in plate)；In plate
Rate of recovery range is respectively 70.0%~106.2%, 78.4%~116.8%(n=6 between plate)；With DNMT3A, the intersection of DNMT3B
Reactivity is respectively 0.31% and 0.34%, and cross reacting rate is low, and specificity is good；It is a certain amount of by being added into serum sample
DNMT1 carries out the calculating of recovery of standard addition, and recovery of standard addition range is between 66.8% ~ 118.8%, in the exploitation of immune reagent kit
In, it is acceptable.Amplify chemiluminescence signal using BIP, improves the sensitivity of chemiluminescence immunoassay detection.
Therefore, the method for above-mentioned detection DNMT1 provided by the invention introduces magnetic nanoparticle as immune solid phase, makes
Obtain HRP-Luminol-H2O2The detection limit of-BIP enhanced chemiluminescence ELISA system detection DNMT1 reduces one again
A order of magnitude, and it is consistent with the intracorporal constant range of people that the range of linearity is limited to a lower concentration range, shows the party
Detection of the method for DNMT1 is feasible.
Finally it should be noted that: the above embodiments are merely illustrative of the technical scheme of the present invention and are not intended to be limiting thereof；To the greatest extent
The present invention is described in detail with reference to preferred embodiments for pipe, it should be understood by those ordinary skilled in the art that: still
It can modify to a specific embodiment of the invention or some technical features can be equivalently replaced；Without departing from this hair
The spirit of bright technical solution should all cover within the scope of the technical scheme claimed by the invention.
Claims (10)
1. a kind of method of the chemiluminescent enzyme-linked immunosorbent immune detection DNMT1 based on Magnetic Isolation, comprising the following steps:
Confining liquid is added in polystyrene micropore plate by closing pretreatment successively to be carried out incubating and the processing of first stage board-washing；
Add magnetic monoclonal antibody that Fe is added into the polystyrene micropore plate handled by first stage board-washing3O4@McAbDNMT1
Dilution simultaneously carries out Magneto separate and the processing of second stage board-washing, so that the polystyrene micropore plate is suspended with the first compound,
First compound has Fe3O4@McAbDNMT1Structure, and the Fe3O4@McAbDNMT1It is surface by DNMT1 mouse anti human list
The Fe of clonal antibody covalent modification3O4Nano immune magnetic bead；
Add antigen that DNMT1 standard antigen solution or to be measured is added into the polystyrene micropore plate for being suspended with first compound
Antigenic solution, and successively incubated, the processing of Magneto separate and phase III board-washing, so that the polystyrene micropore plate is suspended with
Second compound, second compound have Fe3O4@McAbDNMT1@AgDNMT1Structure；
It adds clonal antibody and rabbit anti-human polyclonal antibody is added into the polystyrene micropore plate for being suspended with second compound
PAbDNMT1Dilution, and successively incubated, the processing of Magneto separate and fourth stage board-washing, so that the polystyrene micropore plate is outstanding
Floating to have third compound, which has Fe3O4@McAbDNMT1@AgDNMT1@PAbDNMT1Structure；
ELIAS secondary antibody is added to be added ELIAS secondary antibody dilution into the polystyrene micropore plate for being suspended with the third compound, and according to
It is secondary incubated, the processing of Magneto separate and the 5th stage board-washing, and the 4th compound is suspended in the polystyrene micropore plate, should
4th compound has Fe3O4@McAbDNMT1@AgDNMT1@PAbDNMT1@ELIAS secondary antibody structure；
Add chemiluminescence bottom liquid sequentially added into the polystyrene micropore plate for being suspended with the 4th compound luminous bottom liquid A and
Hydrogenperoxide steam generator carries out chemiluminescence processing, forms chemiluminescent solution, wherein includes luminol in the luminous bottom liquid A
And xenol；
It shines and detects and establish the luminous intensity that regression equation detects the chemiluminescent solution using chemiluminescence detector, root
According to the relationship between the luminous intensity and the DNMT1 standard antigen solution or determined antigen solution concentration of detection, establish
Detect the regression equation of DNMT1.
2. the method for the chemiluminescent enzyme-linked immunosorbent immune detection DNMT1 according to claim 1 based on Magnetic Isolation, special
Sign is, the pretreated step of closing include: by concentration be 0.5% BSA/PBS confining liquid with every 250~350 μ L of hole
It is added in the polystyrene micropore plate, then is placed in 60~120 min of incubation in 37 DEG C of constant incubators, then use PBST again
Solution carries out the board-washing processing of the first stage, and pats dry.
3. the method for the chemiluminescent enzyme-linked immunosorbent immune detection DNMT1 according to claim 1 or 2 based on Magnetic Isolation,
It is characterized in that, the step of addition monoclonal antibody includes: molten with the PBS of 0.008~0.012 mmol/L pH 7~7.5
Liquid dilutes Fe with 1: 35~45 thinner ratio3O4@McAbDNMT1The Fe is made in stoste3O4@McAbDNMT1Dilution, and by institute
State Fe3O4@McAbDNMT1Dilution is added in the polystyrene micropore plate with every 100 μ L of hole, then successively carries out magnetic point
The second stage board-washing processing is carried out from processing and using PBST solution, so that suspending in the polystyrene micropore plate
State the first compound.
4. the method for the chemiluminescent enzyme-linked immunosorbent immune detection DNMT1 according to claim 3 based on Magnetic Isolation, feature
It is, the Fe3O4@McAbDNMT1The preparation method of stoste the following steps are included:
Cleaning: by carboxylated Fe3O4Nano granule suspension used after ultrasound, Magneto separate abandon supernatant processing concentration for 0.01~
The MES buffer that 0.015 mol/L, pH is 6.0 balances 2~5 min to it, and then it is heavy to obtain first for progress Magneto separate abandoning supernatant
Starch；
Activation: it is 8~11 that EDC solution that concentration is 8~10 mg/mL and concentration are sequentially added in the first sediment of Xiang Suoshu
The NHS solution of mg/mL carries out 10~15 min of reaction that are vortexed, and Magneto separate washs it using MES buffer after abandoning supernatant processing
3 times, Magneto separate is then carried out again, abandoning supernatant handles to obtain the second sediment；
Buffer system conversion: second sediment is washed 3~4 times using PBS buffer solution, Magneto separate obtains third precipitating
Object；
Coupling: being added DNMT1 monoclonal antibody in Xiang Suoshu third sediment and PBS buffer solution carries out the 1.5~2h of reaction that is vortexed,
It through Magneto separate, abandons after supernatant is handled and it is cleaned 2 times using PBS buffer solution, Magneto separate, abandoning supernatant obtain the 4th sediment；
Closing: being added BSA confining liquid in the 4th sediment of Xiang Suoshu, carries out 0.5~1 h of reaction that is vortexed, and Magneto separate abandons supernatant
After processing, using PBST buffer solution for cleaning 3~4 times, Magneto separate, abandoning supernatant obtain the 4th i.e. Fe of sediment3O4@McAbDNMT1；
It saves: in coupled product Fe3O4@McAbDNMT1Middle addition PBS buffer solution, light shake shake up, and are made the Fe3O4@
McAbDNMT1Stoste.
5. the method for the chemiluminescent enzyme-linked immunosorbent immune detection DNMT1 according to claim 1 or 4 based on Magnetic Isolation,
It is characterized in that, described the step of adding antigen includes: the buffer solution with the PBS of 0.008~0.012 mmol/L pH 7~7.5
DNMT1 antigen standard is diluted to the DNMT1 standard antigen solution of multiple and different concentration, is buffered with the PBS of identical pH value
Solution is placebo solution；The DNMT1 standard antigen solution and placebo solution are added to every 100 μ L of hole outstanding
It is floating to have in the polystyrene micropore plate of first compound, 60~120 min of incubation in 37 DEG C of constant incubators are placed in,
Then Magneto separate processing is successively carried out and carries out the phase III board-washing using PBST solution to handle, so that the polystyrene
Second compound is suspended in microwell plate.
6. the method for the chemiluminescent enzyme-linked immunosorbent immune detection DNMT1 according to claim 5 based on Magnetic Isolation, special
Sign is that described the step of adding clonal antibody includes: with the rabbit for being 1 mg/mL by concentration containing 0.5% BSA/PBS buffer solution
Anti-human polyclonal antibody PAbDNMT1Solution dilutes 1500~2500 times of obtained rabbit anti-human polyclonal antibody PAbDNMT1Dilution, so
Afterwards by the rabbit anti-human polyclonal antibody PAbDNMT1Dilution is added to every 100 μ L of hole and is suspended with second compound
In the polystyrene micropore plate, 60~120 min of incubation in 37 DEG C of constant incubators are placed in, are then successively carried out at Magneto separate
Reason and fourth stage board-washing processing is carried out using PBST solution, so that being suspended with described the in the polystyrene micropore plate
Triplex object.
7. the method for the chemiluminescent enzyme-linked immunosorbent immune detection DNMT1 according to claim 1 or 6 based on Magnetic Isolation,
It is characterized in that, described the step of adding ELIAS secondary antibody includes: with the enzyme for being 2 mg/mL by concentration containing 0.5% BSA/PBS buffer solution
It marks two corresponding anti-solution and dilutes 3500~4500 times of obtained ELIAS secondary antibody dilutions, by the ELIAS secondary antibody dilution with every 100 μ L of hole
Be added to and be adsorbed in polystyrene micropore plate described in the third compound, be placed in 37 DEG C of constant incubators incubate 60~
Then 120 min successively carry out Magneto separate processing and carry out the 5th stage board-washing using PBST solution to handle, so that described
The 4th compound is suspended in polystyrene micropore plate.
8. the method for the chemiluminescent enzyme-linked immunosorbent immune detection DNMT1 according to claim 7 based on Magnetic Isolation, special
Sign is that described the step of adding chemiluminescence bottom liquid includes: by the luminous bottom liquid and the hydrogenperoxide steam generator respectively with every
100 μ L of hole is added in the polystyrene micropore plate for being suspended with the 4th compound, the shape in the polystyrene micropore plate
At the chemiluminescent solution, wherein the luminous bottom liquid includes the luminol and 5 × 10 that concentration is 0.001 mol/L-5
The luminescence enhancer xenol of mol/L.
9. the method for the chemiluminescent enzyme-linked immunosorbent immune detection DNMT1 according to claim 8 based on Magnetic Isolation, special
Sign is, the luminous detection and the step of establishing regression equation include: first to detect the polyphenyl using chemiluminescence detector
The RLU value or RLU of the chemiluminescent solution in each hole of ethylene microwell plate0, then in antigen concentration be 0.5 ~ 128 ng/mL
In range, the equation of linear regression of detection DNMT1 is established:y=0.50143x+ 1.76886, whereinyFor RLU/RLU0,xFor
logCDNMT1, related coefficientRThe luminous intensity values of DNMT1 determined antigen, RLU are represented for 0.9907, RLU0Represent blank test survey
Fixed luminous intensity values, CDNMT1Represent DNMT1 determined antigen concentration.
10. the method for the chemiluminescent enzyme-linked immunosorbent immune detection DNMT1 according to claim 9 based on Magnetic Isolation, special
Sign is that the lowest detection of the DNMT1 determined antigen concentration is limited to 0.01 ng/mL.
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| CN112626171A (en) * | 2020-12-30 | 2021-04-09 | 郑州大学 | Method for detecting DNMT1 based on self-assembly nucleic acid probe signal amplification method |
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| CN114350633A (en) * | 2021-12-31 | 2022-04-15 | 中国农业科学院上海兽医研究所(中国动物卫生与流行病学中心上海分中心) | Antigen peptide of DNA methyltransferase 1 and polyclonal antibody thereof |
| CN114350633B (en) * | 2021-12-31 | 2024-04-26 | 中国农业科学院上海兽医研究所(中国动物卫生与流行病学中心上海分中心) | Antigen peptide of DNA methyltransferase 1 and polyclonal antibody thereof |
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