CN106046169A - Method for magnetic particle coupling of antibody molecules - Google Patents
Method for magnetic particle coupling of antibody molecules Download PDFInfo
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- CN106046169A CN106046169A CN201610388140.XA CN201610388140A CN106046169A CN 106046169 A CN106046169 A CN 106046169A CN 201610388140 A CN201610388140 A CN 201610388140A CN 106046169 A CN106046169 A CN 106046169A
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- magnetic particle
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K17/00—Carrier-bound or immobilised peptides; Preparation thereof
- C07K17/14—Peptides being immobilised on, or in, an inorganic carrier
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Abstract
The invention belongs to the technical field of biology, and particularly relates to a method for magnetic particle coupling of antibody molecules. The method comprises the following steps of magnetic particle balance, magnetic particle formyl modification and reduction, activation magnetic particle and antibody coupling. The detection efficiency is greatly improved, and the detection time is shortened; the method is easy to implement, the stable antibodies obtained through magnetic particle coupling can be obtained by only two steps, reaction conditions are mild, no raw materials are wasted, and large-scale preparation is easy; the production cost is saved; the great market prospect and economic value are achieved.
Description
Technical field
The invention belongs to biological technical field, a kind of method being specifically related to magnetic particle coupled antibody molecule.
Background technology
Magnetic particle have superparamagnetism, higher specific surface area, can the characteristic such as rhetorical function group.On magnetic particle surface
Coupling is carried out by chemical modification and antibody molecule, under the control of outside magnetic field, by steps such as immunoadsorption, washing, desorbings,
Target molecule can be isolated from the biological specimen of the complexity such as blood, urine, leucorrhea, recycle Enzyme-linked Immunosorbent Assay, chemistry
The technological means such as luminescence can be rapidly completed concentration or the assay of target molecule.Magnetic particle coupled antibody have separation simple,
Magnetic particle after affine absorption high specific and hypersensitivity and the process based prediction model many merits, with antibody coupling such as stably
It is mainly used in the fields such as cell sorting, biomolecule detection, microorganism separation.
Magnetic particle and the coupling of antibody molecule are mainly by active group such as amino, carboxyl, sulfydryl, the hydroxyl on surface at present
Covalent bond, the magnetic that surface amino groups is modified by the Chinese patent of Publication No. CN 101348517A is carried out Deng with protein molecule
Microgranule activates into maleimide and sulfydryl respectively with the amino on protein molecule surface, then by the two coupling, this method coupling
Efficiency is higher, but operation complexity, especially need the pre-treatment of coupling protein matter to require reagent the highest, and protein surface
The sulfydryl of activation is easily oxidized to disulfide bond makes it reunite.It is unfavorable for large-scale production and application.Publication No.
The Chinese patent of CN103357359A uses two aldehyde reagents as coupling agent, generates Schiff by aldimine condensation antibody is even
Being linked on magnetic particle, the method reaction condition is gentle, simple to operate, low to the requirement of reagent.But the imines due to conjugate
Unit is susceptible to back reaction in neutral conditions so that antibody molecule dissociates with magnetic particle, can cause antibody from coupling with
And magnetic particle from coupling, thus result in coupling efficiency reduce, waste raw material, simultaneously make detection accuracy, repeatability
It is substantially reduced.
Therefore, this method is improved, it is ensured that the high activity of antibody after the stability of its coupled product and coupling,
Guarantee the sensitivity of reagent, be one of the core technology of this platform development.
Summary of the invention
To this end, the technical problem to be solved is to overcome the technical bottleneck of prior art, thus one is proposed
The method of the active high magnetic particle coupled antibody molecule of magnetic particle that coupling efficiency is high, sensibility in practice is high, prepare.
For solving above-mentioned technical problem, a kind of method that the invention discloses magnetic particle coupled antibody molecule, described method
Order comprises the steps:
A. the balance of magnetic particle: take the dispersion liquid containing magnetic particle, Magneto separate, remove supernatant, wash with activation buffer；
B. magnetic particle is aldehyde group modified: add to, in dialdehyde solution, be simultaneously introduced selective reduction by the magnetic particle after balance
Agent solid, reaction；Magneto separate, obtains the magnetic particle that the surface aldehydes of activation is modified；
C. activation magnetic particle and the coupling of antibody: the antibody being dissolved in coupling buffer its surface free amine group and aldehyde radical
The magnetic particle of activation passes through aldimine condensation by covalent coupling on magnetic particle surface, is simultaneously introduced selective reduction agent by imine reduction
Obtain stable conjugate.
Preferably, described selective reduction agent is the one in sodium cyanoborohydride, sodium triacetoxy borohydride.
Preferably, the described dialdehyde in dialdehyde solution is the one in butanedial, glutaraldehyde, hexandial.
Preferably, described dialdehyde solution concentration is 0.5%～10%.
Preferably, the pH of described activation buffer is 7.0～8.0, for MES buffer, Tris-HCl buffer, PBS
One in buffer, Triethanolamine buffer.
Preferably, the pH of described coupling buffer is 7.0～7.5, for Tris-HCl buffer or PBS.
Preferably, described storage buffer be pH value be 7.2, for 10mM Tris-HCl, 150mM NaCl, 1mM
EDTA, 0.5%BSA, 0.05% Hydrazoic acid,sodium salt buffer in one in.
Preferably, described magnetic particle surface through amido modified, and should be stably dispersed in aqueous solution.
Preferably, described magnetic particle coupling effect detection method is enzyme linked immunological chemoluminescence method.
It is more highly preferred to, also comprises the steps: after described step c.
D. the preservation of coupling magnetic particle and effect detection: the magnetic particle after coupling is sub-packed in storage buffer preservation.
The technique scheme of the present invention has the advantage that compared to existing technology
1, the antibody magnetic particle complex of coupling of the present invention can substitute being coated for Enzyme-linked Immunosorbent Assay, chemistry of antibody
The detection of luminescence, fluorescence etc., the composite particles of synthesis stable being suspended in buffer can carry out class homogeneous reaction with antigen, greatly
Amplitude improves detection efficiency, shortens the detection time；The present invention is simple to operate, only needs two steps i.e. to obtain stable magnetic particle coupling
Antibody, reaction condition is gentle, to raw material without waste, it is easy to prepare on a large scale.
2, amido modified magnetic particle is added to higher concentration two aldehyde reagent by the present invention, can reduce magnetic largely micro-
Between Li from coupling, concurrently form imine intermediate and can be reduced to secondary amine by the specific reducing agent in reaction system, and draw
The aldehyde radical entered then is not reduced so that magnetic will be made micro-due to the back reaction of imines during follow-up magnetic particle and antibody coupling
Abscission, thus greatly reduce magnetic particle or antibody from coupling, increase substantially efficiency and the target molecule of coupling
The sensitivity of detection and accuracy.
3, the magnetic particle that the middle product that the present invention obtains-surface aldehydes is modified can preserve steadily in the long term as " raw material ",
And available one-step method and antibody, antigen, Avidin etc. carry out coupling, being a kind of general magnetic carrier, therefore it is at test kit
Production can increase substantially efficiency, save production cost.
4, the present invention does not interferes with the biologic activity of the antibody being coupled on magnetic particle: a) raw material itself will not destroy
The activity of biomolecule, the condition such as the temperature of coupling reaction, ionic strength, pH value, the degeneration of antibody will not be caused；B) antibody with
The magnetic particle long-armed and antibody molecule bridging by multiple carbon molecules, effectively avoids the space steric effect pair of solid phase carrier
The impact of antibody activity.
Detailed description of the invention
Embodiment 1 present embodiment discloses the side of a kind of magnetic particle coupled antibody molecule (rabbit anti-estriol polyclonal antibody)
Method, comprises the steps:
1. the balance of magnetic particle: take the magnetic particle colloidal sol that 100mg surface amino groups modifies and settle under the action of a magnetic field that (magnetic divides
From) 10 minutes, removing supernatant, the phosphate activation buffer that magnetic particle 20mM pH is 7.4 of sedimentation cleans 2～3 times, often
Secondary consumption 10ml.
2. magnetic particle surface aldehydes activation:
2.1. 25%～50% glutaraldehyde solution phosphate activation buffer is diluted to 5%, takes 10mL and add flat
Magnetic particle after weighing apparatus, after the suspendible that vibrates under room temperature reacts 1 hour
2.2. add 5.6mg sodium triacetoxy borohydride, continue reaction 2 hours.
2.3. Magneto separate 10 minutes, remove supernatant, and magnetic particle coupling buffer washs 3 times, each consumption 10mL.
2.4. the magnetic particle 1mL coupling buffer after activation heavily disperses latter 4 DEG C to save backup, and can preserve for a long time.
3. magnetic particle and the coupling of antibody:
3.1. antibody pretreatment: take 3mg rabbit anti-estriol polyclonal antibody and load bag filter, use 1000mL under the conditions of 4 DEG C
Antibody activation buffer was dialysed more than 12 hours.After dialysis, concentration measured by antibody ultraviolet spectrophotometry, and concentration should be greater than
1mg/ml, otherwise by concentrating adjustment concentration.
3.2. take antibody after 2mg dialysis, add the magnetic particle of the aldehyde radical activation preserved, oscillating reactions 1 hour under room temperature.
3.3. add 5.6mg sodium triacetoxy borohydride, continue reaction 3～4 hours.
3.4. Magneto separate 10 minutes, are stored in 2～8 DEG C after being carefully removed from by supernatant, detect for coupling efficiency.
3.5. the storage buffer of the magnetic particle coupled antibody after separating is washed 3 times, uses 10ml every time.The most heavily it is scattered in
In storage buffer, final concentration of 10mg/mL, 2～8 DEG C of preservations.
4. magnetic particle and antibody coupling efficiency test: dense with antibody in supernatant after the mensuration magnetic particle coupling of Bradford method
Degree, calculates uncombined antibody mass.Uncombined antibody mass is deducted again divided by antibody total amount by the antibody gross mass participating in coupling
It is antibody coupling rate.
5. magnetic particle Activity determination after antibody coupling:
5.1. enzyme-labelled antigen (the estriol antigen of alkali phosphatase enzyme mark) being diluted to concentration is 0.02 μ g/ml.
5.2. by storage buffer by magnetic particle after coupling (concentration 10mg/ml) doubling dilution, respective concentration is 10mg/
Ml, 5mg/ml, 2.5mg/ml, 1.25mg/ml etc..
5.3. the magnetic particle 30 μ l after dilution is mixed with enzyme-labelled antigen solution 30 μ l, mix rear 37 DEG C of incubations 5 minutes.
5.4. Magneto separate removes supernatant, and magnetic particle washs 3 times, uses 200ul every time.
5.5. alkali phosphatase cataluminescence substrate 3-(2-spiral diamantane (obsolete))-4-methoxyl group-4-(3-phosphorus oxygen is added
Acyl)-phenyl-1,2-dioxane disodium salt (AMPPD) 30ul, measures its luminous value, according to luminescence after 37 DEG C of incubation 5min
Value can determine that the activity of conjugate.
Experimental example
1, the magnetic particle coupling efficiency of method described in embodiment 1 and prior art are not added the coupling effect of specific reducing agent
Rate compares, and the result obtained is as shown in table 1
The comparison (magnetic particle quality is 500mg) of table 1 magnetic particle coupling efficiency
The magnetic 2, magnetic particle obtained by method described in embodiment 1 and prior art not adding specific reducing agent prepared is micro-
Grain carries out Activity determination and compares, and the result obtained is as shown in table 2.
Magnetic particle Activity determination after table 2 antibody coupling
As can be seen from the above table, the method described in embodiment 1, detection efficiency is greatly improved, shortens the detection time；With
The magnetic particle that method described in embodiment prepares, activity is high；Relatively prior art has the most progressive and practical value.
Obviously, above-described embodiment is only for clearly demonstrating example, and not restriction to embodiment.Right
For those of ordinary skill in the field, can also make on the basis of the above description other multi-form change or
Variation.Here without also cannot all of embodiment be given exhaustive.And the obvious change thus extended out or
Change among still in the protection domain of the invention.
Claims (10)
1. the method for a magnetic particle coupled antibody molecule, it is characterised in that described method order comprises the steps:
A. the balance of magnetic particle: take the dispersion liquid containing magnetic particle, Magneto separate, remove supernatant, wash with activation buffer；
B. magnetic particle is aldehyde group modified: adds in dialdehyde solution by the magnetic particle after balance, is simultaneously introduced selective reduction agent solid
Body, reaction；Magneto separate, obtains the magnetic particle that the surface aldehydes of activation is modified；
C. activation magnetic particle and the coupling of antibody: its surface free amine group of the antibody being dissolved in coupling buffer activates with aldehyde radical
Magnetic particle by aldimine condensation by covalent coupling on magnetic particle surface, be simultaneously introduced selective reduction agent and imine reduction obtained
Stable conjugate.
2. the method for magnetic particle coupled antibody molecule as claimed in claim 1, it is characterised in that described selective reduction agent
For the one in sodium cyanoborohydride, sodium triacetoxy borohydride.
3. the method for magnetic particle coupled antibody molecule as claimed in claim 2, it is characterised in that in described dialdehyde solution
Dialdehyde is the one in butanedial, glutaraldehyde, hexandial.
4. the method for magnetic particle coupled antibody molecule as claimed in claim 3, it is characterised in that described dialdehyde solution concentration
It is 0.5%～10%.
5. the method for magnetic particle coupled antibody molecule as claimed in claim 4, it is characterised in that described activation buffer
PH is 7.0～8.0, for the one in MES buffer, Tris-HCl buffer, PBS, Triethanolamine buffer.
6. the method for magnetic particle coupled antibody molecule as claimed in claim 5, it is characterised in that described coupling buffer
PH is 7.0～7.5, for Tris-HCl buffer or PBS.
7. the method for magnetic particle coupled antibody molecule as claimed in claim 6, it is characterised in that described storage buffer is
PH value is 7.2, for 10mM Tris-HCl, 150mM NaCl, 1mM EDTA, 0.5%BSA, the buffer of 0.05% Hydrazoic acid,sodium salt
In one in.
8. the method for magnetic particle coupled antibody molecule as claimed in claim 7, it is characterised in that described magnetic particle surface should
Through amido modified, and it is stably dispersed in aqueous solution.
9. the method for magnetic particle coupled antibody molecule as claimed in claim 8, it is characterised in that described magnetic particle coupling effect
Really detection method is enzyme linked immunological chemoluminescence method.
10. the method for magnetic particle coupled antibody molecule as claimed in claim 9, it is characterised in that also wrap after described step c.
Include following steps:
D. the preservation of coupling magnetic particle and effect detection: the magnetic particle after coupling is sub-packed in storage buffer preservation.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610388140.XA CN106046169A (en) | 2016-06-01 | 2016-06-01 | Method for magnetic particle coupling of antibody molecules |
| PCT/CN2017/084741 WO2017206713A1 (en) | 2016-06-01 | 2017-05-17 | Method for coupling magnetic particles with antibody molecules |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610388140.XA CN106046169A (en) | 2016-06-01 | 2016-06-01 | Method for magnetic particle coupling of antibody molecules |
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| Publication Number | Publication Date |
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| CN106046169A true CN106046169A (en) | 2016-10-26 |
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| CN201610388140.XA Pending CN106046169A (en) | 2016-06-01 | 2016-06-01 | Method for magnetic particle coupling of antibody molecules |
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| CN (1) | CN106046169A (en) |
| WO (1) | WO2017206713A1 (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2017206713A1 (en) * | 2016-06-01 | 2017-12-07 | 深圳市瀚德标检生物工程有限公司 | Method for coupling magnetic particles with antibody molecules |
| CN109061186A (en) * | 2018-08-14 | 2018-12-21 | 深圳天辰医疗科技有限公司 | A kind of Procalcitonin detection kit and Procalcitonin detection method |
| CN109725148A (en) * | 2018-12-30 | 2019-05-07 | 广东环凯微生物科技有限公司 | A kind of immunomagnetic beads preservation liquid |
| CN110133271A (en) * | 2018-02-09 | 2019-08-16 | 北京豪迈生物工程股份有限公司 | A method of antibody or its antigen-binding fragment are covalently bond to particle surface |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110632290B (en) * | 2019-09-27 | 2022-04-12 | 昆山迪安医学检验实验室有限公司 | Anti-double-chain DNA antibody detection reagent |
| CN114264810B (en) * | 2021-12-08 | 2024-03-15 | 南京诺唯赞医疗科技有限公司 | Monitoring method of antibody-coupled latex microsphere and application thereof |
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| CN102350326A (en) * | 2011-07-19 | 2012-02-15 | 武汉大学 | Preparation method of zirconium arsenate-bonded magnetic silicon spheres |
| CN103357359A (en) * | 2013-05-16 | 2013-10-23 | 英科新创(厦门)科技有限公司 | Complex immunity magnetic particle and preparation method thereof |
| CN103492420A (en) * | 2010-12-10 | 2014-01-01 | 浦项工科大学校产学协力团 | Hyaluronic acid-protein conjugate and method for preparing same |
| CN104099317A (en) * | 2014-07-18 | 2014-10-15 | 江南大学 | Method for fixing pullulanase with chitosan magnetic nanoparticles |
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| CN101348517A (en) * | 2008-06-20 | 2009-01-21 | 北京倍爱康生物技术有限公司 | Method for covalent coupling protein on amino magnetic bead surface |
| EP2677316A4 (en) * | 2011-02-15 | 2014-05-14 | Kyowa Medex Co Ltd | Streptavidin-bonded magnetic particles and manufacturing method for same |
| CN106046169A (en) * | 2016-06-01 | 2016-10-26 | 深圳市瀚德标检生物工程有限公司 | Method for magnetic particle coupling of antibody molecules |
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2016
- 2016-06-01 CN CN201610388140.XA patent/CN106046169A/en active Pending
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2017
- 2017-05-17 WO PCT/CN2017/084741 patent/WO2017206713A1/en active Application Filing
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103492420A (en) * | 2010-12-10 | 2014-01-01 | 浦项工科大学校产学协力团 | Hyaluronic acid-protein conjugate and method for preparing same |
| CN102350326A (en) * | 2011-07-19 | 2012-02-15 | 武汉大学 | Preparation method of zirconium arsenate-bonded magnetic silicon spheres |
| CN103357359A (en) * | 2013-05-16 | 2013-10-23 | 英科新创(厦门)科技有限公司 | Complex immunity magnetic particle and preparation method thereof |
| CN104099317A (en) * | 2014-07-18 | 2014-10-15 | 江南大学 | Method for fixing pullulanase with chitosan magnetic nanoparticles |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2017206713A1 (en) * | 2016-06-01 | 2017-12-07 | 深圳市瀚德标检生物工程有限公司 | Method for coupling magnetic particles with antibody molecules |
| CN110133271A (en) * | 2018-02-09 | 2019-08-16 | 北京豪迈生物工程股份有限公司 | A method of antibody or its antigen-binding fragment are covalently bond to particle surface |
| CN109061186A (en) * | 2018-08-14 | 2018-12-21 | 深圳天辰医疗科技有限公司 | A kind of Procalcitonin detection kit and Procalcitonin detection method |
| CN109725148A (en) * | 2018-12-30 | 2019-05-07 | 广东环凯微生物科技有限公司 | A kind of immunomagnetic beads preservation liquid |
| CN109725148B (en) * | 2018-12-30 | 2022-05-03 | 广东环凯微生物科技有限公司 | Immunomagnetic bead preservation solution |
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| Publication number | Publication date |
|---|---|
| WO2017206713A1 (en) | 2017-12-07 |
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Application publication date: 20161026 |