CN109946453A - A kind of coupling method of proliferating cell nuclear antigen - Google Patents
A kind of coupling method of proliferating cell nuclear antigen Download PDFInfo
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- CN109946453A CN109946453A CN201910261654.2A CN201910261654A CN109946453A CN 109946453 A CN109946453 A CN 109946453A CN 201910261654 A CN201910261654 A CN 201910261654A CN 109946453 A CN109946453 A CN 109946453A
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Abstract
The present invention relates to immune fields, in particular to a kind of coupling method of proliferating cell nuclear antigen.A kind of coupling method of proliferating cell nuclear antigen, comprising the following steps: the carboxylated microparticles of activation carry out coupling reaction with proliferating cell nuclear antigen in Acetate Solution.A kind of coupling method of proliferating cell nuclear antigen provided by the invention, it is to find to obtain by inventor, Acetate Solution is selected to be coupled, solve the problems, such as that antigen coupling efficiency is low, is coupled poor repeatability, target antigen is set steadily to be coupled on particulate carrier, convenient for being applied to the detection of target antibody.
Description
Technical field
The present invention relates to immune fields, in particular to a kind of coupling method of proliferating cell nuclear antigen.
Background technique
Proliferating cell nuclear antigen (PCNA) is the core component of eucaryote replication complex, has particularly ring-shaped three-level
Structure.One PCNA molecule is made of 3 end to end autohaploids, and it is two identical that each monomer contains space structure
Structural domain, therefore each PCNA molecule has 6 duplicate structural domains, forms a symmetrical closed circular in six sides.This is complete
PCNA tripolymer be made of different amino acid, the surfaces externally and internally of cyclic structure has different property: its inner surface by
α spiral composition rich in basic amino acid, there are two α spiral, this inside being made of helical structure at each structural domain center
Construction, but also can be in a replication process so that PCNA not only can be with DNA chain double helix closely around together on space structure
It is free to slide.And have nine antiparallel β-pleated sheets on the outside of each structural domain, its hydrophobic structure is sufficiently exposed, different function are made
Energy albumen can be combined thereon by hydrophobic effect, and most important protein binding site is between two structural domains in monomer inside
Connecting portion, that is, be located at the side PCNA coiled structure so that coupling process is more complicated.
About the coupling of antigen or antibody and magnetic particle, have some open source literatures, such as application No. is
201610980451.5 open magnetic microparticle chemiluminescence immunity detection reagent and its detection method, are related to autoimmune disease
Antigen is SS-A, ribosomes P albumen, SS-B, nRNP/Sm, Sm, kinetochore, scl-70, Jo-1, double-stranded DNA, histone, core are small
Body, 2 type of mitochondria (M2), cyclic citrullinated peptide (CCP), myeloperoxidase (MPO), protease 3 (PR3), glomerular basement membrane
(GBM), anti-1 type of liver-kidney microsomes (LKM-1), liver cell solute antigen 1 (LC-1), soluble liver antigen (SLA/LP), PM-
One of SCL, proliferating cell nuclear antigen (PCNA) and rheumatoid factor.
It discloses FITC in example 2 to connect with autoimmunity antigen, connection buffer used is 0.1-0.2mol/L
The carbonic acid buffer of pH 9.0-10.0.
Patent CN103149350A provides the coupling method of a kind of carboxylation nanometer magnetic particle and antibody, which uses 2-
Quinoline ethanesulfonic acid is primarily adapted for use in the coupling of antibody as coupling buffer, the buffer.
The proliferating cell nuclear antigen special for structure, using existing these coupling buffers such as 2-morpholine ethane sulfonic acid,
Carbonic acid buffer etc. has that antigen coupling efficiency is low and unstable.
In view of this, the present invention is specifically proposed.
Summary of the invention
The purpose of the present invention is to provide a kind of coupling method of proliferating cell nuclear antigen, specific selection Acetate Solution into
Row coupling solves the problems, such as that antigen coupling efficiency is low, is coupled poor repeatability, so that target antigen is steadily coupled at magnetic micro-
On grain carrier, in order to be applied to the detection of target antibody.
In order to realize above-mentioned purpose of the invention, the following technical scheme is adopted:
A kind of coupling method of proliferating cell nuclear antigen, comprising the following steps:
The carboxylated microparticles of activation carry out coupling reaction with proliferating cell nuclear antigen in Acetate Solution.
A kind of coupling method of proliferating cell nuclear antigen provided by the invention, is to find to obtain by inventor, selects acetic acid
Salting liquid is coupled, and solves the problems, such as that antigen coupling efficiency is low, is coupled poor repeatability, target antigen is enable to stablize
Ground is coupled on particulate carrier, convenient for being applied to the detection of target antibody.
Present invention is alternatively directed to the coupling conditions of proliferating cell nuclear antigen to have carried out all various attempting (including buffer kind
Class, concentration, volume, pH etc.), the optimal conditions of proliferating cell nuclear antigen coupling is established, and this buffer is in other albumen
Coupling process in applicability it is extremely low, therefore, coupling of the acetate buffer to proliferating cell nuclear antigen and particle such as magnetic particle
There is stronger specificity.
Specific embodiment
The present invention provides a kind of coupling method of proliferating cell nuclear antigen, comprising the following steps:
The carboxylated microparticles of activation carry out coupling reaction with proliferating cell nuclear antigen in Acetate Solution.
Since proliferating cell nuclear antigen (PCNA) is the core component of eucaryote replication complex, have particularly ring-shaped
Tertiary structure, structure is complex, and existing coupling buffer is unable to satisfy the coupling of the antigen and magnetic particle, that is, exists
Antigen coupling efficiency is low, is coupled the problem of poor repeatability.Based on this, the inventors discovered that, Acetate Solution is as coupling liquid energy
Effectively solve the above problems.
Present invention is alternatively directed to the coupling conditions of proliferating cell nuclear antigen to have carried out all various attempting (including buffer kind
Class, concentration, volume, pH etc.), the optimal conditions of proliferating cell nuclear antigen coupling is established, and this buffer is in other albumen
Coupling process in applicability it is extremely low, therefore, acetate buffer has the coupling of proliferating cell nuclear antigen and magnetic particle relatively strong
Specificity.It is specific as follows:
Further, the concentration of the acetate in the Acetate Solution is 10-100mM, and pH is 5 ± 0.2, the acetic acid
Also contain sodium chloride in salting liquid, concentration of the sodium chloride in the Acetate Solution is 0-0.3M.
Coupling buffer concentration is screened, including 10mM, 20mM, 50mM, 100mM, and wherein 20mM, 50mM are suitable.Make
To be preferred, the concentration of the acetate in the Acetate Solution is 20-50mM.
Salt ionic concentration in coupling buffer is screened, including 0M, 0.1M, 0.15M, 0.2M, 0.3M, wherein
0.15M is optimal.Preferably, concentration of the sodium chloride in the Acetate Solution is 0.1-0.2M.
Preferably, the group of the Acetate Solution is divided into acetate and acetic acid.
Acetate in the present invention is the metal cation salt of acetic acid, such as can be sodium acetate, potassium acetate.
Preferably, the acetate is sodium acetate.
In the present invention, acetate buffer is prepared to obtain by sodium acetate and acetic acid.
Coupling buffering liquid product is screened, including 50 μ L, 100 μ L, 200 μ L, 400 μ L, 800 μ L, 1600 μ L, wherein
100 μ L are optimal.Further, in the coupling reaction, the volume of the Acetate Solution is 50-200 μ L, preferably 100-
200μL。
Antigen dosage is optimized, including coating 10ng/ person-portion, 15ng/ person-portion, 20ng/ person-portion, 30ng/ person-portion,
Middle 20ng/ person-portion is optimal.
Further, the additive amount of the proliferating cell nuclear antigen is 10-30ng, preferably 20 ± 5ng.
The coupling reaction time is screened, including coupling reaction 1h, 1.5h, 2h, 2.5h, 3h, wherein 2.5h is optimal.
Further, the coupling reaction are as follows: shake 1-3h, preferably 1.5-2.5h at room temperature.
Further, the condition of the concussion is 1000 ± 200rpm.
Further, through Seal treatment, suspension after coupling reaction terminates.
Further, the confining liquid that the Seal treatment uses is bovine serum albumin solution, the bovine serum albumin(BSA)
The mass concentration of bovine serum albumin(BSA) in solution is 1% ± 0.2%.
Further, the Seal treatment is first to be washed 1-3 times using the confining liquid, and the confining liquid is then added,
Concussion processing 50-80min is carried out with 1000 ± 200rpm.
Further, it is described suspension use containing mass concentration for 1% ± 0.2% bovine serum albumin(BSA) PBST into
Row.
Further, the particle is magnetic particle, silicon particle, silver particles, golden particle or its composite particles.Composite particles are
Refer to that magnetic, silicon, silver, any two in gold above carry out compound obtained particles.
Further, the carboxylated microparticles are activated using following methods: NHS solution is added in microparticle suspending liquid and EDC is molten
Liquid processing.
It is specifically as follows, magnetic grain is resuspended in 152 μ L MES buffers (pH 6.0), and the NHS of 28 μ L Fresh is added
(50mg/mL) is vortexed and mixes, add the EDC (50mg/mL) of 20 μ L Fresh, shake 50min at room temperature
(1000rpm/min).After removing supernatant, magnetic particle is added Acetate Solution and suspends, and then carries out with proliferating cell nuclear antigen even
Connection reaction, etc..
Embodiment of the present invention is described in detail below in conjunction with embodiment, but those skilled in the art will
Understand, the following example is merely to illustrate the present invention, and is not construed as limiting the scope of the invention.It is not specified in embodiment specific
Condition person carries out according to conventional conditions or manufacturer's recommended conditions.Reagents or instruments used without specified manufacturer is
The conventional products that can be obtained by commercially available purchase.
Embodiment 1
Coupling buffer screening:
One, material and instrument
Carboxylation magnetic particle: contain carboxyl in surface.
Proliferating cell nuclear antigen: recombinant antigen, purity are greater than 90%.
Centromere protein B antigen: recombinant antigen, purity are greater than 90%.
The anti-human IgG antibodies of phycoerythrin label: anti-human IgG antibodies derive from mouse.
Serum sample: it is provided by chain hospital.
2-morpholine ethane sulfonic acid (MES), carbodiimide, n-hydroxysuccinimide, trishydroxymethylaminomethane, 2- morpholine second
Sulfonic acid, 4- hydroxyethyl piperazineethanesulfonic acid, potassium dihydrogen phosphate, disodium hydrogen phosphate, sodium carbonate, sodium bicarbonate, acetic acid, sodium acetate, boron
It is pure that sand, boric acid, Tween20, bovine serum albumin(BSA) and other reagents reach analysis.
Constant temperature blending instrument, magnetic frame, constant temperature oscillator, magnetic board, interpretoscope.
Two, the coupling method of proliferating cell nuclear antigen and magnetic particle
(1) 50000 magnetic particles (1000 person-portion) is taken, Magneto separate removes supernatant, is washed with 20mM MES buffer (pH 6.0)
It washs 2 times, then is resuspended using 152 μ L MES buffers (pH 6.0)；
(2) NHS (50mg/mL) of 28 μ L Fresh is added, is vortexed and mixes, add the EDC of 20 μ L Fresh
(50mg/mL) shakes 50min (1000rpm/min) at room temperature；
(3) it is washed 2 times using different buffers, then is resuspended with the corresponding buffer of 100 μ L；
(4) proliferating cell nuclear antigen is added by the dosage of 20ng/ person-portion, shakes 2.5h (1000rpm/min) at room temperature；
(5) it is washed 2 times using confining liquid (bovine serum albumin(BSA) containing 1%BSA), 500 μ L confining liquids is added, shake at room temperature
Swing 1h (1000rpm/min)；
(6) stand-by using suspending after PBST (bovine serum albumin(BSA) containing 1%BSA) washing.
Three, test method
(1) it takes 50uL magnetic particle (containing 50 magnetic particles) in 96 orifice plates of transparent material, 50 μ L is added and (are contained using PBST
1%BSA bovine serum albumin(BSA)) 50 times of pre-dilution of serum sample, vibrates 15 minutes under the conditions of 37 DEG C；
(2) magnetic particle is adsorbed using magnetic board, after removing reaction solution, is washed 2 times with PBST；
(3) anti-human IgG antibodies of 50 μ L phycoerythrin label are added, are vibrated 15 minutes under the conditions of 37 DEG C；
(4) magnetic particle is adsorbed using magnetic board, after removing reaction solution, is washed 2 times with PBST；
(5) it is suspended using PBST and interpretoscope is used to read fluorescence signal value.
Four, test result
The testing result of the different buffers of table 1
The detection repeatability of the different buffers of table 2
Test result shows the magnetic particle being coupled in 20mM acetate buffer (pH5.0) for weak positive, positive, strong
Positive sample discrimination with higher (relative to negative sample, fluorescence signal value is higher), and the buffer is directed to weak sun
Property sample test repeatability (coefficient of variation) within 5%, be much better than other buffers, thus this kind buffering coupling effect is most
It is good.
Conclusion: with 2-morpholine ethane sulfonic acid (pH5.0), 4- hydroxyethyl piperazineethanesulfonic acid (pH8.0), phosphate (pH7.4), boron
Hydrochlorate (pH8.0), carbonate (pH9.6) are compared, and acetate buffer (pH5.0) coupling effect is obviously optimal.
Embodiment 2
The screening of coupling buffer concentration
Acetate buffer is as coupling buffer, and the specific steps are the same as those in embodiment 1, unlike, coupling method (3)
Coupling buffer concentration in step uses 10mM, 20mM, 50mM or 100mM.Testing result is as shown in table 3.
3 reactivity of table is investigated
It is higher that test result shows that 20mM or 50mM acetate (pH5.0) has weak sun, positive, strong positive sample
Discrimination (relative to negative sample, fluorescence signal value is higher), thus the buffer coupling effect under the concentration conditions is most
It is good.
Conclusion: the coupling effect of 20mM or 50mM acetate (pH5.0) is relatively excellent.
Embodiment 3
The screening of coupling buffer volume
Acetate buffer is as coupling buffer, and the specific steps are the same as those in embodiment 1, unlike, coupling method (3) step
In coupling buffer stereomutation be 50 μ L, 100 μ L, 200 μ L, 400 μ L, 800 μ L, 1600 μ L.Testing result such as 4 institute of table
Show.
4 reactivity of table is investigated
When test result shows that coupling buffer volume is 100 μ L, reactivity is optimal, and coupling buffer volume is after of continuing rising
Gao Hou, reactivity decline.
Conclusion: coupling buffer volume preferably 100 μ L.
Embodiment 4
The screening of coupling reaction time
Acetate buffer is as coupling buffer, and the specific steps are the same as those in embodiment 1, unlike, coupling method (4) step
In duration of oscillation be changed to 1h, 1.5h, 2h, 2.5h or 3h.Testing result is as shown in table 5.
5 reactivity of table is investigated
When test result shows that duration of oscillation is 2.5h, reactivity is more excellent, when preferably 2.5h is as coupling reaction
Between.
Conclusion: coupling reaction time preferred 2.5h.
Embodiment 5
The screening of salt ionic concentration
Acetate buffer is as coupling buffer, and the specific steps are the same as those in embodiment 1, unlike, coupling method (3)
It walks, the concentration for changing NaCl in acetate buffer solution is 0.1M, 0.15M, 0.2M or 0.3M.Testing result is as shown in table 6.
6 reactivity of table is investigated
When test result shows that NaCl concentration is 0.15M in coupling buffer, reactivity is optimal.
Conclusion: the preferred 0.15M of NaCl concentration in acetate buffer.
Embodiment 6
The screening of antigen dosage
Acetate buffer is as coupling buffer, and the specific steps are the same as those in embodiment 1, unlike, coupling method (4) step
The dosage for changing proliferating cell nuclear antigen is 10ng/ person-portion, 15ng/ person-portion, 20ng/ person-portion or 30ng/ person-portion.Testing result is such as
Shown in table 7.
7 reactivity of table is investigated
Test result shows that the dosage of proliferating cell nuclear antigen is 20ng/ person-portion, and reactivity is optimal.
Conclusion: the preferred 20ng/ person-portion of the dosage of proliferating cell nuclear antigen.
Embodiment 7
One, material and instrument, with embodiment 1.
Two, the coupling method of proliferating cell nuclear antigen and magnetic particle
(1) 50000 magnetic particles (1000 person-portion) is taken, Magneto separate removes supernatant, is washed with 20mM MES buffer (pH 6.0)
It washs 2 times, then is resuspended using 152 μ L MES buffers (pH 6.0)；
(2) NHS (50mg/mL) of 28 μ L Fresh is added, is vortexed and mixes, add the EDC of 20 μ L Fresh
(50mg/mL) shakes 50min (1000rpm/min) at room temperature；
(3) using 20mM acetate (pH 5.0, NaCl containing 0.15M) washing 2 times, then with 100 μ L 20mM acetate (pH
5.0) it is resuspended；
(4) proliferating cell nuclear antigen is added by the dosage of 20ng/ person-portion, shakes 2.5h (1000rpm/min) at room temperature；
(5) it is washed 2 times using confining liquid (bovine serum albumin(BSA) containing 1%BSA), 500 μ L confining liquids is added, shake at room temperature
Swing 1h (1000rpm/min)；
(6) stand-by using suspending after PBST (bovine serum albumin(BSA) containing 1%BSA) washing.
Three, test method, with embodiment 1.
Four, test result
(1) reactivity is investigated as shown in table 8.
8 reactivity of table is investigated
The background signal that sample by testing 4 parts of different titers can be seen that negative sample is very low, and weakly positive sample has
Stronger fluorescence signal, positive, strong positive sample fluorescence signal are in increasing trend.
(2) precision is investigated as shown in table 9.
9 precision of table is investigated
To same a test sample 10 times, for the coefficient of variation within 5%, test repeatability is more excellent.
Conclusion: the fluorescence signal value of various concentration sample can be significantly distinguished using the magnetic particle that this formula is coupled, and is surveyed
Test mass renaturation is more excellent, shows that this formula coupling effect is preferable.
Embodiment 8
Centromere protein B antigen is coupled using acetate buffer
For step with embodiment 1, the coupling buffer in coupling method (3) step uses acetate buffer or 2- morpholine second
Sulfonic acid, the antigen in (4) step are changed to Centromere protein B by proliferating cell nuclear antigen.
10 reactivity of table is investigated
Test result shows that Centromere protein B antigen is poor using the reactivity after 20mM acetate (pH5.0) coupling,
And use the reactivity after 20mM 2-morpholine ethane sulfonic acid (pH5.0) coupling more excellent, thus it is speculated that synantigen is not in different buffers
Conformation has differences, and specific buffer can not be general.
Conclusion: acetate buffer is suitable for the coupling of proliferating cell nuclear antigen and magnetic particle, but is not necessarily suitble to other
The coupling of antigen.
Although illustrate and describing the present invention with specific embodiment, it will be appreciated that without departing substantially from of the invention
Many other change and modification can be made in the case where spirit and scope.It is, therefore, intended that in the following claims
Including belonging to all such changes and modifications in the scope of the invention.
Claims (10)
1. a kind of coupling method of proliferating cell nuclear antigen, which is characterized in that the carboxylated microparticles of activation in Acetate Solution with
Proliferating cell nuclear antigen carries out coupling reaction.
2. coupling method according to claim 1, which is characterized in that the concentration of the acetate in the Acetate Solution is
10-100mM, pH are 5 ± 0.2, also contain sodium chloride in the Acetate Solution, the sodium chloride is in the Acetate Solution
Concentration be 0-0.3M；
Preferably, the concentration of the acetate in the Acetate Solution is 20-50mM；
Preferably, concentration of the sodium chloride in the Acetate Solution is 0.1-0.2M；
Preferably, the group of the Acetate Solution is divided into acetate and acetic acid；
Preferably, the acetate is sodium acetate.
3. coupling method according to claim 2, which is characterized in that in the coupling reaction, the Acetate Solution
Volume is 50-200 μ L, preferably 100-200 μ L.
4. coupling method according to claim 3, which is characterized in that the additive amount of the proliferating cell nuclear antigen is 10-
30ng, preferably 20 ± 5ng.
5. coupling method according to claim 1, which is characterized in that the coupling reaction are as follows: shake at room temperature
1-3h, preferably 1.5-2.5h.
6. coupling method according to claim 5, which is characterized in that the condition of the concussion is 1000 ± 200rpm.
7. coupling method according to claim 1, which is characterized in that coupling reaction terminate after through Seal treatment, suspension is
It can；
Further, the confining liquid that the Seal treatment uses is bovine serum albumin solution, the bovine serum albumin solution
In bovine serum albumin(BSA) mass concentration be 1% ± 0.2%.
8. coupling method according to claim 7, which is characterized in that the Seal treatment is first to be washed using the confining liquid
It washs 1-3 times, the confining liquid is then added, concussion processing 50-80min is carried out with 1000 ± 200rpm.
9. coupling method according to claim 7, which is characterized in that the suspension uses containing mass concentration as 1% ±
The PBST of 0.2% bovine serum albumin(BSA) is carried out.
10. -9 described in any item coupling methods according to claim 1, which is characterized in that the particle is magnetic particle, silicon is micro-
Grain, silver particles, golden particle or its composite particles；
Further, the carboxylated microparticles are activated using following methods: being added at NHS solution and EDC solution in microparticle suspending liquid
Reason.
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| CN110596374A (en) * | 2019-09-19 | 2019-12-20 | 潍坊市康华生物技术有限公司 | Method for coupling magnetic particles and antigen |
| CN111272999A (en) * | 2020-02-17 | 2020-06-12 | 珠海丽珠试剂股份有限公司 | Antigen-coupled magnetic particle, preparation method thereof, detection method of anti-U1-snRNP antibody and kit |
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Application publication date: 20190628 |