CN104535758B - A kind of coupling method that polypeptide or protein are covalently coupled on microsphere - Google Patents
A kind of coupling method that polypeptide or protein are covalently coupled on microsphere Download PDFInfo
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- CN104535758B CN104535758B CN201510006992.3A CN201510006992A CN104535758B CN 104535758 B CN104535758 B CN 104535758B CN 201510006992 A CN201510006992 A CN 201510006992A CN 104535758 B CN104535758 B CN 104535758B
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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
Abstract
The present invention relates to a kind of coupling method polypeptide or protein being covalently coupled on microsphere, it includes step: provide microsphere；Polypeptide or protein are provided；Microsphere is contacted carbodiimide and N hydroxy thiosuccinimide with polypeptide or protein simultaneously, carries out covalent coupling reaction.Utilize reagent prepared by this method, be effectively improved the oneself's polymerization in coupling process of polypeptide or protein；And have the most linear when drawing standard curve.
Description
The application is on 06 17th, 2013 invention entitled " a kind of generals of Application No. 201310236062.8 filing date
Molecule covalent containing amino is coupled to the coupling method on microsphere " the divisional application of Chinese patent application.
Technical field
The present invention relates to biochemical and Clinical Laboratory field.Specifically, the present invention relates to a kind of covalent coupling method.More
For body, relate to a kind of coupling method being coupled on microsphere by the molecule covalent containing amino.
Background technology
In the middle of traditional immunization turbidimetry, if antigen to be detected in sample or antibody concentration is the lowest or molecular weight
Too small, then antigen-antibody complex extremely difficult formation turbidity, unless placed the long time.As formed bigger complex,
Then the consumption of antigen and antibody increases the most accordingly.In view of drawbacks described above, just develop latex enhancing immune turbidimetry.
Latex enhancing immune turbidimetry utilizes biochemical method to be combined with microsphere by antibody (or antigen).When sample is treated
When the antigen (or antibody) of detection combines with the antibody (or antigen) being tagged on microsphere, just define Ag-Ab-latex
Particle composites.This improves the formation of turbidity undoubtedly, enhances reaction absorbance.Biochemistry analyzer is utilized to carry out than turbid survey
Fixed, whole analysis process only needs a few minutes, and it is higher that the method compares its sensitivity with traditional immunization turbidimetry.
During carrying out latex enhancing immune turbidimetry, need to be combined antibody (or antigen) with microsphere.In conjunction with
Mode can be divided into two big classes: physical absorption or covalent coupling.Wherein physical absorption is hydrophobic by protein molecular structure
Interaction between the hydrophobic group of group and microsphere surface, is adsorbed onto microsphere surface by antigen or antibody.E is
Reacted by the chemical group of protein with microsphere surface, by protein covalent coupling on microsphere.
E, due to features such as specificity preservation good, easy, favorable dispersibilities, has been widely used in in-vitro diagnosis
In reagent.Visible polytype microsphere on the market, the most most commonly seen is polystyrene latex particles.A lot of latex particles
Surface includes but not limited to carboxyl, amino, hydrazides, aldehyde radical, epoxy radicals, sulfydryl, hydroxyl, Metal Substrate, silicon hydroxyl with chemical group
Base.These groups, through suitable reaction, can be with biomolecule various on covalent coupling.Wherein, Carboxylated latex particles can be adopted
Activate with multiple strategy, produce and with the nucleophilic group (such as amino) in protein, the reactive middle of coupling can occur
Body.
So far, for protein and other molecule containing amino are coupled on Carboxylated latex particles modal
Response strategy is the method that water-soluble carbodiimide (EDC, 1-ethyl-(3-dimethylaminopropyl) carbodiimide) mediates.Should
Method can be carried out by both of which: one-step method；Or two-step method.
In one-step method, EDC is applied directly in protein and microsphere mixed liquor, and Carboxylated latex particles is swashed by water solublity EDC
Living and produce intermediate ester, intermediate ester can directly react with the amino on albumen.
In two-step method, first activate latex particle with EDC, further add N-hydroxy-succinamide (NHS) or N-
Hydroxy thiosuccinimide (Sulfo-NHS) generates another secondary intermediate ester (NHS-ester or Sulfo-NHS ester).With
The intermediate ester of one-step method is compared, and NHS-ester or Sulfo-NHS ester are the most stable.Therefore, the yield of two-step method is led to
Chang Genggao (Greg T.Hermanson.Bioconjugate Techniques.Academic Press, 2010).
In coupling process, owing to there are a lot of amino (such as lysine, arginine residues) on most protein simultaneously
With carboxyl (such as serine, threonine residues), between the easy mediating protein of EDC of excess, there is oneself's polymerization, two-step method energy
Enough overcoming this defect, but owing to step is various, not only increase time cost, enterprise also have to write more SOP
File (rule of operation file), have to introduce more Quality Control Links.Obviously, from the angle of the large-scale production of modernization
For degree, two-step method is not a kind of cost-effective mode.
Comparatively speaking, the step simple and fast of one-step method, it is more suitable for large-scale production.But, oneself's polymerism meeting
Relatively serious, this result in raw-material waste undoubtedly, cost increases.Further, the reagent blank value prepared is higher,
It is envisioned that this blank detection limit that will certainly damage product and sensitivity.Additionally, reagent prepared by tradition one-step method is in mapping
During standard curve the most poor, thus eventually affect the measured value of sample to be tested.Therefore, this area remains a need for a kind of improvement
One step coupling method.
Summary of the invention
When the NHS just used in only two-step method is incorporated in the middle of one-step method by inventor, it was unexpectedly found that, pass through
Reagent prepared by this method the most blank low, linearly also significantly improve.Further, Sulfo-NHS shows than NHS
More superior effect.
Therefore, according to an aspect of the present invention, it is provided that a kind of idol that the molecule covalent containing amino is coupled on microsphere
Linked method, it includes step:
1) microsphere is provided；
2) molecule containing amino is provided；
3) by uniform with the molecular mixing containing amino for microsphere, it is thus achieved that microsphere and the mixture of the molecule containing amino；
4) step 3 is made) mixture contacts carbodiimide and N-hydroxy-succinamide simultaneously, carries out covalent coupling reaction；
5) results coupling has the microsphere of the molecule containing amino,
Wherein said step 1) and step 2) order be interchangeable.
In some embodiments, described microsphere is the polystyrene latex particles of carboxyl modified.
Microsphere used by the present invention can be any suitable commercially available microsphere, such as Bangs Laboratories, Sigma,
The suppliers such as Roche, Gibco, Merck, Amresco, Invitrogen, Millipore are provided with various model, specification
The polystyrene latex particles of carboxyl modified.It will be understood by those skilled in the art that the polyphenyl second of the carboxyl modified prepared voluntarily
Alkene latex particle can be used for implementing the present invention.
In some embodiments, described microsphere diameter is 80nm to 250nm.But, those skilled in the art can manage
Solving, the enforcement of the present invention is not rely on the size of microsphere, may be used for implementing this as long as its surface is modified with carboxylic group
Invention.Microsphere supplier provides various various sizes of microsphere, latex enhancing immune than turbid detection field in the middle of, common microsphere
Size be 80nm to 250nm.In a specific embodiment, microsphere diameter used is 150 or 200nm.
In some embodiments, the described molecule containing amino is selected from polypeptide or protein.But, those skilled in the art
It is appreciated that in coupling process, produces after carboxyl microsphere is activated and with nucleophilic group (such as amino), coupling can occur
Reactive intermediate.In consideration of it, in theory, any molecule containing amino can be transferred through the present processes covalent coupling
To carboxyl microsphere.In the middle of the field of immunoturbidimetry detection, it is common practice to protein or polypeptide are coupled on microsphere,
So that detecting the testing molecule in sample whereby.Described protein or polypeptide include but not limited to antigen, antibody, enzyme, part,
Receptor.In a detailed description of the invention, by antibody coupling to microsphere.In a detailed description of the invention, by polypeptide coupling
To microsphere.
In some embodiments, step 1) can carry out according to one-step method of the prior art operation.Such as, by microsphere
It is suspended in suitable buffer and microsphere is provided, it is also possible to the method recommended according to microsphere supplier provides microsphere.Available
Buffer include, but not limited to MES buffer, PBS, HEPES buffer, MOPS buffer or above-mentioned buffering
The combination of liquid.PH of buffer can be in the range of pH 5-8.Should be appreciated that those skilled in the art can be according to latex particle
Type determines buffer type and pH scope thereof voluntarily, it would however also be possible to employ the buffer that microsphere supplier recommends.Concrete at one
Embodiment in, microsphere is suspended in the buffer of MES solution pH 6-7 and microsphere is provided.
In some embodiments, step 2) can carry out according to one-step method of the prior art operation.Such as, ammonia will be contained
The molecular melting of base is in suitable buffer.In a detailed description of the invention, antibody is dissolved in the PBS buffering of pH7.4
Liquid provides the molecule containing amino.It is to be understood, however, that the present processes is not limited to this, available buffer includes,
But it is not limited to, MES buffer, PBS, HEPES buffer, MOPS buffer or the combination of above-mentioned buffer.Buffering
Liquid pH can be in the range of pH 5-8.Should be appreciated that those skilled in the art can determine suitable according to the molecule treating coupling voluntarily
When buffer type and pH scope.
In some embodiments, make microsphere contact carbodiimide and N-hydroxyl with the mixture of the molecule containing amino simultaneously
Butanimide, carries out covalent coupling reaction.Tradition one-step method only with carbodiimide one compound as coupling agent.And biography
Unified footwork is compared, and the present processes has also been simultaneously introduced N-hydroxy-succinamide.N-hydroxy-succinamide is two
Just can use in the middle of footwork.In two-step method, carbodiimide and N-hydroxy-succinamide are to be separated from each other, and successively add to instead
Answer in system.Different from two-step method, the present processes is to make microsphere contact carbon with the mixture of the molecule containing amino simultaneously
Diimine and N-hydroxy-succinamide.
In some embodiments, carbodiimide and N-hydroxy-succinamide are formulated in same solution, add reaction
System.
In some embodiments, step 4) N-hydroxy-succinamide to could alternatively be N-hydroxy succinyl sub-
Amine.It is not limited to this theory, it is believed that N-hydroxy thiosuccinimide is born with more than N-hydroxy-succinamide
Electric charge, this contributes to giving more electrostatic repulsion forces between microsphere, thus for longer periods keeps microsphere during coupling
Stable suspension, this further improves the phenomenon of oneself's polymerization.
In some embodiments, coupling reaction can be carried out under the conditions of tradition one-step method coupling reaction；Can also be by
The reaction condition recommended according to microsphere supplier is carried out；Can also be according to treating that known to coupling molecule, coupling reaction condition is carried out.This
Skilled person can determine suitable reaction vessel, anti-according to production scale, microsphere specification, character containing amino molecule
Answer temperature, response time, whether stir and mixing speed.For some protein, need the reaction temperature at 2-8 degree Celsius
Under carry out；For some protein, can than carry out at a temperature of wider range, such as 2-40 degree Celsius.Response time can
Think 0.5-5 hour, preferably 1-4 hour, more preferably 2-3 hour.Generally improve mixing speed and contribute to the carrying out of reaction, but
Too fast mixing speed, owing to mechanical force can destroy the activity of protein.Therefore, those skilled in the art can determine voluntarily and are
No stirring and the speed of stirring.
In a specific embodiment, coupling reaction is carried out under room temperature (20-25 degree Celsius), and gentle agitation is held
Continuous 2-3 hour.
In some embodiments, the microsphere through coupling can be gathered in the crops according to prior art.Such as, it is centrifuged, filters, sinks
Form sediment.
In a specific embodiment, gather in the crops the microsphere through coupling by being centrifuged.
According to another aspect of the present invention, the present invention provides a kind of has containing amino according to the coupling prepared by said method
The microsphere of molecule.
In a specific embodiment, the invention provides a kind of coupling has the carboxyl modified of anti-Cys-C antibody
Polystyrene latex particles.
In another particular embodiment of the invention, the invention provides a kind of coupling has the carboxyl of streptolysin O to repair
The polystyrene latex particles of decorations.
According to another aspect of the present invention, the invention provides a kind of latex enhancing immune than turbid reagent, it contains
State coupling and have the microsphere of the molecule containing amino.
In a specific embodiment, the invention provides a kind of Cys-C latex enhancing immune than turbid reagent, it contains
Coupling is had to have the polystyrene latex particles of carboxyl modified of anti-Cys-C antibody.
In another particular embodiment of the invention, the latex intensified that the invention provides a kind of antistreptolysin O (ASO) is exempted from
Epidemic disease is than turbid reagent, and it contains the polystyrene latex particles that coupling has the carboxyl modified of streptolysin O.
According to a further aspect of the invention, the invention provides a kind of latex enhancing immune than turbid test kit, described examination
Agent box contains above-mentioned coupling has the microsphere of molecule containing amino or containing above-mentioned latex enhancing immune than turbid reagent.
In some embodiments, the invention provides a kind of latex enhancing immune than turbid test kit, it can be single examination
Agent form or many dosage form.
In some specific embodiments, the invention provides a kind of Cys-C latex enhancing immune than turbid test kit, its
It it is double reagent form.Described test kit contains the first reagent and the second reagent.Wherein the first reagent contains buffer, salt, poly
Thing, surfactant, can also contain stabilizer and/or preservative optionally according to needs.Wherein the second reagent contains coupling
Have polystyrene latex particles and the buffer of the carboxyl modified of anti-Cys-C antibody, optionally according to needs can also contain salt,
Polymer, surfactant, stabilizer and/or preservative.
Wherein said surfactant is nonionic surfactant.Described preservative selected from potassium sorbate, sodium benzoate,
One or more in sodium azide, sodium nitrite, PC300.Described polymer one or many in PEG series, PVP series
Kind.One or more in TWEEN series, SPAN series, TRITON series of wherein said nonionic surfactant.
According to a further aspect of the invention, the invention provides and have containing amino according to the coupling prepared by said method
The microsphere of molecule purposes in preparing diagnostic reagent.
Accompanying drawing explanation
Fig. 1: the standard curve that the Cys-C latex enhancing immune prepared by tradition one-step method is drawn than turbid reagent.
Fig. 2: the Cys-C latex enhancing immune prepared by the method (adding Sulfo-NHS) of the present invention is drawn than turbid reagent
Standard curve.
Fig. 3: the antistreptolysin O (ASO) latex enhancing immune prepared by tradition one-step method is more bent than the standard that turbid reagent is drawn
Line.
Fig. 4: the antistreptolysin O (ASO) latex enhancing immune prepared by the method for the present invention (adding NHS) is than turbid reagent
The standard curve drawn.
Detailed description of the invention
The present invention is expanded on further below in conjunction with specific embodiment to make present invention may be readily understood,.Unless otherwise,
" % " represents mass/volume.
The following provide the concrete material used in embodiment of the present invention and source thereof.It is to be understood that
These are merely exemplary, it is not intended to limit the present invention, with following reagent and the type of instrument, model, quality, character or
The same or analogous material of function may be incorporated for implementing the present invention.
Following example are tested as a example by anti-Cys-C antibody and streptolysin O, but people in the art
Member is it is understood that the molecule containing amino such as other polypeptide, protein etc. can be used to implement the present invention equally.
Term
In the context of the present invention, " polypeptide " refers to that a-amino acid is linked together by peptide chain and the compound that formed,
Usually contain and be made up of 2-50 aminoacid.
In the context of the present invention, " protein " refers to that a-amino acid is linked together by peptide chain and formed, then by one
The macromolecular compound that bar or the polypeptide chain of one or more combine according to ad hoc fashion and formed, generally by the amino of more than 50
The peptide of acid composition is referred to as protein.
In the context of the present invention, " polystyrene latex particles " refers to the polymerization being polymerized with styrene for monomer
Thing；The size of latex particle, different and different with the ratio of each material in polymerization, can synthesize according to different purposes, diameter from
Tens nanometers to several microns；Common latex particle mostly is what surface was modified, because this type of granule can be widely applied to perhaps
Multi-field (such as biochemistry, medical science, pharmacy, chromatographic column filler and Clinical Laboratory etc.), these modification groups such as carboxyl, hydroxyl
Base, amino, vinyl, azo group, hydrazides, aldehyde radical, epoxy radicals, sulfydryl, Metal Substrate, silicone hydroxyl etc..
Material and instrument
Embodiment 1: tradition one-step method coupling anti-Cys-C antibody
1) take 100 μ L 10% latex particle (150nm), add 2mL 10mM MES solution (pH6.1) and mix, 15,000rpm
Centrifugal 50min, after abandoning supernatant, adds 2mL 10mM MES solution (pH6.1) and repeatedly aspirates with aspirator and make latex particle disperse
Mixing, latex particle concentration is 0.5%.
2) take the rabbit anti-human Cys-C antibody that 4.5 μ L concentration are 22mg/mL, be diluted to 100 μ with the PBS of pH7.4
L, mixing.
3) by step 1) microspheres solution and the 40 μ L steps 2 that obtain) the anti-Cys-C antibody-solutions that obtains mixes, and is placed in magnetic
Adding rotor stirring 15min on power agitator makes both fully mix.
4) accurately weigh 0.0050g EDC and be dissolved in the EDC obtaining 5mg/mL in 1mL deionized water, take 25 μ L and be added to mixing
Latex particle and anti-Cys-C antibody mixed solution in, at room temperature, continuously stirred reaction 3h.
5) after reaction terminates, by reactant liquor 15,000rpm is centrifuged 40min, abandons supernatant, it is thus achieved that coupling has anti-Cys-C to resist
The latex particle of body.
Embodiment 2: the inventive method (using Sulfo-NHS) coupling anti-Cys-C antibody
1) take 100 μ L 10% latex particle (150nm), add 2mL 10mM MES solution (pH6.1) and mix, 15,000rpm
Centrifugal 50min, after abandoning supernatant, adds 2mL 10mM MES solution (pH6.1) and repeatedly aspirates with aspirator and make latex particle disperse
Mixing, latex particle concentration is 0.5%.
2) take the rabbit anti-human Cys-C antibody that 4.5 μ L concentration are 22mg/mL, be diluted to 100 μ with the PBS of pH7.4
L, mixing.
3) by step 1) microspheres solution and the 40 μ L steps 2 that obtain) the anti-Cys-C antibody-solutions that obtains mixes, and is placed in magnetic
Adding rotor stirring 15min on power agitator makes both fully mix.
4) take 25 μ LEDC (5mg/mL) together with 25 μ LSulfo-NHS (50mg/mL) be concurrently applied to mixing latex particle with
In anti-Cys-C antibody mixed solution, at room temperature, continuously stirred reaction 3h.
5) after reaction terminates, by reactant liquor 15,000rpm is centrifuged 40min, abandons supernatant, it is thus achieved that coupling has anti-Cys-C to resist
The latex particle of body.
The preparation of embodiment 3:Cys-C reagent
The latex particle that Example 1 (or 2) is gathered in the crops adds 2mL 0.01M PBS (pH7.4) ultrasonic disperse and is carried out；
15,000rpm is centrifuged 40min and abandons supernatant；Add 5mL 0.1M glycine buffer (containing 0.5%BSA, 0.05%NaN3, pH 8.4)
It is the latex particle of 0.2% that ultrasonic disperse obtains concentration；After magnetic agitation 30min, it is placed in 2-8 DEG C and stores until using.
Formula is as follows:
0.1M glycine buffer pH8.4；
0.5%BSA；
0.2% coupling has the microsphere of anti-Cys-C antibody；
0.05%NaN3。
The preparation of embodiment 4:Cys-C diagnostic kit
Cys-C diagnostic kit is prepared according to following formula:
First reagent R1:
0.1M glycine buffer pH8.4；
0.8%NaCl:
0.5%PEG-6000:
0.05%Tween-20；
0.05%NaN3。
Second reagent R2: Cys-C reagent embodiment 3 prepared is as the second reagent R2.
Respectively the first reagent and the second reagent are placed in suitable container, such as when for automatic clinical chemistry analyzer
Time, the first reagent and the second reagent can be placed in instrument bottle.
Embodiment 5: draw standard curve
Diagnostic reagent prepared by embodiment 4 is loaded in Toshiba-40 biochemistry analyzer, to Cys-C standard substance (0.5,1,
2,4,8mg/L) detect according to following parameter, and draw standard curve:
Detection pattern: Two point end assay；
Dominant wavelength/commplementary wave length: 546nm/700nm；
Read point: 60-62/34-36；
The Direction of Reaction: forward；
Type of calibration: Spline；
Sample size: 2.1 μ L；
R1/R2:175 μ L/35 μ L.
Embodiment 6: experimental result
1. the reagent prepared of microsphere obtained by tradition one-step method the results are shown in Table 1 and Fig. 1:
Table 1.Cys-C standard substance measured value (repeats the average of experiment for three times)
| Standard concentration | ΔA | A (main-secondary) | A (leads) |
| 0 | 0.0029 | 0.3328 | 0.6151 |
| 0.5 | 0.0256 | 0.3507 | 0.6585 |
| 1 | 0.0494 | 0.3847 | 0.7392 |
| 2 | 0.0815 | 0.4330 | 0.8655 |
| 4 | 0.1084 | 0.4857 | 1.0115 |
| 8 | 0.1169 | 0.5113 | 1.1024 |
Using said determination value Criterion curvilinear equation, linear equation and correlation coefficient be:
Y=0.0133X+0.0298, r2=0.7591
During 3 hours of embodiment 1 coupling reaction, having arrived the phenomenon of self-polymerization at later observations, granule can not
Stably it is in suspension, has precipitation visible.
2. the result of the reagent that the microsphere obtained by the inventive method (adding Sulfo-NHS) is prepared such as table 2 and Fig. 2 institute
Show:
Table 2.Cys-C standard substance measured value (repeats the average of experiment for three times)
| Standard concentration | ΔA | A (main-secondary) | A (leads) |
| 0 | -0.0033 | 0.2883 | 0.5161 |
| 0.5 | 0.0024 | 0.3008 | 0.5497 |
| 1 | 0.0086 | 0.3127 | 0.5805 |
| 2 | 0.0281 | 0.3483 | 0.6652 |
| 4 | 0.0723 | 0.4205 | 0.8519 |
| 8 | 0.1099 | 0.4963 | 1.0747 |
Using said determination value Criterion curvilinear equation, linear equation and correlation coefficient be:
Y=0.0148X-0.002, r2=0.9709
During 3 hours of embodiment 2 coupling reaction, the omnidistance phenomenon the most not observing oneself's polymerization, granule is steady
Surely suspension it is in.And the when that applicant also attempting extending to the response time such as 4 hours longer time, still
So can keep suspension, have no the gathering of latex particle.
For theoretically, preferable linear equation should be through initial point.By the most visible, as X=0, Y=
0.0298 (traditional method), Y=-0.002 (the inventive method)；As Y=0, X=-2.24 (traditional method), X=0.135
(the inventive method).This means linear equation that the method for the present invention obtained with the cross point of coordinate axes closer to former
Point.
Further, the method for the present invention has more preferable linearly dependent coefficient r2=0.9709 (and the linear phase of traditional method
Close coefficient and be only 0.7591).Skilled artisan knows that, r2The strongest closer to 1 dependency.Visible this excellent being linearly fully able to is expired
The application requirement of foot Clinical Laboratory.
Embodiment 7: tradition one-step method coupling streptolysin O
1) take 100 μ L 10% latex particle (200nm), add 2mL 10mM MES solution (pH6.1) and mix, 15,000rpm
Centrifugal 50min, after abandoning supernatant, adds 2mL 10mM MES solution (pH6.1) and repeatedly aspirates with aspirator and make latex particle disperse
Mixing, latex particle concentration is 0.5%.
2) take in the PBS that 1mg streptolysin O is dissolved in 1ml pH7.4, mixing.
3) by step 1) microspheres solution and the 40 μ L steps 2 that obtain) the streptolysin O solution that obtains mixes, is placed in
Adding rotor stirring 15min on magnetic stirring apparatus makes both fully mix.
4) take 25 μ LEDC (5mg/mL) to be added in latex particle and the streptolysin O mixed solution of mixing, in room temperature
Under, continuously stirred reaction 2h.
5), after reaction terminates, by reactant liquor 15,000rpm is centrifuged 40min, abandons supernatant, it is thus achieved that coupling has streptococcus haemolysis
The latex particle of element O.
Embodiment 8: the inventive method (using NHS) coupling streptolysin O
1) take 100 μ L 10% latex particle (200nm), add 2mL 10mM MES solution (pH6.1) and mix, 15,000rpm
Centrifugal 50min, after abandoning supernatant, adds 2mL 10mM MES solution (pH6.1) and repeatedly aspirates with aspirator and make latex particle disperse
Mixing, latex particle concentration is 0.5%.
2) take in the PBS that 1mg streptolysin O is dissolved in 1ml pH7.4, mixing.
3) by step 1) microspheres solution and the 40 μ L steps 2 that obtain) the streptolysin O solution that obtains mixes, is placed in
Adding rotor stirring 15min on magnetic stirring apparatus makes both fully mix.
4) take 25 μ L EDC (5mg/mL) and be concurrently applied to latex particle and the hammer of mixing together with 25 μ LNHS (20mg/mL)
In bacterium hemolysin O mixed solution, at room temperature, continuously stirred reaction 2h.
5), after reaction terminates, by reactant liquor 15,000rpm is centrifuged 40min, abandons supernatant, it is thus achieved that coupling has streptococcus haemolysis
The latex particle of element O.
Embodiment 9: the preparation of Streptolysin O Test
The latex particle that Example 7 (or 8) is gathered in the crops adds 2mL 0.01M PBS (pH7.4) ultrasonic disperse and is carried out；From
The heart abandons supernatant；Add 5mL 0.1M pH8.4 glycine buffer (containing 0.5%BSA, 0.1%NaN3) ultrasonic disperse acquisition concentration
It it is the latex particle of 0.2%；After magnetic agitation 30min, it is placed in 2-8 DEG C and stores until using.
Formula is as follows:
0.1M pH8.4 glycine buffer；
0.5%BSA:
0.1%NaN3；
0.2% coupling has the microsphere of streptolysin O.
Embodiment 10: the preparation of antistreptolysin O (ASO) diagnostic kit
Antistreptolysin O (ASO) diagnostic kit is prepared according to following formula:
First reagent R1:
0.1M pH8.4 glycine buffer；
300mM NaCl:
0.1%Tween 20；
1%PEG6000；
0.1%NaN3。
Second reagent R2: Streptolysin O Test embodiment 9 prepared is as the second reagent R2.Respectively by first
Reagent and the second reagent are placed in suitable container, such as when for automatic clinical chemistry analyzer, and can be by the first reagent
With second reagent place instrument bottle in.
Embodiment 11: draw standard curve
Diagnostic reagent prepared by embodiment 10 is loaded in Toshiba-40 biochemistry analyzer, to streptolysin O standard
Product (50,100,200,400,800IU/mL) detect according to following parameter, and draw standard curve:
Detection pattern: Two point end assay；
Dominant wavelength/commplementary wave length: 570nm/700nm；
Read point: 48-50/34-36
The Direction of Reaction: just
Type of calibration: Spline；
Sample size: 3 μ L；
R1/R2:240 μ L/60 μ L.
Embodiment 12: experimental result
1. the result of the reagent that the microsphere obtained by tradition one-step method (embodiment 7) is prepared, as shown in table 3 and Fig. 3:
Table 3. streptolysin O standard substance measured value (repeats the average of experiment for three times)
| Standard concentration | ΔA | A (main-secondary) | A (leads) |
| 0 | 0.1893 | 0.4990 | 0.6679 |
| 50 | 0.2375 | 0.5234 | 0.7260 |
| 100 | 0.3350 | 0.5408 | 0.8137 |
| 200 | 0.4373 | 0.5659 | 0.9813 |
| 400 | 0.5046 | 0.6180 | 1.2906 |
| 800 | 0.5918 | 0.7255 | 1.3438 |
Using said determination value Criterion curvilinear equation, linear equation and correlation coefficient be:
Y=0.0005X+0.2597, r2=0.8367
During 2 hours of embodiment 7 coupling reaction, having arrived the phenomenon of self-polymerization at later observations, granule can not
Stably it is in suspension, has precipitation visible.
2. the result of the reagent that the microsphere obtained by the inventive method (embodiment 8) is prepared is as shown in table 4 and Fig. 4:
Table 4. streptolysin O standard substance measured value (repeats the average of experiment for three times)
| Standard concentration | ΔA | A (main-secondary) | A (leads) |
| 0 | -0.0018 | 0.4156 | 0.6613 |
| 50 | 0.0751 | 0.4490 | 0.7348 |
| 100 | 0.1263 | 0.4884 | 0.8283 |
| 200 | 0.2280 | 0.5594 | 0.9958 |
| 400 | 0.4544 | 0.6961 | 1.3348 |
| 800 | 0.6047 | 0.8137 | 1.5641 |
Using said determination value Criterion curvilinear equation, linear equation and correlation coefficient be:
Y=0.0008X+0.0515, r2=0.9407
During 2 hours of embodiment 8 coupling reaction, the omnidistance phenomenon the most not observing oneself's polymerization, granule is steady
Surely suspension it is in.Applicant continues to attempt to the response time extends to such as 3-4 hour longer time, starts to
Fainter latex particle is assembled.Compare with the situation of embodiment 2, it is seen that Sulfo-NHS wants for the effect of anti-oneself's polymerization
It is better than NHS.In any case but, add NHS and still than traditional coupling method, there is more preferable anti-oneself's polymerization effect.
The method of the present invention has more preferable linearly dependent coefficient r than traditional method2=0.9407.Skilled artisan knows that, r2
The strongest closer to 1 dependency.The visible this excellent application requirement being linearly fully able to meet Clinical Laboratory.
Claims (4)
1. coupling method protein being covalently coupled on latex particle, it includes step:
1) taking the latex particle of 100 μ L 10%, add the MES solution mixing that 2mL 10mM pH value is 6.1,15,000rpm are centrifuged
50min, after abandoning supernatant, adding 2mL 10mM pH value is the MES solution of 6.1, repeatedly aspirates with aspirator and makes latex particle disperse
Mixing so that latex particle concentration is 0.5%,
2) take the rabbit anti-human Cys-C antibody that 4.5 μ L concentration are 22mg/mL, be diluted to 100 μ L with the PBS of pH7.4, mixed
It is even,
3) by step 1) latex particle solution and the 40 μ L steps 2 that obtain) the anti-Cys-C antibody-solutions that obtains mixes, and is placed in magnetic
Adding rotor stirring 15min on power agitator makes both fully mix,
4) take the 5mg/mL carbodiimide of 25 μ L and be concurrently applied to step together with the 50mg/mL N-hydroxy thiosuccinimide of 25 μ L
Rapid 3) in gained solution, at 20 to 25 DEG C of continuously stirred reaction 3h,
5) after reaction terminates, by step 4) gained reactant liquor is 15, and 000rpm is centrifuged 40min, abandons supernatant, it is thus achieved that coupling has anti-
The latex particle of Cys-C antibody；
Wherein, described latex particle is the polystyrene latex particles of carboxyl modified, and particle diameter is 150nm,
Described step 1) and step 2) order be interchangeable.
2. coupling has a latex particle for anti-Cys-C antibody, and it is by prepared by method described in claim 1.
3. latex enhancing immune is than a turbid reagent, and it contains the coupling described in claim 2 latex of anti-Cys-C antibody
Grain.
4. latex enhancing immune is than a turbid test kit, and it contains the latex enhancing immune described in claim 3 than turbid reagent.
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| CN105606822A (en) * | 2016-01-26 | 2016-05-25 | 宁波天康生物科技有限公司 | Beta 2-microglobulin detection kit |
| CN106153887A (en) * | 2016-05-27 | 2016-11-23 | 安徽伊普诺康生物技术股份有限公司 | A kind of kit measuring antistreptolysin O |
| CN111707817A (en) * | 2020-05-29 | 2020-09-25 | 吉林基蛋生物科技有限公司 | Preparation and detection method of hepatobiliary acid determination kit |
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